estudios termodinámicos de los procesos de vitrificación en la ...

UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR

DE INGENIEROS AGRÓNOMOS

ESTUDIOS TERMODINÁMICOS DE LOS

PROCESOS DE VITRIFICACIÓN EN LA

CRIOCONSERVACIÓN DE GERMOPLASMA

VEGETAL

- Tesis doctoral -

ALINE SCHNEIDER TEIXEIRA

Licenciada en Biología

2013

DEPARTAMENTO DE BIOLOGÍA VEGETAL

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS

ESTUDIOS TERMODINÁMICOS DE LOS PROCESOS DE

VITRIFICACIÓN EN LA CRIOCONSERVACIÓN DE

GERMOPLASMA VEGETAL

THERMODYNAMIC STUDIES OF VITRIFICATION

PROCESSES IN PLANT GERMPLASM

CRYOPRESERVATION

- Tesis doctoral –

ALINE SCHNEIDER TEIXEIRA

Licenciada en Biología

Directores:

María Elena González Benito (Doctora Ingeniero Agrónomo)

Antonio Diego Molina García (Doctor en Ciencias Químicas)

2013

La doctora María Elena González Benito, Catedrática de Universidad del Departamento de

Biología Vegetal (Universidad Politécnica de Madrid), y el doctor Antonio Diego Molina

García, Científico Titular del ICTAN-CSIC.

CERTIFICAN QUE la presente Tesis Doctoral titulada “Estudios termodinámicos de los

procesos de vitrificación en la crioconservación de germoplasma vegetal/ Thermodynamic

studies of vitrification processes in plant germplasm cryopreservation”, ha sido realizada bajo

su dirección por la licenciada en Biología Dña. Aline Schneider Teixeira.

Fdo María Elena González Benito Fdo. Antonio Diego Molina García

Caminante, no hay camino

Caminante son tus huellas El camino nada más;

caminante no hay camino se hace camino al andar. Al andar se hace camino y al volver la vista atrás

se ve la senda que nunca se ha de volver a pisar.

Caminante, no hay camino sino estelas sobre el mar.

¿Para qué llamar caminos A los surcos del azar...?

Antonio Machado

AGRADECIMIENTOS

Gracias a la Universidad Politécnica de Madrid y al Programa de Doctorado en

Biotecnología y Recursos Genéticos de Plantas y Microorganismos Asociados, y al CSIC por

la beca JAE-Pre y al proyecto �“CRYODYMINT�” (AGL2010-21989-C02-02) del Ministerio

de Ciencia e Innovación del Gobierno de España.

Gracias a los directores de esta tesis, el Dr. Antonio Diego Molina García, del Instituto de

Ciencia y Tecnología de los Alimentos y Nutrición (ICTAN-CSIC), y la Dra. María Elena

González Benito, del Dpto. de Biología Vegetal de la Universidad Politécnica de Madrid

(UPM), por su paciencia, visión crítica y espíritu didáctico. Mi más sincero agradecimiento

por toda su ayuda y por las oportunidades que me han brindado en el ámbito de la

investigación.

El doctorado ha supuesto una gran aventura para mí en muchos aspectos como sumergirme

en el fascinante mundo de la CRIOCONSERVACIÓN, donde además de aprender, conocí y

compartí con gente fantástica a la que no puedo dejar de agradecer.

Una de ellas es el �“Profesor Antonio�”, por los muchos momentos compartidos en el

instituto, por las largas tardes de mate y charlas. Siempre disponible para ordenar una idea en

un dibujo. Y a su esposa Lola, quien siempre encuentra tiempo para hacer se presente, por la

amistad y por varios momentos alegres.

Gracias al personal del ICTAN, de la USTA: Rubén Domínguez, Inmaculada Álvarez y

Estela Vega, por la cobertura técnica en los análisis de las muestras. A los técnicos de

informática: Luis Canet y Roberto Arcoya por la disponibilidad y amabilidad y al personal de

la administración Nuria Serrenes, Víctor Martín y Buenaventura Rodríguez. A Óscar García

Bodelón por las charlas y los cafés. A las investigadoras del CIB y del ICTAN, Dras. Begoña

de Ancos, María Escribano, Maite Serra, Pilar Rupérez, Sara Pérez, Sylvia Rodríguez, Miguel

Ángel Peñalva y José Luis García por la agradable compañía a la hora de comer. Al personal

de seguridad, por estar siempre atentos durante mi permanencia en el despacho por las

noches.

Mi amiga, Mirari Arancibia compañera de instituto, de investigación, de piso, de viajes,

paseos y de muchos findes de películas. Gracias por ser �“mis dos manos más�”.

Mi técnico querido Fernando Pinto, que trabaja en el Instituto de Ciencias Agrarias del

CSIC, en el Servicio de Microscopía Electrónica, que siempre aceptó los desafíos de mis

muestras, siempre dispuesto a ayudarme. Gracias por la agradable convivencia.

Mi más sincero agradecimiento a todos los profesores y a los técnicos que trabajan en el

laboratorio de cultivo in vitro de la E.U.I.T. Agrícola de la UPM, Carlos Ruiz y Marta

Huertas.

Mientras estudiaba en la Universidad Politécnica de Madrid, he tenido el privilegio de

contar con las enseñanzas y la amistad de varios compañeros con los que he compartido tan

buenos momentos. Entre ellos, quiero destacar a Carolina Kremer y Natacha Coelho (por ser

un soplo de aire fresco en el laboratorio); Shanez Zai, Narcizo Mesa, Cesar Tapia y James

Quiroz por el ánimo fuera de la Escuela; Julia Quintana, Juan Fernández y Manuel Rodelo mi

gran familia española; Mónica García la ultima incorporación a la familia pero no menos

querida y Alina Gheorghe que de compañera de clase pasó a compañera de instituto y amiga.

Amigos que fueron fundamentales en el trayecto, Fábia Andrade, Bartolomeu de Souza,

Danniely Campos Ferreira, Augusto Campos Ferreira, Paula Buarque, Melyssa Negri, Nadia

Silva, Bruna Catarina Fonseca gracias: por apoyarme en todo lo que me propuse y por hacer

me ver que lo imposible solo tarda un poco más.

Amigos a que no les importo la distancia y que estuvieron siempre presentes: Roberta

Casanova, Roberta Gomes, Fabiana Rotta, Fernando Lang, Catalina Guzmán, Pinar Singur,

Sophie Lalanne, Pachi Marino, Popi Coppola, Lucia Klein, Joaquin Hasperué, João Vieira,

Anai Loreiro, Simone Barrionuevo, Maria do Carmo Ruaro Peralba, Ady García, Osmar

Conte, Regilene Souza, Renata Azevedo, Giovani Brandão Mafra de Carvalho, Catarina

Fardilha, Susi Poersch, Margarete Cegolini, Cristiane Dalla Vecchia, Giselle Baccan, David

Duran, Ademir Coser, Erika Cavalcante y Rodrigo Morais de Souza.

Gracias al equipo del CIDCA de La Plata, en Argentina, dónde hice mi primera estancia:

mis amigos, las Dras. Miriam Martino, Lorena Deladino, Alba Navarro, Estela Bruno, Natalia

Graiver y Noemí Zaritzky y el M.Sc. Alex Fernando, por su apoyo, optimismo e incentivo.

Al equipo del �“USDA-ARS�”, de Fort Collins, en EEUU donde fue recibida por los Drs.

Christina Walters y Daniel Ballesteros, por los M.Sc. Jennifer Crane, Fernanda Pierruzi y

Patrick Reeves y por Lisa Hill y John Waddell, por su amabilidad y confianza, por creer en mí

y apoyarme en la realización del proyecto. Me llevé la sonrisa de cada uno de ellos, como

señal del buen ambiente y amistad.

Al equipo del �“Crop Research Institute�”, de Praga, en la Republica Checa, donde fui

recibida por los Drs. Milos Faltus, Jiri Záme ník, Renata Kotková, Jane Záme ník y Alois

Bilav ík, por la oportunidad, por la experiencia inolvidable y por las charlas de la cultura

checa, y a la Dra. Helena Stavelikova que me permitió hacer un recorrido de bicicleta por los

campos de cultivo del instituto VÚRV-Olomouc.

Gracias al personal del �“National Centre for Macromolecular Hydrodynamics, School of

Biosciences, University of Nottingham�”, de Gran-Bretaña, Gary Adams and Fahad Almutairi

por recibir mis muestras y amablemente ayudar en la construcción de ese trabajo.

Finalmente, agradezco de todo corazón a mi familia, a la que amo y extraño muchísimo:

mis hermanos (Denise y Fábio), mi cuñada (Valquiria), mi adorable ahijado (Otavio), mis

tíos, primos; pero fundamentalmente a mis padres (Glaci y Diamarante), por su gran amor y

por hacerme sentir que están tan cerca, gracias a todos porque sin sus constantes muestras de

apoyo y cariño nada de esto hubiera sido posible.

INDICE/INDEX

INDICE/INDEX

Página/Page

1.1. INTRODUCCIÓN/INTRODUCTION............................................................................ 1

1. 1.Crioconservación ........................................................................................................... 1

1.2. Técnicas de crioconservación basadas en la vitrificación ............................................ 2

1.2.1. Técnicas de vitrificación (en sentido estricto)...................................................... 3

1.2.2. Técnicas de vitrificación-�“droplet�” ...................................................................... 4

1.2.3. Técnicas de encapsulación-deshidratación........................................................... 4

1.2.4. Técnicas de encapsulación-vitrificación .............................................................. 5

1.3. Aspectos físicos de la crioconservación ....................................................................... 5

1.3.1. Formación de cristales de hielo ............................................................................ 5

1.3.2. La transición vítrea ............................................................................................... 7

1.3.3. La velocidad de cambio de temperatura en crioconservación............................ 10

1.3.4. Soluciones crioprotectoras.................................................................................. 11

1.3.5. Encapsulación en alginato .................................................................................. 12

1.3.6. Quitosano............................................................................................................ 13

1.4. La menta como modelo en el estudio de la crioconservación .................................... 14

2 OBJETIVOS/AIMS............................................................................................................. 16

3. MEASUREMENT OF COOLING AND WARMING RATES IN VITRIFICATION-

BASED PLANT CRYOPRESERVATION PROTOCOLS................................................ 17

3.1. Introduction ................................................................................................................ 17

3.2. Materials and Methods ............................................................................................... 19

3.2.1. Plant materials pre-culture and shoot tips extraction.......................................... 19

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INDICE/INDEX

3.2.2. Dehydration of shoot tips in the vitrification method ........................................ 19

3.2.3. Vitrification......................................................................................................... 20

3.2.4. Droplet-vitrification............................................................................................ 20

3.2.5. Plant recovery and viability................................................................................ 20

3.2.6. Temperature measurement and cooling and warming rates ............................... 21

3.2.7. Thermocouples ................................................................................................... 21

3.2.8. Experimental set-up and temperature change rate measurement design ............ 22

3.3. Results and Discussion............................................................................................... 23

4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS .............. 33

4.1. Introduction ................................................................................................................ 33


4.2.1. Plant material pre-culture and shoot tips extraction ........................................... 36

4.2.2. Incubation and dehydration of shoot tips ........................................................... 36

4.2.3. Plant recovery and viability................................................................................ 37

4.2.4. Low temperature scanning electron microscopy ................................................ 38

4.2.5. Dry matter and water content determination ...................................................... 39

4.2.6. Differential scanning calorimetry ....................................................................... 39

4.2.7. Sucrose content assessment in shoot tips............................................................ 41


4.3.1. Detection of glassy state and ice by cryo-SEM.................................................. 42

4.3.2. Determination of pre-culture effecte by cryo-SEM............................................ 43

4.3.3. Observed plant recovery and viability................................................................ 43

4.3.4. Analysis of the droplet-vitrification protocol steps by cryo-SEM...................... 45

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INDICE/INDEX

4.3.5. Changes in the dry mass and water content during the droplet-vitrification

protocol......................................................................................................................... 45

4.3.6. Measurement of ice heat of fusion using DSC ................................................... 47

4.3.7. Effects of quench-cooling on heat of fusion of ice............................................. 50

4.3.8. Detection of glass transition by DSC.................................................................. 51

4.3.9. Changes in sucrose content during the droplet-vitrification protocol ................ 51

4.4. General discussion...................................................................................................... 53

5. CRYOPRESERVATION OF MINT SHOOT TIPS BY ENCAPSULATION-

DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-

SEM AND DSC................................................................................................................ 57

5.1. Introduction ................................................................................................................ 57


5.2.1. Plant material and shoot tips extraction.............................................................. 59

5.2.2. Shoot tips cryopreservation procedure ............................................................... 59

5.2.3. Water content determination............................................................................... 60

5.2.4. Differential scanning calorimetry ....................................................................... 61

5.2.5. Temperature measurement and cooling and warming rates ............................... 62



5.3.1. Water content reduction during the drying period.............................................. 64

5.3.2. Calorimetric studies on water status ................................................................... 66

5.3.3. Cooling and warming rate determination ........................................................... 68


vi

5.3.5. Cryopreserved shoot tips survival and re-growth............................................... 74

INDICE/INDEX

5.4. General discussion...................................................................................................... 74

6. GLASS TRANSITION AND ANNEALING BEHAVIOUR OF PLAN

VITRIFICATION SOLUTIONS ................................................................................... 77

6.1. Introduction ................................................................................................................ 77


6.2.1. Standard compounds .......................................................................................... 79

6.2.2. Solutions ............................................................................................................. 79

6.2.3. Plant material...................................................................................................... 80

6.2.4. Differential scanning calorimetry conditions ..................................................... 80

6.2.5. Glass transition characterization for PVS........................................................... 81

6.2.6. Cooling rate test .................................................................................................. 81

6.2.7. Warming rate test................................................................................................ 81

6.2.8. Storage test ......................................................................................................... 82

6.2.9. Annealing test ..................................................................................................... 82

6.2.10. Statistical analysis ............................................................................................ 83


6.3.1. Vitrification behaviour of different vitrification solutions ................................. 83

6.3.2. Effect of cooling rate on vitrification behaviour ................................................ 85

6.3.3. Effect of warming rate on vitrification behaviour .............................................. 89

6.3.4. Crystallinity of garlic shoot tips ......................................................................... 89

6.3.5. Effect of the storage time on the vitrification behaviour of PVS ....................... 91

6.3.6. Annealing of PVS1 and PVS3............................................................................ 93

7. THE APPLICATION OF CHITOSAN FILM ON ALGINATE BEADS FOR

CRYOPRESERVATION USES .................................................................................... 95

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INDICE/INDEX

7.1. Introduction ................................................................................................................ 95


7.2.1. Chitosan types and hydrodynamic characterization ........................................... 99

7.2.2. Alginate beads .................................................................................................. 100

7.2.3. Chitosan solution and alginate bead treatment ................................................. 100

7.2.4. water content determination and air dehydration studies ................................. 100

7.2.5. Differential scanning calorimetry and frozen and unfrozen water fractions .... 101

7.2.6. Low temperature scanning electron microscopy observations......................... 101

7.2.7. Environmental scanning electron microscopy.................................................. 102

7.3. Results and Discussion............................................................................................. 102

7.3.1. Chitosan hydrodynamic characterization ......................................................... 102

7.3.2. Water content determination and air dehydration ............................................ 105

7.3.3. Calorimetric studies on water status................................................................. 106

7.3.4. Scanning electron microscopy of alginate beads with and without chitosan ... 107

8. DISCUSIÓN GENERAL/GENERAL DISCUSSION................................................... 110

9. CONCLUSIONES/CONCLUSIONS.............................................................................. 122

10. REFERENCIAS/REFERENCES ................................................................................. 124

11. ANEXOS/ANNEXES ..................................................................................................... 154

Annex I. COMPOSITION OF VITRIFICATION SOLUTIONS ................................... 153

Annex II. PUBLICATIONS............................................................................................ 155

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LISTA DE FIGURAS/LIST OF FIGURES

Página/Page

Figura 1.1. Ilustración sobre la transición vítrea en comparación con una fusión/cristalización de una sustancia pura no polimérica. Se denota el estado de equilibrio mediante un trazo continuo y el de no equilibrio por trazos discontinuos. (a) Volumen específico, b) viscosidad aparente, (c) capacidad calorífica (primeras derivadas), representando la dirección hacia arriba una transición endotérmica. (Figura tomada de Walstra, 2003).................................................9

Figura 1.2. Ilustración sobre la transición vítrea de una solución de vitrificación (PVS3) en comparación con una fusión/cristalización de ápices de ajo. Se denota el estado de equilibrio mediante un trazo continuo y el de no equilibrio por trazos discontinuos .................................9

Figura 1.3. Ilustración sobre la transición vítrea en comparación con la fusión en un termograma de DSC (A) y el acercamiento de las temperaturas, por aumento de TG y disminución de Tf (B) ...............................................................................................................10

Figure 3.1. Experimental set up for thermal change rate measurement for (a) vitrification, or (b), droplet-vitrification protocols ...........................................................................................22

Figure 3.2. Cooling (a) and warming (b) evolution determined in samples treated after the vitrification protocol. Data are the average of at least three independent determinations. Control data were obtained with the thermocouple tip inserted in an empty cryovial. The lines shown represent the individual data points measured .............................................................24

Figure 3.3 a and b. Measurements of cooling rate obtained following the droplet-vitrification protocol: (a) AFS inside cryovial and (b) naked AFS. Data are the average of at least three repeats ......................................................................................................................................25

Figure 3.4 a and b. Measurements of warming rates obtained following the droplet protocols. Data are the average of at least three repeats............................................................................26

Figure 3.5. Mint shoot tip survival and re-growth percentages after different cryopreservation techniques and a 4-week recovery period. Means of survival or re-growth with the same letter are not significantly different according to the Duncan�’s Multiple Range Test at alpha = 0.05. Bars: standard error .................................................................................................................27

Figure 4.1. Scheme of the steps of the droplet cryopreservation protocol employed, see text for more details on media and solutions employed .................................................................37

Figure 4.2. Cartoon showing the three mint tips on the microscopy holder before insertion in the microscope (a) and after equatorial freeze fracture (b) ... ..................................................38

Figure 4.3. Cryo-SEM micrographs of shoot tips cooled in LN in the microscope cryo-unit (see Methods). Control (A); stage a, cold treatment (B); stage b, preculture (C and D); stage c, loading (E and F), and stage d, dehydration (G and H). The bar corresponds to 10 µm (A, B, D, F, H) or 50 µm (C, E, G) ...............................................................................................44

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INDICE/INDEX

Figure 4.4. Typical DSC thermograms corresponding to re-warming processes of shoot tips in different stages of the droplet cryopreservation protocol: cold treatment (stage a), preculture (stage b), loading (stage c) and dehydration (stage d). Each experiment was performed with five shoot tips. The insert shows an expanded thermogram section which allows the glass transition, obtained with 30 tips, to be appreciated. Scanning rate was 10ºC min-1. (See Materials and Methods and Figure 4.1. for more details). The ordinates scale of the thermograms baseline is arbitrar ........................................................................................48

Figure 4.5. Typical DSC thermograms corresponding to re-warming processes of the cryopreservation solutions employed in different starges of the droplet cryopreservation protocol: preculture (stage b): liquid MS medium containing 0.3M sucrose, loading (stage c): loading solution and dehydration (stage d): PVS2. The insert shows an expanded thermogram section which allows the glass transition to be appreciated. Scanning rate was 10ºC min-1. (See Materials and Methods and Figure 14 for more details). The ordinates scale of the thermograms baseline is arbitrary ............................................................................................49

Figure 4.6. DSC scans showing warming at 10ºC min-1 for shoot tips treated at the preculture stage (b) of the droplet cryopreservation protocol. Cooling rate was 10ºC min-1 or quenching (direct immersion of the DSC pan in LN) ...............................................................................50

Figura 5.1. Scheme of the steps of the encapsulation-dehydration protocol employed, see text for more details on media and solutions used ..........................................................................60

Figura 5.2. Experimental set up for thermal change rate measurement for encapsulation-dehydration ..............................................................................................................................63

Figura 5.3. Cartoon showing the three mint tips-containing beads on the microscopy holder before insertion in the microscope (a) and after equatorial freeze fracture (b) .......................64

Figura 5.4. Typical DSC thermograms corresponding to re-warming processes of shoot tips with beads of the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar-flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h and T6 = 6 h. Data are the mean of at least three repeats. Each experiment was performed with three beads. The inserts show an expanded thermogram section which allows seeing the glass transition. Scanning rate was 10ºC m-1. The ordinates scale of the thermograms baseline is arbitrary ........................................................................66

Figure 5.5. Ratio Wf(dm) Wu(dm)-1 for mint shoot tips (control and preculture) and shoot tip-

containing beads (for drying times 0 to 6 hours). Bars: standard error ...................................67

Figure 5.6. Temperature evolution, during cooling by immersion in LN, of cryovials with 10 alginate beads containing mint shoot tip and treated after the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. Data are the mean of at least three repeats .................70

Figure 5.7. Temperature evolution, during warming in a 45ºC water bath for 1 min and then in 25ºC for 1 min of cryovials with 10 alginate beads containing mint shoot tip and treated after the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3

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INDICE/INDEX

h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. Data are the mean of at least three repeats ....................................................................................................................70

Figura 5.8. Cryo-SEM micrographs of alginate beads. View of beads external surface at drying time 0 (A) and 6 hours (B), and internal cryofracture surface, at drying time 0 (C) and 6 hours (D). The bar corresponds to 10 µm .............................................................................71

Figura 5.9. Cryo-SEM micrographs of mint shoot tips in different stages of the encapsulation-dehydration protocol. Drying times (hours): A (0), B (1), C (2), D (3), E (4), F (5), G (6). H, tip growing for one week, after warming. The bar corresponds to 10 µm ........73

Figura 5.10. Mint shoot tip survival and re-growth percentages after dehydration for different times following the encapsulation-dehydration protocol. Observations were made after a 4-week recovery period. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = before cooling in LN. Data are the mean of at least three repeats. Means of survival or re-growth with the same letter are not significantly different according to the Duncan�’s Multiple Range Test at alpha = 0.05. Bars: standard error ...............................75

Figura 6.1. Scheme of the steps of the annealing protocol used; see text for more details on solutions employed ..... .............................................................................................................83

Figure 6.2. PVS3 thermograms showing the glass transition, performed at a warming rate of 10ºC min-1 and after cooling at different rates. Quenching: direct immersion of the DSC pan in LN; cryovial: pan included in a cryovial and then in LN. White arrows mark the glass transition inflection point ........................................................................................................86

Figure 6.3. Glass transition temperature (a) and glass transition heat capacity changes (b) for the different PVS and garlic shoot tips (tips in the last step of the droplet-vitrification method), obtained in warming (10ºC min-1) DSC experiments, after being cooled at different rates. * Rate not tested for garlic samples. Bars: standard error .............................................88

Figure 6.4. Glass transition temperature (a and b) and heat capacity (c and d) obtained in DSC during warming at different rates (5, 10 or 20°C min-1). DSC pans had been cooled either at 10°C min-1 in the calorimeter or by quenching in LN. Bars: standard error ..... ........90

Figura 6.5. Typical scan of shoot tips of garlic after treatment with PVS3 for 2 h. Both cooling and warming rates were 10ºC min-1. The black arrows mark to the glass transition inflection point in cooling and warming scans, while gray arrows signal the freezing and melting events...........................................................................................................................91

Figure 6.6. Typical thermogram of PVS1 on the step E-F of Figure 6.1. The annealing areas were comprised between the curve and the extended baseline ...............................................93

Figura 6.7. Annealing area versus temperature (D in Figure 6.1) of the solutions PVS1( ) and PVS3(�•) .............................................................................................................................94

Figure 7.1. Schematic representation of the chemical structures of the chitin and chitosan (adapted from Goy et al., 2009) ..............................................................................................96

Figure 7.2. Plots for extrapolation of the sedimentation coefficient of the different chitosan types to infinite dilution: a = MMW, b = LMW and c = C chitosan .....................................103

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INDICE/INDEX

Figure 7.3. Evolution of the water content over total sample mass, Wc(s), with air flow drying time, for alginate beads covered with chitosan or not, prepared with 2 and 3% sodium alginate concentration .........................................................................................................................105

Figure 7.4. Cyro-SEM micrographs showing the external AB (right) and ACB (left) bead surface after quench cooling to liquid nitrogen temperature. The bar corresponds to 5 µm .....................................................................................................................................................107

Figure 7.5. Environmental SEM micrographs showing bead surfaces after different air-flow dehydration times: a) AB and c) ACB (0 hours -not dehydrated beads); b) AB, d) and e) ACB (5 hours dehydration). The bar corresponds to 50 m for a) and e) and to 100 m for b), c) and d) .....................................................................................................................................108

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LISTA DE TABLAS/LIST OF TABLES

Página/Page

Table 3.1. Cooling and re-warming rate measurements obtained following the vitrification protocols ..................................................................................................................................30

Table 3.2. Cooling and re-warming rate measurements obtained following the droplet-vitrification protocols ..............................................................................................................31

Table 4.1. Compositional and thermal parameters (mean standard deviation) of the thawing events obtained from DSC thermograms for mint shoot tips specimens at different stages (a, b, c and d) of the droplet cryopreservation protocol ...............................................................46

Table 4.2. Solute content (mean standard deviation) of mint shoot tips at the different stages of the droplet cryopreservation protocol.... ..............................................................................53

Table 5.1. Compositional and thermal parameters of the thawing events obtained from gravimetry and DSC thermograms for mint shoot tips specimens at different stages of the encapsulation-dehydration cryopreservation protocol .............................................................64

Table 5.2. Cooling and warming rate measurements obtained following the encapsulation-dehydration protocol ................................................................................................................68

Table 6.1. Composition of vitrification solutions ...................................................................80

Table 6.2. Glass transition parameters of different plant vitrification solutions (cooling and warming rates were both 10°C min-1) .....................................................................................84

Table 6.3. Crystallization parameters (mean standard deviation) of garlic shoot tips in the last stage of the droplet-vitrification protocol (in PVS3). Onset of the melting endotherm and crystallinity as crystallization percentage, both measured in the warming scan. Cooling scans were performed at 10ºC min-1 and warming scans at different rates .......................................91

Table 6.4. Cryopreservation solutions glass transition temperature (mean standard deviation) versus storage time .................................................................................................92

Table 6.5. Cryopreservation solutions heat capacity change (mean standard deviation) versus storage time ..................................................................................................................92

Table 7.1. Analytical ultracentrifugation-derived and chitosan size parameters (mean ± standard deviation) ................................................................................................................103

Table 7.2. Water and dry matter content (mean ± standard deviation) for alginate beads (AB) and chitosan-covered alginate beads (ACB). See Materials and Methods for parameter description .............................................................................................................................105

Table 7.3. Calorimetric parameters (mean ± standard deviation) obtained for 3% alginate beads (AB) and chitosan-covered alginate beads (ACB). See Materials and Methods for parameter description ............................................................................................................106

xiii

RESUMEN/ABSTRACT

RESUMEN

En la actualidad, las técnicas de crioconservación poseen una importancia creciente para el

almacenamiento a largo plazo de germoplasma vegetal. En las dos últimas décadas, estos

métodos experimentaron un gran desarrollo y se han elaborado protocolos adecuados a

diferentes sistemas vegetales, utilizando diversas estrategias como la vitrificación, la

encapsulación-desecación con cuentas de alginato y el método de “droplet”-vitrificación. La

presente tesis doctoral tiene como objetivo aumentar el conocimiento sobre los procesos

implicados en los distintos pasos de un protocolo de crioconservación, en relación con el estado

del agua presente en los tejidos y sus cambios, abordado mediante diversas técnicas biofísicas,

principalmente calorimetría diferencial de barrido (DSC) y microscopía electrónica de barrido a

baja temperatura (crio-SEM). En un primer estudio sobre estos métodos de crioconservación, se

describen las fases de enfriamiento hasta la temperatura del nitrógeno líquido y de

calentamiento hasta temperatura ambiente, al final del periodo de almacenamiento, que son

críticas para la supervivencia del material crioconservado. Tanto enfriamiento como

calentamiento deben ser realizados lo más rápidamente posible pues, aunque los bajos

contenidos en agua logrados en etapas previas de los protocolos reducen significativamente las

probabilidades de formación de hielo, éstas no son del todo nulas. En ese contexto, se analiza

también la influencia de las velocidades de enfriamiento y calentamiento de las soluciones de

crioconservación de plantas en sus parámetros termofísicos referente a la vitrificación, en

relación su composición y concentración de compuestos. Estas soluciones son empleadas en la

mayor parte de los protocolos actualmente utilizados para la crioconservación de material

vegetal. Además, se estudia la influencia de otros factores que pueden determinar la estabilidad

del material vitrificado, tales como en envejecimiento del vidrio. Se ha llevado a cabo una

investigación experimental en el empleo del crio-SEM como una herramienta para visualizar el

estado vítreo de las células y tejidos sometidos a los procesos de crioconservación. Se ha

comparado con la más conocida técnica de calorimetría diferencial de barrido, obteniéndose

resultados muy concordantes y complementarios. Se exploró también por estas técnicas el

efecto sobre tejidos vegetales de la adaptación a bajas temperaturas y de la deshidratación

inducida por los diferentes tratamientos utilizados en los protocolos. Este estudio permite

observar la evolución biofísica de los sistemas en el proceso de crioconservación. Por último,

se estudió la aplicación de películas de quitosano en las cuentas de alginato utilizadas en el

protocolo de encapsulación. No se observaron cambios significativos en su comportamiento

RESUMEN/ABSTRACT

frente a la deshidratación, en sus parámetros calorimétricos y en la superficie de las cuentas. Su

aplicación puede conferir propiedades adicionales prometedoras.

RESUMEN/ABSTRACT

ABSTRACT

Currently, cryopreservation techniques have a growing importance for long term plant

germplasm storage. These methods have undergone great progress during the last two decades,

and adequate protocols for different plant systems have been developed, making use of diverse

strategies, such as vitrification, encapsulation-dehydration with alginate beads and the droplet-

vitrification method. This PhD thesis has the goal of increasing the knowledge on the processes

underlying the different steps of cryopreservation protocols, in relation with the state of water

on tissues and its changes, approached through diverse biophysical techniques, especially

differential scanning calorimetry (DSC) and low-temperature scanning electron microscopy

(cryo-SEM). The processes of cooling to liquid nitrogen temperature and warming to room

temperature, at the end of the storage period, critical for the survival of the cryopreserved

material, are described in a first study on these cryopreservation methods. Both cooling and

warming must be carried out as quickly as possible because, although the low water content

achieved during previous protocol steps significantly reduces ice formation probability, it does

not completely disappear. Within this context, the influence of plant vitrification solutions

cooling and warming rate on their vitrification related thermophysical parameters is also

analyzed, in relation to its composition and component concentration. These solutions are used

in most of the currently employed plant material cryopreservation protocols. Additionally, the

influence of other factors determining the stability of vitrified material is studied, such as glass

aging. An experimental research work has been carried out on the use of cryo-SEM as a tool for

visualizing the glassy state in cells and tissues, submitted to cryopreservation processes. It has

been compared with the better known differential scanning calorimetry technique, and results

in good agreement and complementary have been obtained. The effect on plant tissues of

adaptation to low temperature and of the dehydration induced by the different treatments used

in the protocols was explored also by these techniques. This study allows observation of the

system biophysical evolution in the cryopreservation process. Lastly, the potential use of an

additional chitosan film over the alginate beads used in encapsulation protocols was examined.

No significant changes could be observed in its dehydration and calorimetric behavior, as well

as in its surface aspect; its application for conferring additional properties to gel beads is

promising.

INDICE/INDEX

ACRÓNIMOS Y ABREVIATURAS

AB: �“alginate beads�”, cuentas de alginato

ABC: �“chitosan-treated alginate beads�”, cuentas de alginato recubiertas de quitosano

AFS: tira de papel alumínio

Aw: peso molecular del agua

aw: actividad del agua

Cpi: capacidad calorífica a presión constante del hielo

Cpw: capacidad calorífica a presión constante del agua

cryo-SEM: microscopía electrónica de barrido de baja temperatura

DA: grado de acetilación

dm: contenido de materia seca

DMSO: dimetil sulfóxido

DSC: calorimetría diferencial de barrido

HPLC: cromatografía de líquidos de alto rendimiento

IBPGR: �“International Board for Plant Genetic Resources�”

IC: cromatografía iónica de alto rendimiento

LMW chitosan: tipo comercial de quitosano, de bajo peso molecular

LN: liquid nitrogen (nitrógeno líquido)

M: peso molecular

MMW chitosan: tipo comercial de quitosano, de peso molecular medio

MS: medio Murashige y Skoog (1962)

NaCleq: concentración molar de cloruro sódico equivalente

NL: nitrógeno líquido

PAD: detección amperométrica por pulsos

PVS: soluciones de vitrificación de plantas

xiv

INDICE/INDEX

PVS1: �“Plant vitrification solution nº 1�”, solución de vitrificación de plantas nº 1 (Uragami et al., 1989)

PVS2: �“Plant vitrification solution nº 2�”, solución de vitrificación de plantas nº 2 (Sakai et al., 1990)

PVS2 mod: solución de vitrificación de plantas nº 2, modificada

PVS3: �“Plant vitrification solution nº 3�”, solución de vitrificación de plantas nº 3 (Nishizawa et al., 1993).

PVS3 mod: solución de vitrificación de plantas nº 3, modificada.

So20,W: coeficiente de sedimentación a dilución infinita, en agua y a 20ºC

Suc: contenido de sacarosa

Suc(dm): concentración de sacarosa respecto a la masa seca de la muestra.

Suceq: concentración molar de sacarosa equivalente

Suc(s): concentración de sacarosa respecto a la masa total de muestra

Tf: temperatura o punto de congelación (o fusión) de equilibrio

Tf*: temperatura de fusión de equilibrio del agua pura

Tf(onset): temperatura de inicio (onset) de la fusión de hielo detectada mediante DSC

Tf(peak): temperatura del pico de la fusión de hielo detectada mediante DSC

TG: Temperatura de transición vítrea

Wc: contenido de agua

Wc(dm): contenido de agua con respecto a la masa seca de muestra

Wc(s): contenido de agua con respecto a la masa total de muestra

Wf: contenido de agua congelada

Wf(dm): contenido de agua congelada respecto a la masa seca de muestra

Wf(s): contenido de agua congelada respecto a la masa total de muestra

Wf(w): contenido de agua congelada respecto al contenido total de agua

Wu: contenido de agua no congelada

Wu(dm): contenido de agua no congelada respecto a la masa seca de muestra

Wu(s): contenido de agua no congelada respecto a la masa total de muestra

xv

INDICE/INDEX

Wu(w): contenido de agua no congelada respecto al contenido total de agua

Hf: calor latente de cambio de fase o entalpía de fusión

Hf(dm): entalpía de cambio de fase respecto a la masa de materia seca

Hf(s): entalpía de cambio de fase respecto a la masa de muestra total

Hf(w)*: entalpía específica de fusión del agua pura

Hf(w): entalpía de cambio de fase respecto al contenido de agua

T: gradiente térmico

Tf: depresión de la temperatura de fusión

s: factor pre-exponencial para el cálculo del peso molecular a partir del coeficiente de sedimentación

: osmolalidad

: Densidad

xvi

1. INTRODUCCIÓN/INTRODUCTION

1. INTRODUCCIÓN

1.1. Crioconservación

Las técnicas de crioconservación, ampliamente empleadas en la actualidad para la

conservación de muy diversos tipos de materiales biológicos, tienen su origen en

investigaciones iniciales llevadas a cabo con células y tejidos animales. Gracias al

descubrimiento de las cualidades del glicerol y del dimetilsulfóxido (DMSO; Mounib et al.,

1968; Graybill & Horton, 1969) capaces de disminuir los efectos negativos que podría

ocasionar la inmersión de tejido vivo en nitrógeno líquido, se obtuvieron los primeros éxitos

significativos en la crioconservación y recuperación de espermatozoides y células sanguíneas.

Más adelante, la técnica se aplicó a estructuras vegetales, tales como semillas y tejidos

preparados para soportar situaciones de estrés (baja temperatura), a consecuencia de presentar

un bajo contenido hídrico y acumular substancias de reserva y compuestos con capacidad

crioprotectora, como aminoácidos (glicina, betaína y prolina) y azúcares, principalmente,

manitol (Reed, 1988). De entre los distintos sistemas organizados, semillas y embriones se

consideran los más adecuados, siempre que sea posible, para la conservación de la diversidad

de los recursos fitogenéticos (Henshaw et al., 1980). Mediante el empleo de las técnicas

desarrolladas específicamente para diversos tejidos y especies, los materiales crioconservados

pueden recuperar satisfactoriamente su funcionalidad tras el almacenamiento (Benson et al.,

1996; Sakai & Engelmann, 2007).

La mayoría de las células vegetales contienen elevadas cantidades de agua por lo que son

extremamente sensibles a las temperaturas por debajo de 0ºC. Para impedir la formación de

cristales de hielo, evitando daños a las membranas y a otros elementos celulares, se recurre a

inducir procesos de deshidratación y aumento de la concentración intracelular de solutos y

adicionar sustancias de actividad crioprotectora. Además, los cambios de temperatura

(enfriamiento y calentamiento) tienen un papel determinante en la formación de hielo y en su

localización y efecto fisiológico. Estas dos etapas de los protocolos de crioconservación,

deshidratación y cambios térmicos, deben ser optimizadas.

La formación de hielo intracelular se considera generalmente letal, si bien el mecanismo

del daño por congelación (freeze injury) resulta controvertido en la literatura (Benson, 2008).

1


El sitio primario de la lesión por congelación en sistemas biológicos parece ser la membrana

celular (Steponkus, 1992). Estudios morfológicos apoyan esa idea demostrando la estrecha

relación entre los cambios en la ultraestructura de la membrana plasmática y las tensiones de

enfriamiento y calentamiento (Singh, 1979; Fujikawa, 1980, 1981; Pearce, 1988). La

formación de hielo extracelular, que puede tener lugar incluso tras una exposición prolongada

de las células a soluciones concentradas o su deshidratación, si el enfriamiento es

especialmente lento, también puede provocar daños a la membrana y otros componentes

celulares (Mazur, 1970).

Según el �“International Board for Plant Genetic Resources�”, IBPGR (1982), la

crioconservación es una tecnología indicada para el mantenimiento de especies con

propagación vegetativa, plantas con semillas recalcitrantes, o especies amenazadas de

extinción. Es considerado un método eficiente, práctico y de bajo coste para la preservación

germoplasma vegetal, y adicionalmente, posee la capacidad de mantener el material viable

por un tiempo considerado indefinido (Touchell & Dixon, 1994).

1.2. Técnicas de crioconservación basadas en la vitrificación

La conservación de material biológico a baja temperatura aprovecha la reducción de

velocidades de difusión y reacciones asociadas al descenso térmico para evitar cambios y

deterioro del material, pero al descender por debajo de la temperatura de equilibrio de

congelación, la formación de cristales de hielo suele constituir un importante riesgo para la

integridad celular. Las técnicas de crioconservación se basan en la estabilización del sistema,

por la práctica anulación de los procesos metabólicos que tiene lugar por debajo de la

transición vítrea. Así, los sistemas vitrificados, mediante diferentes metodologías, serían

básicamente estables frente al tiempo, pues los procesos basados en movilidad molecular no

podrían tener lugar por debajo de esta transición. Estos procesos incluyen la formación de

hielo misma, que requiere de una masiva reorganización de la estructura del agua líquida, y

por tanto, estaría completamente impedida en este estado. Una vez alcanzada la temperatura

final, de nitrógeno líquido, la formación de hielo no es posible. Sin embargo, existen dos

momentos fundamentales a considerar para evitar daños en el material que se desea

crioconservar: el riesgo de formación de cristales de hielo al inicio (enfriamiento) y al final

(calentamiento) del proceso.

2


Las técnicas de crioconservación pueden dividirse, según la velocidad de enfriamiento

empleada, en métodos clásicos, donde se logra una deshidratación inducida por el descenso

controlado de la temperatura (mediada por la formación de hielo extracelular), y métodos más

novedosos, donde se produce una vitrificación global de las soluciones contenidas en los

tejidos, es decir, tanto de la soluciones intra- como extracelulares (Benson, 2008).

El método clásico se basa en el descenso lento de la temperatura. En este caso, el agua del

medio intracelular es extraída hacia el medio extracelular, donde se forman preferentemente

los cristales de hielo, por tener una concentración de solutos menor, por lo que las soluciones

del citoplasma se concentran, facilitando su entrada en el estado vítreo. El resultado es una

región extracelular con hielo y crecientes cantidades de agua, y un citoplasma cada vez más

concentrado y viscoso, libre de hielo y sin daños celulares.

Se suele operar en dos etapas. Tras una etapa de enfriamiento lento (por ejemplo, a una

velocidad de 0,5-2,0ºC min-1, hasta -40ºC), el espécimen se sumerge en nitrógeno líquido

(LN) (-195,9ºC), lo que constituye un enfriamiento mucho más rápido. Se pueden usar

congeladores programables que realizan estas etapas a velocidad de enfriamiento controlada

(González-Benito et al., 2004).

Los nuevos métodos se basan en la vitrificación de todas las soluciones del sistema

mediante un descenso rápido de la temperatura obtenido por la inmersión directa del material

en nitrógeno líquido. Las soluciones intra- y extracelulares, cuyo contenido en agua y solutos

ha sido previamente modulado, pasan directamente al estado vítreo y el enfriamiento ocurre

sin formación alguna de hielo ni daño para las células. Estas técnicas se dividen en:

vitrificación en sentido estricto, vitrificación-droplet, encapsulación-deshidratación,

encapsulación-vitrificación.

1.2.1. Técnicas de vitrificación (en sentido estricto)

Como se ha indicado, la vitrificación conlleva la práctica solidificación del líquido, sin

cristalización de hielo, sino mediante una elevación extrema de la viscosidad durante el

enfriamiento. Para alcanzarlo, los sistemas biológicos deben ser sometidos a diversos

procesos previos. Los tejidos son primeramente cultivados en medios con altas

concentraciones de sacarosa u otros agentes osmóticos, con la finalidad de reducir su

contenido en agua e inducir la síntesis de sustancias de defensas naturales frente a estrés.

3


Posteriormente se transfieren a una solución glicerol-sacarosa, llamada solución de carga

(Towill, 1990; Sakai et al., 2000). A continuación se reduce el contenido en agua de las

soluciones intra y extracelulares, exponiendo los tejidos a soluciones crioprotectoras

altamente concentradas (solución de vitrificación); posteriormente el espécimen (en el interior

de un criovial) se sumerge rápidamente en nitrógeno líquido. Tras el calentamiento (rápido),

la solución de vitrificación se retira del criovial y se añade una solución de, generalmente,

1,2M sacarosa y se sustituye una vez con solución fresca. Por último el material vegetal se

transfiere al medio de cultivo.

La reducción del contenido acuoso y la disminución de la movilidad molecular

conseguidos mediante estos tratamientos permiten que, mediante un proceso de enfriamiento

lo suficientemente rápido (inmersión directa en nitrógeno líquido) las soluciones vitrifiquen.

1.2.2. Técnicas de vitrificación-droplet

La técnica de vitrificación-droplet puede considerarse una modificación de la anterior, si

bien deriva de la técnica de congelación de gotas desarrollada por Kartha et al. (1982). En

este protocolo, los sistemas vegetales son tratados con una solución de vitrificación. Gotas de

esta solución conteniendo el material a crioconservar son dispuestas sobre pequeñas tiras de

papel de aluminio y el conjunto es rápidamente sumergido en nitrógeno líquido. En el

posterior proceso de recuperación, el calentamiento se realiza sumergiendo el soporte de papel

de aluminio en medio líquido, a temperatura ambiente.

La ventaja principal de ésta técnica es la posibilidad de alcanzar una velocidad muy alta de

enfriamiento y calentamiento, debido al volumen reducido de la solución de vitrificación

donde están sumergidas las estructuras vegetales y a la escasa masa del conjunto.

1.2.3. Técnicas de encapsulación-deshidratación

La encapsulación-deshidratación, desarrollada por Fabre & Dereuddre (1990), consiste en

la inclusión de estructuras vegetales en cuentas de alginato, seguida de su cultivo en

soluciones de sacarosa altamente concentradas y, posteriormente, de una etapa de

deshidratación física, que elimina parte del agua congelable a 0ºC, e inmersión directa en NL.

4


La deshidratación física se lleva a cabo empleando gel de sílice o el flujo de aire de una

campana de flujo laminar (Paulet et al., 1993).

La técnica de vitrificación permite el procesado de los sistemas biológicos para su

conservación en protocolos relativamente rápidos. Bajo condiciones óptimas, produce

mayores niveles de recuperación y reduce enormemente el tiempo necesario para la

deshidratación de muestras (Sakai et al., 1990). Sin embargo, los principales puntos débiles de

esta técnica son la dificultad de tratar un gran número de especímenes a la vez y la duración

de los pasos del protocolo, muy reducida y necesariamente exacta, lo que dificulta la

manipulación de pequeños explantes.

Por el contrario, los protocolos de encapsulación-deshidratación suelen ser más

prolongados comparados con los de vitrificación. Sin embargo, los explantes encapsulados

son más fáciles de manipular, gracias al tamaño relativamente grande del recubrimiento de

alginato. Desventajas de ésta técnica son, en general, su menor porcentaje de supervivencia y

de recuperación, y el mayor grado de deshidratación que ocasiona en las estructuras vegetales

(Matsumoto & Sakai, 1995) comparada con la técnica de vitrificación.

1.2.4. Técnicas de encapsulación-vitrificación

La encapsulación-vitrificación consiste en una combinación de las ventajas de la técnica de

encapsulación-deshidratación y vitrificación. Se reunieron en un mismo protocolo la facilidad

de manipulación de los explantes encapsulados y el tratamiento de las muestras mediante

sustancias crioprotectoras (rapidez de ejecución), seguido por la deshidratación con

soluciones de vitrificación, previamente a la etapa de enfriamiento (Sakai & Engelmann,

2007). En el posterior proceso de recuperación, el calentamiento se realiza sumergiendo el

criovial en un baño de agua a temperatura controlada.

1.3. Aspectos físicos de la crioconservación

1.3.1. Formación de cristales de hielo

La formación de cristales de hielo, es decir, la cristalización del agua, se produce en tres

etapas, denominadas nucleación, propagación y maduración. Así, cada cristal parte de una

5


estructura microscópica inicial o núcleo, que se forma por acción de los movimientos

Brownianos, al azar, de las moléculas de agua. El cristal se desarrolla creciendo por adición

de más moléculas de agua al núcleo original. A tiempos más prolongados, los cristales ya

formados se reorganizan y maduran, creciendo los mayores a costa de los más pequeños. Por

lo tanto, la formación de hielo es un fenómeno que depende fuertemente de la movilidad

molecular y el tiempo, y está directamente relacionado con la temperatura y el contenido de

agua (Roos & Karel, 1991). Otro parámetro importante para la formación de cristales de hielo

es la viscosidad de la solución, muy directamente relacionada con la movilidad a escala

molecular. Mayores concentraciones de solutos y menores contenidos de agua suelen estar

asociados a altas viscosidades y movilidades reducidas, que ralentizan tanto la nucleación del

hielo como el crecimiento posterior de sus cristales.

La nucleación de hielo ocurre siempre por debajo de la llamada temperatura de equilibrio

de congelación, o de fusión (Tf, también del inglés freezing), que caracteriza al cambio de fase

termodinámico entre el agua líquida y sólida. Sin embargo, debido a la necesidad de

formación de núcleos estables, en la práctica, la formación de hielo no suele ocurrir justo por

debajo de Tf, sino frecuentemente bastantes grados por debajo. La probabilidad de formación

de hielo está controlada de manera compleja por la temperatura, la movilidad molecular (a su

vez dependiente de la temperatura) el tamaño del sistema y el tiempo, siendo un fenómeno

básicamente estocástico, controlado cinética y no termodinámicamente (Walstra, 2002). La

temperatura de congelación depende directamente del contenido en solutos, siendo el

descenso de Tf proporcional al de la concentración (Levine & Slade, 1992).

La extensión de los daños causados por el crecimiento de cristales de hielo depende de su

localización y del tamaño del cristal. Las perturbaciones observadas en las células están

frecuentemente relacionadas con el estado de las membranas celulares, que pueden sufrir

diversos daños funcionales, incluyendo la pérdida de contenido celular, evidenciado durante

el proceso de calentamiento en la recuperación. A pesar de la imagen intuitiva del cristal de

hielo rompiendo la membrana, por presión mecánica, en su crecimiento, los daños derivados

de la formación intracelular de hielo suelen estar asociados a la deshidratación de membranas

y sistemas enzimáticos, que por otra parte, son también responsables del daño observado en

otros contextos similares, tales como en alimentos congelados e incluso en plantas cultivadas

sometidas a bajas temperaturas pero por encima del punto de congelación (Levitt, 1980;

Steponkus & Webb, 1992; Thomashow, 1998; Salinas, 2002).

6


Así, para conseguir que la formación de hielo sea más improbable durante un enfriamiento

rápido, se deben inducir cambios en las propiedades físico-químicas celulares, reduciendo el

punto de congelación del sistema y la probabilidad de formación de hielo, por medio de la

deshidratación, aumento de la microviscosidad citoplasmática y reducción la movilidad

molecular (Blanshard & Franks, 1987; Angell 2000; Wesley-Smith et al., 2001), lo que

favorece la entrada en estado vítreo.

1.3.2. La transición vítrea

Los líquidos, a temperaturas suficientemente bajas, pierden movilidad molecular,

traslacional y rotacional, y se convierten en sólidos amorfos, sin orden cristalino, duros y

frágiles, siendo denominados vidrios (Sperling, 1986). Con el aumento de la temperatura, la

estructura del material pierde rigidez, permitiendo así que exista alguna movilidad molecular.

Este proceso puede ser descrito como una transición entre el estado vítreo y el estado de goma

o líquido, y se produce a una temperatura conocida como temperatura de transición vítrea (TG,

del inglés glass transition; Levine & Slade, 1992; Angell, 2000).

A pesar de que la movilidad está muy reducida en el estado vítreo, alguna lenta

reorganización puede tener lugar en el ámbito del agua, los gases disueltos y algunas

pequeñas moléculas (Ubbink & Krüger, 2006). La transición entre el estado vítreo y el líquido

no debe ser considerada como una transición de fase termodinámica propiamente, ya que las

temperaturas de transición vítrea no son precisamente constantes y el proceso tiene lugar, en

realidad, en un intervalo de temperatura (Walstra, 2002). Los solutos y el contenido en agua

tienen gran influencia sobre el valor de TG de los sistemas acuosos. Distintas sustancias

poseen efectos especialmente intensos sobre esta transición. Estudios realizados con azúcares

muestran que existe una relación entre el peso molecular de los mismos y su correspondiente

temperatura de transición vítrea (Roos & Karel, 1991; Levine & Slade, 1992).

Los cambios más importantes que ocurren en la transición vítrea están relacionados con el

volumen específico, la viscosidad aparente y la capacidad calorífica. Un ejemplo de estas tres

propiedades, tanto en relación con la temperatura de transición vítrea, como con la

temperatura de fusión, se puede observar en la Figura 1.1. La variación del volumen

específico (el inverso de la densidad, ) frente a la temperatura tiene lugar de manera

diferente según sea la velocidad del proceso. Si el líquido se enfría de manera suficientemente

7


lenta, al llegar a la temperatura de cambio de fase tiene lugar el fenómeno de cristalización y

se observa una disminución abrupta de 1/ . Si el sistema sigue siendo enfriado, la reducción

del volumen específico continúa, pero a una velocidad mucho más lenta. Sin embargo, si el

líquido se enfría muy rápidamente, se evita la cristalización y el volumen específico continúa

disminuyendo al mismo ritmo que antes, hasta alcanzar la TG. En este punto, el volumen

específico disminuye con la misma dependencia con la temperatura que en el sólido cristalino

(Walstra, 2002).

La transición vítrea afecta las propiedades mecánicas del material como consecuencia del

aumento de la movilidad molecular, es decir, el coeficiente de difusión de las moléculas. Al

aumentar la temperatura por encima de TG, la viscosidad disminuye drásticamente (Roos,

1995). La entalpía del sistema (Figuras 1.1 y 1.2) se comporta como una derivada de primer

orden. El pico estrecho que se produce en Tf corresponde a la fusión de la estructura

cristalina, mientras que el pequeño salto que se observa corresponde a la TG. Cuando se

representa la segunda derivada, se observa un máximo que corresponde a la temperatura de

transición vítrea, por lo que se considera que se puede hablar de una transición de segundo

orden (Walstra, 2002). En las temperaturas superiores a TG, se observa un aumento en la

movilidad molecular y una disminución de la viscosidad que posibilitan la cristalización del

agua, si se dispone de tiempo suficiente para la realización del proceso (Roos & Karel, 1991).

Cuando la temperatura está por debajo de la de transición vítrea, el sistema se puede

considerar estable, porque no es posible la formación de hielo, ya que en estas condiciones la

movilidad molecular está muy reducida. Con frecuencia, la dificultad está en el control de la

TG, ya que es un parámetro muy sensible a la proporción de agua y otras moléculas pequeñas,

y pequeñas cantidades de agua puede causar grandes variaciones en TG (Roos & Karel, 1991;

Roos, 1995; Zamecnik et al., 2007).

Por lo tanto, en el intervalo de temperatura entre los parámetros mencionados, Tf y TG, es

donde puede haber riesgo de formación de hielo, tanto durante el enfriamiento como en el

calentamiento (Figura 1.3A). Así, es de interés el recudir este intervalo (disminuyendo el

valor de Tf y aumentando el de TG) (Figura 1.3B). Además, como se trata de

comportamientos cinéticos, el sistema debe permanecer el menor tiempo posible en este

intervalo, para minimizar la probabilidad de formación de hielo.

8


TG Tf

Figura 1.1. Ilustración sobre la transición vítrea en comparación con una fusión/cristalización de una sustancia pura no polimérica. Se denota el estado de equilibrio mediante un trazo continuo y el de no equilibrio por trazos discontinuos. (a) Volumen específico, b) viscosidad aparente, (c) capacidad calorífica (primeras derivadas), representando la dirección hacia arriba una transición endotérmica (Figura tomada de Walstra, 2002).

9


Figura 1.2. Ilustración sobre la transición vítrea de una solución de vitrificación (PVS3) en comparación con una fusión/cristalización de ápices de ajo. Se denota el estado de equilibrio mediante un trazo continuo y el de no equilibrio por trazos discontinuos.

Tf TG

Tf TG

Figura 1.3. Ilustración sobre la transición vítrea en comparación con la fusión en un termograma de DSC (A) y el acercamiento de las temperaturas, por aumento de TG y disminución de Tf (B).

1.3.3. La velocidad de cambio de temperatura en crioconservación

Las velocidades de enfriamiento y calentamiento están determinadas por la masa térmica

del material (su capacidad calorífica total) y las propiedades de transferencia de calor del

sistema (Bald, 1987). La dificultad de la inducción de cambios de temperatura (enfriamiento o

calentamiento) muy rápidos, es mayor cuanto mayor es la masa del sistema, especialmente si

el contenido en agua (de alta capacidad calorífica específica) es elevado, ya que es necesario

transferir una mayor cantidad de calor (Franks, 1986). Esta transferencia de calor debe

alcanzar al centro de la muestra y para eso debe, frecuentemente, atravesar capas de

materiales malos conductores térmicos, como son las paredes de contenedores plástico y

crioviales (Song et al., 2010).

Para diseñar procedimientos de crioconservación adecuados, en los cuales la rapidez de las

etapas de enfriamiento y calentamiento es esencial, resulta necesario controlar los diversos

10


factores concurrentes: tamaño de la masa de muestra (y demás componentes del sistema),

contenido en agua y transferencia de calor. El resultado es una interacción compleja entre las

propiedades físicas citadas de los diversos componentes del sistema (muestra, contenedores,

soportes, soluciones), que tiene como resultado directo las velocidades de variación de

temperatura aquí determinadas y, una vez comprendido dentro de un proceso de

crioconservación real, la obtención de mayores o menores porcentajes de viabilidad

(Pennycooke & Towill, 2000).

1.3.4. Soluciones crioprotectoras

Blanshard & Franks (1987) llevaron a cabo numerosos estudios sobre el control de la

cristalización del agua: inhibición y/o control de la nucleación, control del crecimiento de los

cristales de hielo y empleo del estado vítreo. Se ha comprobado que la adición de polímeros y

azúcares puede retrasar la cristalización (Iglesias & Chirife, 1978). Este efecto se ha

observado también con adición de fructosa (Roos & Karel, 1991) y relacionado con la

variación de la viscosidad y el correspondiente efecto en la movilidad en la estructura

molecular (Roos, 1995).

A partir de estas observaciones se han desarrollado tratamientos alternativos con

soluciones crioprotectoras, como agentes osmóticos (ej.: manitol), que causan deshidratación

reduciendo el contenido de agua intracelular, y crioprotectores (ej.: DMSO), que penetran en

las células para estabilizar proteínas y membranas (Engelmann & Takagi, 2000; Benson,

2008). Sin embargo, el modo de acción de cada componente de la solución crioprotectora es

específico, siendo modulado por las condiciones de incubación, y por otra parte, su presencia

puede interferir en el desarrollo de estrategias de defensa de cada sistema biológico frente a la

congelación.

Los protocolos seguidos en los distintos métodos de crioconservación se basan en

combinaciones de diversas sustancias, desarrolladas básicamente mediante metodología

empírica (prueba y error). El tiempo de exposición y condiciones deben ser, por tanto,

optimizadas para obtener la protección suficiente contra la formación de hielo sin dañar las

células por estrés osmótico o toxicidad química de las soluciones de vitrificación (Volk &

Walters, 2006).

11


Diferentes soluciones de vitrificación han sido desarrolladas por varios equipos de

investigación en todo el mundo. Sin embargo, las soluciones más utilizadas son las soluciones

en las que uno de los componentes es el glicerol, como por ejemplo PVS1, PVS2 y PVS3

(Plant vitrification solutions 1, 2 y 3). La solución PVS1 contiene 19% p/v de glicerol, 13%

p/v de etilenglicol, 13% p/v de propilenglicol, 6% p/v de DMSO en medio líquido MS y 0,5

M de sorbitol (Uragami et al., 1989). La PVS2 contiene 30% p/v de glicerol, 15% p/v de

etilenglicol, 15% p/v de DMSO y 0,4 M de sacarosa. La PVS3 se prepara con 40% p/v de

glicerol y de sacarosa en medio de cultivo básico (Sakai & Engelmann, 2007).

Soluciones acuosas del polímero sintético de alcohol polivinílico, su copolímero de bajo

peso molecular y el polímero poliglicerol están siendo utilizadas como soluciones de

vitrificación alternativas, pues reducen la actividad de nucleación y el crecimiento de los

cristales de hielo (Kami et al., 2008).

1.3.5. Encapsulación en alginato

El empleo de cuentas de gel formadas por el polímero alginato cálcico se ha generalizado,

como medio auxiliar en los protocolos de crioconservación de germoplasma vegetal, ya que

confiere una protección frente a los tratamientos de deshidratación empleados.

Las cuentas de gel suelen ser generadas en dos etapas. Primeramente los especímenes pre-

tratados son introducidos en una solución de alginato sódico (soluble en agua) que contiene

nutrientes y azúcares (por ejemplo, medio MS líquido + 0,35 M sacarosa + 3% alginato).

Después, los especímenes son absorbidos con la ayuda de una pipeta y dispensados, en gotas

de la solución de alginato, en el seno de otra solución con el medio de encapsulación o

polimerización (una sal soluble de calcio). Las gotas de solución de alginato con el material

biológico incluido forman, entonces, esferas de consistencia creciente, a medida que la

difusión del calcio externo permite la formación del gel de alginato cálcico. Tras un periodo

de gelificación adecuado (minutos) las cuentas pueden ser extraídas de la solución y tratadas,

ya sea mediante soluciones concentradas o deshidratadas al aire, porque el gel es permeable a

los solutos y al agua.

El alginato es un polisacárido extraído de tres especies de algas pardas (Laminaria

hyperborea, Ascophyllum nodosum y Macrocystis pyrifera), en las cuales representa más del

40% del peso seco. Se encuentra como sal de varios cationes comunes en el mar como Mg2+,

12


Sr2+, Ba2+ y Na+. Químicamente es un polisacárido lineal compuesto por bloques alternados

de residuos de ácido -L-gulurónico (G) y ß-D-manurónico (M) unidos (Draget, 2000;

George & Abraham, 2006; Coviello et al., 2007). Las distintas regiones presentan geometrías

diferentes, en hélice y plana. Cuando dos regiones de bloques gulurónicos se alinean lado a

lado, forman un hueco en forma de diamante el cual es ideal para alojar iones divalentes

unidos cooperativamente. Las propiedades físicas de los alginatos están determinadas por la

composición y extensión de las secuencias de ácido manurónico, gulurónico y de la estructura

alternada de ambos, así como por su peso molecular (George & Abraham, 2006).

La propiedad más importante de los alginatos es la de formar geles con cationes divalentes

como el Ca2+. La afinidad de los alginatos por los metales alcalino térreos aumenta en el

orden Mg < Ca < Sr < Ba; la selectividad por éstos aumenta marcadamente con el mayor

número de residuos -L-gulurónicos en las cadenas (Draget, 2000; George & Abraham,

2006). La gelificación y el entrecruzamiento del polímero se logran por el intercambio de los

iones sodio de los ácidos gulurónicos con los cationes divalentes y el apilamiento de estos

grupos gulurónicos para formar la estructura.

Cuando la solución de alginato de sodio entra en contacto con la solución gelificante que

contiene iones calcio, ocurre la gelificación instantánea en la interfase, con el tiempo los iones

calcio difunden en la solución e interaccionan con la solución de alginato. La máxima dureza

del gel se registra en la superficie, ya que la concentración de alginato es mayor en ésta y

disminuye hacia el centro del gel (King, 1983; Draget, 2000).

1.3.6. Quitosano

Las cuentas de alginato cálcico son empleadas en otros contextos como protectores o

vehículos para nutrientes u otros compuestos. Sus propiedades de permeabilidad a gases, agua

y solutos, así como su resistencia mecánica han sido moduladas mediante la adición de otros

polisacáridos (Anbinder et al., 2011). Tal es el caso del quitosano, que añade a sus

características de interés, la de ser un efectivo agente antimicrobiano de amplio espectro

(Eaton et al., 2008).

El quitosano se obtiene directamente a partir de la quitina, por desacetilación. Es el

principal componente estructural del exo-esqueleto de los crustáceos y también se encuentra

en moluscos, insectos y hongos. La forma más común de obtención es el -quitosano, a partir

13


de la quitina de los desechos de caparazón de cangrejos y camarones, representando alrededor

del 70% de sus componentes orgánicos. En su obtención, los caparazones molidos son

desproteinizados y desmineralizados por tratamiento sucesivos con bases y ácidos, y a

continuación se extrae la quitina la cual es desacetilada a quitosano por hidrólisis alcalina a

alta temperatura (George & Abraham, 2006).

La quitina es un co-polímero muy ordenado de 2-acetoamido-2-deoxi- -D-glucosa (como

componente principal) y 2-amino-2-deoxi- -D-glucosa. A diferencia de otros polisacáridos

abundantes, como la celulosa, la quitina contiene nitrógeno (Terbojevich & Muzzarelli, 2000).

El quitosano presenta propiedades biológicas favorables para el uso en la biotecnología

como nula toxicidad, biocompatibilidad, biodegradabilidad (Sinha & Kumria, 2001) y

actividad antimicrobiana contra una amplia cantidad de hongos filamentosos, levaduras y

bacterias (No et al., 2002; Eaton et al., 2008). Sin embargo, el estudio de encapsulados

obtenidos a partir de esto polímero en el campo de la crioconservación es un campo aún por

explorar.

1.4 La menta como modelo en el estudio de la crioconservación

La familia Lamiaceae (Labiatae) está formada por aproximadamente 200 géneros y 3200

especies de distribución cosmopolita, pero son especialmente abundantes en la región del

Mediterráneo y Centro-Este asiático. Más de la mitad de las especies pertenecen a ocho

géneros: Salvia (500), Hyptis (350), Scutellaria (200), Coleus (200), Plectranthus (200),

Stachys (200), Nepeta (150) y Teucrium (100). Otros géneros de la familia con un elevado

número de especies son Lavandula, Marrubium, Mentha y Thymus (Cronquist, 1981). La

familia se caracteriza por la presencia de flavonoides y terpenoides (Cole, 1992), metabolitos

secundarios con importantes aplicaciones en medicina y como condimento alimentario.

El género Mentha L. tiene amplia distribución en todos los continentes, a excepción de

Sudamérica y la Antártida. Sus centros de diversidad son Europa, Australia y Asia Central. En

Europa hay una gran diversidad de especies, encontrándose unas 22 entre especies e híbridos

(Flora Europaea, versión on line, Chambers, 1992).

La menta es una planta de interés económico, principalmente en la industria farmacéutica y

cosmética (Gupta, 1991; Munsi, 1992), y es cultivada en los Estados Unidos, Italia, Francia,

14


Hungría y otros países (Fahn, 1979; Lawrence, 1985). Por la gran cantidad de aceites

esenciales que contienen, entre ellos mentol, mentona y mentofurano, con acción

antimicrobiana, antiespasmódica, antiulcerosa y antiviral, forma parte de los productos

estratégicos industriales en Biotecnología. Estos compuestos se encuentran en mayor cantidad

en las hojas y son ampliamente investigados tanto desde el punto de vista agronómico como

químico (Schilcher, 1988; Martins, 1998). La infusión de sus hojas puede ser utilizada para

facilitar la digestión, eliminación de gases del aparato digestivo (Lorenzi & Matos, 2002;

Simões & Spitzer, 2007) y en medicina popular (Piccaglia et al., 1993).

En el trabajo aquí desarrollado, se utilizó Mentha ×piperita Linn., siendo un híbrido estéril

entre Mentha aquatica L. y Mentha spicata L. (Arvy & Gallouin, 2006). Esta es una planta

herbácea, perenne, aromática, de aproximadamente 30 cm de altura, con ramas de color verde

oscuro a morado, tallo ramificado, flores lilas a azuladas y las hoja opuestas pecioladas, oval

y borde cerrillado (Matos, 1998).

La elección de esta planta como modelo para el estudio de los procesos de

crioconservación responde a su frecuente uso comercial con multiplicación vegetativa,

presencia en diversos métodos y protocolos publicados, así como a su buen comportamiento

frente a la crioconservación (Bhat et al, 2001; Wang & Reed, 2003; Islam et al., 2005; Volk

& Walters, 2006; Volk et al., 2006; Sakai & Engelmann, 2007; Senula et al., 2007; Uchendu

& Reed, 2008).

15

2. OBJETIVOS/AIMS

2. OBJETIVOS

La crioconservación de germoplasma vegetal es una alternativa eficiente a la conservación

clásica (semillas o colecciones en campo), que adicionalmente permite conservar a largo

plazo material vegetal utilizado u obtenido mediante biotecnología. En la actualidad, resulta

necesario poner a punto los protocolos adecuados para cada especie, e incluso, genotipo,

mediante ensayo y error. Entender los procesos subyacentes a los diversos pasos de un

protocolo de crioconservación facilitará el desarrollo de nuevos protocolos. En el presente

trabajo se estudiaron tres técnicas diferentes de crioconservación: vitrificación, vitrification-

droplet, y encapsulación-deshidratación. El objetivo principal de este proyecto de tesis es

aumentar el conocimiento sobre los procesos implicados en los distintos pasos de un

protocolo de crioconservación, en relación con el estado del agua presente en los tejidos y sus

cambios, abordado mediante diversas técnicas biofísicas, principalmente calorimetría

diferencial de barrido y la microscopía electrónica de barrido a baja temperatura, estudiando

la dinámica de formación de hielo y el estado vítreo. La especie objeto de estudio en la mayor

parte de los capítulos ha sido la menta (Mentha ×piperita M186). En dicha especie existían ya

puestos a punto protocolos de crioconservación basados en todas las técnicas mencionadas

anteriormente (Senula et al., 2007; Uchendu & Reed, 2008; Volk & Walters, 2006).

Los objetivos concretos de la tesis fueron:

1) Estudio comparativo de las velocidades de enfriamiento y calentamiento en las diferentes

técnicas de crioconservación.

2) Estudio de formación de cristales de hielo y los procesos de vitrificación en las distintas

etapas del protocolo de vitrificación-droplet, mediante crio-SEM y DSC en menta.

3) Estudio de los distintos estados de deshidratación en el protocolo de encapsulación-

deshidratación, mediante crio-SEM y DSC, en menta.

4) El efecto de la velocidad de enfriamiento en la temperatura de transición vítrea y capacidad

calorífica en diversas soluciones de vitrificación de plantas.

5) Modulación del papel del encapsulado de alginato mediante la adición de otros

polisacáridos.

16

3. MEASUREMENT OF COOLING AND WARMING RATES


IN VITRIFICATION-BASED PLANT CRYOPRESERVATION

PROTOCOLS

3.1. Introduction

Cryopreservation of living tissues for its functional recovery is a widely employed

procedure, for the multi-purpose preservation of medical, veterinary and crop and wild plant

species materials. The basis of cryopreservation is the drastic reduction of most chemical and

physical activities in the cells. While temperature reduction over the physiological fluids

freezing point can only slow these processes, freezing confers a higher degree of activity

stoppage, due to the associated reduction of water activity. Unfortunately, intracellular ice

formation has been found to be lethal for most tissues and specimens. Satisfactorily stable

storage conditions are found for most cases when storage is carried out at temperatures well

below the freezing point: in the glassy state. When viscosity of liquids increases, molecular

mobility is reduced. This reduction can be so extreme that most processes are virtually

detained (Mazur, 1984).

The change between liquid and vitreous state is a second order phase change, not

determined by an enthalpy increment as first class changes, but by a step in some of the

physical properties of the system, such as the heat capacity, specific volume and apparent

viscosity (Walstra, 2002). Glassy state is kinetically controlled, so it can be considered to be

metastable, though the very slow mobility guarantees the actual stoppage of most processes.

Moreover, ice formation, a process requiring the assembly of an initial nucleus of water

molecules driven by Brownian movement, is completely impaired in glasses (Slade & Levine,

1995).

The molecular mobility reduction required for entering the glassy state is achieved by

either a temperature drop or by an increase of the solute concentration. The glassy state

conditions can be achieved by dehydration (which causes the effective solute concentration to

rise) and it can be employed for preservation of systems surviving extreme dehydration, such

as most seeds (orthodox), even at room temperature (Sacandé et al., 2000). Most systems

would be damaged by such dehydration degree, and, therefore, a reduction of temperature is

17


also required, to reach the glass transition temperature TG, which is lower the higher the

solvent (water) content is. However, cooling below the equilibrium freezing temperature Tf,

gives rise to a problem: the wide temperature gap between Tf and TG must be crossed and ice

can be formed in this region.

Ice crystal formation takes place always under the equilibrium freezing temperature, but its

initial nucleation step is stochastic in nature, depending on the random rearrangement of water

molecules to achieve the initial nucleus conformation able to spontaneously grow. That

implies that there is an increasing probability of ice being formed the longer the permanence

between Tf and TG. For this reason thermal change (cooling and warming) rates are

considered critical for the success of cryopreservation (Blanshard & Franks, 1987; Angell,

2000; Wesley-Smith et al., 2001).

Cryopreservation is customarily applied to animal, plant and microbial specimens. Plant

tissues are often preserved after vitrification protocols (Towill, 1990; Mazur et al., 2007;

Nadarajan et al., 2008), which imply a previous set of steps designed to increase solute

content and reduced water in cells, as well as to promote the plant natural defences towards

stress. Cooling by plunging into liquid nitrogen (LN) completes the procedure. The recovery

process is nearly symmetric, and first re-warming is carried out by immersion in a warm

water bath, followed by a rehydration and solute unloading stage before specimen culture. A

method frequently used in the cryopreservation of plant germplasm is that named directly as

vitrification (Sakai & Engelmann, 2007). Another usual procedure is droplet-vitrification, and

the main difference with the former is the mass (of specimen plus cryoprotectant solution and

container) to be cooled or warmed (Sakai & Engelmann, 2007; Senula et al., 2007).

The need for a fast temperature change in the vitrification and de-vitrification processes is

deemed as being of crucial importance, and is closely associated to the success of the

procedure (Wesley-Smith et al., 2001). In spite of this, measurement of actual cooling and

warming rates in these protocols has rarely being recorded and reported. Actually, most

temperature rate changes reported for cryopreservation processes correspond to animal or

human origin samples, where the applied protocols are often of different nature. The few

reports of cooling/warming rates in plant cryopreservation lack precision and their

methodology is scarcely described (Pennycooke & Towill, 2000; Towill & Bonnart, 2003).

The cause for this lack of measurements is, most likely, related to the difficulty of performing

reliable fast thermal measurements. In the present work, the rates of cooling and warming in

18


cryopreservation experiments, performed with mint shoot tips and following the vitrification

and droplet-vitrification protocols, were determined using a fast temperature determination

system. The viability of apices after cryopreservation was also studied, to ascertain the

validity of the protocol followed.

3.2. Materials and Methods

3.2.1. Plant material pre-culture and shoot tip extraction

Shoot tips were extracted from in vitro shoots of Mentha ×piperita (genotype �“MEN 186�”

obtained from the IPK Genebank, Gatersleben, Germany). In vitro plants were monthly

subcultured on medium MS (Murashige & Skoog, 1962) with 3% sucrose, and incubated at

constant temperature (25°C) with a photoperiod of 16 h, and an irradiance of 50 µmol m-2 s-1

from fluorescent tubes. One-node segments were obtained from these shoots, transferred to

fresh medium and incubated at alternating temperatures of 25°C (day) and -1°C (dark),

always with 16 h photo- and thermoperiod, 50 µmol m-2 s-1 irradiance, provided by

fluorescent tubes (Senula et al., 2007). After 3-weeks of culture under these conditions, shoot

tips (1-2 mm) were excised from axillary buds.

3.2.2. Dehydration of shoot tips in the vitrification method

All excised shoot tips were pre-cultured for 24 h on filter paper in liquid MS medium

containing 0.3 M sucrose, at 25ºC. Thereafter, the explants were transferred to a Petri dish

with 2 mL of loading solution (2 M glycerol + 0.4 M sucrose) over filter paper, for 20 min, at

room temperature. Finally, they were osmotically dehydrated in 2 mL PVS2 (plant

vitrification solution 2: 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v DMSO and 0.4

M sucrose in half-strength MS liquid medium; Sakai et al, 1990) in a Petri dish, on filter

paper, for 30 min at 0ºC. All constituents of PVS2 were autoclaved except DMSO, which was

filter-sterilised. All solutions stated here or thereafter were prepared in liquid medium MS.

19


3.2.3. Vitrification

Modifications of the protocols for mint from Volk & Walters (2006) and Uchendu & Reed

(2008) were used. After dehydration in PVS2, ten shoot tips were placed in a 1.0 mL cryovial

containing 0.5 mL or 1.0 mL PVS2. Then, this container was submerged into liquid nitrogen

(LN) and, after a short period in LN conditions (approx. 1 h), the cryovial was removed and

rewarmed in 40°C water for 2 min, stirring gently. Then shoot tips were washed with 1.2 M

sucrose for 20 min at room temperature and subsequently cultured.

3.2.4. Droplet-vitrification

Modifications of the protocol developed by Senula et al. (2007) for mint were studied.

After dehydration in PVS2, shoot apices were transferred into droplets of 2 µL PVS2 placed

on aluminium foil strips (AFS; 5 droplets per strip, each containing a shoot tip). The strips

with the adhered droplets were immersed into LN, either directly (naked) or placed inside

cryovials. After a short period in LN conditions (approx. 1 h), cryovials/AFS were retrieved

and warmed. Cryovials were plunged into a water bath at 40°C for 3 to 5 s; the AFS were then

removed from the cryovial and the apices were incubated in a 1.2 M sucrose solution for 20

min at room temperature. When naked aluminium strips had been immersed in LN, they were

directly plunged into the 1.2 M sucrose solution at room temperature and shoot tips incubated

for 20 min.

3.2.5. Plant recovery and viability

Shoot tips exposed to the vitrification or droplet-vitrification techniques were cultured on

re-growth medium (semi-solid MS medium supplemented with 0.5 mg/L BAP and 3%

sucrose), incubated in the dark for 24 h and thereafter under 16 h photoperiod, at 25ºC. For

control samples (-LN), all steps were carried out except immersion in LN and rewarming

Survival and re-growth were calculated as percentages over the total number of shoot tips

used 4 weeks after culture. Survival was defined including all forms of visible viability

(evidence of green structures or callus). Re-growth was defined as the formation of small

plantlets. The number of replicates was 3 or 6 for the vitrification and droplet-vitrification

20


protocols, respectively. Data were subjected to analysis of variance and Duncan�’s Multiple

Range Test (Alpha = 0.05) was used for comparison of means.

3.2.6. Temperature measurement and cooling and warming rates

The specimen temperature changes were acquired using a fast data acquisition

multichannel temperature measuring system, MW100-UNV-H04 (Yokogawa, Tokio, Japan).

Several reading speeds were tested and finally a reading speed of 20 data per second

(measuring interval 50 s) was selected as optimal. Liquid nitrogen and ice were used for

calibration points (-196 and 0ºC, respectively), and calibration was performed frequently

between measurements. Cooling and warming rates were defined as the ratio of a

predetermined temperature interval T1-T2, over the time elapsed between the reported

measurement and of T1-T2.

The determination of cooling and warming rates was carried out, at least in triplicate,

during the cooling and warming phases of the described protocols.

3.2.7. Thermocouples

Several types of thermocouples and wire gauges were tested and finally, T-type

thermocouples (copper-constantan) were selected for its good performance in the temperature

region studied (45 to -150ºC). Thin (diameter 0.5 mm) but steel-shielded and mineral-isolated

thermocouples (TC Medida y Control, S.A., Madrid, Spain) were chosen, in order to

minimize external electromagnetic influences on measurements. They were placed either on

the surface of AFS (commercial household aluminium foil, strips of approx. 20 x 5 mm, 5

mg) or in the centre of cryovials, introduced through a hole in the lid, depending on the

cryopreservation technique studied.

Controls were set for empty cryovials and thermocouples without specimens. Additional

controls were set for evaluating the effect of heat losses through the thermocouple wire,

comparing results with those obtained with additional thermocouple tips inserted in the

sample. No significant differences were found that could justify a heat loss correction.

Thermocouples were frequently calibrated as a part of the measurement set-up, as described

above, and especially after any thermocouple exchange or reconnection.

21


3.2.8. Experimental set-up and temperature change rate measurement design

A scheme of the experimental set up used for temperature change rate is shown in Figure

3.1. The aim of the experiment was to reproduce the conditions used in customary

cryopreservation, which involves a certain degree of variability due to not completely

controlled factors and the operator action.

Figure 3.1. Experimental set up for thermal change rate measurement for (a) vitrification, or (b), droplet-vitrification protocols.

The sample holders (either a criovial or an AFS) were handled by means of the attached

thermocouple wire, by means of isolated tongs, in order to reduce the heat exchange sources.

For cooling rate determination cryovials/AFS were tempered at 1-4ºC before immersion in

LN, and data collection started at 0ºC, and continued till a plateau near -196ºC was reached.

For warming, data collection started when the specimens were immersed and thermally

equilibrated in LN and continued till room temperature equilibrium. Rates were calculated

after adopting an ice risk temperature window, by dividing the thermal difference between the

window extremes by their time difference.

Temperature change rates were determined on specimens being submitted to the

vitrification protocol, using a cryovial containing 0.5 or 1 mL of PVS2 solution (containing

10 mint shoot tips), as well as an empty cryovial for blank comparison. Rates were also

measured on specimens treated with the droplet-vitrification protocol, introducing variations

in several parameters. Two experimental conditions were tested (with different masses and

specimen distributions): an AFS with 5 droplets of 2 L (containing each one a mint shoot

tip), naked or inside a cryovial. Two controls were added: an AFS with no shoot tip sample,

inside or not a cryovial.

22


3.3. Results and Discussion

Figure 3.2 shows cooling and warming experiments corresponding to the vitrification

protocol. Figure 3.2a shows the temporal evolution during cooling of the sample, consisting

of 10 shoot tips in a 1.0 mL cryovial containing 0.5 mL or 1.0 mL PVS2. Figure 3.2b shows

data for the same samples during warming. A control performed with an empty cryovial is

also shown in both cases. Given the relatively slow rates for this case, the data points obtained

at 50 µs interval form a continuous line. In spite of the visible dependence of rate with the

thermal difference with the surrounding fluid, data cannot be properly fitted to a function, as

many smaller scale phenomena have a variable influence on both cooling and warming

processes. This includes the changes in heat capacity and conductivity of the containers, fluids

and sample components during this wide temperature span, the possible phase changes

(including to and from glassy state) in all components, involving, when not a heat effect, an

additional heat capacity step change, the heat interchanges derived from mechanical forces

when the volume of components are altered, etc. Also the complex convention of the external

fluid (and Leidenfrost effect in LN), would generate differences in the instantaneous heat

exchange rates (Leindenfrost, 1966; Song et al., 2010).

The cryoprotecting solution content seems to be a major determinant of the rate, both for

cooling and warming: the empty cryovial, with less total heat capacity than those with

solution, shows faster rates. Those of 0.5 and 1.0 mL samples do present a very similar

behaviour, probably compensating the higher heat capacity of the larger volume sample and

its higher heat conductivity, due to the higher heat exchange surface. The trends observed are

quite symmetric for cooling and warming, both for samples and control measurements.

Control observations carried out using several thermocouple wires indicated that any effect

on thermal change rates was negligible, allowing discarding reductions of the measured rate

due to the wire contribution to thermal mass or to heat fluxes through it (data not shown).

Figures 3.3 and 3.4 show cooling and warming experiments corresponding to the droplet-

vitrification protocol. Figure 3.3 a and b show temperature temporal evolution determined

during cooling by immersion in LN, for different masses and sample distributions (naked AFS

or inside a cryovial and with different number of droplets).

23


Figure 3.2. Cooling (a) and warming (b) evolution determined in samples treated after the vitrification protocol. Data are the average of at least three independent determinations. Control data were obtained with the thermocouple tip inserted in an empty cryovial. The lines shown represent the individual data points measured.

Figure 3.4 a and b show temperatures obtained during the warming process as described

in the droplet-vitrification protocol, for the same samples as in Figure 3.3. Data obtained

were fairly reproducible and, in spite of the additional error introduced by specimen

variability and operator manipulation, their scattering was much smaller than the differences

observed among different experimental conditions. Comparison of these results showed a

diverse behaviour in both cooling and warming rates for the different conditions. The

presence of PVS2 cryoprotecting solution drops in the droplet technique induces a reduction

of both cooling and warming rates, when compared with the same methodology but without

the drops.

In those systems based on an aluminium foil strip, the small additional mass of this strip

(aprox. 3-4 mg) can be compensated by the increased heat diffusion granted by the larger

interchange surface, and it can be observed that both cooling and heating rates of these strips

24


(with the measuring thermocouple attached) were larger than those determined with the

thermocouple alone.

Figure 3.3 a and b. Measurements of cooling rate obtained following the droplet-vitrification protocol: (a) AFS inside cryovial and (b) naked AFS. Data are the average of at least three repeats.

The rates in the warming process for this protocol show larger differences from those of

cooling than in the case of the vitrification protocol, due to the particularities of the warming

process. The introduction of the AFS + 5 x 2 µL PVS2 drops sample in the sucrose solution at

room temperature has the effect of a reduction of the warming rate, probably due to different

interface contact and heat exchange behaviour than that resulting of the immersion in LN.

Because of this slower rate, the warming process of this sample has been shown in a different

sub-figure than the cooling, faster, one.

In Figure 3.4 b), a slope change in the warming process can be appreciated for the two

samples considered, at 0.3-0.4 s. This was related to the physical transfer from nitrogen to air

25


and then to the warming medium. It could not be appreciated in the data sets of Figure 3.4 a)

because it was obscured by the larger duration of the warming process. The end of the 3-5 s

warming step in the water bath (and exit to room temperature air), for the two samples treated

in that way and shown in Figure 3.4 a), per contra, does not give rise to any warming rate

irregularity. This may be a result of both these samples being included in a cryovial, whose

isolation could mask fast thermal effects. As expected, when comparing the results from both

vitrification and droplet-vitrification protocols, it can be seen that the inclusion of specimens

in a cryovial considerably reduces both cooling and warming rates.

Figure 3.4 a and b. Measurements of warming rates obtained following the droplet protocols. Data are the average of at least three repeats.

The data dispersion in the thermal change rates observed comprise both the effect of real

experimental errors and those derived from the possible small variations of the

cryopreservation procedure. The experimental methodology followed was intended to

reproduce the usual operations in cryopreservation techniques, rather than to minimize the

causes of variability, so that the sources of variation associated to manual manipulations when

transferring samples, the holding with tongs and the degree of stirring have not been

26


standardized. In the same way, an extreme important factor, as it is the position of the active

thermocouple tip within the experimental set-up, especially inside cryovials, has not been

fixed. A controlled position in the cryovial centre would have produced more homogeneous

data, but it would not have been representative of the real temperature values in positions

closer to its wall. All these factors add to an increase in the dispersion of the obtained data.

Figure 3.5 shows how control specimens, not immersed in liquid nitrogen (LN), present a

100% survival, proving that the dehydration and chemical treatments applied (including

exposure to PVS2) have no toxic effect that could damage apices. Survival and regrowth

values were fairly similar for all cases. The highest regeneration percentage was observed

(96%) when the droplet-vitrification procedure was employed with direct immersion in LN, a

value decreasing with the inclusion in a cryovial. The vitrification protocol, yielded similar

regeneration values for the treatments with 0.5 or 1 mL PVS2 solution inside the cryovial, but

much lower (about 43%) than the droplet-vitrification protocol.

Figure 3.5. Mint shoot tip survival and re-growth percentages after different cryopreservation techniques and a 4-week recovery period. Means of survival or re-growth with the same letter are not significantly different according to the Duncan�’s Multiple Range Test at alpha = 0.05. Bars: standard error.

The observed percentages can be directly related to physical characteristics of specimens

and experimental systems employed (containers and cryoprotecting solutions), especially their

mass, heat capacity and heat conductivity. For example, those systems with a larger mass will

27


have a higher inertia towards temperature change. The more relevant factor would be the heat

amount that the system requires to absorb or to yield for causing a temperature change, i.e. its

total heat capacity. The contribution of the different heat capacities of the system components

must be accounted for. A higher thermal inertia can be expected when the water content (with

a large specific heat capacity) is high, while the mass of cryovials and aluminium foil would

represent a lesser influence, due to its lower heat capacity.

On the other hand, the temperature change is determined not only by the heat amount to be

exchanged, but by the rate of this transfer. The different experimental systems employed

present many diverse characteristics on this respect. The presence of containers always

represents an additional barrier for heat exchange. The position of the specimens inside the

cryovial partly determines this behaviour, as depending on the extent of its contact with its

walls, heat transfer could be very different. This may explain the complex effect of the

presence of vitrification solutions inside the cryovial. On one hand, the thermal mass

increases and the total heat amount to be transferred is higher. But this solution could

constitute a thermal bridge between the cryovial walls and the specimen, improving heat

exchange and so fastening the temperature change process.

The data obtained and showed in figures 2-4 show continuous temperature variation on

time. As it can be seen, the slope of the curves (instantaneous rate) changes continuously,

decreasing as the driving heat gradient declines. In order to provide a single figure rate to

enable comparisons, a �“temperature window�” must be defined. This also produces a

parameter more interesting than the temperature change rate, for cryopreservation purposes:

the time of permanence in the ice formation risk area.

Although the purpose of cryopreservation is to keep samples free from ice formation risk

in the glassy state, and the procedures previous to plunging into LN are endeavouring to

reduce the likelihood of ice formation in any case (mainly by increasing viscosity and

reducing the water available), the risk of ice formation during the cooling or warming

processes crossing the area below the freezing point and over the glass transition temperature,

is ever existing (Angell, 1982; Franks, 1986).

A good knowledge of a given system to be cryopreserved would allow setting a more

adjusted temperature window. For comparison purposes, here we have defined two windows:

from 0 to -150ºC and from -20 to -120ºC. The first one, spanning from pure water equilibrium

freezing point (the higher temperature where ice can exist, in any aqueous solution) to -150ºC,

28


well below any expected glass transition in a living system, is largely overestimated over the

real ice formation risk zone for systems in cryopreservation, as its Tf is always much lower

than 0ºC, due its high solute and low water contents. The variability in the glass transition

temperature is high, being very sensitive to the content of water and other small size

molecules (Roos & Karel, 1991; Zamecnik et al., 2007), but, for plant viable germplasm, is

usually well over -150ºC. Moreover, the thermal gradient ( T, the difference between sample

and surrounding media temperatures) is similar in the initial and final windows extremes, for

both cooling and warming (using a 40ºC bath). For example, for the 0 to -150ºC window, T

would evolve from 196ºC at the initial point, to 46ºC at the window end-point, for cooling,

and from an initial value of 190ºC to a final one of 40ºC, for warming. This contributes to

generate similar cooling and warming rates, as other contributing phenomena will have a

more reduced weight, due to the high speed of the process (such as convention layer changes

or geometrical alterations of sample and containers).

The narrower temperature window, from -20 to -120ºC, was fixed taking into consideration

the freezing point observed for mint shoot tips in previous stages of the droplet

cryopreservation protocol (Teixeira et al., 2013). The specimen at the final stage of the

protocol, actually those plunged into LN, was reported to present no freezing event in

calorimetric conditions (at a cooling rate of 10ºC min-1), but we have considered that -20ºC

would be a safe limit for ice formation, considering also the usual reduction of the nucleation

temperature with respect to the equilibrium freezing one. The -120ºC limit follows the

reported TG for this system on -119ºC (Teixeira et al., 2013).

Table 3.1 and 3.2 show the calculated cooling and warming rates and permanence times

calculated for these two windows for experiments of Figures 3.2 to 3.4. In all cases, the

narrower (-20 to -120ºC) window gives higher rates. This is due to the larger temperature

gradient, as the region closer to the end point (always showing slower rate) is left out of the

calculation. The more adjusted the window is to the real ice formation probabilities, the more

relevant the rate and times calculated would be. Anyhow, it must be noted that the probability

of ice formation is not constant for the whole window. Its dependence with temperature is

complex and difficult to study, but it increases as temperature descends from the equilibrium

freezing point, being counterweighted by the reduction on molecular mobility, also driven by

lower temperatures. Near the TG, for time spans of the order of seconds, this probability is

most likely very small. A region of about 20ºC over the TG could be considered also safe for

these purposes (for short permanence, not for long term storage), as it has been reported that

29


its high viscosity grants that any scale change (such as ice nucleation) would take place very

slowly (Mishima & Stanley, 1998).

Table 3.1. Cooling and re-warming rate measurements obtained following the vitrification protocols.

Cooling Warming Method Window

(ºC) Time1

(s) Rate1

(ºC s-1) Time1

(s) Rate1

(ºC s-1) 0/-150 21.0 ± 0.2 7.14 ± 0.01 27.8 ± 0.4 5.4 ± 0.6 Control: cryovial

LN bath 40ºC -20/-120 10.0 ± 0.1 10.0 ± 0.1 16.3 ± 0.3 6.1 ± 0.7

0/-150 52 ± 1 2.88 ± 0.02 95 ± 1 1.58 ± 0.01 Cryovial with 0.5 mL PVS22 LN bath 40ºC -20/-120 31.43 ± 0.5 3.18 ± 0.04 55.1 ± 0.9 1.81 ± 0.02

0/-150 65 ± 1 2.31 ± 0.02 86 ± 1 1.74 ± 0.01 Cryovial with 1 mL PVS21 LN bath 45ºC -20/-120 41.9 ± 0.9 2.39 ± 0.03 54.0 ± 0.8 1.85 ± 0.02

1Average values and standard deviation for at least three repeats. 2Experiment performed with shoot apices in the cryovial. Window: interval selected for time and rate calculation. Time: that spanning between the recording of the window temperature interval (either during cooling of warming). Rate: calculated in the window temperature interval.

The main difference between the methods of cryopreservation used in this work is the

amount of PVS2. The advantage of using the droplet is the possibility of achieving very high

cooling/warming rates due to the very small volume of cryoprotective medium in which the

explants are placed. The signification of the highest rates reported must be taken with

moderation. The directly measured parameter is (apart from temperature), the time between

two temperature readings. In the faster processes, the fraction of this time that could

correspond to manipulations of the operator when transferring samples can be non-negligible,

and when the rate is calculated by division of these two quantities, very high differences can

be so generated, perhaps unduly.

Resorting to fast temperature changes in cryopreservation has been broadly studied and

employed to reduce ice crystal size. Luyet and co-workers (1965) pioneered the use of rapid

(non-equilibrium) cooling as a means of restricting the size of ice crystals during

cryopreservation of hydrated samples. For example, cooling rates in excess of 100°C s-1 are

reported to facilitate the preservation of microorganisms (Wesley-Smith et al., 2001). In spite

of the crucial role of thermal change rate on cryopreservation, few studies include detailed

measurements of the evolution of temperature in these conditions (Sakai et al., 1968;

30


Vertucci, 1989, 1990; Wesley-Smith et al., 1992, 2001). High viabilities in recovered tissues

have been reported at elevated cooling rates (40-400ºC s-1; Pennycooke & Towill, 2000; Kim

et al., 2006b). The rates reported by these workers with potato shoot tips show good

agreement (6ºC s-1) with our results for cooling inside a cryovial, while for cooling on

exposed AFS, a lower value (130ºC s-1) was reported, when compared to our 350-500ºC s-1

data.

Table 3.2. Cooling and re-warming rate measurements obtained following the droplet-vitrification protocols.

Cooling Warming Method Window

(ºC) Time1

(s) Rate1

(ºC s-1) Time1

(s) Rate1

(ºC s-1) 0/-150 2.32 ± 0.05 65 ± 1 0.09 ± 0.03 1700 ± 500 Thermocouple

LN bath 40ºC (3-5 s) -20/-120 1.38 ± 0.05 72.5 ± 2 0.05 ± 0.03 1887 ± 700

0/-150 0.43 ± 0.03 350 ± 25 1.59 ± 0.05 94 ± 2 AFS LN 1.2M sucrose room temperature -20/-120 0.20 ± 0.03 500 ± 70 0.32 ± 0.03 310 ± 30

0/-150 25.10±0.30 6.00 ± 0.05 40.0 ± 0.4 3.75 ± 0.03 AFS inside cryovial LN bath 40ºC

(3-5 s) -20/-120 11.80 ± 0.10 8.5 ± 0.1 26.5 ± 0.4 3.77 ± 0.04

0/-150 1.35± 0.05 110 ± 3 4.58 ± 0.08 32.8 ± 0.4 AFS with 5 x 2 µl PVS2 droplets2 LN 1.2 M sucrose room temperature

-20/-120 0.94 ± 0.04 106 ± 3 2.03 ± 0.04 49 ± 1

0/-150 20.6 ± 0.3 7.28±0.06 26.4 ± 0.5 5.68 ± 0.03 AFS with 5 x 2 µl PVS2 droplets2 in a cryovial LN bath 40ºC (3-5 s)

-20/-120 11.00 ± 0.02 9.1 ± 0.1 15.4 ± 0.2 6.49 ± 0.08 1Average values and standard deviation for at least three repeats. 2Experiment performed with shoot tips. Window: interval selected for time and rate calculation. Time: that spanning between the recording of the window temperature interval (either during cooling of warming). Rate: calculated in the window temperature interval.

The need to employ a high rate to avoid ice formation during cooling (and warming) has

especial importance when the specimens�’ water content is high. However, the rates required

to avoid ice formation cannot be practically reached when water content is very high, for a

sample of the size required for plant germplasm successful manipulation and cultivation. It is

often necessary to act on the specimen to reduce the probabilities of ice formation, generally

decreasing water content, with the multiple effect of reducing the ice formation risk window

31


(both by lowering Tf and, at the same time, increasing TG), while the increase in intracellular

viscosity limits the ice formation probability, even within this window. So, partial

dehydration reduces the requirements of very fast cooling.

Relatively slow temperature variations, as those reported in this study for procedures using

cryovials (1-7ºC s-1) would be inadequate for application to high water content systems.

However, the higher rates (100-500ºC s-1) obtained in some methods using aluminium foil

strips, would be appropriated for specimens with higher water contents. The rate differences

associated to the shoot tip water content, would be, however, small in cryovials-based

methods (differences between 2 and 4ºC s-1), being the limiting effect the thermal diffusion

and the own cryovial mass.

32

4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS

4. GLASSY STATE AND CRYOPRESERVATION OF MINT

SHOOT TIPS

4.1. Introduction

Biologically material, most frequently that being used for plant propagation (i.e.,

germplasm) can be stored at low temperature for long periods. Plant germplasm, particularly

seeds, pollen or reproductive tissues, is often kept at liquid nitrogen (LN) temperature to

avoid deterioration (Towill, 1990). Cryopreservation is currently an important tool for

germplasm long-term storage, requiring minimal space and maintenance, and the number of

species and cultivars cryopreserved quickly increases (Sakai & Engelmann, 2007). Low-

temperature storage of living tissues and cells must avoid ice crystal formation (especially

intracellular) during the whole process (cooling, storage and final re-warming), as ice results

lethal for cells, impairing specimens viability (Muthusamy et al., 2005; Nadarajan et al.,

2008).

Most techniques employed include an initial step designed to reduce the tissues water

content (with the limit of avoiding dehydration damage) and increase the cytoplasm internal

viscosity. This step may include the incorporation of extrinsic viscosity enhancing substances

(such as sugars, glycerol or ethylene glycol) and the induction of cold adaptation and defence

mechanisms by the plant cells. A second step consists in quick cooling by plunging into LN,

to avoid ice formation. The chances of ice crystallization are limited by both a decrease in the

amount and mobility of intracellular water and an increase in cooling rates, hindering the

rearrangement of water molecules into ice, a necessary previous nucleation step. Cooling to

LN temperature places the system well into the glassy state. Once vitrified, despite some

time-depending phenomena still taking place, relatively large molecular reorganizations, as

those required for ice formation, are not possible and specimens can be stored for

hypothetically indefinite periods, without ice being formed (Zamecnik et al., 2007). A third

step consists in the re-warming process, which must be performed quickly enough to avoid ice

nucleation while crossing by the temperature region comprised between the glass transition

temperature (TG) and the equilibrium freezing point (Tf) (Niino et al., 1992; Reinhoud, 1996;

Volk & Walters, 2006). Progressive dehydration and rapid cooling has been found to avoid

the formation and growth of lethal intracellular ice in plant cells (Wesley-Smith et al., 2001).

33


Many apparently independent physical properties undergo large changes associated to the

glass transition within similar temperature ranges. For example, the TG values determined by

calorimetry, thermal expansivity or viscosity are often nearly identical (Kauzmann, 1948).

This is a result of all these properties being parallelly influenced by changes in molecular

mobility occurring during the glass transition.

The initial works on mint cryopreservation by vitrification included gradual dehydration

using a mixture of ethylene glycol, dimethyl sulfoxide (DMSO) and polyethylene glycol

(Towill, 1990). A successful method among those usually employed for plant germplasm

cryopreservation is that named droplet-vitrification (Senula et al., 2007). It is derived from

the droplet-freezing technique (Kartha et al., 1982) developed for preserving shoot tips, which

are placed in droplets of cryoprotective medium and cooled slowly in a programmable

freezer. In the droplet-vitrification method, the specimen is initially incubated in solutions

with increasing concentration of cryoprotecting and dehydrating agents, for different periods.

Then it is treated and included in a small drop of cryoprotecting agents solution (containing

high concentrations of glycerol, ethylene glycol, DMSO and sucrose (Sakai et al., 1990)), and

deposited on a small strip of aluminium foil. Finally, the strip with the droplets is quickly

plunged into LN, and then stored in a cryovial in a LN container. Warming is carried out

similarly, plunging the strip plus droplets into warm liquid medium containing 1.2 M sucrose

and, after 20 min unloading, shoot tips are retrieved and placed on recovery medium. The

results of this method, in terms of viability, have been good for a variety of systems (Sakai &

Engelmann, 2007; Senula et al., 2007)

The main advantage of this technique is enabling very high cooling and warming rates, due

to the small volume of medium in which the explants are placed (Sakai & Engelmann, 2007).

Many protocols involve exposure of plant shoot apices to PVS2 at 0°C (Sakai et al., 1990;

Reed, 2008). To prevent cellular damage, cryoprotectant solutions both dehydrate and

penetrate cells to stabilize proteins and membranes. However, the mode of action of each

component may differ with cell type, species, temperature, and other solution components

(Dereuddre et al., 1990; Jouve et al., 2004) A particular component of the cryoprotectant

solutions employed, DMSO, enhances chemical penetration into cells and, in spite of its own

cytotoxicity -for which testing the treatment duration is essential- it has been found to

promote survival of cryoexposed cells (Fahy et al., 1984; Benson, 1999; Volk & Walters,

2006).

34


The interplay between exogenous and endogenous carbohydrates plays an important role

on plant resistance to stresses. For example, a two-step preculture treatment of gentian buds

with increasing sucrose concentrations produced a raise of abscisic acid, followed by an

increase of endogenous sucrose concentration, all of which resulting in a higher dehydration

tolerance (Suzuki et al., 2006). Various high-performance liquid chromatography (HPLC)

techniques have been developed to facilitate carbohydrates analyses in an effort to better

understand their role in biochemical processes. High-performance ion chromatography (IC)

coupled with pulsed amperometric detection (PAD) is an efficient method to quantify

carbohydrates in natural samples and food products (Carpenter & Dawson, 1991; Lee, 1996;

Masuda et al., 2001; Schiller et al., 2002; Cheng & Kaplan, 2003).

Differential scanning calorimetry (DSC) is a well-known technique used for studying

thermal properties of thermally driven processes. It has been used for long in cryopreservation

studies, as it yields interesting information on freezing and vitrification processes (Yamada et

al., 1991; Matsumoto et al., 1994; Volk & Walters, 2006; González-Benito et al., 2007;

Zamecnik et al., 2007). Water freezing latent heat is large, so calorimetry is a sensitive

method to monitor ice formation or thawing, even in small quantities. Besides the amount of

ice formed, after knowledge of the system�’s water content, the ratio of frozen-unfrozen water

may be also obtained. DSC yields also Tf, and its reduction speaks of increasing solute

concentrations. TG is also acquired, if scans are performed at sufficiently low temperature,

although DSC is not particularly sensitive for this purpose.

Scanning electron microscopy is a powerful and user-friendly microscopic technique, able

to provide a wealth of structural information on a wide range of samples. When combined

with a cryo-stage (cryo-SEM), it is especially interesting to study the microstructure of

biological systems, not altered in sample preparation (Craig & Beaton, 1996). Low pressure

LN frozen samples preserve most structural relations present in the original sample, with little

or no artefacts. Cryo-SEM allows sample observation without need of prior chemical fixing or

drying. Ice crystals formed are small and not altering tissue structures. Ice formed inside cells

can be observed, after fracture in the cryo-stage and a suitable etching process, as black

�“void�” regions surrounded by grey ridges formed by highly cryo-concentrated solution either

frozen as euthectic, unfrozen or even vitrified.

In this work, these three powerful techniques, DSC, Cryo-SEM and HPLC, have been

applied to follow the behaviour towards LN cooling of specimens of plant germplasm -mint

35


(Menta ×piperitha) shoot tips- in different steps of the droplet-vitrification protocol. To date,

no publication provides data obtained during this cryopreservation protocol, but only of its

final outcome. Actually, very little physical information exists on actual cryopreserved or

vitrified plant specimens. Besides, the combined use of information resulting from these

physicochemical techniques allows us to better understand the phenomena taking place

between the normal and the vitrified states of plant tissues and back, to restore its viability as

germplasm. Interesting relations among sucrose and cryoprotectant content in cells and its

freezing and vitrification behaviour are found, as well as with the other factors ruling over the

physical control of ice formation: water content and cooling rate. The data here presented are

expected to help to design improved cryopreservation procedures, involving less damaging

cryoprotectants and/or suitable for problematic species.

4.2. Materials and methods

4.2.1. Plant material pre-culture and shoot tips extraction

Shoot tips were extracted from in vitro shoots of Mentha ×piperita (genotype �“MEN 186�”

obtained from the IPK Genebank, Gatersleben, Germany). In vitro plants were monthly

subcultured on medium MS (Murashige & Skoog, 1962) with 3% sucrose, and incubated at

constant temperature (25°C) with a photoperiod of 16 h, and an irradiance of 50 µmol m-2 s-1

from fluorescent tubes. One-node segments were obtained from these shoots, transferred to

fresh medium and incubated at alternating temperatures of 25°C (day) and -1°C (night),

always with 16 h photo- and thermoperiod, 50 µmol m-2 s-1irradiance, provided by fluorescent

tubes (stage a, see Figure 4.1). After 3-weeks of culture under these conditions, shoot tips (1-

2 mm) were excised from axillary buds.

4.2.2. Incubation and dehydration of shoot tips

A protocol based in that of Senula et al. (2007) was followed. Five excised shoot tips were

pre-cultured overnight with 2 mL of liquid MS medium containing 0.3 M sucrose at 25ºC,

over filter paper (stage b, see Figure 4.1). Thereafter, the explants were transferred to a Petri

dish with 2 mL of loading solution (2 M glycerol + 0.4 M sucrose) over filter paper, for 20

min, at room temperature (stage c). Finally, they were osmotically dehydrated in 2 mL PVS2

36


(plant vitrification solution 2: 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v DMSO

and 0.4 M sucrose in plant growth medium (Sakai et al., 1990)) in a Petri dish, on filter paper,

for 30 min at 0ºC (stage d). Immersion in LN was carried out in the cryo-SEM or inside the

aluminium pans of DSC, depending on the experiment. A scheme of these steps can be seen at

Figure 4.1.

Figure 4.1. Scheme of the steps of the droplet cryopreservation protocol employed, see text for more details on media and solutions employed.

4.2.3. Plant recovery and viability

Treated and control shoot tips were warmed from LN temperature by plunging into warm

liquid medium with 1.2 M sucrose for 20 min. After the unloading of cryoprotectants, shoot

tips were cultured on re-growth medium, composed of solid MS medium supplemented with

0.5 mg/L BAP + 3% sucrose, cultivated in the dark for 24 h and finally placed into the culture

room at 22-25ºC and 16 h illumination. For control samples (-LN), all steps were carried out,

including the incubation in PVS2 for 30 min. After that, the explants were rinsed in 1.2 M

sucrose solution for 20 min and then placed on the re-growth medium.

Survival and re-growth were calculated as rates over the total number of shoot tips used.

Survival was defined including all forms of visible viability (evidence of green structures or

callus) observed 1-2 weeks after re-warming. Re-growth was defined as the formation of

small plantlets, 4 weeks after re-warming.

37


4.2.4. Low temperature scanning electron microscopy

Low temperature scanning electron microscopy (cryo-SEM) observations were performed

with a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold plate

(Oxford, UK). Three shoot tips in the same stage of the cryopreservation protocol were fitted

on a special bracket with their axes vertically aligned. The geometry of sample and sample

holder required optimization for both freezing and fracturing procedures: these steps require

stable mechanical mounting of the sample on a support. This piece was plunged into liquid

nitrogen (LN) under low pressure, physically fixing tissues for microscopic observation. This

cooling step was considered equivalent to the cooling process taking place in the actual

cryopreservation process, by LN immersion. The holder was, then, inserted in the pre-

chamber of the Cryotrans cold plate, at -180ºC, and specimens were fractured perpendicularly

to their axis, to obtain a suitable observation surface (a cartoon of the tips before introduction

in the microscope and after fracture can be seen in Figure 4.2.

a

b

Figure 4.2. Cartoon showing the three mint tips on the microscopy holder before insertion in the microscope (a) and after equatorial freeze fracture (b).

Later, samples were inserted in the microscope and etching (partial ice sublimation,

induced to provide contrast) was performed for three minutes at -90ºC. Once etched and re-

cooled, the sample was retrieved into the cryo-preparation chamber, and coated with high

38


purity Au, which acts as a conductive contact for electrical charge. The etched and coated

sample was reinstated into the cooled SEM stage and observed using an accelerating voltage

of 15 kV, at -150/-160ºC.

4.2.5. Dry matter and water content determination

Six sets of five tips were used for determining the dry mater content (dm) in shoot tips at

the different stages of the cryopreservation protocol. It resulted from weighing after oven-

drying (~85°C, 72 h) and was expressed as relative to the total tip mass. The water content

(Wc) of shoot tips could be calculated for stages a and b, directly as the difference between its

total mass and dm. It was expressed as relative to the total shoot tip mass (Wc(s)) or the dry

mass (Wc(dm)).

4.2.6. Differential scanning calorimetry

Differential scanning calorimetry (DSC) experiments were performed with a Mettler-

Toledo DSC 30 instrument (Mettler-Toledo, Griefsen, Suiza). Cooling was performed using

the calorimeter control (10°C min-1), or for higher rates, by quenching the samples in LN (see

below). The response of shoot tips apices to each step in the mint protocol was evaluated.

Five shoot tips in the same stage of the cryopreservation protocol were introduced in a DSC

pan, which was sealed and weighed. Shoot tips had been sequentially treated with MS

medium, loading solution and PVS2. Samples were submitted to a cooling scan from room

temperature to -150ºC and a short 5 min equilibration at this temperature, followed by a

warming scan (at 10ºC min-1), back to room temperature. Calorimetric data were collected

from two replicates per treatment.

For the quenching method shoot tips, sealed into a DSC pan, were quickly plunged into

LN. Pans were subsequently cold-loaded into a pre-chilled DSC (-150ºC) for analysis.

Later, pans were punctured and dried in an oven at 85ºC, for 72 hours, when they showed

constant weigh. Thermograms were analysed using the standard procedures provided in the

Mettler-Toledo STARe software. Ice thawing thermal events (more precise than freezing

events), were used for extraction of Tf. The routine produced the temperature corresponding

39


to the event onset, Tf(onset), corresponding to the equilibrium freezing temperature and that to

the peak, Tf(peak).

The equilibrium freezing temperature can be related to the water activity (aw) of the

system, to its osmolality ( ) and to its solute content (Fullerton et al, 1994; Elliott et al.,

2007). For comparative purposes, a �“sucrose equivalent�” concentration (Suceq) and a �“sodium

chloride equivalent�” (NaCleq) were determined from Tf, assuming that the only solute present

were sucrose or sodium chloride, respectively. Tf(peak) data were used for the determination of

these parameters, as it was considered to better represent the behaviour of the whole phase

change. Eq. 1 (Fullerton et al., 1994) was followed for aw determination:

Eq. 1 ln aw = Hf(w) Aw (Tf - Tf *) / (Tf Tf *)

where Hf(w) is the pure water freezing enthalpy (specific, per water gram), Aw water

molecular weight, and Tf * the equilibrium freezing point of pure water. The osmolality was

calculated after eq. 2 (Elliott et al., 2007):

Eq. 2 = Tf /1.86

where Tf is the freezing point depression, which for water at atmospheric pressure, being the

pure substance freezing point 0ºC, equals to Tf with a positive sign.

Suceq and NaCleq could not be calculated from Raoult�’s law, using analytical equations

(Guignon et al., 2008), only valid for more diluted solutions. Experimental data from

literature (Wolf et al., 1985; Fullerton et al., 1994) and calorimetric determinations performed

on concentrated sucrose solutions (data not shown) were employed to relate these hypothetic

solute concentrations with the observed cryoscopic temperature decrease.

The process associated enthalpy, phase change enthalpy Hf, proportional to the event

area, was also obtained. It was expressed as relative to either the total shoot tip mass ( Hf(s)),

its dry mass ( Hf(dm)) or its water content Hf(w), for allowing easier data comparison.

Water fractions: frozen water (Wf) and unfrozen water (Wu), were calculated by

comparison of Hf and the pure water freezing enthalpy ( Hf(w)* = 333.4 J/g at 0ºC), together

with the total water contents previously determined, after eq. 3:

40


Eq. 3 Wf= ( Hf/ Hf(w)*); Wu=Wc-Wf

Specific enthalpy values (per sample gram) were always used, after the corresponding

oven dry pan weighs. Enthalpy values measured at temperatures different from the

corresponding phase change equilibrium temperature, 0ºC, are underevaluated, as a result of

the difference between water and ice heat capacities (Cpw and Cpi, respectively). So, the

value of Hf(w)* was corrected after eq. 4, before applying eq. 3. Single freezing temperatures,

Tf(peak), were used, and Cpw and Cpi data were obtained from a data calculation routine

produced in this laboratory (Otero et al., 2002), after interpolation when required, and other

sources (Angell et al., 1973; Angell et al., 1982).

Eq. 4 - (

Frozen (Wf) and unfrozen (Wu) water contents were expressed as relative to the total shoot

tip mass (Wf(s),Wu(s)), its dry mass (Wf(dm), Wu(dm)) or its total water content, when available,

(Wf(w), Wu(w)).

The glass transition can be observed as a small step in the heat capacity thermogram

baseline. The corresponding heat capacity increment is very small and, in our experiments,

only could be appreciated by using 30 shoot tips in the same DSC pan.

For comparative purposes, calorimetric experiments were performed with samples of the

cryoprotectant solutions employed in each stage of the droplet protocols (i.e., liquid MS

medium with 0.3 M sucrose, loading solution and PVS2). Experiments were performed in the

same conditions than those carried out with shoot tip specimens.

4.2.7. Sucrose content assessment in shoot tips

Specimens were analysed after each stage of the cryopreservation procedure for its sucrose

content. Five shoot tips were weighed (average fresh weight 3.0 ± 0.1 x 10-3 g) and mortar-

ground into a fine powder in LN. As grinding proceeded, the vessel was slowly warmed.

Immediately, 1.5 mL Milli-Q water was added and the mixture was filtered through a syringe-

driven filter (Millex-GS, 0.22 m).

= * )

41


The determination of sucrose was carried out by an Ion Chromatography 817 Bioscan

(Metrohm, Herisau, Switzerland) system equipped with a Metrosep Carb 1-250 column and a

pulsed amperometric detector (PAD) with a gold electrode. A three-step PAD protocol was

used with the following time intervals (ms) and potentials (mV): t1, 400/E1 = +0.05

(detection); t2, 200/E2 = +0.75 (cleaning); t3, 400/E3 = 0.15 (regeneration). Samples (1.5

mL) were injected using an autosampler (model, 838 Advanced Sample Processor, Metrohm,

Herisau, Switzerland) and the flow rate through the column was 1mL min-1, the eluent was

100 mM NaOH.

Identification of sucrose was performed by comparison with the retention time of the pure

standard sucrose (Merck) and the data was acquired using IC Net 2.3 software. Sucrose

content was expressed as ppm and the data were the mean of three replicates. Results (Suc)

are expressed as p.p.m. of the total tip mass.


4.3.1. Detection of glassy state and ice by cryo-SEM

Two different observation approaches were compared: secondary electron and

backscattered electron image. Basically the same information was obtained with both,

however, the cellular structures could be better appreciated using backscattered electrons, and

this was employed in the observations and micrographs here presented.

Figure 4.3 shows micrographs showing shoot tips cooled in LN in the microscope cryo-

unit as described in the Methods section, in different moments of the droplet cryopreservation

protocol: control (A); stage a, cold treatment (B); stage b, preculture (C, D); stage c, loading

(E, F), and stage d, dehydration (G, H). C, E and G show lower magnification images of the

shoot tips axial plane. The concentric cell distribution in the equatorially fractured tips could

be appreciated. There were no noticeable differences among central or more external cells,

indicating that the fracture was close to the apical dome, so effective homogeneity for

temperature and solute and water concentrations could be assumed for tip cells.

Ice crystals formed in the more concentrated solutions were smaller, due to the limitations

to crystal growth caused by the higher solution viscosity. On the other hand, conversion of

water into ice during cooling increased the concentration of solutes in the more concentrated

42


regions. The etching procedure applied gave rise to a contrast in the SEM micrograph,

removing part of the ice formed by sublimation and leaving the darker regions separated by

sector or �“ridges�” of freeze-concentrated solution that can be appreciated in micrographs A-F.

This behaviour allowed visualization of individual crystals. Sublimation is only taking place

at an appreciable rate out of pure ice crystals, while water in glassy state has a sublimation

rate almost negligible (Umrath, 1983; Franks, 1986). So, when solutions become vitrified,

they cannot be etched in this way. Micrographs G and H show a complete lack of the

structural details revealed by etching.

4.3.2. Determination of pre-culture effect by cryo-SEM

Figure 4.3 A and B show, respectively, typical cryo-SEM micrographs after 3 weeks of

culture at 25ºC (control apices) or at 25º/-1ºC. Widespread cellular disruption occurred in

control apices, as it can be observed in micrograph 3 A, where ice can be seen as dark areas

inside the cell�’s cytoplasm and vacuole, meanwhile clearer parts would correspond to the

supercooled cryo-concentrated solution matrix. Cold-treated shoot tips (4.3 B) showed the

formation of smaller ice crystals, mostly in the vacuole space, which indicates a higher solute

cytoplasmic concentration with respect to control apices. The observed size crystal reduction

is understood as due to an increase in the amount of endogenous cryoproctecting agents,

developed by the plant during the cold hardening procedure, confirming the positive influence

of tips cold hardening prior to cryopreservation observed by other workers (Senula et al.,

2007).

4.3.3. Observed plant recovery and viability

Both survival and recovery reached a 96% value for specimens treated with the complete

droplet cryopreservation protocol (i.e., quenched in LN after stage d, dehydration in PVS2).

Meanwhile, survival and recovery of previous stages, a to c, were 0% for all cases. The

control samples (-LN) showed a 100% survival and recovery.

43


A B

C D

E

G

F

H

Figure 4.3. Cryo-SEM micrographs of shoot tips cooled in LN in the microscope cryo-unit (see Methods). Control (A); stage a, cold treatment (B); stage b, preculture (C and D); stage c, loading (E and F), and stage d, dehydration (G and H). The bar corresponds to 10 µm (A, B, D, F, H) or 50 µm (C, E, G).

44


4.3.4. Analysis of the droplet-vitrification protocol steps by cryo-SEM

Micrographs of specimens in the stages b (preculture) (C, D), c (loading) (E, F) and d

(dehydration) (G, H) of the droplet-vitrification protocol can be seen in Figure 4.3.

Specimens in both b and c stages showed a clearly visible tissular and cellular structure, with

some organelles and vacuolar membrane elements present. Specimens in stage b showed

larger ice crystals and a higher degree of alteration of cellular structures by ice than those in

stage c. Tips treated with alternating temperatures (see previous section) had a similar aspect

to those in step b (preculture). The ice crystal size difference could be due to concentration

differences among samples, as the crystals formed in the more diluted solution can grow to a

larger final size, while those occurring in concentrated solutions, where molecular mobility is

impaired, would be smaller.

During the treatment in the loading solution, the cells of specimens in stage c of the

protocol accumulated exogenous glycerol and other solutes of endogenous origin. These cells

were clearly partly plasmolyzed as a result of the osmotic dehydration imposed by the loading

treatment, which was related to the progressive acquisition of tolerance toward freezing

during the protocol.

PVS2, the cryopreservation solution employed in the last step, affected cellular freezing

properties (Volk & Walters, 2006). It has components that are penetrating (dimethyl

sulfoxide, glycerol and ethylene glycol) and non-penetrating (sucrose) for the cellular

membrane of most cell types (Benson, 2008). This complex solution served to dehydrate

shoot tips and to change the behaviour of the water remaining within shoot tips.

Micrographs of specimens in the stage d of the protocol, after osmotic dehydration in

PVS2 solution, presumably with a lower water content and more concentrated in solutes

induced by permeation from the cryopreservation solution, showed a grey continuous, little

detail surface, indicating vitrification (4.3 G and H).

4.3.5. Changes in the dry mass and water content during the droplet-vitrification

protocol

The dry mass and water content of shoot tips, derived of its weight-difference after drying,

are shown in Table 4.1. For step a and b, where water was the only volatile substance

45


(overseeing very small quantities of organic compounds), Wc could be obtained by this

procedure, and is presented in Table 4.1, expressed as related to the total sample mass Wc(s) or

to the dry mass Wc(dm).

The water content of tips undertaked a small decrease with the first steps of the

cryopreservation protocol, associated to sucrose incubation in stage b. Water content for

stages c and d could not be derived from oven-drying data, as the amount of volatile

substances apart from water was high. Nevertheless it could be assumed that there was a large

decrease in the water content associated to step c, as inferred from the increase in dm, which

was in good agreement with the reported microscopic observations showing a smaller ice

crystal size in these samples and with the sucrose content data (see below).

Table 4.1. Compositional and thermal parameters (mean standard deviation) of the thawing events obtained from DSC thermograms for mint shoot tips specimens at different stages (a, b, c and d) of the droplet cryopreservation protocol. Cold treatment

(stage a) Preculture (stage b)

Loading (stage c)

Dehydration (stage d)

dm (gdm gsample-1) 0.117 0.009 0.151 0.009 0.344 0.049 0.252 0.013

Wc(s) (gwater gsample-1) 0.883 0.009 0.849 0.009 -------- --------

Wc(dm) (gwater gdm-1) 7.56 0.65 5.64 0.39 -------- --------

Tf (onset) (ºC) -2.96 0.06 -4.9 1.8 -7.93 0.79 --------

Tf (peak) (ºC) -0.48 0.08 -0.58 0.21 -5.5 1.1 --------

Hf(s) (J g sample

-1) 241 16 209.85 0.12 93.0 7.2 --------

Hf(dm) (J gdm-1) 1800 650 1394 83 274 + 60 --------

Hf(w) (J gw-1) 272 14 247.1 2.5 -------- --------

Wf(s) (gwater gsample

-1) 0.724 0.47 0.632 0.000 0.290 0.023 0 Wf(dm) (gwater gdm

-1) 6.20 0.88 4.20 0.25 0.86 0.19 0 Wf(w) (gwater gwater

-1) 0.820 0.045 0.744 0.007 --------- 0

Wu(s) (gwater gsample-1) 0.159 0.038 0.217 0.009 -------- --------

Wu(dm) (gwater gdm-1) 1.34 0.22 1.44 0.14 -------- --------

Wu(w) (gwater gwater-1) 0.180 0.045 0.256 0.007 -------- --------

aw 0.9954 0.001 0.9944 0.002 0.9470 0.001 ---------

(Osm) 0.26 0.04 0.31 0.12 2.97 0.60 ---------

Stage d, following the same arguments, would show the complex result of water

osmotically driven out of the cell by the strong potential of the very concentrated PVS2

solution, rich in the non-penetrating sucrose, entrance of the penetrating agents DMSO,

ethylene glycol and glycerol, and water retention inside the cell by the increased internal

presence of these cryoprotectors (Schäfer-Menhur et al., 1997; Hubálek, 2005; Panis &

46


Lombardi, 2006; Benson, 2008; Volk & Walters, 2008). Notwithstanding the increasing

presence of endogenous cryoprotecting (and often water binding) agents, as a result of the

plant response to the induced stress.

4.3.6. Measurement of ice heat of fusion using DSC

Figure 4.4 shows typical re-warming DSC thermograms for the same specimens types (at

stages a, b, c and d), previously cooled at a rate of 10ºC min-1. Table 4.1 shows the thermal

parameters derived from DSC experiments: Tf(onset) and Tf(peak), respectively, the onset

temperature of the melting endotherm (corresponding to the equilibrium freezing temperature)

and its peak temperature. The freezing enthalpy ( H) is also included. The information that

both enthalpy and equilibrium freezing temperature can provide is of the highest interest, as it

yields information on the water content within cells and, in a context where it is difficult to

know the detailed cytoplasm composition and interacting effects of endogenous and

exogenous solutes, to draw conclusions on the availability of this water, to form ice, to permit

diffusion-driven processes and even to keep the stability of protein and membranes.

Specimens both in stages b and c of the cryopreservation protocol showed freezing (not

shown) and melting events in the respective cooling and re-warming scans.

The equilibrium freezing temperature is a good raw data to determine accurately solution

characteristics, such as water activity and frozen and unfrozen water (Guignon et al., 2008).

However, the precision yielded by DSC for melting temperatures, especially in complex

systems, such as natural tissues, is limited. The onset temperature is considered usually as the

best estimation of the equilibrium freezing point. But, in complex solutions,

compartmentalized in different isolated pools, with markedly different composition and water

contents (cytoplasm, vacuoles, intercellular space, different cells and tissues) the onset

temperature would rather correspond to the melting of the most concentrated pool, which

would thaw at the lowest temperature.

We have considered that, for these sample types, the peak temperature was giving a better

estimation of the average melting of the specimens studied. Moreover, the DSC-measured

temperature was affected by the thermal differences (as well as the compositional ones)

within the sample, not negligible at a relatively fast scanning speed, as 10ºC min-1.

47


The temperatures determined were decreasing steeply with the progress of the

cryopreservation protocol, which spoke about the increase in the �“number�” solute molecule

concentration. The last stage showed no thawing event, and there would be no frozen water at

all, in these conditions. From Tf, the aqueous solution parameters aw and were derived and

shown in Table 4.1.

Figure 4.4. Typical DSC thermograms corresponding to re-warming processes of shoot tips in different stages of the droplet cryopreservation protocol: cold treatment (stage a), preculture (stage b), loading (stage c) and dehydration (stage d). Each experiment was performed with five shoot tips. The insert shows an expanded thermogram section which allows the glass transition, obtained with 30 tips, to be appreciated. Scanning rate was 10ºC min-1. (See Materials and Methods and Figure 4.1. for more details). The ordinates scale of the hermograms baseline is arbitrary.

t

The thawing enthalpy is, in practice, a much more accurate parameter than freezing

enthalpy, depending less on kinetic, geometric or compartmentalization issues. The phase

change enthalpy, that is presented in Table 4.1 as relative to either the total sample mass,

Hf(s), the dry mass, Hf(dm), or the total water content, Hf(w), decreased as the protocol

progresses. After correction for the different value of the pure water melting enthalpy at the

actual phase change temperature for each of these processes, the amount of thawed water in

48


each process could be calculated, by simple comparison with this quantity. The resulting

frozen water content, presented in Table 4.1 relative to either the total sample mass, Wf(s), the

dry mass, Wf(dm), or the total water content, Wf(w), again markedly decreased as the protocol

progressed. The last step could also be comprised in this trend, as Wf would equal to zero.

The unfrozen water fraction (derived from the frozen water fraction) can help to visualize

the changes undergoing in the cryopreserved tissues, and is also showed in Table 4.4 relative

to the total sample mass, Wu(s), the dry mass, Wu(dm), or the total water content, Wu(w).

Traditionally, frozen water was understood and often named as free water, while the unfrozen

fraction was considered as bound water (Robinson, 1931). These denominations are much

criticised nowadays (Franks, 1986), as the differences found in the actual binding of these two

water fractions are very small, if any at all (Hills et al., 1990; Wiggins, 1990; Hills et al.,

1991; Slade et al., 1991; Hills et al., 1998; Wolfe et al., 2002).

For comparative purposes, Figure 4.5 shows typical calorimetric thermograms obtained

during re-warming for the cryoprotective solutions employed (at stages b, c and d), in the

same conditions than those applied in the experiments of Figure 4.4.

Figure 4.5. Typical DSC thermograms corresponding to re-warming processes of the cryopreservation solutions employed in different starges of the droplet cryopreservation protocol: preculture (stage b): liquid MS medium containing 0.3M sucrose, loading (stage c): loading solution and dehydration (stage d): PVS2. The insert shows an expanded thermogram section which allows the glass transition to be appreciated. Scanning rate was 10ºC min-1. (See Materials and Methods and Figure 14 for more details). The ordinates scale of the thermograms baseline is arbitrary.

49


Hf(s) for stage b (liquid MS medium with 0.3 M sucrose) was 228 ± 2 J g sample-1, while for

stage c (loading solution) it was 138 ± 2 J g sample-1. Stage d (PVS2) showed no melting

exotherm. Hf(w) for stage b amounted to 250 ± 3 J gw-1. The Tf (onset) were -3.97 ± 0.2ºC for

stage b and -4.68 ± 0.3ºC for stage c.

4.3.7. Effects of quench-cooling on heat of fusion of ice

In Figure 4.6, thermograms of the re-warming DSC stage of mint shoot tips, cooled by

quenching are compared with those at a rate of 10ºC min-1 in specimens of step a. It can be

seen that, in the quench cooled sample, the thawing temperature decreased and the enthalpy

increased slightly. These results, though consistent in all repeats and for experiments in

different conditions (data not shown) were unexpected, and of difficult explanation. A

possibility is that the fast cooling might trap solute molecules within the ice phase, which

would later contribute to an average effective melting at lower temperatures (Obbard et al.,

2003; Ohnoa et al., 2005; Hashimoto et al., 2011).

Figure 4.6. DSC scans showing warming at 10ºC min-1 for shoot tips treated at the preculture stage (b) of the droplet cryopreservation protocol. Cooling rate was 10ºC min-1 or quenching (direct immersion of the DSC pan in LN).

50


Nevertheless, this behaviour, although may be further from equilibrium than the observed

in the slow cooling, would correspond more closely to the real events taking place in

cryopreservation practice.

4.3.8. Detection of glass transition by DSC

Specimens at the last stage of the cryopreservation protocol did not show any freezing

event, in any case, either at the cooling (not shown) or melting runs of DSC studies.

Nevertheless, in many experiments, the step in the heat capacity baseline showing the actual

glass transition in DSC thermograms could not be appreciated. Cooling rates of 10ºC min-1,

controlled by the calorimeter itself, or externally driven, by quick introduction into LN

(quenching) were tested, but the glass transition could only be observed (for both cooling

rates) for samples containing 30 shoot tips. Calorimetry is often claimed to be a rather

insensitive method to observe the glass transition (Verdonck et al., 1999), and this is

especially applicable here, as the dehydrated tips have a very small weight (total average

sample mass for each DSC experiment -5 tips- for d-stage specimens was approximately 3.5

mg and the total mass of water in these samples 2.5 mg).

As shown in the insert of Figure 4.5, PVS2 solution is also showing a glass transition in

these conditions, centred at -120ºC.

4.3.9. Changes in sucrose content during the droplet-vitrification protocol

The actual composition of the cellular contents in cryopreservation conditions is critical for

the formation of ice or the vitrification of the system. Most studies rather equate this

composition (or the physical behaviour of the cytoplasm) to that of the cryopreservation

solutions applied, which is not necessarily correct. A simple extraction procedure and a

general chromatographic method for small carbohydrate molecule quantification calibrated

for sucrose were applied, as a first approach to work out this composition. The sucrose

content data obtained are presented in Table 4.2, expressed as related to the total sample

mass, Suc(s), or to the dry mass, Suc(dm), reflecting the increase in sucrose content within

tissues, not only as a result of the actual increase of the content of this compound, but of the

decrease in that of water.

51


There was a strong increase in its level between stages b and c (Table 4.2). Meanwhile the

sucrose incubation of the preculture leading to stage b only implied a moderate increase

(about 10%) of the internal sucrose level, the loading with glycerol gave rise to a dramatic

increase of this concentration (7 fold). The stage d, dehydration, after PVS2 treatment,

underwent a further increase, but much more moderate. This could be interpreted as a

reflection of the actual cellular water content, and was in good agreement with the data of

water contents and its frozen and unfrozen fraction showed in Table 4.1.

Other sources for changes in sucrose levels, different from the alteration in the water

content, were possible, such as the actual penetration of sucrose, which could be facilitated by

the presence of DMSO in the PVS2 solution, an agent known for promoting the penetration of

other solutes through membranes (Sakai & Engelmann, 2007).

Equivalent solute concentrations (isoosmotic) can be obtained from the freezing

equilibrium temperatures, obtained by DSC (Buckley et al., 1958). The observed decrease in

this temperature can be related by Raoult�’s law to the molal concentration of the solution. In

the case of tissues it would rather be an average of the different solution pools in the different

cell types and sub-cellular compartments of the specimens studied. The analytical equations

are only valid for solutions much more diluted than those considered here, and their

application would overestimate the solvent concentration required to induce the observed

cryoscopic decrease. The Tf(peak) obtained were, so, compared with tabulated data sets,

complemented when necessary with experimental DSC determinations for concentrated

solutions (data not shown). Equivalent sucrose concentration, Suceq, and equivalent sodium

chloride concentrations, NaCleq, were calculated and presented in Table 4.2. These coarse

estimations could not describe the complex composition of cellular fluids. Nevertheless, as

the observed decreases in Tf were caused by the increased number of molecules in solution,

the amount of small molecular weight compounds (either dissociated, as NaCl, or non-

dissociated, as sucrose), would have a more determinant role than the presence of larger

macromolecules and other specific plant-generated substances.

When comparing the chromatographic sucrose content with these isoosmotic solute

estimated concentrations, it could be seen that the total solute content of cellular

compartments must be much higher than the sucrose amount reported. Especially, a high

increase in the estimated solute concentrations could be appreciated after incubation with the

loading solution, may be reflecting the entrance of significant amounts of glycerol in cells,

52


apart form the already commented dehydration. It must be noted, when comparing Table 4.2

parameters that, while the sucrose content chromatographically determined refers to the total

or dry sample mass of the shoot tips, the estimated sucrose and sodium chloride contents, as

originated from the cryoscopic decrease, refer to the solution mass (only considering solutes

and solvent).

Table 4.2. Solute content (mean standard deviation) of mint shoot tips at the different stages of the droplet cryopreservation protocol. Cold

treatment (stage a)

Preculture (stage b)

Loading (stage c)

Dehydration (stage d)

Suc(s) (p.p.m. total

sample weight) 5900 600 6500 300 46900 700 53000 4000

Suc(dm) (g gdm

-1) 0.059 0.006 0.044 0.002 0.137 0.002 0.209 0.015

1Suceq (p.p.m.) 8800 10800 470000 ---------

1NaCleq (p.p.m.) 8000 10000 91000 ---------

1These concentrations, as derived from the freezing point depression, refer to the solution mass (mass of solutes plus mass of solvent), not taking into consideration any non-dissolved substances.

4.4. General discussion

The combined results of DSC and cryo-SEM are in good agreement, implying that part of

the water contained in the system for specimens on steps a and b becomes frozen when

cooled in LN (with less amount of ice formation in the latter) while specimens in step c would

avoid ice formation, presumably getting vitrified in the cooling process, as they showed no ice

trace, either microscopically or calorimetrically. This is also in good agreement with our data

for viability and recovery and those of literature, resulting of applying the droplet method to

several systems, including mint apices (Keller & Dreiling, 2003; Halmagyi et al., 2005;

Leunufna & Keller, 2005; Panis et al., 2005; Kim et al., 2006a; Kryszczuk et al., 2006;

Senula et al., 2007; Sant et al., 2008; Halmagyi et al., 2010; Vujovic et al., 2011).

The different experimental approaches employed agree in a reduction of the cellular water

content with the cryopreservation protocol progress. The mayor dehydration would take place

during the loading solution incubation (stage c, showing much lower water content than b).

This agrees well with the dry weight evolution, as well as the calorimetric frozen water

content and the observed decrease of the equilibrium freezing temperature. The decrease in

53


the size of ice crystals observed by cryo-SEM and the increases in the sucrose content

obtained by ionic chromatography would confirm this result.

Regarding the last and most interesting stage d, there is less information available, as on

one hand, the presence of large quantities of volatile substances precludes the measurement of

a reliable water content by differential weighing, and on the other, there were no freezing

event observed that could yield information about water fractions or cryoscopic decrease.

Nevertheless, the sucrose content data could imply a further dehydration degree over stage c,

unless a significant amount of the sucrose contained in PVS2 solution could have penetrated

cells facilitated by DMSO. The evidences of vitrification derived of the cryo-SEM

observations and the glass transition detected in DSC (as well as the very lack of freezing or

thawing events at any scanning rate) justify a higher cytoplasm viscosity in stage d than in

stage c specimens, evidently not vitrified. This viscosity increase could be originated either by

a further water content reduction or by the entrance in cells of the viscosity enhancing PVS2

components (or both).

The incubation with glycerol, a known penetrating agent (Schäfer-Menhur et al., 1997;

Hubálek, 2003; Volk & Walters, 2006; Panis & Lombardi, 2006; Benson, 2008) has been

reported to promote water exit from cells, driven by the osmotic gradient, but also a

substitution of part of this water by glycerol inside cells (Benson, 2008). The comparison of

total shoot tips specimen and dry mass (Table 4.1) with water content data from other

workers (Volk & Walters, 2006), whose mint shoot tip protocol is not equivalent but similar

enough, allow calculate that for stage c specimens, at a water content of approximately 1.8

gwater gdm-1, the amount of glycerol possible incorporated to shoot tips would be small (about

0.1 gwater gdm-1). While in this case most of the effect of the protocol step would be, then,

simple osmotic dehydration, the same type of calculation applied to stage d yields, for a water

content of approximately 0.7 gwater gdm-1, an entrance of extrinsic compounds corresponding to

nearly 60% of the final specimen mass (about 2.3 gwater gdm-1).

A general reflection on these data may acknowledge the natural variability of the

properties here determined. In spite of the efforts to prepare equivalent start point materials

and to treat them in a similar way, differences in size, permeability, microscopic scale damage

and all short of small compositional and microstructural differences would exists, in a way not

easy to monitor. Comparison with other similar experiments (data not shown), it can be

observed that this variability would have a larger impact on the equilibrium freezing

54


temperature data, while the enthalpy and associated water fractions would be more constant.

And, an absolute observation is that for all repeats, sets of experiments, and even cooling rates

applied, the final stage of the cryopreservation protocol is never producing calorimetrically

detectable formation of ice. This is in good agreement with the viability results found for this

protocol (Senula et al., 2007), viability which is incompatible with the formation of ice.

The calorimetric properties (melting enthalpies and equilibrium freezing and glass

transition temperatures) of the cryoprotecting solutions are not too different of those of the

specimens treated with them. In addition to some differences being evident, this calorimetric

similarity cannot be taking as a proof of equal composition and/or behaviour in other respects.

The probabilities of ice formation in specimens under TG and over TG but at temperatures

not too elevated (roughly, no more than 20ºC over TG) are similar, if a short frame time is

considered, such as those corresponding to cooling or warming processes in cryopreservation.

But this may be not so for longer time periods, such as those corresponding to storage.

Although storage is normally carried out below -150ºC, well under these systems TG, it must

be remembered that, when over TG, at long storage periods, ice formation is always a real

possibility (Angell, 1982; Franks, 1986).

A further consideration can be derived from the lack of ice observations in DSC at the last

stage of the cryopreservation protocol, when the cooling is carried out at 10ºC min-1, a rather

slow speed. This, generally observed for this samples and this protocol (data not shown),

would be indicative of a possible excessive amount of cryoprotecting agents, although, for the

biological material here employed, we find that there is no appreciable damage, at least in

terms of viability and recovery. Cooling at 10ºC min-1 would mean that the samples are in the

ice formation window (roughly, without considering the differences in ice formation

probabilities for different temperature regions, between -20ºC to -110ºC), for about 9-10 min.

Quench cooling would mean a permanence in this ice formation possible temperature window

for only a few seconds (data not shown). Considering only the effects of the cryopreservation

protocol in the formation of ice (not valuating here the possible membrane or protein

protection towards low temperature or dehydration, for example) a reduction in the

concentration of the vitrification solution employed could produce specimens that may be ice-

formation prone at low cooling rates but, would not form ice at quenching rates, which are

those actually used, in practical cryopreservation. This observation should be taken into

consideration, especially when the effect of some of the cryoprotecting components of these

55


solutions, such as DMSO, are criticised for its possible mutagenic or cytotoxic effects

(Harding, 2004).

56

5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC

5. CRYOPRESERVATION OF MINT SHOOT TIPS BY

ENCAPSULATION-DEHYDRATION: DETERMINATION OF

THE GLASSY STATE BY CRYO-SEM AND DSC

5.1. Introduction

Mint is an economically important crop presenting, together with its related wild species,

both types of material susceptible to be preserved as germplasm: seeds and vegetative

material. Although the initial studies on mint were mainly focused to its chemical

composition, cultivation systems and essential oil extraction and product quality, currently the

preservation, especially long term, of mint genetic resources is getting an outstanding

attention. This trend is comprised in the current general interest for preservation and the boost

of cultivation of medicinal and aromatic plants. Mint belongs to the genus Mentha L.

(Lamiaceae), a plant native of mild climatic areas but showing a cosmopolitan character

(Towill, 2002).

Cryopreservation offers long-term storage capability, maximal stability of phenotypic and

genotypic characteristics of stored germplasm, minimal storage space and maintenance

requirements, and has been considered to be an ideal means for long-term conservation of

genetic resources of vegetatively propagated plants (Engelmann, 1997, 2000; Shibli, 2000). It

is usually performed in liquid nitrogen (LN) at -196ºC, and it is the only method currently

available ensuring the safe, efficient, and cost-effective storage of germplasm of many plant

species. Cryopreservation results in arrested metabolic and biochemical processes, such as

cell division and growth (Towill, 1996; Engelmann, 2000). Other biophysical processes

leading to protein and nucleic acid degradation would also be halted. Thus, the plant material

can be stored without deterioration or modification for unlimited periods (Ashmore, 1997),

maintaining its genetic stability and regeneration potential (Rajasekaran, 1996; Matsumoto et

al., 1998; Towill, 2002).

To be successful, cryopreservation must avoid the formation of ice crystals inside cells

during immersion in LN. This is achieved by reducing cell water content prior to rapid

cooling. Therefore, treatments resulting in low cell water content without causing cell death

are key factors for successful cryopreservation (Matsumoto et al., 1998). Several pre-

57 57


treatments, including cold acclimation, exposure to ABA, immersion in concentrated sugar

solutions, and extensive dehydration, have been reported to enhance the survival after

cryopreservation in plant cells and tissues (Shibli et al., 1999; Tahtamouni & Shibli, 1999;

Shibli, 2000). The result is an increase of the cytoplasmic microviscosity, slowing the water

reorganization process required for ice nucleation. Viscosity, which is also increasing steeply

as the system temperature decreases, can reach such high values that movement is virtually

stopped. In this situation, named glassy or vitreous state, most chemical reactions and

physical processes are considered to be detained, and ice formation is deemed as impossible

(Sakai & Engelmann, 2007).

Cryopreservation techniques include vitrification (Moukadiri et al., 1999), encapsulation-

vitrification (Hirai & Sakai, 1999), and encapsulation-dehydration (González-Arnao et al.,

1996, 1999; Sakai et al., 2000; Shibli et al., 1999: Shibli, 2000). Among these methods,

encapsulation-dehydration is widely used because it is applicable to many species (Shibli,

2000). Encapsulation-dehydration cryopreservation methods, used with a variety of plant

germplasm (Rao et al., 1998), are based on the successive osmotic and evaporative

dehydration of plant cells, previous to the LN cooling step (Swan et al., 1999). Dehydration

techniques allow more flexibility when handling large sample numbers because the

processing is less time-critical than the vitrification techniques sensu stricto (Sakai et al.,

2000). Encapsulation-dehydration also avoids the use of toxic cryoprotectants as compared to

other methods (Shibli et al., 2006).

DSC has been frequently employed to study ice freezing and vitrification (Rajasekaran,

1996; Ashmore, 1997). Calorimetric data include information on the amount of frozen and

unfrozen water, as well as a clear proof of vitrification and its corresponding glass transition

temperature, TG. Thermal analysis of samples during pre-treatment prior to cryopreservation

in LN has only been employed previously to examine the processes occurring in alginate-

encapsulated Ribes nigrum shoot tips (Benson et al., 1996). The larger volume of alginate

relative to the encapsulated sample, together with the physical changes and processes within

the bead itself during pre-treatment and cryopreservation, may exert a significant influence on

the success of the cryopreservation protocol, and hence on the survival of plant samples.

In spite of the recognized importance of performing both cooling and warming steps

extremely fast for success of the cryopreservation process (Wesley-Smith et al., 2001), there

have been no previous reports of the measurement of cooling and warming rates for alginate

58 58


encapsulated samples. The closer data available refer to other plant cryopreservation protocols

(Kaczmarczyk et al., 2011; see Chapter 3).

Low-temperature scanning electron microscopy (cryo-SEM) allows observation of

microstructural features with no alteration due to preparation procedures, such as fixation and

dyeing (Craig & Beaton, 1996). Intra- and extracellular ice can be observed, after fracture and

etching, and be differentiated from vitrified tissues.

The aim of this paper is to study the effects of different dehydration times of alginate beads

on mint shoot tip viability (survival and re-growth) after cryopreservation and on the water

content and vitrification events, the latter studied using thermal analysis techniques (DSC)

and cryo-SEM.


5.2.1. Plant material and shoot tips extraction

Plant material was prepared as described elsewhere (Teixeira et al., 2013). Shoot tips were

extracted from in vitro shoots of Mentha ×piperita (genotype �“MEN 186�” obtained from the

IPK Genebank, Gatersleben, Germany). In vitro plants were monthly subcultured on medium

MS (Murashige & Skoog, 1962) with 3% sucrose, and incubated at constant temperature

(25°C) with a photoperiod of 16 h, and an irradiance of 50 µlmol m-2 s-1 from fluorescent

tubes. One-node segments were obtained from these shoots, transferred to fresh medium and

incubated at alternating temperatures of 25°C (day) and -1°C (dark), always with 16 h photo-

and thermoperiod, 50 µlmol m-2 s-1irradiance, provided by fluorescent tubes. After 3-weeks of

culture under these conditions, shoot tips (1-2 mm) were excised from axillary buds.

5.2.2. Shoot tip cryopreservation procedure

Ten excised shoot tips were precultured overnight with 2 mL of liquid MS medium

containing 0.3M sucrose at 25ºC, over filter paper (Figure 5.1). Thereafter, the explants were

placed in 3% (w/v) low viscosity alginic acid (Sigma, USA) in liquid MS without calcium, at

pH 5.7. Beads were formed by suspending shoot tips in alginate and dripping them, with the

help of a Pasteur pipette, into a calcium chloride solution (MS medium with 100 mM CaCl2

59 59


and 0.4 M sucrose). Beads were allowed to polymerize for 30 min. Encapsulated shoot tips

were cultured in liquid MS medium with 0.75 M sucrose on a rotary shaker (130 rpm)

overnight. Beads were blotted dry on sterile filter paper, transferred to an open glass Petri dish

and dehydrated for 1, 2, 3, 4, 5 and 6 h in a laminar-flow hood. Dehydrated beads (30 per

treatment) were placed in 1.0 mL cryovial (10 beads per cryovial) and plunged into NL

(Figure 1). The cryovials were rewarmed in water bath at 45ºC for 1 min and then at 25ºC for

one more minute (Uchendu & Reed, 2008). Subsequently, beads were placed on the re-growth

medium (MS, with 3% sucrose and 0.5 mg/L BAP), incubated in the dark for 24 h and

thereafter under 16 h photoperiod, at 25ºC. For control samples (-LN), all steps were carried

out except immersion in LN and rewarming.

Survival and re-growth were calculated as percentages over the total number of beads used,

4 weeks after culture. Survival was defined including all forms of visible viability (evidence

of green structures or callus). Re-growth was defined as the formation of small plantlets.

Three replicates (of 10 beads each) were used per treatment. Data were subjected to analysis

of variance and Duncan�’s Multiple Range Test (alpha = 0.05) was used for comparison of

means.

Figure 5.1. Scheme of the steps of the encapsulation-dehydration protocol employed, see text for more details on media and solutions used.

5.2.3. Water content determination

Four sets of three specimens were used for determining the dry mater content (dm) of

either shoot tips (control and after 0.3 M sucrose incubation step) or beads after the different

drying times. It resulted from weighing after oven-drying (~85°C, 72 h). The water content

(Wc) was calculated as the difference between the total mass and dm. It was expressed as

relative to the fresh weight mass (Wc(s)) or the dry mass (Wc(dm)).

60 60


5.2.4. Differential scanning calorimetry

DSC experiments were performed with a Mettler-Toledo DSC 30 instrument (Mettler-

Toledo, Griefsen, Suiza). Cooling was performed using the calorimeter control (10°C min-1),

or for higher rates, by quenching the samples in LN (see below). The response of beads after

each step of the cryopreservation protocol was evaluated; three beads were included in a DSC

pan, which was sealed and weighed. Samples were submitted to a cooling scan from room



from two replicates per treatment. For study of the quenching method, beads, sealed into a

DSC pan, were quickly plunged into LN. Pans were subsequently cold-loaded into a pre-

chilled DSC (-150ºC) for analysis. Later, pans were punctured and dried in an oven at 85ºC,

for 72 hours. Thermograms were analysed using the standard procedures provided in the

Mettler-Toledo STARe software. Ice thawing thermal events (more precise than freezing

events), were used for determination of Tf. The routine produced the temperature

corresponding to the event onset, Tf(onset) (corresponding to the equilibrium freezing

temperature) and the peak temperature, Tf(peak).

The melting enthalpy Hf, proportional to the event area, was also obtained. It was

expressed as relative to either the total sample mass ( Hf(s)), its dry mass ( Hf(dm)) or its water

content Hf(w), for allowing easier data comparison. Water fractions: frozen water (Wf) and

unfrozen water (Wu), were calculated by comparison of Hf and the pure water freezing

enthalpy ( Hf(w)* = 333.4 J/g at 0ºC), together with the total water contents previously

determined, after eq. 1:

Eq. 1 Wf= ( Hf/ Hf(w)*); Wu=Wc-Wf


oven dry pan weighs. Enthalpy values measured at temperatures different from the

corresponding phase change equilibrium temperature, 0ºC, are underevaluated, as a result of

the difference between water and ice heat capacities (Cpw and Cpi, respectively). So, the

value of Hf(w)* was corrected after eq. 2, before applying eq. 1. Single freezing temperatures,

Tf(peak), were used, and Cpw and Cpi data were obtained from a calculation routine produced

61 61


in this laboratory (Otero et al., 2002), after interpolation when required, and other sources

(Angell et al., 1973, 1982).

Eq. 2 - (

Frozen (Wf) and unfrozen (Wu) water contents were expressed as relative to the total

sample mass (Wf(s),Wu(s)), its dry mass (Wf(dm), Wu(dm)) or its total water content (Wf(w),

Wu(w)).

5.2.5. Temperature measurement and cooling and warming rates

Thin T-type thermocouples (copper-constantan) (TC Medida y Control, S.A., Madrid,

Spain), with good performance at LN temperatures, were employed to measure cooling and

warming rates, using a fast data acquisition equipment MW100-UNV-H04 (Yokogawa,

Tokio, Japan), with a frequency of 20 data per second, basically as described previously (see

Chapter 3). Liquid nitrogen and ice were used as controls (-195.8 and 0.2ºC, respectively),

calibration being performed frequently between measurements. Cooling and warming rates

were defined as the ratio of a predetermined temperature interval T1-T2, over the time elapsed

between the reported measurement of T1 and T2. The determination of cooling and warming

rates was carried out, at least in triplicate, during the cooling and warming phases of the

described protocols. Thermocouples were calibrated as a part of the measurement routine.

A cartoon of the experimental set employed for cooling and warming rate determination is

shown in Figure 5.2. Cryovials, containing 10 alginate beads, were handled by means of the

attached thermocouple wire, with isolated tongs, to reduce heat losses. For cooling rate

determination cryovials were tempered at 1-4ºC before immersion in LN, and data collection

took place from 0 ºC till near -196ºC. For warming, cryovials were immersed and stirred in a

40ºC water bath (for 1 min) followed by immersion and stirring in another bath at 25ºC (for 1

min). Measuring took place from the exit from LN till room temperature equilibrium. Rates

were calculated after adopting a suitable ice risk temperature window.

= * )

62 62


Figure 5.2. Experimental set up for thermal change rate measurement for encapsulation-dehydration.


Low temperature scanning electron microscopy (cryo-SEM) observations were performed,

basically, as previously described (Teixeira et al., 2013), with a Zeiss DSN 960 scanning

microscope equipped with a Cryotrans CT-1500 cold plate (Oxford, UK). Cryo-SEM allows

sample observations without the need of prior chemical fixing or drying processes.

Three shoot tips or beads containing tips in the same stage of the cryopreservation protocol

were fitted on a special bracket with their axes vertically aligned (a cartoon of the tip-

containing beads, before placing inside the microscope and after fracture can be seen in

Figure 5.3), and this piece was immersed into liquid nitrogen (LN) under low pressure,

physically fixing tissues for microscopic observation. This cooling step was considered

equivalent to the cooling process taking place in the actual cryopreservation process, by LN

immersion. The holder was, then, placed in the pre-chamber of the Cryotrans cold plate, at

-180ºC, and specimens were fractured perpendicularly to their axes, to obtain a suitable

observation surface. Later, samples were inserted in the microscope and etching (partial ice

sublimation, induced to provide contrast) was performed for three min at -90ºC. Once etched

and re-cooled, the samples were retrieved into the cryo-preparation chamber and coated with

high purity Au, which acts as a conductive contact for electrical charge. Finally, the etched

and coated samples were reinstated into the cooled SEM stage and observed under secondary

electron mode, at -150/-160ºC, using an accelerating voltage of 15 kV.

63 63


Figure 5.3. Cartoon showing the three mint tips-containing beads on the microscopy holder before insertion in the microscope (a) and after equatorial freeze fracture (b).

The initial water content of beads was lower than that of precultured tips, as a result of the

culture of beads in 0.75 M sucrose. For increasing drying times water content decreased

asymptotically, with the highest water reduction taking place between 1 and 3 hours of drying

time. Longer drying periods were associated to slight or non-significant water content

variations, when compared with the experimental error. An associated bead size decrease and

hardening could also be observed.

The dry mass (dm) and water content of shoot tips (control and 0.3 M sucrose precultured

tips) and beads containing shoot tips (after different drying times: 0 to 6 hours), derived from

weight-difference after extensive drying, are shown in Table 5.1. Water content was

expressed in relation to the total sample mass (Wc(s)) and the dry mass (Wc(dm)). The water

content of tips underwent a small decrease with the preculture in medium with 0.3 M sucrose.

5.3.1. Water content reduction during the drying period


a

b

64 64


65 65

Table 5.1. Compositional and thermal parameters (mean standard deviation) of the thawing events obtained from gravimetry and DSC thermograms for mint shoot tips specimens at different stages of the encapsulation-dehydration cryopreservation protocol. Preculture Dehydration (h) Control 0.3M suc. T0 T1 T2 T3 T4 T5 T6 dm (gdm gsample

-1) 0.12 ± 0.01 0.18 ± 0.10 0.25 ± 0.01 0.35 ± 0.02 0.57 ± 0.02 0.77 ± 0.12 0.77 ± 0.01 0.78 ± 0.01 0.79 ± 0.01 Wc(s) (gwater gsample

-1) 0.88 ± 0.01 0.82 ± 0.01 0.75 ± 0.08 0.66 ± 0.20 0.43 ± 0.02 0.23 ± 0.12 0.27 ± 0.13 0.22 ± 0.01 0.21 ± 0.01 Wc(dm) (gwater gdm

-1) 7.30 ± 0.60 4.70 ± 0.40 3.00 ± 0.10 1.90 ± 0.10 0.74 ± 0.06 0.30 ± 0.20 0.30 ± 0.01 0.28 ± 0.02 0.27 ± 0.02

Tf (onset) (ºC) -3.10 ± 0.30 -3.80 ± 0.06 -8.90 ± 0.30 -12.50 ± 1.90 -28.20 ± 0.40 --- --- --- --- Tf (peak) (ºC) 4.10 ± 0.60 -0.17 ± 0.06 2.40 ± 0.80 -3.00 ± 4.00 -12.60 ± 3.10 --- --- --- ---

Hf(s) (J g sample

-1) 237 ± 8.00 210 ± 11.00 180 ± 3.00 148 ± 10.00 64 ± 3.00 --- --- --- --- Hf(dm) (J gdm

-1) 1940 ± 160.00 1200 ± 150.00 714 ± 33.00 420 ± 50.00 112 ± 9.00 --- --- --- --- Hf(w) (J gw

-1) 264 ± 1.00 255 ± 9.00 242 ± 3.00 228 ± 10.00 151 ± 1.00 --- --- --- --- Wf(s) (gwater gsample

-1) 0.69 ± 0.01 0.63 ± 0.03 0.540 ± 0.01 0.46 ± 0.04 0.21 ± 0.01 --- --- --- --- Wf(dm) (gwater gdm

-1) 5.80 ± 0.50 3.50 ± 0.20 2.14 ± 0.09 1.30 ± 0.20 0.37 ± 0.02 --- --- --- --- Wf(w) (gwater gwater

-1) 0.79 ± 0.01 0.77 ± 0.02 0.73 ± 0.02 0.70 ± 0.05 0.49 ± 0.01 --- --- --- ---

Wu(s) (gwater gsample-1) 0.19 ± 0.01 0.19 ± 0.02 0.21 ± 0.01 0.20 ± 0.02 0.22 ± 0.01 0.30 ± 0.20 0.30 ± 0.01 0.28 ± 0.02 0.27 ± 0.02

Wu(dm) (gwater gdm-1) 1.50 ± 0.20 1.20 ± 0.02 0.86 ± 0.05 0.60 ± 0.05 0.37 ± 0.03 0.30 ± 0.20 0.30 ± 0.01 0.28 ± 0.02 0.27 ± 0.02

Wu(w) (gwater gwater-1) 0.21 ± 0.05 0.23 ± 0.02 0.27 ± 0.01 0.30 ± 0.06 0.51 ± 0.01 1.00 1.00 1.00 1.00

TG (ºC) --- --- --- --- --- -68.70 ± 0.30 -51.00 ± 2.00 -43.00 ± 2.00 -44.00 ± 2.00


5.3.2. Calorimetric studies on water status

Calorimetric measurements were carried out for shoot tips (control and precultured) and

tip-containing beads for 0 to 6 hours drying times. Figure 5.4 shows typical scans obtained

for beads at the different drying times. Inserts help visualizing the small glass transitions,

when present. The parameters derived from DSC data analysis (melting exotherm

temperatures and enthalpies and the derived frozen water contents) appear in Table 5.1.

Clearly, samples could be divided in two sets, corresponding to higher and lower water

contents. Experiments with naked shoot tips and for beads desiccated for 0, 1 or 2 hours

presented a melting exotherm, implying that part of the water in these systems became frozen

upon cooling. Meanwhile, samples at drying times 3 to 6 hours showed no trace of melting

events, meaning no water was frozen. Instead, these samples exhibited glass transitions,

proving these beads became vitrified upon cooling.

Figure 5.4. Typical DSC thermograms corresponding to re-warming processes of shoot tips with beads of the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar-flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h and T6 = 6 h. Data are the mean of at least three repeats. Each experiment was performed with three beads. The inserts show an expanded thermogram section which allows seeing the glass transition. Scanning rate was 10ºC m-1. The ordinates scale of the thermograms baseline is arbitrary.

66


Data analysis (Table 5.1) allowed drawing a perfect parallelism between water content and

the calorimetric behaviour. The temperatures of the melting process (when taking place) were

decreasing as water content did, showing that the solute concentration was increasing. The

area of the curves, i.e., the melting enthalpy, was also decreasing with water content, as well

as the derived frozen water content. While Wf(w) for control tips was approx. 80 %, this

amount decreased to nearly 50% in beads, after 2 h desiccation, which was the longer drying

time showing freezing processes. This, added to the approx. 40% reduction in total water

content at T2, implies that the mass of frozen water was only 20% of the sample total mass.

The ratio of frozen to unfrozen water decreased from control and precultured tips and with

increasing drying times, to reach a value of 1 at the highest drying time showing a freezing

event, i.e. 2 h desiccation (Figure 5.5).

Glass transition temperatures (Table 5.1) showed an increase with the progress of the

drying process. It increased from 3 h to 5 h desiccation, while there was no difference from 5

to 6 h, which corresponded to the very small or constant water content reduction at these

times.

Figure 5.5. Ratio Wf(dm) Wu(dm)-1 for mint shoot tips (control and precultured on 0.3 M

sucrose) and shoot tip-containing beads (for drying times 0 to 6 hours). Bars: standard error.

Beads and tips studied by DSC in a similar way but after LN quench cooling presented a

very similar behaviour (data not shown), but their freezing events displaced to lower

67


temperatures. Three to 6 h dried beads showed no freezing processes and the glass transitions

observed in these cases were unchanged, within experimental error.

5.3.3. Cooling and warming rate determination

An ice-risk formation window of 0ºC to -150ºC was considered in this work, for cooling

and warming rate calculation. This window comprises the regions were ice formation can take

place, though with a very low probability or in a very long time (i.e., in very slow cooling or

warming processes). The selected window is widely over-dimensioned compared to the actual

ice formation risk for cryopreserved systems, as its equilibrium freezing temperature is always

well under 0ºC, due to its high solute and low water contents. Per contra, the glass transition

temperature, under which the possible ice formation area ends, although highly variable, tends

to be located well over the -150ºC limit. Other narrower windows would yield higher rates

(see Chapter 3), while considering the duration of the process all the way down to LN

temperature (-196ºC) would yield meaningless and exceedingly long times.

Cooling (Figure 5.6) and warming (Figure 5.7) processes followed an approximately

symmetrical behaviour with not too strong differences among samples. It must be noted that

natural differences in samples and manipulation variations during normal cryopreservation

procedures were responsible for a higher dispersion in data (see Chapter 3).

The cooling rates obtained with beads dried for different times (Table 5.2) showed an

increase easy to be related to the water content decrease (Table 5.1). Rates could be related to

the samples physical characteristics: mass, heat capacity and thermal conductivity. Besides

the dependence with mass, larger water contents imply larger global heat capacities. In this

case, both sample mass and its heat capacity decreased with drying time. Heat conductivity

also plays a role in determining cooling and warming rates. Factors as the position of beads in

the cryovials and the contact between the wall and beads are important. These thermal

contacts are favoured by the higher water content on beads.

The larger variations in rates were found between 2 and 3 hours drying time, and at longer

periods the variation was quite small, also correlating with the asymptotic water content

reduction. Warming rates behaved in a similar way, although the observed warming rates

were smaller than the cooling ones.

68


Table 5.2. Cooling and warming rate measurements obtained following the encapsulation-dehydration protocol.

Cooling Warming

Sample Rate

(ºC min-1)

Time

(s)

Rate

(ºC min-1)

Time

(s)

Sample temperature at

the end of protocol3

(ºC)

Control 3.83 ± 0.60 39.90 ± 6.65 3.41 ± 0.30 44.28 ± 3.80 9.93 ± 1.20

T0 1.64 ± 0.10 91.83 ± 5.60 1.28 ± 0.01 111.87 ± 0.08 -7.30 ± 0.70

T1 1.60 ± 0.04 94.02 ± 2.10 1.27 ± 0.01 110.87 ±1.12 -9.80 ± 1.35

T2 1.83 ± 0.08 82.10 ± 3.55 1.29 ± 0.01 112.93 ±0.14 -3.93 ± 0.85

T3 2.78 ± 0.11 53.93 ± 2.16 1.97 ± 0.02 76.08 ± 0.81 8.50 ± 0.28

T4 2.47 ± 0.13 60.93 ± 3.23 1.95 ± 0.08 77.25 ± 3.20 8.13 ± 0.57

T5 2.76 ± 0.04 54.40 ± 0.87 2.06 ± 0.06 73.07 ± 2.08 8.80 ± 0.72

T6 2.73 ± 0.05 54.95 ± 0.92 2.49 ± 0.10 60.42 ± 2.47 11.70 ± 0.60

Average values and standard deviation for at least three repeats. Experiments performed with shoot tips included in beads, inside a cryovial. Rates and permanence times calculated for the ice formation risk window 0ºC to -150ºC. Beads were desiccated under the air flow of a laminar flow bench T0 = 0 hours (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. 3Temperature of beads after warming for 1 min at 45ºC and 1 min at 25ºC.

Warming process was performed after the encapsulation-dehydration protocol, which

included a 1 min warming in a 45ºC water bath and another minute in a 25ºC bath. This time

was, for beads with the highest water contents, insufficient for them to reach temperatures

over 0ºC (Table 5.2). To complete the warming process at room temperature would lengthen

the time under 0ºC and could increase the risk of ice formation.

69


Figure 5.6. Temperature evolution, during cooling by immersion in LN, of cryovials with 10 alginate beads containing mint shoot tip and treated after the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. Data are the mean of at least three repeats.

Figure 5.7. Temperature evolution, during warming in a 45ºC water bath for 1 min and then in 25ºC for 1 min of cryovials with 10 alginate beads containing mint shoot tip and treated after the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. Data are the mean of at least three repeats.

70



The microstructure of alginate gel beads submitted to the extreme conditions of the drying

process (6 h) was observed by cryo-SEM, and compared to that of non-desiccated ones.

Observation with secondary electrons and backscattered electrons was compared, and

although the same basic information was obtained with both, cellular structures appear clearer

with backscattered electrons, which were used in the micrographs presented.

Figure 5.8. Cryo-SEM micrographs of alginate beads. View of beads external surface at drying time 0 (A) and 6 hours (B), and internal cryofracture surface, at drying time 0 (C) and 6 hours (D). The bars correspond to 10 µm.

Figure 5.8 shows pictures corresponding to these conditions (sections not including shoot

tips). The water content reduction from A-C to B-D beads implied a significant increase of

solute concentration in the remaining water. Although not directly measured, it could be

roughly estimated, considering an initial 0.75 M sucrose concentration in the T0 beads, which,

for an approximate 10 fold decrease in Wc(dm) between T0 and T6, would give a final 7.5M

sucrose. This would give rise to a strong reduction in molecular mobility for high drying

71


times, which caused the system to vitrify when quickly cooled at LN temperatures (in the

microscope stage).

The etching procedure employed for creating contrast in cryo-SEM removes part of the ice

formed in the cooling of the sample in the microscope (always small crystals, due to the high

cooling rate and the small size of the sample) by sublimation, leaving darker �“void�” regions

separated by cleared �“ridges�” of freeze-concentrated solution. Glassy state has a near null

sublimation rate, so vitrified solutions do not present enhanced contrast after the etching

process (Angell et al., 1982; Umrath, 1983). This was considered a suitable method for

studying vitrified tissues and their microstructure and was employed in previous works of this

group (Teixeira et al., 2012, 2013).

Figure 5.9 shows cryo-SEM micrographs corresponding to mint shoot tips at different

stages of the encapsulation-dehydration protocol. A to G correspond to the progressive drying

times. It can be seen that A to D show microstructural intracellular details resulting from the

etching ice sublimation procedure, and showing that in the conditions of microscopic

observation, assumed to be equivalent to those of the rapid cooling in LN used in normal

cryopreservation protocols, there would be intracellular ice and hence, damage to cell

function. On the other hand, E to G, corresponding to the longer drying times, show no

structural details, implying that water could not sublimate during etching and so that these

tissues were in glassy state.

The four first pictures (A to D) show intracellular ice crystals of decreasing size as the

cytoplasm concentration increased with water reduction. Apices after 1 and 2 h desiccation

(T1 and T2; B and C) exhibited extracellular solutions partially vitrified that could be also

associated to the higher sucrose concentration.

Meanwhile, cells at T3 (Figure 5.9 D) show a mix of intracellular ice with vitrified

regions. The ice crystals size reduction is a direct effect of the increase in viscosity, allowing

more ice nuclei to grow instead of favouring the growth of only a few crystals.

The cells shown in Figure 5.9. H correspond to a recovered specimen, after

cryopreservation, warming and one week of successful recovery. Its water and solute contents

would be similar to that of material in the starting point of the process (as those found in

studies conducted by Teixeira et al., 2012), and ice crystals, larger than in picture A, can be

observed, as corresponds to a higher water content.

72


Figure 5.9. Cryo-SEM micrographs of mint shoot tips in different stages of the encapsulation-dehydration protocol. Drying times (hours): A (0), B (1), C (2), D (3), E (4), F (5), G (6). H, tip growing for one week, after warming. The bars correspond to 10 m.

D

A B

C

E F

G H

73


5.3.5. Cryopreserved shoot tips survival and re-growth

Survival and re-growth after 4 weeks culture on recovery medium were studied for shoot

tips cryopreserved following the encapsulation�–dehydration protocol with different air-

dehydration times and subsequent cooling in liquid nitrogen (Figure 5.10). In most cases

viability and re-growth rates were fairly similar within treatment. Control specimens, not

immersed in liquid nitrogen (LN), present nearly a 100% survival, proving the good initial

performance of shoot tips.

The T0 and T1 high water content samples showed complete lack of survival or re-growth.

Intermediate water contents (T2 and T3) had rates of less than 50% for both these parameters,

while the dryer samples (T4, T5 and T6) showed high and similar survival and re-growth

figures. Re-growth percentages for beads exposed for six hours to dehydration were as high as

93.3%.

5.4. General discussion

The general behaviour of mint shoot tips following the encapsulation-dehydration

cryopreservation protocol at different air-drying times is clear and consistent, in spite of the

inherent variability of plant samples and of the whole process. Water content in beads (and

hence in the encapsulated tips) decreased with drying time, reaching soon (T3) to

approximately stable values (Wc(s) ~ 0.25 gwater gsample-1, Wc(dm) ~ 0.30 gwater gdm

-1). These water

contents are in the range of critical values reported for encapsulated shoot tips (Dereuddre et

al., 1990; Uragami et al., 1990). Damages associated to water contents of these orders have

been reported in different plant germplasm systems (González-Benito et al., 1998; Sun, 1999;

González-Benito et al., 2004).

74


a a a a a a a a

b b b c

c d c d

Figure 5.10. Mint shoot tip survival and re-growth percentages after dehydration for different times following the encapsulation-dehydration protocol. Observations were made after a 4-week recovery period. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = before cooling in LN. Data are the mean of at least three repeats. Means of survival or re-growth with the same letter are not significantly different according to the Duncan�’s Multiple Range Test at alpha = 0.05. Bars: standard error.

Most of the other parameters and observations behaved in parallel to water content. Both

cooling and warming rates increased as the drying time did (Figure 5.6 and 5.7). Fast cooling

and warming processes reduce the probability of ice formation and increase that of

vitrification. Nevertheless, the increase in these rates for the different drying times is

relatively small and quite likely not the main source of the different success of the

cryopreservation process.

A water content reduction would mean an increased solute concentration in the remaining

water. This results, for both bead and shoot tip, in higher viscosity (reduced molecular

mobility) and hence, an increased probability of vitrification as they get quickly cooled down.

Low temperature scanning electron microscopy showed (Figure 5.8 and 5.9) how samples at

higher water content suffered ice crystallization but after 4 hours drying vitrification took

place, avoiding any ice trace (with the T3 samples in a intermediate situation).

The water freezing parameters obtained from DSC (Table 5.1) agreed with the previous

observations by showing melting processes fully compatible with increasing solute

concentrations (decrease of freezing temperature and reduction of frozen water content) up to

3 h desiccation when no ice formation could be appreciated. The calorimetric glass transitions

75


that can be visualized after this drying time confirm the cryo-SEM observations and, as their

temperature increased up to 5 h desiccation, indicate the progress of the dehydration process.

Viability of cryopreserved specimens (survival and recovery, as a global physiological

evaluation of the success of the procedure), is in good agreement with the results reported for

this protocol (Uchendu & Reed, 2008) and confirmed the thermodynamic results and the

images observed.

The encapsulation-dehydration procedure submits specimens to prolonged dehydration and

high sucrose concentration stresses. This may be reflected in damages to recovered plants, not

evaluated in the viability analysis, such as genetic alterations (Martín et al., 2011). A

conclusion of this study is that the asymptotic reduction of water content and the associated

variation of most parameters makes unnecessary to employ too prolonged times to protect

samples from ice formation. DSC observations would imply that 3 hours drying could be

enough to avoid the formation of ice (even operating at a slow cooling rate: 10ºC min-1).

Nevertheless, cryo-SEM showed how the shoot tips cytoplasm at 3 hours drying time might

be not completely ice-free, as some crystal evidence is observed. This is confirmed by the

viability data, in which samples desiccated for 3 h showed an intermediate response, implying

that for some of the specimens, lethal ice formation took place.

A more complex picture is completed by the glass transition temperature evolution in the

lower water content samples. Although water content data indicated no further changes from

3 h desiccation onwards, there was a distinct increase of TG from 3 h to 5 h desiccation, where

a stable situation was reached.

76

6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS

6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF

PLANT VITRIFICATION SOLUTIONS

6.1. Introduction

Glass, the state of matter where molecular mobility is so reduced that most

physicochemical processes (including ice formation) are actually detained, is a basic state of

matter for cryopreservation. The temperature at which glass initially forms from supercooled

liquid is known as the glass transition temperature (TG). It is rather a range of temperatures

than a single defined one. The glass transition is also characterized by a change in heat

capacity ( Cp), which is measured as the baseline difference at the transition inflection point.

At temperatures above TG, there is an increase in molecular mobility and a decrease in

viscosity which enables the crystallization of water, if sufficient time to carry out the process

is granted (Roos & Karel, 1991). Per contra, when temperature is below the glass transition,

the system can be considered stable. Most physical and chemical processes (especially those

that are diffusion driven) are impeded, in a physically accessible time frame. Actually, there is

movement in the glassy state: many reports describe the existence of a significant degree of

short range mobility, associated to solvent molecules (water, in this case) (for example Ablett

et al., 1993; Mestre et al., 2002). However, the relatively large-scale molecular

reorganizations required for ice crystal nucleation are certainly not possible in these

conditions of greatly reduced molecular mobility (Angell et al., 1978).

Consequently, glassy state allows living tissues preservation with no or very limited

physicochemical changes and completely free of ice formation, which is associated with lethal

freeze injury (Muthusamy et al., 2005; Nadarajan et al., 2008). Since the knowledge that cells

treated with specific cryoprotecting solutions survive exposure to cryogenic temperatures was

achieved, numerous variations on solution composition were developed for plant cells (Volk

& Walters, 2006). Different vitrification solutions were developed by various research teams

worldwide (Sakai et al., 1990; Steponkus et al., 1992). Sakai�’s group developed the PVS

(Plant Vitrification Solution) series. PVS1 has been employed for work with asparagus cell

suspensions (Uragami et al., 1989, 1990). PVS2, originally developed for treating citrus cell

suspensions (Sakai et al., 1990), has been successfully used for different explants of around

200 species (Sakai, 2003), including garlic (Niwata, 1995). PVS3 has been notably employed

77


with wasabi (Matsumoto et al., 1995), asparagus (Nishizawa et al., 1993) and garlic

(Makowska et al., 1999) shoot tips. The cryopreservation of garlic germplasm, an

economically important crop, has been profusely studied by several authors using different

approaches (Niwata, 1995; Makowska et al., 1999; Keller 2002; Volk & Walters 2006, 2007).

The glass transition temperature of aqueous systems is very sensitive to the proportion of

water and other small molecules, and small changes in compositions can give rise to large

variations in TG. When taking advantage of the glassy state for preservation purposes, often

the difficulty lies in the prediction and control of this temperature. Another problem is how to

reach temperatures below TG, from room temperature, without ice being formed during the

cooling process (the same problem arises when warming specimens to room temperature)

(Roos & Karel, 1991; Zamecnik et al., 2007). The probability of ice formation increases with

the time that the system is at a temperature comprised between Tf (the equilibrium freezing

temperature) and TG. Actually, the ice nucleation temperature should be considered, instead

of Tf. This nucleation temperature has not a fixed value, being dependent on Brownian

movement, but is often placed well below Tf. On the other hand, the region �“close�” above TG

is considered too viscous for anything like ice nucleation to happen, in a short time frame. A

zone of 20ºC above TG is often accepted as having a very low probability of ice formation

(Mishima & Stanley, 1998).

Glass is known to undergo relaxation when stored at temperatures below TG and this

process might affect the preservation quality of biological materials embedded in the glassy

matrix (Gao et al., 2005). Most available literature reports are concerned with the glass

forming or nucleation properties of polyalcohols/water mixtures (Alberola et al., 1983; Shaw

et al., 1997; Murthy, 1997), and little attention was given to their relaxation behavior. Some

authors reported the glass transition and (or) enthalpy relaxation of pure polyalcohol

compounds, such as glycerol (Chang & Baust, 1991; Takeda et al., 1998), propylene glycol

(Mehl, 1996; Takeda et al., 1998), ethylene glycol (Angell & Smith, 1982; Takeda et al.,

1998) and their binary mixture systems (Takeda et al., 1998), but there is a lack of

information about relaxation of plant vitrification solutions below TG.

The glass transition is not a phase transformation in the full thermodynamic sense. Its

occurrence is determined by the history of the material, and is dependent on the exact

conditions of the experiment (Yavin & Arav, 2007). The change between liquid and vitreous

state has also been described as a second order phase change, not characterized by an enthalpy

78


increment as first class changes are, but by a step in some of the physical properties, such as

the heat capacity, specific volume and apparent viscosity (Walstra, 2002). Differential

scanning calorimetry (DSC) is a powerful tool to investigate glass transition, heat capacity

and annealing behaviors of plant vitrification solutions (Leprince & Walters-Vertucci, 1995).

The present study aimed the characterization of the calorimetric properties of the most

common plant vitrification solutions, and of garlic shoot tips treated with one of such

solutions, under a wide range of cooling and warming rates and the investigation of the

stability of their glasses under conditions relevant to their use as cryopreservation agents.

6.2. Materials and methods

6.2.1. Standard compounds

Dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) of

chromatographic grade were purchased from Sigma Aldrich (Heidelberg, Germany); glycerol,

sorbitol and sucrose used were manufactured by Penta (Chrudim, Czech Republic) and

isothiazolinone by Schulke & Mayr (Norderstedt, Germany). All other chemicals used were

of the highest commercially available purity. See Annex I for the molecular formulas of the

compounds.

6.2.2. Solutions

The vitrification solutions were (Table 6.1): (1) PVS1 (Plant Vitrification Solution 1: 19%

w/v glycerol, 13% w/v EG, 13% w/v PG, 6% w/v DMSO in half-strength MS liquid medium

+ 0.5 M sorbitol; Uragami et al., 1989), (2) PVS2 (30% w/v glycerol, 15% w/v EG, 15% w/v

DMSO, and 0.4 M sucrose in half-strength MS liquid medium; Sakai et al., 1990), (3)

modified PVS2 (PVS2 mod: 37.8% w/v glycerol, 16.7% w/v EG, 16.5% w/v DMSO and 0.4

M sucrose in half-strength MS liquid), (4) PVS3 (50% w/v glycerol, 50% w/v sucrose in

water; Nishizawa et al., 1993), (5) modified PVS3 (PVS3 mod: 50% w/v glycerol, 50% w/v

sucrose, 5% DMSO in water; Nishizawa et al., 1993).

79


Table 6.1. Composition of vitrification solutions. Component (% w v-1) Solution Sorbitol EG DMSO PG Glycerol Sucrose Water

PVS1 9.1 13 6 13 - - 64.1 PVS2 - 15 15 - 30 13.7 40.3

PVS2 mod - 16.5 16.5 - 37.8 13.7 31.3 PVS3 - - - - 50 50 28.6

PVS3 mod - - 5 - 50 50 24.1

6.2.3. Plant material

Experiments were carried out with Alium sativum L. sativum cv. ´Djambul 2´. Microbulbils

were rinsed with 1% isothiazolinone solution for two hours. Subsequently, the shoot apex (1

mm) in each microbulbil was excised with a scalpel blade and forceps. Shoot apices were

placed in 75% isothiazolinone solution for 2 s and then transferred to 1% isothiazolinone

solution for 10 min. Finally, batches of 20 apices were directly placed onto MS medium

(Murashige & Skoog, 1962) with 10% sucrose overnight.

Excised shoot tips were immersed into the loading solution (13.7% w/v sucrose + 18.4%

w/v glycerol; Sakai et al., 1990) for 20 min at 25ºC. Shoot tips were immersed in PVS3 at

25ºC for 2 h (Sakar & Naik, 1998).

6.2.4. Differential scanning calorimetry conditions

Thermal processes in vitrification solutions were measured using a TA 2920 DSC (TA

Instruments, New Castle, DE, USA). Hermetic aluminum pans (TA) were used in all DSC

experiments and an empty pan was used as reference. The furnace block of DSC was flushed

with dry nitrogen gas to avoid condensation of moisture from the air. Helium gas (99.999%)

was used as sample purge at a rate of about 33 ml min-1. The temperature scale of the

instrument was calibrated with cyclohexane (-87°C), mercury (-38.87°C), water (0.01°C) and

indium (156.6°C) and with heat fusion of water (334 J g-1). All calibrations were performed

by using scanning rates of 5, 10 and 20°C min-1. Calorimetric data were collected from two

replicates per treatment.

Samples within pans were either approx. 10 mg of vitrification solutions or three garlic

shoot tips, immediately after treatment with the vitrification solution PVS3. In work with

shoot tips, pans were weighed before and after DSC; in the second case pans were punctured

80


and oven-dried (~85°C, 72 h), to obtain the water content. Calorimetric data were collected

from two replicates per treatment.

6.2.5. Glass transition characterization for PVS

The glass transition parameters (TG and Cp) were determined for the different

vitrification solutions considered, in both cooling and warming scans. Pans with samples were

cooled at standard rates (10ºC min-1) from 30 ºC to -145ºC. After 5 min equilibration at this

temperature, they were warmed back to 30ºC, also at 10ºC min-1. This cycle was repeated four

times. Two different samples were studied for each solution.

6.2.6. Cooling rate test

For this test, cooling was performed at different rates: either using the calorimeter control

(5, 10 and 20°C min-1), or, for higher rates, by quickly immersing the closed pan with the

sample in LN (either naked or previously included inside a cryovial). The pan was then

transferred to the calorimeter sample chamber, pre-cooled to -150°C, where a short

equilibration time was allowed. Glass transition temperature and the corresponding heat

capacity change were observed upon warming from -145°C to room temperature, at a

standard warming rate of 10°C min-1. The samples were either vitrification solutions or garlic

shoot tips, after treatment with the vitrification solution PVS3.

6.2.7. Warming rate test

For investigating the effect of the warming rate, cooling was performed at two fixed rates:

10°C min-1 (using the calorimeter control) or by quenching in LN (without cryovial).

Quenched pans were transferred to the calorimeter sample chamber, pre-cooled to -150°C,

where a short equilibration time was allowed. Glass transition temperature and the

corresponding heat capacity change were measured upon warming pans with samples from

-145°C to room temperature, at different warming rates: 5, 10 and 20°C min-1. The samples

were either vitrification solutions or garlic shoot tips, after treatment with the vitrification

solution PVS3.

81


Crystallinity, for garlic shoot tips was evaluated using TA proprietary software (TA

Instruments, undated), using a value for the specific fusion heat of water of 334 J g-1. The

routine basically evaluates the thermal event enthalpy and, by comparing with the water

specific fusion heat and considering the water content of the sample (obtained by differential

weighing, after drying in an oven after the DSC experiment), calculates the fraction of water

that had crystallized into ice.

6.2.8. Storage test

To test the effect of storage time, DSC pans with solution samples were plunged directly

into LN and kept immersed (at -196ºC) for five different time lapses (0, 1, 7, 30 and 60 days).

Pans in LN were, after the storage period, transferred to the calorimeter sample chamber, pre-

cooled to -150°C, where a short equilibration time was allowed. Glass transition temperature

and the corresponding heat capacity change were measured upon warming pans with samples

from -145°C to room temperature, at a standard warming rate of 10°C min-1.

6.2.9. Annealing test

The annealing thermal behavior of PVS1 and PVS3 was studied. These two solutions were

chosen as their vitrification parameters were distinct (see Results). The samples were (Figure

6.1.) first cooled at 10°C min-1 (standard cooling rate) from room temperature (A) to -145°C

(well below the vitreous transition of the solutions), and kept at this temperature for 5 min

(B). Then, the samples were heated to a temperature over its TG, (-90ºC for PVS1 and -70°C

for PVS3) at a rate of 20°C min-1 and kept for 5 min at these temperatures (C). Later, the

samples were cycle cooled again at 20°C min-1 to a target temperature decreasing one degree

per cycle (from -100 to -120ºC for PVS1 and from -77 to -100°C for PVS3), and kept at these

temperatures for 30 min (D). Finally, the samples were cooled again at 20°C min-1 to -145°C,

kept for 5 min (E) and warmed to 30°C, at 20°C min-1 (F). The DSC thermogram of the last

step was recorded and the annealing areas measured. In order to reduce experimental scatter

due to differences in heat transfer between the calorimeter and the sample, it was left

undisturbed inside the furnace for a set of experiments. All experiments were repeated twice.

82


Figure 6.1. Scheme of the steps of the annealing protocol used; see text for more details on solutions employed.

6.2.10. Statistical analysis

The results were analyzed by analysis of variance (ANOVA), and means were compared

by Duncan�’s multiple range test (P 0.05) using STATISTICA version 10, (StatSoft, Inc.,

2011).


6.3.1. Vitrification behavior of different vitrification solutions

Table 6.2 shows the results of determination of the glass transition parameters for the five

PVS employed, at standard rates (10ºC min-1). Glass transitions temperatures were fairly

reproducible and showed clear and consistent differences among vitrification solutions

(confirmed by the statistical analysis), which were related to different composition and water

contents (Table 6.1). TG values calculated for each solution were identified by the statistical

analysis as distinct. The value of TG generally decreased as the water content of the solutions

increased. This behavior was not followed for PVS1 and PVS2, as the latter solution TG was

the lowest, while PVS1 had the highest water content.

83


Table 6.2. Glass transition parameters of different pla-1

nt vitrification solutions (cooling and warming rates were both 10°C min ).

Type of PVS TG Cp

(°C) (J g °C-1)

PVS1 -112.15 ± 0.38b 1.22 ± 0.03a

PVS2 -114.31 ± 0.38a 1.12 ± 0.04a,b

PVS2 m c

b

od -109.42 ± 0.11 d

1.07 ± 0.04 b

b

PVS3 -89.85 ± 0.29 e

0.87 ± 0.01

PVS3 mod -86.84 ± 0.65 0.83 ± 0.01

Average values and standard deviation TG and Cp, in a column, with the sa er are tly ording to the Duncan�’s Multiple Range Tes 0.05

olecules available per each solute molecule was low

and even lower the num

t -89ºC (PVS3 and PVS3 mod). The heat

capacity changes measured were less distinct than the corresponding T values, but a

0) reported a TG value of -115ºC (equal for both cooling and

warm

for at least four repeats. Means of not significanme lett different acc

t at alpha = .

These vitrification solutions contained large amounts of solutes able to form hydrogen

onds with water. The number of water mb

ber of water molecules per potential hydrogen bond in solute

molecules. Nevertheless, in such concentrated and complex mixtures not all these potential

bonds could be expected to bind water, as many intersolute (and intramolecular) bonds would

arise (see Annex I for molecular formulas of the components of the cryopreservation

solutions and tables of their molar composition).

The different TG found could be divided into two groups: one centered around -112ºC

(PVS1, PVS2 and PVS2 mod) and the other a

G

decrease with the water content in solutions could be appreciated. However, PVS solutions

could be distributed according to Cp in the same groups. The prediction of glass transition

behavior for complex mixtures as those studied here, is, currently, not a solved problem (see,

for example, Angell et al., 2000). Nevertheless, water content seems to be the dominant factor

over the solute composition.

Very few data on glass transition parameters of PVS have been reported. The initial PVS2

publication (Sakai et al., 199

ing scans), within experimental error of our data. It must be noted that, in the same

publication, the existence (upon warming at 10ºC min-1, following 80ºC min-1 cooling) of a

devitrification event (at -75ºC), subsequently followed by melting (at -36ºC) was reported.

84


After extensive, repeated and reproducible DSC experiments, we have been unable to find any

ice formation event, at any of the combinations of cooling and warming rates considered, and

for any of the PVS studied. The reported devitrification event took place upon warming at

10ºC min-1, twice as fast as our 5ºC min-1 slowest experiment (where ice formation would

have been more likely). PVS3 was also previously reported to have a TG in good agreement

with those reported here (Záme ník et al., 2012).

Pure water glass transition temperature, though difficult to physically measure, is believed

to take place at 135K (-138ºC) (Johari et al., 1990; Johl et al., 2005), actually not so far away

fro

on vitrification behavior

Vitrification in cryopreservation protocols is achieved, without sophisticated cooling

ph

rec

G

nce observed among different solutions. The TG of a

sol

m the TG found here for PVS solutions. Other authors even rise this temperature to 165 K

(-108ºC) (Velikov et al., 2001; Angell, 2008), or even higher (Swenson & Teixeira, 2010;

McCartney & Sadtchenko, 2013). Clearly, the question is far from being settled.

6.3.2. Effect of cooling rate

equipment, by simply plunging specimens into liquid nitrogen (LN) after a set of

ysicochemical treatments, designed to increase their cytoplasmatic microviscosity and

enhance tissue resistance to cold and dehydration. Fast cooling is required to achieve

vitrification avoiding the ice formation. In a similar way, warming after the storage period

must be carried out quickly, and it is practically performed by plunging samples (naked or

inside cryovials) directly from LN into a water bath or warm culture medium.

Figure 6.2 shows details of the thermograms obtained from testing the effect of cooling

time on the vitrification behavior of PVS3, after the method described. The glass transition

orded in the warming scan (always carried out at 10°C min-1) was similar both in

temperature and heat capacity change. The width of the glass transition process was estimated

to be on an average of 7ºC.

Figure 6.3 shows TG and Cp for the five solutions studied. T variation with cooling

rates was much smaller than the differe

ution did not significantly change within a wide range of cooling rates (from 5 to 20°C

min-1, under the DSC control, and the faster resulting from plunging the sample pan into LN,

with the aluminum pan naked or inside a cryovial). A total change in TG inflexion point of

1.2°C, as was observed between the �“slow�” (5-20ºC min-1) methods and the �“faster�” one

85


(direct into LN), did not influence considerably the glass transition region, as compared with

the width of the whole transition interval.

PVS3 thermograms showing the glass transition, performed at a warm and after cooling at different rates. Quenching: direct immersion of the DSC pan in

quenching

cryovial

20ºC min-1

5ºC min-1

10ºC min-1

Figure 6.2. ing rate of 10ºC min-1

LN; cryovial: pan included in a cryovial and then in LN. White arrows mark the glass

ow a larger variation, no clear trend with the cooling rate could be

observed (Figure 6.3). Besides, statistical analysis on both TG and Cp, showed that the

dif

expected value (3-5ºC per rate magnitude order corresponds to 9-15ºC, for the more

transition inflection point.

Although Cp values sh

ferences observed among cooling rates were not significant. This was an unexpected

finding, as both TG and the Cp are generally considered to be dependent on cooling rate (e.g.

Angell et al., 1978; Debenedetti & Stillinger, 2001). After this, the slower a liquid is cooled,

the longer time would be available for configuration of samples at each temperature, and

hence the colder it could become before falling out of liquid-state equilibrium. This would be

reflected in TG increasing with cooling rate (Moynihan et al., 1976; Brüning & Samwer,

1992), as the properties of the glass depend on the process by which it is formed. In practice,

the reported dependence of TG on the cooling rate is weak: TG changes by 3�–5°C when the

cooling rate changes by an order of magnitude (Ediger et al., 1996; Debenedetti & Stillinger,

2001).

Possible explanations for the discrepancy between the small variation (1.2ºC) reported here

and the

86


tha

eters measured at fast cooling rates were evaluated always at the ensuing

wa

measured. They range from the very slow processes of

tho

n three orders between 5 and 6600ºC min-1 of our experiments) may lie on that the

experimental observations in which TG undergoes these changes with cooling rate refer in

most cases to much simpler systems, either pure liquids or binary mixtures. These ternary,

quaternary and pentary solutions may present a compensating behavior, with different

components having contributions of different sign to the glass transition displacement with

cooling rate.

Another explanation may lay on the fact that, in the present work, the reported glass

transition param

rming scan. Actually, very few experimental techniques can perform a proper glass

transition measurement while cooling at fast rate. Faster cooling would cause the glass

transition to take place at earlier, higher temperatures (giving rise to a less stable glass). Upon

warming, the glass transition would occur as soon as the lower TG (of the more stable glass,

found at slow warming processes) was reached. In that way, the influence of the previous

cooling rate would be ignored.

Cooling rates present a wide variation in practical plant cryopreservation, though they have

not been very frequently accurately

se methods relying on extracellular freezing for induced intracellular vitrification (using

temperature control equipment, with rates as low as 0.3ºC min-1; Kaczmarczyk et al, 2011), to

those of he vitrification method (approx. 2.3°C s-1), the encapsulation-dehydration protocol

(approx. 2.7°C s-1) and the faster droplet-vitrification method (110ºC s-1) (Kaczmarczyk et al,

2011; see Chapters 3 and 5). Other faster approaches have being tested, such as those

avoiding the Leidenfrost effect that limits the cooling rate in LN by using other cryogenic

fluids, such as liquid propane or ethane, or even using mechanical implements to make faster

the immersion of the sample in the cryogenic medium. The two quench cooling procedures

used here had rates similar to those of the droplet-vitrification (naked DSC pan) and the

encapsulation-dehydration method (inside the cryovial), i.e. approximately 110ºC s-1 (6600ºC

min-1), and 2.7ºC s-1 (160ºC min-1), respectively.

87


Figure 6.3. Glass transition temperatures (a) and glass transition heat capacity changes b) for the different PVS and garlic shoot tips (after treatment with PVS3), obtained in warming

he behavior of the vitrification parameters of garlic shoot tips in the last stage of the

cryopreservation protocol (after treatment with PVS3) was different from that of the

vitr

a

b

*

*

(

(10ºC min-1) DSC experiments, after being cooled at different rates. * Rate not tested for garlic samples. Bars: standard error.

T

ification solutions (Figure 6.3). Shoot tips present a much more marked dependence of

88


both TG and Cp with the cooling rate than solutions. This may be due to the fact that the

shoot tips have higher cytoplasmic water content or to the presence of other cellular

components, apart form their structural and compartmentalization elements.

6.3.3. Effect of warming rate on vitrification behavior

ing rates after

usi

e order of those reported

for

6.3.4. Crystallinity of garlic shoot tips

ication procedure exhibited both freezing and glass

tra

such as water content or permeability to solutes, may sustain alternative explanations.

Figure 6.4 shows the glass transition parameters obtained for different warm

ng two different cooling rates: 10°C min-1 (a and c) and LN quenching (b and d). The

variations in both TG and Cp were not significant and, again, showed no dependence of the

warming rate, but a clear relation with the solution composition and for all the cooling and

warming conditions studied, with a similar response of the group PVS1-PVS2-PVS2 mod, on

one hand and the group PVS3-PVS3 mod, on the other.

Practical warming rate values for cryopreservation are of the sam

cooling rates, as most cases heat exchange is driven by stirring the sample (naked,

encapsulated or inside a cryovial) in a warm water bath or culture medium, being the

temperature gradients induced comparable to those created by plunging room temperature

specimens into LN. Nonetheless, warming rates slightly lower than those found for cooling

have been reported (see Chapters 3 and 5): approx. 1.8, 2 and 40°C s-1, for mint shoot tips

following the vitrification, encapsulation-dehydration and droplet-vitrification protocols,

respectively.

Garlic shoot tips subjected to the vitrif

nsition events when cooled slowly (10ºC min-1) (Figure 6.5). Other systems treated by the

vitrification method did not produce water freezing when, in the last step of the protocol, were

cooled at this slow rate. This is the case of mint shoot tips (Teixeira et al., 2013) and this

might be due to their smaller size, when compared with garlic shoot tips. Mint shoot tip

average weight after the vitrification dehydration step was 0.7 mg (Teixeira et al., 2013),

while garlic shoot tips in the same stage weighed 3.9 ± 0.4 mg. Sample size is one of the most

important determinants of vitrification probability (Yavin & Arav, 2007). Other differences,

89


Figure 6.4. Glass transition temperature (a and b) and heat capacity (c and d) obtained in DSC during warming at different rates (5, 10 or 20°C min-1). DSC pans had been cooled either at 10°C min-1 in the calorimeter (a and c) or by quenching in LN (b and d). Bars: standard error.

e of this (always very small) melting endotherm allows calculation of the

action of crystallized water. The onset of the melting process and the crystallinity calculated

after the TA proprietary software, are presented in Table 6.3 for different warming rates. The

crysta

The presenc

fr

llinity decreased with increasing warming rates, while the transition onset grew. The

corresponding glass transition temperatures were almost constant with warming rate. In

similar systems, it has been reported that Cp decreases while crystallinity increases (Gao et

al., 2005), which agrees with the observations of Figure 6.5 and Table 6.3. Although, at

quenching rates (those employed in practical cryopreservation) there was no formation of ice,

these findings could imply a potential amount of freezable water that could result in ice

formation.

90


Figure 6.5. Typical scan of shoot tips of garlic after treatment with PVS3 for 2 h. Both cooling and warming rates were 10ºC min-1. The black arrows mark to the glass transition inflection point in cooling and warming scans, while gray arrows signal the freezing and melting events.

d warming scans at different rates. Warming rate Onset Crystallized

Table 6.3. Crystallization parameters (mean ± standard deviation) of garlic shoot tips after treatment with PVS3 for 2 h. Onset of the melting endotherm and crystallinity as crystallization percentage, both measured in the warming scan. Cooling scans were performed at 10ºC min-1 an

(°C min-1) (°C) (%) 5 -19.87 ± 9.40 1.96 ± 2.51 10 -16.49 ± 3.73 1.21 ± 0.25 20 -14.61 ± 0.62 0.37 ± 0.51

6.3.5. Effect of the stora time on on b VS

Table 6.4 and 6.5 w no nges TG or Cp, respectively,

ssociated to the period of storage in LN, up to 60 days. Although the storage period of

e maximum storage

period used here, it was considered to be a reasonable period and should enable the

ge the vitrificati ehavior of P

sho significant cha either in

a

cryopreserved material, in many practical instances, exceeds well th

observation of any change with time in the vitrified sample. The fact that quenched cooled

samples did not show any variation in these properties with storage time confirmed that the

system had reached a stable vitreous state, suitable for cryopreservation.

91


Table 6.4. Cryopreservation solutions glass transition temperature (mean ± standard deviation) versus storage time.

TG (°C)

Storage time (days) Type of PVS

1 7 30 60

PVS1 -112.38 ± 0.10 -112.35 ± 3.01 -111.33 ± 0.001 -111.81 ± 0.11

PVS2 -112.59 ± 0.86 -112.72 ± 1 3.88 ± 0.52 -114.19 ± 0.66

-108.81 ± 1.64 -108.7 ± 0.06 -109.97 ± 0.09

PVS3 -88.63 ± 2.17 -88.92 ± 0.04 -88.52 ± 0.98 -88.38 ± 0.13

PV d

.55 -11

PVS2 mod 1 ± 0.73 -108.88

S3 mo -85.47 ± 0.93 -85.80 ± 1.53 -85.43 ± 0.15 -86.29 ± 0.01

able 6.5. Cryopreservation solutions heat capacity change (mean ± standard deviation) ersus storage time.

Cp (J g°C-1)

Tv

Storage time (days) Type of PVS

1 7 30 60

PVS1 1.06 ± 0.06 1.08 ± 0.38 0.95 ± 0.15 1.22 ± 0.05

PVS2 0.65 ± 0.14 1.10 ± 1.16 ± 0.05

0.78 ± 0.09 1.26 0.09 1.28 ± 0.01

PVS3 0.83 ± 0.24 0.97 ± 0.02 0.69 ± 0.20 0.98 ± 0.11

PV d

0.20 1.07 ± 0.33

PVS2 mod ± 0.03 1.01 ±

S3 mo 0.68 ± 0.15 0.97 ± 0.12 0.63 ± 0.02 0.94 ± 0.05

.3.6. Annealing of PVS1 and PVS3

Considering the previously reported grouping of the vitrification solutions, the annealing

ehavior of PVS1 and PVS3 were studied, as representative of each group. Figure 6.6 shows

typical thermogram of PVS1 with a detail of the step E-F of the annealing method depicted

6

b

a

92


in Figure 6.1, where areas over the curve were measured. These areas are represented in

Figure 6.7, versus the corresponding target temperature (D), for PVS1 and PVS3. The

behavior observed is clearly distinct for the two solutions: for PVS1 the curve slope increased

with decreasing target temperature, implying quickly growing annealing areas. On the other

hand, the slope was much shallower for PVS3, and large temperature variations were reflected

in a reduced area increase.

Figure 6.6. Typical thermogram of PVS1 on the step E-F of Figure 6.1. The annealing areas were comprised between the curve and the extended baseline.

93


Figure 6.7. Annealing area versus temperature (D in Figure 6.1) of the solutions PVS1( ) and PVS3(�•).

Increases in the annealing areas are considered as proof of a glass to be unstable,

susceptible to evolve to form more stable vitreous structures at temperatures close to TG (Gao

et al., 2005). In this case, PVS3 would be forming more stable glasses and this could indicate

a better behavior for storage of cryopreserved samples. This would be relevant for storage at

temperatures well over that of LN. The difference between the LN temperature (-196ºC) and

the annealing temperatures employed here is nearly 100ºC, and stability at LN conditions

would be guaranteed. A different case would be for storage in -80ºC freezers, at temperatures

too close to TG, of these order (-87 to -114ºC). Interestingly, it has also been reported an

influence of the annealing time on the glass transition temperature (Chan & Baust, 1991). In

our experiments, an annealing time as long as four hours did not produce a noticeable

difference over the customary 30 min employed.

94

7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION

7. THE APPLICATION OF CHITOSAN FILMS ON

ALGINATE BEADS FOR CRYOPRESERVATION USES

7.1. Introduction

Chitosan, a natural-origin product with a wide range of interesting properties, comprising

antimicrobial, protective, antioxidant, plant defense promoting and ligand binding activities,

has become in the last few decades an ubiquitous molecule and the number and variety of its

applications are continuously increasing. They range from its now common use as a

pharmaceutical inert phase, either involved in the modulated delivery of drugs or as a

protecting cover (Uhrich et al., 1999; Morris et al., 2010), to medical uses, for example, as a

wound healing promoter (Ravi-Kumar, 2000), and to its extended inclusion as food

component, with a variety of functions (Knorr, 1984).

Chitosan is a natural linear bio-polyaminosaccharide derived from chitin, which is the

principal component of protective cuticles of crustaceans (No et al., 2003; Rinaudo, 2006).

Chitosan (Figure 7.1) is obtained from chitin by N-deacetylation (Andres et al., 2007) and

consequently is rich in amino groups, which makes chitosan a cationic polyelectrolyte (pKa

~6.5). In aqueous acidic media chitosan has high positive charge on �–NH3+ groups, having the

ability to aggregate to negatively charged molecules (Krajewska et al., 2011). Chitosan is

most frequently characterized by its molecular weight (M) and its degree of acetylation (DA)

(Goy et al., 2009) but there are other important variables, such as the nature of the salt

counterion, pH, ionic strength and the addition of a non-aqueous solvent.

Chitosan (and also chitin) have been shown to have several useful activities for biological

and biotechnological applications, such as null toxicity, biocompatibility and biodegradability

(Sinha & Kumria, 2001). Its properties are especially relevant in relation to plant biology:

they include antiviral, antibacterial and antifungal, as well as mineral chelating activities and

plant natural defences enhancement (El Hadrami et al., 2010).

The mechanisms underlying the effects of chitosan against plant disease are little known,

but would include direct microbial toxicity and metal chelation, as well as the formation of

physical barriers. Additionally, it would trigger signalling cascades leading to defence

activation. The plant defence enhancing properties of chitosan include the induction of

95


lignification, pH cytoplasm changes, chitinase activation, reactive oxygen species scavenging

and other biochemical and genetic responses (Barber et al., 1989; Kuchitsu et al., 1995;

Minami et al., 1996). Chitosan has a role in early elicitation of plant defence (Amborabé et

al., 2008), promoting the accumulation of its related metabolites. A possible way of action is

the recognition of chitosan, among other cell wall polysaccharides, by specific intruder

receptors (Dangl et al., 2001).

Figure 7.1. Schematic representation of the chemical structures of the chitin and chitosan (adapted from Goy et al., 2009).

Chitosan has been used to promote the germination of plants of several species, such as

maize, peanut or rice (Reddy et al., 1999; Ruan & Xue, 2002; Zhou et al., 2002).

Additionally, it promotes wound healing by means of its binding properties (Hirano et al.,

1999) and its applicability to fresh product storage is promising. Chitosan also has been

reported to have an interesting antioxidant activity (Xie et al., 2001; Sun et al., 2006, 2008),

and it can function as an oxidative molecule scavenger.

96


The antimicrobial activity of chitosan against different groups of microorganisms has

received considerable attention in recent years (Goy et al., 2009; El Hadrami et al., 2010).

This polysaccharide owns antimicrobial activity against both Gram-positive and Gram-

negative bacteria (Krajewska et al., 2011). Chitosan is employed to limit microbial

proliferation on many substrates, for example, as a food protecting layer, while also

modulates permeability to chemicals. Its antimicrobial activity has been shown to depend on

its molecular weight (El Hadrami et al., 2010).

Besides the effect of chitosan on fungi by promoting the host plant defence responses, it

has been shown to directly inhibit growth of many fungi such as Fusarium oxysporium and

Botrytis cinerea (Leuba & Stössel, 1986; Benhamou, 1992; El Ghaouth et al., 1994;

Benhamou et al., 1998; Romanazzi et al., 2002; Ait Barka et al., 2004; El Hassni et al., 2004).

Exceptions to the wide antimicrobial and antifungal activity of chitosan (Muzzarelli et al.,

1990) are some fungi with chitosanolytic activity (Palma-Guerrero et al., 2008). Chitosan has

been successfully employed as a treatment to prevent fungi damage to seeds (Reddy et al.,

1999; Rabea et al., 2003) and it has been applied to control plant diseases by foliar application

and soil amendment. As foliar spray, it reduced mildew infection severity (Faoro et al., 2008),

while as soil amendment, acted against fungi of the genera Fusarium (Rabea et al., 2003) and

Aspergilus (El Ghaouth et al., 1992). In spite of this wide-spectrum antimicrobial properties,

the understanding the underlying mechanism is poor.

Alginate is another naturally occurring polysaccharide of sea origin, also with growing and

widespread applications. It is produced by three brown sea algae species (Laminaria

hyperborea, Ascophyllum nodosum and Macrocystis pyrifera). Alginic acid and its salts are

anionic linear polysaccharides containing 1,4-linked D-mannuronic acid and L-glucuronic

acid residues. It can form irreversible hydrogels in the presence of some multivalent metal

cations, such as the divalent calcium ions (Coviello et al., 2007; Draget et al., 2011). A

common use of alginate is as gel beads that are easily formed by releasing drops of a solution

of suitable concentration of alginic acid into the counterion solution (drops of a cation

solution dispersed in the alginic acid solution also produce gel beads). Cations diffuse into the

alginate drops promoting its gelation, and the gel strength and the beads consistency increases

with time. The counterion employed is often calcium, as this divalent ion allows the formation

of strong and flexible gels for low alginate concentrations (2-3%), at reduced diffusion times.

97


The consistency of the alginate beads grows with time, as the calcium diffusion within the

alginate drop progresses, allowing cross-linking. The maximum gel hardness occurs in the

bead surface, as the ions concentration is higher there (King, 1983; Draget et al., 1997;

Draget, 2000).

The biocompatible and inert nature of alginate has allowed the use of its gelling properties

in several contexts, such as the development of new food forms and the delivery of nutrients

(Coviello et al., 2007; Deladino et al., 2008; Anbinder et al., 2011) and pharmaceutical

products (Pasparakis & Bouropoulos, 2006), or the encapsulation of plant germplasm as a

way of protection against environmental fluctuations (Phunchindawan et al., 1997;

Engelmann et al., 2008; Rai et al., 2009). The sometimes called �“artificial seeds�” are

elaborated by simply enclosing a somatic embryo in a drop of the alginate solution, and

dispensing it in the calcium solution (Senaratna, 1992; Khor & Loh, 2005).

The gelation of alginate is compatible with a variety of dissolved substances, so the

required nutritive media can be added. Once the gel is formed, it behaves mostly as an inert

solution sustaining network, so it can be permeated by other substances, and its water content

can be altered by evaporation. These properties are currently used in cryopreservation

protocols.

Cryopreservation of plant germplasm often includes steps in which the viscosity of the

solutions of the different tissue compartments, including cytoplasm, is reduced in order to

decrease the probability of ice formation and increase the glass transition temperature. This is

achieved either by evaporative dehydration, induced by air flow, and/or by incubation in

highly concentrated sucrose solutions. Alginate beads allow easy equilibration by both means

(Engelmann et al., 2008).

The permeability of alginate beads to water and solutes as well as their mechanical

resistance has been modulated by addition of other polysaccharides (Deladino et al., 2008;

Anbinder et al., 2011). The combination of alginate and chitosan polymers, preserving their

intrinsic biocompatibility, non-toxicity, and biodegradability characteristics, is increasingly

employed for uses such as drug delivery and enzyme and cell immobilization (George &

Abraham, 2006; Pasparakis & Bouropoulos, 2006; Kim et al., 2008; Rajendran, 2009).

Chitosan, being a cationic polysaccharide, can externally bind to alginate beads or diffuse into

three-dimensional alginate gel network, which has an anionic character (Hyland et al., 2011).

98


With the purpose of studying the possible application of chitosan to improve the

performance of the encapsulating substrates employed, the permeability and water interaction

properties of chitosan-covered alginate beads were investigated. Additionally, scanning

electron microscopic techniques were applied to observe the chitosan layer on the beads.


7.2.1. Chitosan types and hydrodynamic characterization

Three different chitosan commercial forms were obtained from Sigma Aldrich (USA):

chitosan C (from shrimp shells, Sigma No. C3646), medium molecular weight chitosan

(MMW) (Sigma No. 448877) and low molecular weight chitosan (LMW) (Sigma No.

448869). The producer specifications indicated that all of them were 75-85% deacetylated and

had been extracted from crustaceous shells. All samples were prepared at a concentration of

3.0 mg/mL dissolved in 0.2 M acetate buffer pH 4.3.

Sedimentation velocity determinations were performed with chitosan solutions using an

Optima XL-I analytical ultracentrifuge (Beckman Instruments, Palo Alto, USA). Samples

were centrifuged at 45000 rpm, at a temperature of 20.0°C. Data were analyzed using the

least-squares c(s) method included in the SEDFIT software (Dam & Schuck, 2005).

Sedimentation coefficients, s, were extrapolated to zero concentration, to correct for non-

ideality effects (Patel et al., 2007).

The extended Fujita approach (Fujita, 1962; Harding et al., 2011) was applied to calculate

approximate molecular weights for chitosan, from the determined sedimentation coefficients,

using the eq. 1:

(Eq. 1) so20,W = s Mb

where M is the molecular weight and so20,W, the sedimentation coefficient at infinite dilution

and 20oC. The pre-exponential factor s, a constant for a particular polymer under a defined

set of conditions, and the exponent b, defining the hydrodynamic behavior (ranging from 0.4�–

0.5 for a coil, ~0.15�–0.2 for a rod and ~0.67 for a sphere), were taken as 0.1 and 0.24,

99


respectively, from data fitted for chitosan samples of similar acetylation degree (Harding et

al., 2011).

7.2.2. Alginate beads

Calcium alginate beads were made with 2 or 3 % (w/v) low viscosity alginic acid (Sigma,

USA) in liquid MS (Murashige & Skoog, 1962) without calcium, at pH 5.7. Droplets of

alginate were pipetted onto 100 mM calcium chloride (CaCl2) solution prepared also in MS

liquid medium, using a 2 mL plastic disposable Pasteur pipette, in a flow chamber, at room

temperature. Beads were allowed to stand in the CaCl2 solution for 30 min. Then, this solution

was filtered to remove the formed alginate beads.

7.2.3. Chitosan solution and alginate bead treatment

2% C type chitosan solutions were prepared in 1% acetic acid (Protanal, Norway), diluted

in MilliQ water (Millipore, Billerica, USA) and stirred overnight. Then, pH was adjusted to

5.6 with NaOH (Sánchez-Dominguéz et al., 2007). Chitosan-covered alginate beads were

prepared by immersion of the preformed calcium alginate beads in a container with the

selected chitosan solution and stirred at 150 rpm, for 30 min. The chitosan solution was

filtered to recover the beads.

7.2.4. Water content determination and air dehydration studies.

Four sets of three beads were used for determining the dry mater and water content. Dry

matter resulted from differential weighing before and after oven-drying (~85°C, 72 h) and

was expressed as relative to the total mass (dm). The beads water content (Wc) could be

calculated for dehydration stages, directly as the difference between its total mass and dm. It

was expressed as relative to the total sample mass -fresh weight- (Wc(s)).

To study the beads air-dehydration kinetics, freshly made beads were transferred to an

open glass Petri dish and dehydrated for 1, 2, 3, 4, 5 and 6 h in a laminar-flow hood.

Afterwards, bead weight was determined as described above.

100


7.2.5. Differential scanning calorimetry and frozen and unfrozen water fractions

DSC experiments were performed with a Mettler-Toledo DSC 30 instrument (Mettler-

Toledo, Griefsen, Switzerland). Three beads with the same treatment were placed in a DSC

pan, which was sealed and weighed. Samples were submitted to a cooling scan from room



from two replicates per treatment. Later, pans were punctured and dried in an oven at 85ºC,

for 72 hours, when they showed constant weight.

Thermograms were analyzed using the standard procedures provided in the Mettler-Toledo

STARe software. Ice thawing thermal events (more precise than freezing events), were used

for extraction of Tf. The routine produced the temperature corresponding to the event onset,

Tf(onset), corresponding to the equilibrium freezing temperature and that to the peak, Tf(peak).

The melting enthalpy Hf, proportional to the event area, was also obtained. It was expressed

as relative to either the total bead mass ( Hf(s)), its dry mass ( Hf(dm)) or its water content

Hf(w), to allow easier data comparison. Water fractions, frozen water (Wf) and unfrozen

water (Wu), were calculated by comparison of Hf and the pure water freezing enthalpy

( Hf(w) = 333.4 J/g at 0ºC), together with the total water contents previously determined, after

eq. 2:

Eq.2 Wf= ( Hf/ Hf(w)); Wu=Wc-Wf


oven dry pan weights. Frozen (Wf) and unfrozen (Wu) water contents were expressed as

relative to the total bead mass (Wf(s), Wu(s)), its dry mass (Wf(dm), Wu(dm)) or its total water

content (Wf(w), Wu(w)).

7.2.6. Low temperature scanning electron microscopy observations.

Cryo-SEM studies were performed, basically as previously described (Teixeira et al.,

2013), using a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold

plate (Oxford, UK). Cryo-SEM allows sample observations without the need of prior

chemical fixing or drying processes. Three beads were fitted on a special bracket and this

101


piece was immersed into liquid nitrogen (LN) under low pressure, physically fixing tissues for

microscopic observation. The holder with the samples was placed in the microscope and

etching (partial ice sublimation, induced to provide contrast) was performed for three minutes

at -90ºC. Afterwards, the samples were coated with high purity Au, which acts as a

conductive contact for electrical charge. Finally, they were inserted in the Cryotrans cold plate

and observed under secondary electron mode, at a temperature of -150/-160ºC, using an

accelerating voltage of 15 kV and a working distance of 10-25 mm.

7.2.7. Environmental scanning electron microscopy

Environmental SEM analysis was performed using a Jeol JSM-6360 (Japan) microscope.

Beads were attached to stubs using a two-sided adhesive tape, then coated with a layer of gold

(40 nm-50 nm) and examined using an acceleration voltage of 10 kV. The observation

temperature was 5-8ºC and the pressure at the sample chamber was 6 torr. Some beads were

air dehydrated for five hours before observation.


7.3.1. Chitosan hydrodynamic characterization

The sedimentation coefficients for each chitosan type were determined and represented as a

function of the concentration (Figure 7.2), to obtain the infinite dilution extrapolated

sedimentation coefficients, so20,W, which are shown in Table 7.1. The molecular weights

calculated using the extended Fujita approach, as described (eq. 1), are also presented in this

Table. The most common commercially available chitosan presentations are often highly

polydisperse and their molecular weight can present large differences among batches.

Employing this relatively easy approach, the size of the chitosan polymers studied can be

estimated (also presented in Table 7.1), with the help of fitting parameters obtained for

similar samples (Harding et al., 2011).

102


0.0 0.5 1.0 1.5 2.0 2.50.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (MMW)

(1/s 2

0,W) (

sec-1

)

Concentration (mg/ml)0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (LMW)

(1/s 2

0,W) (

sec-1

)

Concentration (mg/ml)

0.0 0.5 1.0 1.5 2.0 2.50.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (C)

(1/s 2

0,W) (

sec-1

)


a b

c

0.0 0.5 1.0 1.5 2.0 2.50.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (MMW)

(1/s 2

0,W) (

sec-1

)


0.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (LMW)

(1/s 2

0,W) (

sec-1

)


0.0 0.5 1.0 1.5 2.0 2.50.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (C)

(1/s 2

0,W) (

sec-1

)


a b

0.0 0.5 1.0 1.5 2.0 2.50.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (MMW)

(1/s 2

0,W) (

sec-1

)


0.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (LMW)

(1/s 2

0,W) (

sec-1

)


0.0 0.5 1.0 1.5 2.0 2.50.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (C)

(1/s 2

0,W) (

sec-1

)


0.0 0.5 1.0 1.5 2.0 2.50.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (MMW)

(1/s 2

0,W) (

sec-1

)


0.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (LMW)

(1/s 2

0,W) (

sec-1

)


0.0 0.5 1.0 1.5 2.0 2.50.0

0.2

0.4

0.6

0.8

1.0

1.2

Chitosan (C)

(1/s 2

0,W) (

sec-1

)


a b

c

Figure 7.2. Plots for extrapolation of the sedimentation coefficient of the different chitosan types to infinite dilution: a = MMW, b = LMW and c = C chitosan.

7.1. Analytical ultracentrifugation-derived and chitosan size parameters (mean ± standard deviation).

Type of chitosan So20,W (s) ks M (x 103) No.

monomers length (nm)

Chitosan (MMW) 2.32 ± 0.07 608 ± 35 490 ± 50 2600 ± 300 1200 ± 700 Chitosan (LMW) 1.85 ± 0.02 297 ± 7 190 ± 8 1020 ± 40 450 ± 20 Chitosan (C) 2.50 ± 0.20 650 ± 72 670 ± 200 3580 ± 1000 1600 ± 500

The sedimentation coefficient concentration dependence factor, ks, accounts for non-

ideality, i.e. interparticle interactions (Morris et al., 2009). This effect arises from the

increased viscosity of the solution at higher concentrations, and from the fact that sedimenting

solute particles must displace solvent backwards in the process. Both effects become

vanishingly small as concentration decreases. It contains information on the particle size,

shape and conformation, as well as on their degree of intermolecular interactions. ks is small

103


for globular particles, but becomes much larger for elongated molecules (Ralston, 1993). It is

extracted from the slope of the plots in Figure 7.2 and it is also show in Table 7.1.

The data obtained here for sedimentation coefficients and molecular weight of chitosans

are of a similar order to those reported, for different origin samples, by other authors (Berth et

al., 2002; Morris et al., 2009; Harding et al., 2011). These authors found that data treatment

derived from semi-flexible rod particles fitted well the experimental determinations from

chitosan. At the experimental conditions at which these experiments were performed (pH 4.3),

the ionisable groups of chitosan would be positively charged and the repelling charges would

keep the molecule in a nearly rigid, straight conformation.

From the molecular weights obtained the number of monomeric units can be also derived.

Taking as monomer weights 221 for N-acetyl glucosamine (the acetylated monomer) and 179

for glucosamine (the deacetylated subunit), a pondered average monomer weight of 187 can

be considered, for chitosans of this deacetylation degree (approximately 80 %). The number

of monomers obtained for each chitosan type is also showed on Table 7.1.

Although further information would be required to calculate a flexibility degree or a

persistence length, the simple contour length, not so different here from the length of the

extended chain, can be calculate with a mass per unit length value of 420 g mol-1 nm-1 (Vold,

2004; Morris et al., 2009). Results show that chitosan molecules are quite large, almost

macroscopic (approximately 1 µm).

In spite the lack of clear understanding of the mechanisms by which chitosan produces its

different effects and activities, such as antimicrobial, antioxidant, plant defence responses

eliciting (El Hadrami et al., 2010; Kong et al., 2010), references to the size-dependence are

frequent. Most reports ascribe a more pronounced effect to larger size chitosan molecules, for

the same acetylation degree (No et al., 2002; El Hadrami et al., 2010), while some others

report activities, such as chitosan plant antiviral action, that seem to be inversely correlated to

molecular weight (Kulikov et al., 2006). However, most of these differences are among low

condensation-degree oligomers, rather than referring to chains as large as those compared

here (No et al., 2002; Cabrera et al., 2006). Although the size differences among the three

chitosan types employed are not too extreme, the higher molecular mass type (�“C�”) was

chosen to perform further experiments. Besides, C chitosan is a cheaper product with

extensive use.

104


7.3.2. Water content determination and air dehydration

Table 7.2 shows the dry mass (dm) and water content of 2 or 3% calcium alginate beads,

naked (AB) or covered by chitosan (ACB). Water content was expressed relative to total mass

(Wc(s)). Both dry matter and water contents showed little differences between AC and ACB.

Table 7.2. Water and dry matter content (mean ± standard deviation) for alginate beads (AB) and chitosan-covered alginate beads (ACB). See Materials and Methods for parameter description.

AB ACB 2% alginate 3% alginate 2% alginate 3% alginate

dm (gdm gsample-1) 0.03 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 0.03 ± 0.01

Wc(s) (gwater gsample-1) 0.97 ± 0.03 0.95 ± 0.03 0.96 ± 0.03 0.97 ± 0.01

The water content decay upon air flow dehydration was similar for all bead types except

for 2% alginate beads, whose dehydration was slower during the first hour. The plateau

where most water has been eliminated was reached between 2 and 3 hours drying time.

Figure 7.3. Evolution of the water content over total sample mass, Wc(s), with air flow drying time, for alginate beads covered with chitosan or not, prepared with 2 and 3% sodium alginate concentration. Bars: standard error.

105


7.3.3. Calorimetric studies on water status

Calorimetric measurements designed to obtain information on the water status on beads

were performed for 3% alginate AB and ACB. Table 7.3 shows the resulting parameters.

Calorimetric parameters showed data corresponding to the fully hydrated beads, again

showing little differences between them. Melting temperatures were found to be similar. The

melting enthalpy obtained, which allowed the calculation of water frozen and unfrozen

fractions, was roughly similar for both bead types. The slightly higher unfrozen water content

of ACB may reflect the intake of an additional water amount on the chitosan covering step,

but the presence of this water content after equilibration would imply additional interactions

with the electrostatically charged chitosan. This could mean that this additional water amount

would not be frozen when temperature was reduced, which is not sustained by these

observations. Alternatively, the slightly higher frozen water content of ACB may be due to a

lower amount of water interacting with alginate, resulting from a saturation of the alginate

charged groups by the chitosan-alginate electrostatic interaction.

Table 7.3. Calorimetric parameters (mean ± standard deviation) obtained for 3% alginate beads (AB) and chitosan-covered alginate beads (ACB). See Materials and Methods for parameter description.

AB ACB Tf (onset) (ºC) -5.1 ± 0.1 -4.5 ± 0.1 Tf (peak) (ºC) 4.9 ± 1 5.8 ± 0.2

Hf(s) (J g sample-1) 255 ± 2 277 ± 2

Hf(w) (J gw-1) 284 ± 5 296 ± 7

Wf(s) (gwater gsample-1) 0.76 ± 0.02 0.83 ± 0.02

Wf(dm) (gwater gdm-1) 7.5 ± 0.8 12.9 ± 2.6

Wf(w) (gwater gwater-1) 0.85 ± 0.01 0.89 ± 0.02

Wu(s) (gwater gsample-1) 0.13 ± 0.01 0.11 ± 0.02

Wu(dm) (gwater gdm-1) 1.3 ± 0.3 1.7 ± 0.6

Wu(w) (gwater gwater-1) 0.15 ± 0.01 0.11 ± 0.02

106


7.3.4. Scanning electron microscopy of alginate beads with and without chitosan

Figure 7.4 shows the aspect of the external surface of AB and ACB under cryo-SEM. As

the purpose of the experiment was to examine the external bead surface under liquid nitrogen

temperature, etching was not performed. No evident differences could be appreciated in bead

examination: chitosan would form a homogeneously distributed film, completely covering the

surface of alginate beads, and the aspect of both AB and ACB would be similar. There were

no evident changes, such as patches or cracks, on the chitosan layer, although cooling to

liquid nitrogen temperature was performed by quenching, a process that could induce

mechanical stress on the bead surface.

Figure 7.5 compares the surfaces of AB and ACB, as observed under an environmental

scanning electron microscope, i.e., at room temperature and reduced pressure. Again, the

surface aspect of both bead types was not especially different for non-dehydrated beads.

Nevertheless, beads after an advanced dehydration time (5 hours) showed a wrinkled surface

in ACB, probably resulting from the chitosan layer adaptation to the bead reduced volume

after drying.

Figure 7.4. Cyro-SEM micrographs showing the external AB (right) and ACB (left) bead surface after quench cooling to liquid nitrogen temperature. The bar corresponds to 5 µm.

Chitosan exhibits favourable characteristics for its use in biological and biotechnological

applications, such as its null toxicity, its biocompatibility and biodegradability (Sinha &

Kumria, 2001) and a wide spectrum antimicrobial, antipathogen and antioxidant activity (No

107


et al., 2002; El Hadrami, 2010). Alginate beads enclosing plant structures help to buffer

environmental conditions and specially water content while the preliminary procedures to

cryopreservation take place (Benson, 2008). These may include air dehydration and

incubation on concentrated solutions, which diffuse into the gel matrix and also promote its

dehydration and that of the enclosed tissues (Fabre & Dereuddre, 1990; Paulet et al., 1993;

Sakai & Engelmann, 2007).

The transfer properties of alginate beads would be critical for its use, either for its stability

and gel integrity, or for the convenient treatment of contained tissues for cryopreservation.

The addition of an external layer of chitosan was considered that would have an influence on

the matter transfer properties of AB. Chitosan has been applied, for example, to modulate

plant water interchanges and transpiration, by creation of an anti-transpirant film (Bittelli et

al., 2001; Iriti et al., 2009). However, in the formulation employed here, chitosan appears not

to significantly alter the alginate beads water interaction behaviour: neither water initial

retention, water loss upon air-flow dehydration or water freezing behaviour have presented

important changes when the chitosan layer has been added to alginate beads.

a b

c d e

Figure 7.5. Environmental SEM micrographs showing bead surfaces after different air-flow dehydration times: a) AB and c) ACB (0 hours -not dehydrated beads); b) AB, d) and e) ACB (5 hours dehydration). The bar corresponds to 50 m for a) and e) and to 100 m for b), c) and d).

108


The alginate beads employed in this study behave quite differently from the beads actually

employed in cryopreservation practice. In the encapsulation-dehydration protocol (Chapter 5)

the alginate beads had been cultured in 0.75 M sucrose. Meanwhile, the beads in this work

had a much lower solute concentration, only that corresponding to the salts and other nutrients

of MS medium, with a total ionic strength of 94.25 mM (Murashige & Skoog, 1962; McCown

& Sellmer, 1987). This causes for example, that water content decreases to almost nought

upon 6 hours drying, in contrast with the less dramatic reduction in solute-loaded beads

(Chapter 5). Also, the low solute content caters for the lack of detection of a vitrification

calorimetric signal here, in spite of the extreme water content reduction.

Although, following other preparation procedures, alginate and chitosan can give raise to a

three-dimensional network (Hyland et al., 2011), the process followed here caused chitosan to

form an external layer around alginate beads. The chitosan layer of ACB beads behaved well

towards liquid nitrogen cooling, not showing any sign of cracking or other type of alteration

when inspected by cryo-SEM. However, environmental SEM revealed that the chitosan

external layer gets wrinkled, in association to the changes in water content and bead volume

caused by dehydration. A stripped or wrinkled bead surface was reported also by Anbinder et

al. (2011), when observing ACB.

Alginate beads can, additionally, be considered as a model of food (and other systems)

behavior. Microbial proliferation may often be considered a surface affair and polyssacharide

gel beads can constitute a suitable model for the study of the interface microbial growth and,

in this case, be helpful to study the mode of action of chitosan towards microorganisms.

Results obtained suggest the applicability of chitosan to the alginate beads employed in

cryopreservation as the most important gel properties, such as its water interaction behaviour,

were found to be scarcely altered. The antimicrobial protection conferred by chitosan and

their antioxidant properties can be also of great interests, as often cultivation and growth after

cryopreservation can be impaired by microbial contamination or cellular oxidative damages.

109

8. DISCUSIÓN GENERAL/GENERAL DISCUSSION

8. DISCUSIÓN GENERAL

La crioconservación constituye un conjunto de técnicas de aplicación práctica relativamente

sencilla, que permiten, con un coste comparativamente pequeño, en términos tanto de mano de

obra especializada, como de material o espacio, mantener colecciones de celulares, ápices y

embriones somáticos durante periodos indefinidos, sin que se observen importantes problemas

de deterioro de su viabilidad (Engelmann, 2004; González-Arnao et al., 2008) y manteniendo la

estabilidad genética del material (Harding, 2004).

Existen en la actualidad protocolos apropiados para una gran diversidad de especies y tipos

de tejido (Sakai, 1997; Engelmann & Takagi, 2000; González-Arnao & Engelmann, 2006;

Reed, 2008). Se han desarrollado también protocolos con técnicas específicas, como la

vitrificación (Langis et al., 1989; Sakai et al., 1991; Yamada et al., 1991) y la encapsulación-

deshidratación (Scottez et al., 1992; Bouafia et al., 1996). Además, la elaboración de

protocolos que permitan la crioconservación de nuevos materiales es un campo de intensa

actividad investigadora (Saragusty & Arav, 2011). Sin embargo, muchos tejidos y especies de

interés no pueden aún ser conservados de esta manera. Las causas de la mortalidad o deterioro

de estos especímenes pueden ser atribuidas ya sea al daño derivado de la formación de hielo en

el proceso, o aquel ocasionado por los procedimientos llevados a cabo para evitar la

congelación (Engelmann, 2008).

En las condiciones de almacenamiento empleadas (temperatura muy baja, estado vítreo)

cualquier actividad biológica, incluso las reacciones bioquímicas que llevarían al

envejecimiento o a la muerte celular, resultan efectivamente paralizadas (Mazur et al., 1984).

Tanto es así, que se ha considerado con frecuencia la posibilidad de que este almacenamiento

pudiera prolongarse por periodos indefinidos (si se evitan los daños producidos por radiación)

(Meryman, 1966a). Sin embargo, los procesos de enfriamiento y calentamiento pueden dar

lugar a daño y muerte celular, o inducir mutaciones en los individuos supervivientes, lo que

puede cuestionar el éxito del proceso.

La formación de hielo en los tejidos vegetales es frecuentemente causa de severos daños,

denominados globalmente “daños por frío” (“cold injury”). Las implicaciones de la nucleación

de hielo intra y extracelular fueron discutidas por Muldrew et al. (2004) y Mazur (2004). El

hielo intracelular es generalmente considerado letal, especialmente en plantas (Levitt, 1956;

Meryman, 1966a; Wesley-Smith et al., 1992; Benson, 2008), si bien en el reino animal, algunos

110


insectos y nemátodos soportan cierto de grado de formación de cristales de hielo intracelulares,

de crecimiento frecuentemente limitado por la acción de proteínas anticongelantes (Ramløv et

al., 1996; Wharton, 2011). También resulta letal, en muchos casos, el crecimiento de cristales

de hielo en el espacio intercelular, en especial, para complejos tejidos multicelulares (Benson,

2008). Esta localización es, en realidad, la más proclive a la formación de hielo, pues la

concentración de los fluidos extracelulares es siempre menor que la del citoplasma. Esto facilita

la formación y crecimiento del hielo doblemente, ya que, cuanto menor es la concentración de

solutos, mayor es la temperatura de equilibrio de congelación (y también la temperatura de

nucleación de cristales de hielo) y menor la viscosidad de los fluidos, lo que, a su vez, facilita

el crecimiento de los núcleos de hielo inicialmente formados. Se ha mencionado incluso un

papel activo de agentes nucleadores de hielo extracelulares, como coadyuvantes de este

fenómeno (Tomashow, 1998).

La deshidratación celular puede ser ocasionada por la formación de cristales de hielo tanto

intra como extra-celulares, ocasionando el correspondiente daño fisiológico (Meryman, 1966a,

b). Si bien se puede producir ruptura de las membranas celulares por la acumulación de hielo

(Levitt, 1980), la extrema deshidratación celular que la congelación ocasiona se considera que

es la principal responsable del daño celular (Levitt, 1980; Steponkus & Webb, 1992). A

temperaturas suficientemente negativas, el potencial químico del agua sólida es menor que el

de la líquida y esto ocasiona que el agua migre y se adicione a los cristales en crecimiento. Aún

si la formación de hielo es externa a las células, la diferencia de potencial hídrico ocasiona la

salida del agua (Mazur, 1963; Mazur et al., 1984) y la extrema deshidratación celular: la

formación de hielo extracelular, a temperaturas tan poco extremas como -10ºC, ocasiona la

salida del 90% del agua celular (Tomashow, 1998).

El agua que se incorpora al hielo procede, entre otras localizaciones, de aquella necesaria

para estabilizar proteínas y solutos, que pueden desnaturalizarse o precipitar en su ausencia, y

especialmente, para mantener la conformación de las membranas celulares. Dependiendo del

tejido y de la temperatura se han encontrado diversos tipos de lesiones en las membranas,

responsables del daño observado (Steponkus & Webb, 1992; Steponkus et al., 1993;

Tomashow, 1998). Es de destacar que el daño por deshidratación es también responsable de las

lesiones por frío en plantas a temperaturas bajas, pero a temperaturas sobre cero (Salinas,

2002).

111


La formación de hielo no ocurre inmediatamente cuando se alcanza la temperatura de

equilibrio de congelación, sino que este proceso está controlado cinética y no

termodinámicamente. Así es necesario un paso previo de nucleación que ocurre con creciente

probabilidad (en la región cercana al punto de congelación) a medida que la temperatura

disminuye (Meryman, 1966b; Franks, 1972; Mazur, 2004). La formación de cristales de hielo

intracelulares puede ocurrir por nucleación homogénea (sin que los cristales de hielo se formen,

en el enfriamiento del citoplasma, alrededor de partículas preexistentes, nucleadoras) o por

nucleación heterogénea (mediada por partículas nucleadoras). La temperatura de nucleación

homogénea del agua pura es de -38ºC. Es decir, que por debajo de esta temperatura la

formación de hielo sería espontánea. Más aún, en el citoplasma celular, multitud de agregados

moleculares y elementos microestructurales pueden dar lugar a la formación de núcleos de

hielo. Por consiguiente, esta temperatura sería, en la práctica, aún mayor. Así, el enfriamiento

de células y tejidos hasta la temperatura de vitrificación requiere un comportamiento

significativamente diferente del que exhibe el agua pura.

La transición vítrea de los sistemas acuosos puede ser concebida como el proceso

correspondiente del agua pura, desplazada hacia temperaturas más altas por el efecto de

reducción de la movilidad inducida por las sustancias en solución. De hecho, la misma

temperatura de transición vítrea del agua es difícil de medir, debido a las complicaciones

asociadas a realizar medidas experimentales en la región líquida metaestable cercana a TG que

llega hasta los 240 K (-33ºC) y es usualmente denominada como “inaccesible” (Angell, 2008,

2011). Gyan Johari, Erwin Mayer y Andreas Hallbrucker (Johari et al., 1990; Johari, 2003,

2005; Kohl et al., 2005 a,b) propusieron en diversos estudios y publicaciones el valor de 136 K

(-137ºC) para la TG del agua pura. Sin embargo, posteriormente, el prestigioso investigador del

estado vítreo Charles Austen Angell, propuso un valor más alto: 165 K (-108ºC) (Velikov et al,

2001; Angell, 2004; Yue & Angell, 2004, 2005). Esta controversia ha sido replanteada por

otros autores, pero el valor de 136 K parece el más probable, según las consideraciones más

recientes (Capaccioli & Ngai, 2011). A pesar de los intentos de Angell para reconciliar estos

diferentes puntos de vista (Angell, 2008), también han sido propuestos valores tan altos como

228 K (-45ºC) (Swenson & Teixeira, 2010) y 205 K (-68ºC), obtenido recientemente, de la

extrapolación de datos de soluciones de solutos orgánicos (McCartney & Sadtchenko, 2013).

Este baile de temperaturas de transición vítrea del agua pura, publicados en unos pocos años,

dan una imagen del furioso debate existente sobre el estado vítreo del agua. Por una parte, se

112


puede observar que es una cuestión que está aún por resolver de una manera completamente

satisfactoria. Por otra parte, se observa que es un tema candente en la actualidad, ya que las

publicaciones de estudios sobre esta materia tienen lugar en fechas contemporáneas.

En relación con los estudios presentados en este trabajo, llaman la atención los valores de TG

propuestos para el agua pura. Como se ha indicado, la interpretación más extendida implica que

la transición vítrea de sistemas acuosos ocurriría a temperaturas crecientes, a medida que la

concentración de solutos, relativa a la del agua, aumenta (por ejemplo, Roos & Karel, 1991;

Arvanitoyannis et al., 1993). Así, las TG de todos los sistemas biológicos y soluciones aquí

estudiados deben, necesariamente, tener valores más altos que la del agua pura. Los valores de

TG medidos y reportados en este trabajo, varían entre los -120ºC y -88ºC para las soluciones de

crioconservación, y entre los -118ºC y -88ºC encontrados para tejidos biológicos preparados

para la crioconservación (Capítulos 4 y 6). Estos valores son muy cercanos a los -137ºC más

aceptados, lo que es algo sorprendente, ya que las concentraciones de solutos de estos sistemas

son muy altas. Más sorprendentes aún son las propuestas para el agua pura de valores tan altos

como -108ºC, -68ºC ó -45ºC, claramente incompatibles con nuestros datos, según la

interpretación aceptada de este fenómeno.

El valor de TG para el agua representa una temperatura límite, bajo la cual el

almacenamiento indefinido de material crioconservado sería seguro. A pesar de ello, la

probabilidad que fenómenos tales como la formación de hielo puedan suceder, en un periodo de

tiempo limitado, se considera muy baja hasta 20ºC por encima de TG (Mishima & Stanley,

1998). Actualmente se considera seguro, fuera de toda discusión, el almacenamiento en NL, a

-196ºC. Por el contrario, el almacenamiento en ultracongeladores tradicionales (a -80ºC), es

controvertido, pues no se garantizaría el estado vítreo de los sistemas almacenados. La

aceptación de mayores temperaturas para la transición vítrea del agua entraría en conflicto con

estas consideraciones.

El empleo de temperaturas intermedias, alrededor de -150ºC (propias del vapor de NL o de

algunos congeladores de temperaturas extremas), sería considerado también como seguro, por

debajo de la TG del agua (y desde luego, de las concentradas soluciones citoplasmáticas), si

bien, como se refleja también en este trabajo (Capítulo 6), esta temperatura podría ser

demasiado alta para evitar reorganizaciones moleculares dentro del estado vítreo, a prolongados

tiempos de almacenamiento. En estudios realizados con semillas ortodoxas, se ha determinado

que la temperatura “segura” de conservación (a aquella que no habrá deterioro a largo plazo)

113


estaría a 70ºC por debajo de la TG (see Pritchard & Dickie, 2003). El efecto de estas

transiciones o reorganizaciones sobre la viabilidad y propiedades del material almacenando es

básicamente desconocido, si bien podría ser responsable del deterioro y la dificultad de

almacenamiento en crioconservación observado en algunos sistemas (Walters et al., 2004).

Desde que la acción crioprotectora del glicerol fue descubierta por Polge (Polge et al., 1949;

Lovelock & Polge, 1954), y posteriormente el efecto del dimetil sulfóxido (DMSO) (Lovelock,

1954; Lovelock & Bishop, 1959; Snedeker & Gaunya, 1970), una gran variedad de compuestos

potencialmente crioprotectores han sido investigados, si bien glicerol y DMSO siguen siendo

los más utilizados (Morris, 1980; Kartha, 1984). Debe tratarse de sustancias hidrosolubles y

presentar nula o baja toxicidad (Lovelock, 1954; Lovelock & Bishop, 1959). Por su modo de

acción, los crioprotectores pueden ser divididos en permeantes (que atraviesan la membrana

celular y pueden actuar intracelularmente) y los no permeantes (que no pueden cruzar la

membrana, y solo actúan extracelularmente).

Los crioprotectores permeantes, tales como glicerol, metanol, DMSO, etilenglicol y

propilenglicol, son sustancias de bajo peso molecular que penetran en la célula y actúan

protegiendo el citoplasma de la formación de hielo y sus estructuras internas de sus efectos

derivados (Benson, 2008). Estas sustancias disminuyen la temperatura de congelación

intracelular y aumentan la viscosidad de la solución, impidiendo la formación de cristales de

hielo (Merymn & Williams, 1980, 1985; Woods et al., 1999). Adicionalmente, son sustancias

que actúan como tampón osmótico durante el enfriamiento y calentamiento, contribuyendo a la

protección de las membranas celulares (Pegg, 1984). Los crioprotectores no permeantes son

sustancias frecuentemente de mayor peso molecular que, a pesar de no penetrar en el interior de

las células, tienen una acción protectora extracelular. Tal es el caso de la sacarosa (Benson,

2008). Se ha recomendado emplear una mezcla de sustancias crioprotectoras penetrantes y no

penetrantes, para aprovechar los efectos de ambos tipos de comportamientos (Fahy et al.,

1984).

El aumento de la concentración de solutos extracelulares desciende el potencial osmótico y,

para mantener la igualdad del potencial hídrico a ambos lados de la membrana, la

correspondiente cantidad de agua debe salir de las células, lo que da lugar a deshidratación

intracelular. La reducción de la cantidad de agua en el citoplasma se traduce en un aumento de

la concentración de los solutos intracelulares, con un efecto similar al de la penetración de

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agentes crioprotectores, en cuanto a aumento de la viscosidad celular, con el consiguiente

impedimento de la nucleación de hielo e inducción de la vitrificación (Benson, 2008).

Esta estrategia puede llevarse a cabo de manera aislada, por ejemplo, en los protocolos

donde se emplea la solución PSV3, que solo contiene sacarosa (Nishizawa et al., 1993;

Matsumoto et al., 1995; Makowska et al., 1999), o de manera combinada con crioprotectores

penetrantes. Tal es el caso en los protocolos donde se emplea PVS1 (Uragami et al., 1989,

Uragami, 1990), PVS2 (Sakai et al., 1990; Niwata, 1995; Sakai, 2003), y PVS4 (Sakai, 2000).

El caso del DMSO es especial, pues no solo atraviesa la membrana celular, sino que facilita

el que otras sustancias la atraviesen (Lovelock, 1954; Lovelock & Bishop, 1959). Esto no

siempre es deseable, en el contexto de la crioconservación, pues un efecto secundario de su uso

es la posibilidad de que alguna sustancia con capacidad mutagénica pueda atravesar la

membrana celular y nuclear con su ayuda, y causar alteraciones genómicas (Maclean & Hall,

1987, Chetverikova, 2011). El DMSO también podría interaccionar electrostáticamente con las

regiones hidrofílicas de los fosfolípidos de la membrana, evitando que su fluidez disminuyera a

temperaturas por debajo de 5ºC, lo que se ha relacionado con lesiones celulares provocadas por

frío (Kumamaru et al., 2010).

La viscosidad de las soluciones está directamente relacionada con la probabilidad de su

vitrificación. El empleo de diferentes crioprotectores (permeantes y no permeantes) en altas

concentraciones tiene como resultado tanto el aumento de la viscosidad como de la temperatura

de transición vítrea de las soluciones, como se ha visto en el estudio termodinámico recogido

en el Capítulo 6 (Towill & Jarret, 1992; Saragusty & Arav, 2011), características que aumentan

la probabilidad de vitrificación.

Algunos agentes crioprotectores, como el glicerol, tienen otro efecto adicional: la protección

directa de proteínas y membranas celulares frente a la deshidratación. Estos agentes, de tamaño

y características moleculares similares al agua, son capaces de sustituirla en las posiciones

donde, en condiciones de hidratación normales, se requerirían moléculas de agua para mantener

la conformación nativa (Charron et al., 2002; Mendes et al., 2004).

Los crioprotectores son esenciales para el éxito de la crioconservación de materiales

biológicos pero también son, de una manera u otra, prejudiciales para las células cuando son

utilizados en altas concentraciones. El uso combinado de diferentes crioprotectores para

conseguir este efecto permite reducir la concentración individual de cada uno, lo que disminuye

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la toxicidad global (Fahy, 1986). Los mismos procesos de añadir y eliminar las sustancias

crioprotectoras a los tejidos generan un estrés osmótico al material biológico y a las membranas

celulares que puede repercutir en su viabilidad.

Así, la reducción del contenido de agua (o el aumento de la concentración de solutos) es uno

de los principales factores determinantes del éxito de los procesos de crioconservación, como

se ha mostrado en los estudios presentados en los Capítulos 4 y 5. La presencia de hielo

intracelular, aun cuando la velocidad de enfriamiento empleada fue alta, en tejidos en que el

contenido acuoso era relativamente elevado, es evidenciada por las micrografías de cryo-SEM

presentadas en los mencionados capítulos, y confirmada por los experimentos de DSC

realizados en condiciones similares. La cantidad total de hielo y el tamaño de sus cristales en el

citoplasma son menores a medida que disminuye la cantidad de agua o, lo que es equivalente,

aumenta la concentración de solutos. Cuando este contenido de agua alcanza un determinado

umbral, la presencia de hielo no puede ser detectada por cryo-SEM o por DSC.

Otros dos factores, altamente interrelacionados, han sido destacados como de suma

importancia (Yavin & Arav, 2007): la velocidad de enfriamiento y calentamiento, y el volumen

de los especímenes.

Al respecto de la velocidad de cambio de temperatura, los procesos de crioconservación se

podrían dividir en aquellos que se llevan a cabo a velocidades relativamente lentas y los que

requieren velocidades lo más altas posibles (Sakai & Engelmann, 2007; Benson, 2008). En el

primer caso, se trata de procedimientos que aprovechan la formación de hielo extracelular para

extraer el agua intracelular (Rall et al., 1978; Mazur et al., 1981). Para evitar que, durante este

proceso, se forme hielo en el citoplasma, el enfriamiento debe ser inicialmente lento. Sin

embargo, como este proceso suele ir asociado a la adición de crioprotectores potencialmente

tóxicos, una velocidad excesivamente reducida daría lugar a una larga permanencia de estos

agentes en las células, a temperaturas a las que esta toxicidad puede ser más acusada (Muldrew

& McGann, 1994). La velocidad óptima de enfriamiento es específica para cada tipo de célula

y está directamente determinada por parámetros como el tamaño de las células, relación

superficie/volumen, permeabilidad de la membrana plasmática al agua y los crioprotectores,

grado de deshidratación celular y la relación entre la temperatura y la permeabilidad de la

membrana. Una vez alcanzado el grado de deshidratación requerido, en una segunda etapa el

proceso de enfriamiento se realiza a alta velocidad.

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Sin embargo, los métodos de crioconservación que han sido estudiados en este trabajo no

contemplan la formación de hielo extracelular. La crioconservación sin presencia de hielo fue

desarrollada inicialmente para células animales (Fahy, 1986) y posteriormente extendida a las

vegetales por Sakai (2004). Los métodos en los que se induce la vitrificación mediante

deshidratación celular y enfriamiento rápido son empleados ventajosamente en tejidos

heterogéneos complejos, para los que sería difícil o imposible encontrar velocidades de

enfriamiento óptimas para los distintos tipos de células (Meryman, 1966a; Benson, 2004,

2008). Se ha comprobado que tejidos de muy diversas especies vegetales toleran la

deshidratación, responden bien a los estreses osmóticos y soportan el enfriamiento rápido en

NL, con poca o ninguna pérdida de viabilidad (Yamada et al., 1991; Niino et al., 1992;

Matsumoto et al., 1994; Reinhoud, 1996; Senulka et al., 2007).

En los tres métodos que han sido considerados aquí (vitrificación-droplet -Capítulo 3 y 4-,

vitrificación -Capítulo 3- y encapsulación-deshidratación -Capítulo 5), la reducción del

contenido en agua es llevado a cabo mediante deshidratación osmótica mediante

crioprotectores o mediante evaporación por flujo de aire, previamente al inicio del

enfriamiento. Así, la velocidad óptima para este proceso sería la más rápida posible (Sakai &

Engelmann, 2007; Kaczmarczyk et al., 2011).

Es de destacar que en estos métodos, como se ha visto en el Capítulo 3, el enfriamiento y el

calentamiento son procesos “simétricos”, y tan imprescindible para la supervivencia es el

enfriamiento rápido como el calentamiento rápido (Meryman, 1966b). Tanto la fase de

deshidratación como la de rehidratación tienen lugar a temperatura ambiente y los procesos de

cambio de temperatura tienen un mayor grado de éxito cuanto más rápidamente ocurren

(Uchendu & Reed, 2008). Esto es debido a que, a pesar de la elevada deshidratación inducida y

de sus efectos sobre la temperatura de equilibrio de congelación, la movilidad molecular y la

temperatura de transición vítrea, aún existen zonas de temperatura que deben ser atravesadas

(tanto durante el proceso de enfriamiento como en el de calentamiento) en las cuales la

formación de hielo es posible. Así, un factor importante es la permanencia del sistema en esas

condiciones donde la probabilidad de formación de hielo aumenta con el tiempo, durante el

menor intervalo posible.

Los fenómenos de formación de hielo en condiciones de alta concentración de solutos y

elevada viscosidad y a temperaturas intermedias entre Tf y TG están aún bajo estudio y la

variación de la probabilidad de formación de hielo con la temperatura es poco conocida. Sin

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embargo, se considera que la fase de enfriamiento podría implicar un alto riesgo de nucleación

de hielo, a temperaturas más cercanas a TG, pero el crecimiento de estos cristales sería difícil en

esas condiciones. Sin embargo, sería en el proceso posterior de calentamiento donde estos

núcleos ya formados podrían, a temperaturas ya más cercanas a Tf, crecer, causando daños a las

células (Rall & Fahy, 1985; Sakai, 1997; Sakai & Engelmann, 2007).

Como se ha visto en el Capítulo 3, el procedimiento más habitual es el que emplea la

inmersión en nitrógeno líquido, para el enfriamiento, y en baños a temperaturas entre 20 y 40ºC

para el calentamiento, dando lugar a velocidades similares (pero de signo opuesto), pues los

gradientes térmicos que dan lugar a estas velocidades son equivalentes. Sin embargo, existen

una gran variedad de procedimientos para acelerar estos procesos. Por ejemplo, el factor

limitante de la velocidad de enfriamiento es el llamado efecto Leidenfrost, la formación de una

capa de nitrógeno gas, de baja conductividad térmica, y que reduce significativamente el

intercambio de calor con el nitrógeno líquido. Los procedimientos explorados para de evitar

este problema incluyen la inmersión en nitrógeno líquido en equilibrio con la fase sólida

(González-Benito et al., 2003), pero también el empleo de otros fluidos criogénicos que no

presentan este problema, tales como freón, o propano o etano líquido. La eliminación de la capa

gaseosa y la aceleración del intercambio térmico se han facilitado mediante técnicas de

inmersión forzada en el nitrógeno líquido (incluso mediante disparo de la muestra), y también

se recurre al peculiar diagrama de fase del agua para, aumentando la presión hidrostática hasta

2000 atmósferas, conseguir un enfriamiento acelerado. Sin embargo, estos procedimientos no

suelen ser empleados en la crioconservación rutinaria, pues implican complicaciones técnicas,

equipos o suministros costosos o no siempre accesibles, y en general solo se recurre a ellos en

determinadas tareas de experimentación científica, por ejemplo, en la obtención de muestras

vitrificadas para la observación por microscopía electrónica sin la formación de artefactos

debidos al hielo formado.

Si bien la velocidad de enfriamiento y calentamiento fue estudiada desde muy temprano

(Luyet & Gehenio, 1940; Mazur et al., 1957; Meryman & Kafig, 1955), la dedicación

experimental a esta tarea ha sido escasa teniendo en cuenta su importancia. Además la

complejidad de los efectos observados dio lugar a dificultades en la interpretación de la

relación velocidad-viabilidad. En ocasiones se ha considerado que las velocidades de cambio

térmico muy altas podrían dar lugar a daños en los especímenes, por la generación de

gradientes térmicos con bruscas variaciones de volumen. Sin embargo, los procesos que aquí

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son considerados tienen lugar en muestras de tamaño lo suficientemente reducido para que el

equilibrio térmico ocurra de manera rápida y los gradientes térmicos son minimizados. De

hecho no hay referencias de daños por esta causa en sistemas vegetales en crioconservación,

aun cuando se han recurrido a técnicas para aumentar la velocidad de enfriamiento, como es el

mencionado empleo de mezclas de nitrógeno líquido y sólido (González-Benito et al., 2003).

Esto nos lleva al tercer factor de importancia, el tamaño del espécimen a ser crioconservado.

El éxito de los procesos de crioconservación es mayor a menor tamaño de éste, como se puede

ver en los Capítulos 4, 5 y 6, por múltiples razones, algunas en relación con el proceso de

deshidratación y de tratamiento con crioprotectores. La difusión de éstos hacia el interior de las

células (y su salida posterior) es más rápida cuanto menor sea el tamaño de muestra. Y lo

mismo ocurre con los procesos de deshidratación y rehidratación, sea cuál sea el método

empleado. La relación superficie/volumen (que, variando la superficie de manera cuadrática y

el volumen, cúbica, es claramente, inversamente proporcional al diámetro equivalente) permite

racionalizar este comportamiento. Esto implica que las situaciones de posible daño fisiológico

ocasionadas por la presencia de crioprotectores potencialmente tóxicos o mutagénicos, de

estreses asociados a la deshidratación y a la alta concentración de solutos, pueden ser limitadas

a menores intervalos de tiempo. Hay que recordar que estos procesos de estrés y posible daño

estarían también detenidos en condiciones de vitrificación, con lo que el periodo en el que

pueden deteriorarse los sistemas a ser crioconservados incluye solo aquel del inicio y el final

del proceso, y no durante el almacenamiento, por largo que éste sea (Meryman, 1966; Sakai &

Engelmann, 2007; Kaczmarczyk et al., 2011).

El tamaño también influye en la velocidad de cambio térmico. De hecho, la manera más

eficiente de aumentar la velocidad de los procesos tanto de enfriamiento como de

calentamiento, es reducir el espécimen a tratar al volumen lo más pequeño posible (incluyendo

cualquier contenedor, encapsulado o solución circundante) (Arav, 1992; Arav et al., 2002;

Yavin & Arav, 2007). Como se ha podido observar en los Capítulos 3 y 5, aquellos

procedimientos donde los especímenes y sus contenedores tenían un tamaño global menor,

podían ser enfriados y calentados a mayores velocidades. La difusión de calor es un proceso

proporcional, entre otros factores, al espesor del material que debe ser atravesado por el flujo

térmico. Así, un el aumento del volumen del espécimen se traduce en el drástico incremento del

tiempo requerido para que su temperatura pueda ser alterada.

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El tamaño de muestra, finalmente, tiene una relación directa con la probabilidad de

formación de hielo. Como se ha explicado, se trata de un proceso estocástico, en el que las

moléculas de agua se reorganizan para formar núcleos de hielo propulsadas por movimientos

Brownianos. La probabilidad de que este movimiento “al azar” dé lugar a un núcleo de hielo

que pueda generar un cristal es proporcional al número de moléculas de agua. Así, los

especímenes pequeños son menos proclives a la formación de hielo, en las mismas condiciones

que los mayores.

La crioconservación de organismos de gran tamaño, tales como relativamente grandes

órganos animales o animales completos, a parte de otros diversos obstáculos, se encuentra,

como uno de los principales impedimentos, con las dificultades derivadas del tamaño mucho

mayor de los especímenes, en comparación por ejemplo, con los estudiados en este trabajo, que

varían desde los 0,7 mg de ápices de menta deshidratados (Capítulo 4), a los 3,9 mg de los

igualmente deshidratados ápices de ajo (Capítulo 4), y a los 10 mg de cuentas de alginato,

incluyendo a ápices, completamente hidratadas (Capítulo 5). La gran diversidad de tipos

celulares a ser conservados y sus diversos requerimientos, así como la necesidad de alcanzar un

alto grado de recuperación en todos estos tipos celulares, se añaden a las mencionadas

dificultades (Meryman, 1966a).

Una de las cuestiones a tener en cuenta en el diseño de procedimientos para la

crioconservación es que los diversos tejidos y células de distintas especies y cultivares

presentan muy diversas respuestas frente a los estreses inducidos en estos procesos. Por una

parte, se procura diseccionar el material a crioconservar, reduciendo su tamaño al mínimo

estrictamente necesario para su posterior recuperación, en aras de trabajar con especímenes de

tamaño reducido, por las ventajas arriba indicadas. Sin embargo, esto implica desproteger estos

tejidos y exponerlos a fuertes estreses, agravados por los derivados de la manipulación de

grandes números de muestras, dilatados tiempos de espera y fluctuaciones de condiciones

ambientales. Como una solución de compromiso, se emplean el encapsulado mediante los

versátiles y completamente biocompatibles geles de alginato cálcico. Los efectos deletéreos de

las fluctuaciones incontroladas de contenido en agua, concentración de solutos y cambios

térmicos son apantallados por esta capa, básicamente formada por una red de polisacáridos

reteniendo agua.

En la actualidad, la crioconservación debe ser considerada como una técnica de gran

potencial para la conservación de recursos fitogenéticos. A pesar de ello, existe la necesidad de

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profundizar en la investigación, tanto para mejorar los métodos existentes como para encontrar

otros nuevos, que se adapten a las necesidades de las diferentes especies que deben ser

conservadas.

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9. CONCLUSIONES/CONCLUSIONS

9. CONCLUSIONS

- Mint shoot tip survival results suggest that the observed physiological response is

dependent upon the cryopreservation method and the cooling and warming rates associated.

This study demonstrates that faster thermal rates give rise to higher survival and recovery

percentages.

- Specimens of mint shoot tips at the final step of the droplet-vitrification cryopreservation

protocol become vitrified and no ice is formed, which is associated to the lack of damage in

the process and the resulting high viability. Meanwhile, shoot tips at previous steps of the

protocol show the formation of ice by both microscopic and calorimetric observations, which

is a determinant cause of damage leading to the observed null viability of these specimens.

The agreement between the results obtained by both cryo-SEM and DSC techniques is

relevant and the potential of its associated use is clearly shown.

conclude that the TG of commonly employed PVSs did not change with the wide

range of cooling and warming rates. The behavior of cryopreserved tissues might be, though,

different. Storage at very low temperature (LN, at -196ºC) would mean no risk due to

reorganization of the vitreous state. But higher temperatures, such as that of freezers or even

in LN vapor phase (at approx. -150ºC), might imply some reorganization after quench

cooling.

- Results obtained suggest the applicability of chitosan to the alginate beads employed in

cryopreservation, as the most important gel properties, such as its water interaction behaviour,

was found to be scarcely altered. The antimicrobial protection conferred by chitosan and their

9. CONCLUSIONES/CONCLUSIONS

antioxidant properties can be also of great interests, as often cultivation and growth after

cryopreservation can be impaired by microbial contamination or cellular oxidative damages.

10. REFERENCIAS/REFERENCES

10. REFERENCIAS

Ablett S, Darke AH, Izzard MJ, Lillford PJ. (1993) Studies of the glass transition in malto-

oligomers. In: The glassy state in foods. (JMV Blanshard & PJ Lillford). Nottingham

University Press. Nottingham, pp. 189-206.

Ait Barka E, Eullaffroy P, Clément C, Vernet G. (2004) Chitosan improves development, and

protects Vitis vinifera L. against Botrytis cinerea. Plant Cell Reports, 22:608-614.

Alberola N, Perez J, Tatibouet J, Vassoille R. (1983) Stability of the vitreous phase in water-

ethylene glycol mixtures studied by internal friction. The Journal of Physical Chemistry,

87:4264-4267.

Amborabé BE, Bonmort J, Fleurat-Lessard P, Roblin G. (2008) Early events induced by

chitosan on plant cells. Journal of Experimental Botany, 59:2317-2324.

Anbinder PS, Deladino L, Navarro AS, Amalvy JI, Martino MN. (2011) Yerba Mate Extract

Encapsulation with Alginate and Chitosan Systems: Interactions between Active

Compound Encapsulation Polymers. Journal of Encapsulation and Adsorption Sciences,

1:80-87.

Andres Y, Giraud L, Gerente C, Le-Cloiréc P. (2007) Antibacterial effects of chitosan

powder: Mechanisms of action. Environmental Technology, 28:1357-1363.

Angell CA. (1982) Supercooled water. In: Water-A comprehensive treatise. 7. (F Franks, ed.).

Plenum Press. New York, pp. 215-338.

Angell CA. (2000) Liquid fragility and the glass transition in water and aqueous solutions.

Chemical Reviews, 102:2627-2650.

Angell CA. (2004) Amorphous water. Annual Review of Physical Chemistry, 55:559-583.

Angell CA. (2008) Insights into phases of liquid water from study of its unusual glass-

forming properties. Science, 319:582-587.

Angell CA, Ngai KL, McKenna GB, McMillan PF, Martin SW. (2000) Relaxation in glass

forming liquids and amorphous solids. Journal of Applied Physics, 88:3113-3157.

Angell CA, Ogunl M, Slchlna WJ. (1982) Heat capacity of water at extremes of supercooling

and superheating. Journal of Physical Chemistry, 86:998-1002.

124


Angell CA, Sare JM, Sare EJ. (1978) Glass transition temperatures for simple molecular

liquids and their binary solutions. The Journal of Physical Chemistry, 82:2622-2629.

Angell CA, Shuppert J, Tucker JC. (1973) Anomalous properties of supercooled water. Heat

capacity, expansivity, and proton magnetic resonance chemical shift from 0 to -38º. The

Journal Physical Chemistry, 77: 3892-3899.

Angell CA, Smith DL. (1982) Test of the entropy basis of the Vogel-Tammann-Fulcher

equation. Dielectric relaxation of polyalcohols near Tg. The Journal of Physical Chemistry,

86:3845-3852.

Arav A. (1992) Vitrification of oocytes and embryos. In: Trends in embryo transfer. (A Lauria

& F Gandolfi, eds.). Portland Press. Cambridge, pp. 255-264.

Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H. (2002) New trends in gamete's

cryopreservation. Molecular and Cellular Endocrinology, 187:77-81.

Arvanitoyannis I, Blanshard JMV, Izzard MJ, Lillford PJ, Ablett S. (1993) Calorimetric study

of the glass transition occurring in aqueous glucose: fructose solutions. Journal of the

Science of Food and Agriculture, 63:177-188.

Arvy MP, Gallouin F. (2006) Especias, aromatizantes y condimentos. Ediciones Mundi-

Prensa. Madrid.

Ashmore SE. (1997) Status report on the development and application of in vitro techniques

for the conservation and use of plant genetic resources. International Plant Genetic

Resources Institute. Rome, pp. 11-18.

Bald WB. (1987) Quantitative Cryofixation. Hilger. Bristol, pp. 76-103.

Barber MS, Bertram RE, Ride JP. (1989) Chitin oligosaccharides elicit lignification in

wounded wheat leaves. Physiological and Molecular Plant Pathology, 34:3-12.

Benhamou N. (1992) Ultrastructural detection of -1,3-glucans in tobacco root tissues

infected by Phytophthora parasitica var. nicotianae using a gold-complexed tobacco -1,3-

glucanase. Physiological and Molecular Plant Pathology, 41:351-357.

125


Benhamou N, Kloepper JW, Tuzun S. (1998) Induction of resistance against Fusarium wilt of

tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and

cytochemistry of the host response. Planta, 204:153-168.

Benson EE. (1999) An introduction to plant conservation biotechnology. In: Plant

conservation biotechnology. (EE Benson, ed.). Taylor & Francis. London, pp. 3-10.

Benson EE. (2004) Cryoconserving algal and plant diversity: Historical perspectives and

future challenges. In: Life in the Frozen State. (B Fuller, N Lane & EE Benson, eds.). CRC

Press. London, pp. 299-328.

Benson EE. (2008) Cryopreservation theory. In: Plant cryopreservation: A practical guide.

(BM Reed, ed.). Springer. New York, pp. 15-32.

Benson EE, Reed BM, Brennan RM, Clacher KA, Ross DA. (1996) Use of thermal analysis

in the evaluation of cryopreservation protocols for Ribes nigrum L. germplasm.

CryoLetters, 17:347-362.

Berth G, Cölfen H, Dautzenberg H. (2002) Physicochemical and chemical characterisation of

chitosan in dilute aqueous solution. Progress in Colloid and Polymer Sciences, 119:50�–57.

Bhat S, Gupta SK, Tuli R, Khanuja SPS, Sharma S, Bagchi GD, Kumar A, Kumar S. (2001)

Photoregulation of adventitious and axillary shoot proliferation in menthol mint, Mentha

arvensis. Current Science, 80:878-881.

Bittelli M, Flury M, Campbell GS, Nichols EJ. (2011) Reduction of transpiration through

foliar application of chitosan. Agricultural and Forest Meteorology, 107:167-175.

Blanshard JM, Franks F. (1987) Ice crystallization and its control in frozen-food systems. In:

Food Structure and Behavior. (JMV Blanshard & PJ Lillford, eds.) Academic Press.

London, pp. 51-65.

Block W. (2003) Water status and thermal analysis of alginate beads used in cryopreservation

of plant germplasm. Cryobiology, 47:59-72.

Bouafia S, Jelti N, Lairy G, Blanc A, Bonnel E, Dereuddre J. (1996) Cryopreservation of

potato shoot tips by encapsulationdehydration. Potato Research, 39:69-78.

126


Brüning R, Samwer K. (1992) Glass transition on long time scales. Physical Review B,

46:318-322.

Buckley KA, Conway EJ, Ryan HC. (1958) Concerning the determination of total

intracellular concentrations by the cryoscopic method. Journal of Physiology, 143:236-245.

Cabrera JC, Messiaen J, Cambier P, van Cutsem P. (2006) Size, acetylation and concentration

of chitooligosaccharide elicitors determine the switch from defence involving PAL

activation to cell death and water peroxide production in Arabidopsis cell suspensions.

Physiologia Plantarum, 127:44-56.

Capaccioli S, Ngai KL. (2011) Resolving the controversy on the glass transition temperature

of water?. The Journal of Chemical Physics, 135:104504.

Carpenter JF, Dawson PE. (1991) Quantification of dimethyl sulfoxide in solutions and

tissues by high performance liquid chromatography. Cryobiology, 28:210-215.

Chambers HL. (1992) Mentha: genetic resources and the collection at USDA-ARS NCGR-

Corvallis. Lamiales News, 1:3-4.

Chang ZH, Baust JG. (1991) Physical aging of glassy state: DSC study of vitrified glycerol

systems. Cryobiology, 28:87-95.

Charron C, Kadri A, Robert M-C, Giegé R, Lorber B. (2002) Crystallization in the presence

of glycerol displaces water molecules in the structure of thaumatin. Acta

Crystallographica, D58:2060-2065.

Cheng X, Kaplan LA. (2003) Simultaneous analyses of neutral carbohydrates and amino

sugars in freshwaters with HPLC-PAD. Journal of Chromatographic Science, 41:434-438.

Chetverikova EP. (2011) Consequences of plant tissue cryopreservation (phenotype and

genome). Biophysics, 56:309-315.

Chua SP, Normah MN. (2011) Effect of preculture, PVS2 and vitamin C on survival of

recalcitrant Nephelium ramboutan-ake shoot tips after cryopreservation by vitrification.

Cryoletters, 32:506-515.

Chung YC, Wang HL, Chen YM, Li SL. (2003) Effect of abiotic factors on the antibacterial

activity of chitosan against waterborne pathogens. Bioresource Technology, 88:179-184.

127


Cole MD. (1992) The significance of the terpenoids in the Labiatae. In: Advances in labiate

science (RM Harley & T Reynolds, eds.). Royal Botanic Gardens. Kew, pp. 315-324.

Coviello T, Matricardi P, Marianecci C, Alhaique F. (2007) Polysaccharide hydrogels for

modified release formulations. Journal of Controlled Release, 119:5-24.

Craig S, Beaton CD. (1996) A simple cryo-SEM method for delicate plant tissues. Journal of

Microscopy, 182:102-105.

Cronquist A. (1981) An integrated system of classification of flowering plants. Columbia

University Press. New York.

Dam J, Schuck P. (2005) Sedimentation Velocity Analysis of Heterogeneous Protein-Protein

Interactions: Sedimentation Coefficient Distributions c (s) and Asymptotic Boundary

Profiles from Gilbert-Jenkins Theory. Biophysical Journal, 89:651-666.

Dangl JL, Jones JDG. (2001) Plant pathogens and integrated defence responses to infection.

Nature, 411:826-833.

Debenedetti PG, Stillinger FH. (2001) Supercooled liquids and the glass transition. Nature,

410:259-267.

Deladino L, Anbinder PS, Navarro AS, Martino MN. (2008) Encapsulation of natural

antioxidants extracted from Ilex paraguariensis. Carbohydrate Polymers, 71:126-134.

Dereuddre J, Scottez C, Arnaud Y, Duron M. (1990) Résistance d'apex caulinaires de

vitroplants de Poirier (Pyrus communis L. cv Beurré Hardy), enrobés dans l'alginate, à une

déshydratation puis à une congélation dans l'azote liquide: effet d'un endurcissement

préalable au froid. Comptes Rendus de l'Académie des Sciences, 10:317-323.

Draget KI. (2000) Alginates. In: Handbook of hydrocolloids (G Phillips & P Williams, eds.).

Woodhead. Cambridge, pp. 379-395.

Draget KI, Skjåk-Bræk G, Smidsrød O. (1997) Alginate based new materials. International

Journal of Biological Macromolecules, 21:47-55.

Draget KI, Taylor C. (2011) Chemical, physical and biological properties of alginates and

their biomedical implications. Food Hydrocolloids 25:251-256.

128


Dumet D, Block W, Worland R, Reed BM, Benson EE. (2000) Profiling cryopreservation

protocols for Ribes ciliatum using differential scanning calorimetry. CryoLetters, 21:367-

378.

Eaton P, Fernandes JC, Pereira E, Pintado ME, Xavier Malcata F. (2008) Atomic force

microscopy study of the antibacterial effects of chitosans on Escherichia coli and

Staphylococcus aureus. Ultramicroscopy, 108:1128-1134.

Ediger MD, Angel CA, Nagel SR. (1996) Supercooled liquids and glasses. Journal of

Physical Chemistry, 100:13200-13212.

El Ghaouth A, Arul J, Asselin A, Benhamou N. (1992) Antifungal activity of chitosan on

postharvest pathogens: induction of morphological and cytological alterations in Rhizopus

stolonifer. Mycological Research, 96:769-779.

El Ghaouth A, Arul J, Wilson C, Benhamou N. (1994) Ultrastructural and cytochemical

aspects of the effect of chitosan on decay of bell pepper fruit. Physiological and Molecular

Plant Pathology, 44:417-432.

El Hadrami A, Adam LR, El Hadrami I, Daayf F. (2010) Chitosan in Plant Protection. Marine

Drugs 8:968-987.

El Hassni M, El Hadrami A, Daayf F, Chérif M, Ait Barka E, El Hadrami I. (2004) Chitosan,

antifungal product against Fusarium oxysporum f. sp. albedinis and elicitor of defence

reactions in date palm roots. Phytopathologia Mediterranea, 43:195-204.

Elliott JAW, Prickett RC, Elmoazzen HY, Porter KR, McGann LE. (2007) A multisolute

osmotic virial equation for solutions of interest in biology. Journal of Physical Chemistry

B, 111:1775-1785.

Engelmann F. (1997) In vitro conservation methods. En Biotechnology and Plant Genetic

Resources: Conservation and Use. (JA Callow, BV Ford-Lloyd & HJ Newbury, eds.).

CAB International. Wellingford, pp. 119-162.

Engelmann F. (2004) Plant cryopreservation: progress and prospects. In vitro Cellular &

Developmental Biology-Plant, 40:427-433.

129


Engelmann F. (2008) Importance of cryopreservation for the conservation of plant genetic

resources. In: Cryopreservation of tropical plant germplasm (F Engelmann & H Takagi,

eds.). International Plant Genetic Resources Institute. Rome, pp. 8-20.

Engelmann F, González-Arnao MT, Wu Y, Escobar R. (2008) The development of

encapsulation dehydration. In: Plant Cryopreservation: A Practical Guide. (BM Reed, ed.).

Springer. New York.

Engelmann F, Takagi H. (2000) Cryopreservation of Tropical Plant Germplasm - Current

Research Progress and Applications. Tsukuba & International Plant Genetic Resources

Institute. Rome, p. 496.

Fabre J, Dereuddre J. (1990) Encapsulation-dehydration: A new approach to cryopreservation

of Solanum shoot- tips. CryoLetters, 11:413-426.

Fahn A. (1979) Secretory tissues in plants. Academic Press. London, pp. 158-222.

Fahy GM. (1986) Vitrification: a new approach to organ cryopreservation. Progress in

Clinical and Biological Research, 224:305-335.

Fahy, GM, MacFarlane DR, Angell CA, Meryman HT. (1984) Vitrification as an approach to

cryopreservation. Cryobiolology, 21:407-426.

Faoro F, Maffi D, Cantu D, Iriti M. (2008) Chemical-induced resistance against powdery

mildew in barley: the effects of chitosan and benzothiadiazole. BioControl, 53:387-401.

Flora Europaea (on line version) http://rbg-web2.rbge.org.uk/FE/fe.html (13/01/2013)

Franks F. (1972) Water, a comprehensive treatise. Plenum Press. New York.

Fujikawa S. (1980) Freeze-fracture and etching studies on membrane damage on human

erythrocytes caused by formation of intracellular ice. Cryobiology, 17:351-362.

Fujikawa S. (1981) The effect of various cooling rates on the membrane ultrastructure of

frozen human erythrocytes and its relation to the extent of haemolysis after thawing.

Journal of Cell Science, 49:369-382.

Fujita H. (1962) Mathematical Theory of Sedimentation Analysis. Academic Press. New

York.

130

http://rbg-web2.rbge.org.uk/FE/fe.html


Franks, F. (1986) Unfrozen water - yes - unfreezable water - hardly - bound water - certainly

not. CryoLetters, 7:207-207.

Fullerton GD, Keener CR, Cameron IL. (1994) Correction for solute/solvent interaction

extends accurate freezing point depression theory to high concentration range. Journal of

Biochemistry Biophysical Method, 29:217-235.

Gao C, Zhou GY, Xu Y, Hua TC. (2005) Glass transition and enthalpy relaxation of ethylene

glycol and its aqueous solution. Thermochimica Acta, 435:38-43.

George M, Abraham TE. (2006) Polyionic hydrocolloids for the intestinal delivery of protein

drugs: alginate and chitosan-a review. Journal of Controlled Release, 114:1-14.

González-Arnao MT, Engelmann F. (2006) Cryopreservation of plant germplasm using the

encapsulation-dehydration technique: review and case study on sugarcane. CryoLetters,

27:155-168.

González-Arnao MT, Moreira T, Urra C. (1996) Importance of pre growth with sucrose and

vitrification for the cryopreservation of sugarcane apices using encapsulation-dehydration.


González-Arnao MT, Panta A, Roca WM, Escobar RH, Engelmann F. (2008) Development

and large scale application of cryopreservation techniques for shoot and somatic embryo

cultures of tropical crops. Plant Cell, Tissue and Organ Culture, 92:1-13.

González-Arnao MT, Urra C, Engelmann F, Ortiz R, Fe C. (1999) Cryopreservation of

encapsulated sugarcane apices: effect of storage temperature and storage duration.


González-Benito ME, Clavero-Ramírez I, López-Aranda JM. (2004) Review. The use of

cryopreservation for germoplasm conservation of vegetatively propagated crops. Spanish

Journal of Agricultural Research, 2:341-351.

González-Benito ME, Mendoza-Condori VH, Molina-García AD. (2007) Cryopreservation of

in vitro shoot apices of Oxalis tuberosa Mol. CryoLetters, 28:23-32.

González-Benito ME, Núñez-Moreno Y, Martín C. (1998) A protocol to cryopreserve nodal

explants of Antirrhium microphyllum by encapsulation-dehydratation. CryoLetters,

19:225-230.

131


González-Benito, M.E., Salinas, P., Amigo, P. 2003. Effect of seed moisture content and

cooling rate in liquid nitrogen on legume seed germination and seedling vigour. Seed

Science and Technology, 31:423-434.

Graybill JR, Horton HF. (1969) Limited fertilization of steelhead trout eggs with

cryopreserved sperm. Journal of the Fisheries Board of Canada, 26:1400-1404.

Goy RC, Britto D, Assis OBG. (2009) A review of the antimicrobial activity of chitosan.

Polímeros, 19:241-247.

Guignon B, Torrecilla JS, Otero L, Ramos AM, Molina-García AD, Sanz PD. (2008) The

initial freezing temperature of foods at high pressure. Critical Reviews in Food Science and

Nutrition, 48:328-340.

Gupta R. (1991) Agrotechnology of medicinal plants. In: The medicinal plant industry. (ROB

Wijessekera, ed.). CRC Press. Boca Raton, pp. 43-57.

Halmagyi A, Deliu C, Coste A. (2005) Plant regrowth from potato shoot tips cryopreserved

by a combined vitrification-droplet method. CryoLetters, 26:313-322.

Halmagyi A, Deliu C, Isac V. (2010) Cryopreservation of Malus cultivars: comparison of two

droplet protocols. Science Horticulture, 124:387-392.

Harding K. (2004) Genetic integrity of cryopreserved plant cells: A review. CryoLetters,

18:217-230.

Harding SE, Schuck P, Abdelhameed AS, Adams G, Kök MS, Morris GA. (2011) Extended

Fujita Approach to the Molecular Weight Distribution of Polysaccharides and other

Polymeric Systems. Methods, 54:136-144.

Hashimoto T, Tasaki Y, Harada M, Okada T. (2011) Electrolyte-doped ice as a platform for

atto- to femtoliter reactor enabling zeptomol detection. Analitical Chemistry, 83:3950-

3956.

Henshaw GG, Stamp JA, Westcott JJ. (1980) Tissue culture and germoplasm storage. In:

Plant cell cultures: results and perspectives. (F Sala, R Parisi, R Cella & O Cifferi, eds.).

Elsevier. Amsterdam, pp. 277-282.

132


Hills BP, Cano C, Belton PS. (1991) Proton NMR relaxation studies of aqueous

polysaccharide systems. Macromolecules, 1:2944-2950.

Hills BP, Takacs SF, Belton PS. (1990) A new interpretation of proton NMR relaxation time

measurements of water in food. Food Chemistry, 37:95-111.

Hills BP, Tang H, Belton PS, Khaliq A, Harris RK. (1998) NMR Oxygen-17 studies of the

state of water in a saturated sucrose solution. Journal Molecular Liquids, 75:45-59.

Hirai D, Saki A. (1999) Cryopreservation of in vitro-grown axillary shoot-tip meristems of

mint (Mentha spicata L) by encapsulation vitrification. Plant Cell Reports, 19:150-155.

Hirano S, Nakahira T, Nakagawa M, Kim SK. (1999) The preparation and applications of

functional fibres from crab shell chitin. Journal of Biotechnology, 70:373-377.

Hubálek Z. (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology,

46:205-229.

Hyland LL, Taraban MB, Hammouda B, Bruce YY. (2011) Mutually reinforced

multicomponent polysaccharide networks. Biopolymers, 95:840-851.

Iglesias HA, Chirife J. (1978) Delayed crystallization of amorphous sucrose in humidified

freeze dried model systems. Journal of Food Technology, 13:137-144.

International Board for Plant Genetic Resources. (1982) The design of seed storage facilities

for genetic conservation. IBPGR. Rome.

Iriti M, Picchi V, Rossoni M, Gomarasca S, Ludwig N, Garganoand M, Faoro F. (2009)

Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure.

Environmental and Experimental Botany, 66:493-500.

Islam M, Dembele D, Keller ER. (2005) Influence of explant, temperature and different culture

vessels on in vitro culture for germplasm maintenance of four mint accessions. Plant Cell,

Tissue and Organ Culture, 81:123-130.

Johari GP. (2003) Water�’s T-endotherm, sub-T peak of glasses and T of water. The Journal of

Chemical Physics, 119:2935.

Johari GP. (2005) Water�’s size-dependent freezing to cubic ice. The Journal of Chemical

physics, 122:194504.

133


Johari GP, Astl G, Mayer E. (1990) Enthalpy relaxation of glassy water. Journal of Chemical

Physics, 92:809-810.

Jouve L, Hoffmann L, Hausman JF. (2004) Polyamine, carbohydrate, and proline content

changes during salt stress exposure of aspen (Populus tremula L.): involvement of

oxidation and osmoregulation metabolism. Plant Biology, 6:74-80.

Kaczmarczyk A, Rokka VM, Keller ERJ. (2011) Potato shoot tip cryopreservation. A review.

Potato Research, 54:45-79.

Kami D, Kasuga J, Arakawa K, Fujikawa S. (2008) Improved cryopreservation by diluted

vitrification solution with supercooling-facilitating flavonol glycoside. Cryobiology,

57:242-245.

Kartha KK. (1984) Culture of shoot meristems: Pea. In: Cell Culture and Somatic Cell

Genetics of Plants. Vol 1: Laboratory Procedures and their Applications. (IK Vasil, ed.).

Academic Press. Orlando, pp. 106-110.

Kartha KK, Leung NL, Mroginski LA. (1982) In vitro growth-responses and plant-

regeneration from cryopreserved meristems of cassava (Manihot esculenta Crantz),

Zeitschrift fur Pflanzenphysiologie, 107:133-140.

Kauzmann W. (1948) The nature of the glassy state and the behavior of liquids at low

temperatures. Chemistry Review, 43: 219-256.

Keller ERJ. (2002) Cryopreservation of Allium sativum L. (Garlic). In: Cryopreservation of

Plant Germplasm II. (LE Towil & PS Bajaj, eds.). Springer. Berlin, pp. 37-47.

Keller ERJ, Dreiling M. (2003) Potato cryopreservation in Germany - using the droplet

method for the establishment of a new large collection. Acta Horticulturae, 623:193-200.

Khor E, Shiong Loh C. (2005) Artificial seeds. In: Applications of Cell Immobilisation

Biotechnology. (V Nedovic & R Willaert, eds.). Springer. Amsterdam, pp. 527-537.

Kim HH, Lee JK, Yoon JW, Ji JJ, Nam SS, Hwang HS, Cho EG, Engelmann F. (2006a)

Cryopreservation of garlic bulbil primordia by the droplet-vitrification procedure.


134


Kim HH, Yoon JW, Park YE, Cho EG, Sohn JK, Kim TS, Engelmann F. (2006b)

Cryopreservation of potato cultivated varieties and wild species: critical factors in droplet

vitrification. CryoLetters, 27:223-234.

Kim W-T, Chung H, Shin II-S, Yam KL, Chung D. (2008) Characterization of calcium

alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate.

Carbohydrate Polymers, 71:566-573

King AH. (1983) Brown seaweed extracts (Alginates). Food Hydrocolloids, 2:115-188.

Knorr D. (1984) Use of chitinous polymers in food. Food Technology, 38:85-94.

Kohl I, Bachmann L, Hallbrucker A, Mayer E, Loerting T. (2005a) Liquid-like relaxation in

hyperquenched water at <= 140 K. Physical Chemistry Chemical Physics, 7:3210-3220.

Kohl I, Bachmann L, Mayer E, Hallbrucker A, Loerting T. (2005b) Water Behaviour: Glass

transition in hyperquenched water?. Nature, 435:E1-E1.

Kong M, Chen XG, Xing K, Park HJ. (2010) Antimicrobial properties of chitosan and mode

of action: A state of the art review. International Journal of Food Microbiology, 144:51-63.

Krajewska B, Wydro P, Ja czyk A. (2011) Probing the modes of antibacterial activity of

chitosan. Effects of pH and molecular weight on chitosan interactions with membrane

lipids in Langmuir films. Biomacromolecules, 12:4144-52.

Kryszczuk A, Keller J, Grübe M, Zimnoch-Guzowska E. (2006) Cryopreservation of potato

(Solanum tuberosum L.) shoot tips using vitrification and droplet method. Food

Agriculture Environment, 4:196-200.

Kuchitsu K, Kosaka H, Shiga T, Shibuya N. (1995) EPR evidence for generation of hydroxyl

radical triggered by N-acetylchitooligosaccharide elicitor and a protein phosphatase

inhibitor in suspension-cultured rice cells. Protoplasma, 188:138-142.

Kulikov SN, Chirkov SN, Il�’ina AV, Lopatin SA, Varlamov VP. (2006) Effect of the

Molecular Weight of Chitosan on Its Antiviral Activity in Plants. Applied Biochemistry

and Microbiology, 42:200�–203.

135


Kumamaru T, Uemura Y, Inoue Y, Takemoto Y, Siddiqui SU, Ogawa M, Satoh H. (2010)

Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin

in rice seed. Plant and Cell Physiology, 51:38-46.

Langis R, Schnabel B, Earle ED, Steponkus PL. (1989) Cryopreservation of Brassica

campestris L. Cell suspensions by vitrification. Cryoletters, 10:421-428.

Lawrence BM. (1985) A review the world production of essential oils. Perfumer and Flavorist,

10:1-16.

Lee YC. (1996) Carbohydrate analyses with high-performance anion-exchange

chromatography. Journal of Chromatography A, 720:137-149.

Leindenfrost JG. (1966) On the Fixation of Water in Diverse Fire. International Journal of

Heat Mass Transfer, 9:1154-1166 (translation of the original 1756 publication).

Leprince O, Walters-Vertucci C. (1995) A calorimetric study of the glass transition behaviors

in axes of Phaseolus vulgaris L. seeds with relevance to storage stability. Plant Physiology,

109:1471-1481.

Leuba JL, Stössel P. (1986) Chitosan and other polyamines: Anti-fungal activity and

interaction with biological membranes. In: Chitin in Nature and Technology. (RAA

Muzzarelli, C Jeuniaux & GW Gooday, eds.). Plenum Press. New York, pp. 215-222.

Leunufna S, Keller ERJ. (2005) Cryopreservation of yams using vitrification modified by

including droplet method: effects of cold acclimation sucrose. CryoLetters, 26:93-102.

Levine H, Slade L. (1992) Glass transition in foods. In: Physical Chemistry of Foods. (HG

Schwartzberg & RW Hartel, eds.) Marcel Dekker. New York, pp. 83-221.

Levitt J. (1956) The hardiness of plants. Academic Press. New York, p. 278.

Levitt J. (1980) Responses of Plants to Environmental Stress. Chilling, Freezing and High

Temperature Stresses. 2nd Ed. Academic Press. New York.

Loewenfeld C, Back F. (1980) Guia de hierbas y especias. Ediciones Omega. Barcelona.

Lorenzi HE, Matos FJ de A. (2002) Plantas medicinais no Brasil/ Nativas e exóticas. Instituto

Plantarum. Nova Odessa.

136


Lovelock JE. (1954) The protective action of neutral solutes against haemolysis by freezing

and thawing. Biochemical Journal, 56:265.

Lovelock JE, Bishop MWH. (1959) Prevention of freezing damage to living cells by dimethyl

sulfoxide. Nature, 183:1394-1395.

Lovelock JE, Polge C. (1954) The immobilization of spermatozoa by freezing and thawing

and the protective action of glycerol. Biochemical Journal, 58:618.

Luyet BJ. (1965) Phase transitions encountered in the rapid freezing of aqueous solutions.

Annals of the New York Academy of Sciences, 12:502-521.

Luyet BJ, Gehenio PM. (1940) Life and death at low temperatures. Biodynamica Press.

Normandy.

McCartney SA, Sadtchenko V. (2013) Fast scanning calorimetry studies of the glass transition

in doped amorphous solid water: Evidence for the existence of a unique vicinal phase.

Journal of Chemical Physics, 138:084501.

McCown BH, Sellmer JC. (1987) General media and vessels suitable for woody plant culture.

In: Cell and tissue culture in forestry, General Principles and Biotechnology. (JM Bonga &

DZ Durzan, eds.). Martinus Nijhoff Publishers. Dordrecht, pp. 4-16.

Maclean N, Hall BK. (1987) Cell commitment and differentiation. Cambridge University

Press. New York.

Makowska Z, Keller J, Engelmann F. (1999) Cryopreservation of apices isolated from garlic

(Allium sativum L.) bulbils and cloves. Cryoletters, 20:175-182.

Martín C, Cervera MT, González-Benito ME. (2011) Genetic stability analysis of

chrysanthemum (Chrysanthemum x morifolium Ramat) after different stages of an

encapsulation�–dehydration cryopreservation protocol. Journal of Plant Physiology,

168:158-166.

Martins MBG. (1998) Biometria tecidual e identificação dos tricomas secretores presentes em

Mentha spicata L. e em Mentha spicatata x suaveolens (Lamiaceae). Revista Hispeci e

Lema, 3:43-48.

137


Masuda T, Kitahara K, Aikawa Y, Arai S. (2001) Determination of carbohydrates by HPLC-

ECD with a novel stationary phase prepared from polystyrene-based resin and tertiary

amines. Journal of the American Chemical Society, 17:895-898.

Matos FJA. (1998) Farmacias vivas: sistemas de utilização de plantas medicinais projetado para

pequenas comunidades. Universidade Federal do Ceará. Fortaleza.

Matsumoto T, Saki A, Nako Y. (1998) A novel preculturing for enhancing the survival of in

vitro-grown meristems of wasabi (Wasabia japonica) cooled to -196°C by vitrification.


Matsumoto T, Sakai A, Takahashi C, Yamada K. (1995) Cryopreservation in vitro grown

apical meristems of wasabi (Wasabi japonica) by encapsulation-vitrification method.


Matsumoto T, Sakai A, Yamada K. (1994) Cryopreservation of in vitro grown apical

meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant-

regeneration. Plant Cell Reports, 13:442-446.

Mazur P. (1963) Kinetics of water loss from cells at subzero temperatures and the likelihood

of intracellular freezing. Journal of General Physiology, 47:347-369.

Mazur P. (1970) Cryobiology: the freezing of biological systems. Science. New York.

Mazur P. (1984) Freezing of living cells: mechanisms and implications. American Journal of

Physiology-Cell Physiology, 247:125-142.

Mazur P. (2004) Principles of cryopreservation. In: Life in the frozen state (B Fuller, N Lane

& E Benson, eds.). CRC Press. London, pp. 3-66.

Mazur P, Pinn IL, Kleinhans FW. (2007) The temperature of intracellular ice formation in

mouse oocytes vs. the unfrozen fraction at that temperature. Cryobiology, 54:223-233.

Mazur P, Rall WF, Leibo SP. (1984) Kinetics of water loss and the likelihood of intracellular

freezing in mouse ova. Influence of the method of calculating the temperature dependence

of water permeability. Cell Biophysics, 6:197-213.

138


Mazur P, Rall WF, Rigopoulos N. (1981) Relative contributions of the fraction of unfrozen

water and of salt concentration to the survival of slowly frozen human erythrocytes.

Biophysical Journal, 36:653-675.

Mazur P, Rhian MA, Mahlandt BG. (1957) Survival of Pasteurella tularensis in gelatin-saline

after cooling and warming at subzero temperatures. Archives of Biochemistry and

Biophysics, 71:31-51.

Mendes MA, Souza BM, Marques MR, Palma MS. (2004) The effect of glycerol on signal

suppression during electrospray ionization analysis of proteins. Spectroscopy, 18:339-345.

Meryman HT. (1966a) Foreword. In Cryobiology. Academic Press, London.

Meryman HT. (1966b) Review of Biological Freezing. In Cryobiology. Academic Press.

London, pp. 1-114.

Meryman HT, Kafig E. (1955) The freezing and thawing of whole blood. Proceedings of the

Society for Experimental Biology and Medicine, 90:587-589.

Meryman HT, Williams RJ. (1985) Basic principles of freezing injury to plant cells: natural

tolerance and approaches to cryopreservation. In: Cryopreservation of plant cells and

organs (LA Withers, ed.). CRC Press. Boca Raton, pp. 13-47.

Meryman HT, Williams RJ, Withers LA, Williams JT. (1980) Mechanisms of freezing injury

and natural tolerance and the principles of artificial cryoprotection. In: Crop genetic

resources. The conservation of difficult material (LA Withers & JT Williams, eds.).

International Union of Biological Sciences. Paris, pp. 5-37.

Meste ML, Champion D, Roudaut G, Blond G, Simatos D. (2002) Glass transition and food

technology: A critical appraisal. Journal of Food Science, 67:2444-2458.

Minami E, Kuchitsu K, He DY, Kouchi H, Midoh N, Ohtsuki Y, Shibuya N. (1996) Two

novel genes rapidly and transiently activated in suspension-cultured rice cells by treatment

with N-acetylchitoheptaose, a biotic elicitor for phytoalexin production. Plant and Cell

Physiology, 37:563-567.

Mishima O, Stanley HE. (1998) The relationship between liquid, supercooled and glassy

water. Nature, 396:329-335.

139


Morris GJ. (1980) Plant cells. In: Plant cells and low temperature preservation in medicine

and biology. (MJ Ashwood & J Farrant, eds.). Pitman Medical. Kent, pp. 253-284.

Morris GA, Castile J, Smith A, Adams GG, Harding SE. (2009) Macromolecular

conformation of chitosan in dilute solution: A new global hydrodynamic approach.

Carbohydrate Polymers, 76:616�–621.

Morris GA, Kok MS, Harding SE, Adams GG. (2010) Polysaccharide drug delivery systems

based on pectin and chitosan. Biotechnology and Genetic Engineering Reviews, 27:257-

284.

Mounib MS, Hwang PC, Idler DR. (1968) Cryogenic preservation of Atlantic cod (Gadus

morhua) sperm. Journal of the Fisheries Board of Canada, 25:2623-2632.

Moynihan CT, Easteal AJ, Bolt MA, Tucker J. (1976) Dependence of the fictive temperature

of glass on cooling rate. Journal of the American Ceramic Society, 59:12-16.

Muldrew K, Acker JP, Elliott AW, McGann LE. (2004) The water to ice transition:

implications for living cells. In: Life in the Frozen State (B Fuller, N Lane & EE Benson,

eds.), CRC Press, London, pp. 67-108.

Muldrew K, McGann LE. (1994) The osmotic rupture hypothesis of intracellular freezing

injury. Biophysical journal, 66:532-541.

Munsi PS. (1992) Nitrogen and phosphorus nutrition response in Japanese mint cultivation.

Acta Horticulturae, 306:436-443.

Murashige T, Skoog F. (1962) A revised medium for rapid bioassays with tobacco tissue

cultures. Physiologia Plantarum, 15:473-497.

Murthy SSN. (1997) Study of the phase behaviour of the supercooled aqueous solutions of

dimethyl sulfoxide, ethylene glycol and methanol. Journal Physical Chemistry, 101:6043-

6049.

Muthusamy J, Staines HJ, Benson EE, Mansor M, Krishnapillay B. (2005) Investigating the

use of fractional replication and taguchi techniques in cryopreservation: a case study using

orthodox seeds of a tropical rainforest tree species. Biodiversity and Conservation,

14:3169-3185.

140


Muzzarelli RAA, Tarsi R, Filippini O, Giovanetti E, Biagini G, Varaldo PE. (1990)

Atimicrobial properties of N-carboxybutyl chitosan. Antimicrobial Agents Chemotherapy,

34:2019-2023.

Nadarajan J, Mansor M, Krishnapillay B, Staines HJ, Benson E, Harding K. (2008)

Applications of differential scanning calorimetry in developing cryopreservation strategies

for Parkia speciosa, a tropical tree producing recalcitrant seeds. CryoLetters, 29:95-110.

Niino T, Sakai A, Yakuwa H, Nojiri K. (1992) Cryopreservation of in vitro-grown shoot tips

of apple and pear by vitrification. Plant Cell, Tissues and Organ Culture, 28:261-266.

Nishizawa S, Sakai A, Amano Y, Matsuzawa T. (1993) Cryopreservation of asparagus

(Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration

by vitrification. Plant Science, 1:67-73.

Niwata E. (1995) Cryopreservation of apical meristems of garlic (Allium sativum L.) and high

subsequent plant regeneration. CryoLetters, 16:102-107.

No HK, Lee KS, Kim ID, Park MJ, Meyers SP. (2003) Chitosan treatment effects yield,

ascorbic acid content, and hardness of soybean sprouts. Journal of Food Science, 68:680-

685.

No HK, Young Park N, Ho Lee S, Meyers SP. (2002) Antibacterial activity of chitosan and

chitosan oligomers with different molecular weights. International Journal of Food

Microbiology, 74:65-72.

Obbard R, Iliescu D, Cullen D, Chang J, Baker I. (2003) SEM/EDS comparison of polar and

seasonal temperate ice. Microscopy Research and Technology, 62:49-61.

Ohnoa H, Igarashib M, Hondota T. (2005) Salt inclusions in polar ice core: location and

chemical form of water-soluble impurities. Earth and Planetary Science Letters, 232:171-

178.

Otero L, Molina-García AD, Sanz PD. (2002) Some interrelated thermophysical properties of

liquid water and ice. I. A user-friendly modelling review for food high-pressure

processing. Critical Reviews in Food Science and Nutrition, 42:339-352.

141


Palma-Guerrero J, Jansson HB, Salinas J, Lopez-Llorca LV. (2008) Effect of chitosan on

hyphal growth and spore germination of plant pathogenic and biocontrol fungi. Journal of

Applied Microbiology, 104:541-553.

Panis B, Piette B, Swennen R. (2005) Droplet vitrification of apical meristems: a

cryopreservation protocol applicable to all Musaceae. Plant Science, 168:45-55.

Panis B, Lombardi M. (2006) Status of cryopreservation technologies in plants (food and

forest crops). In: The role of biotechnology in exploring and protecting agricultural genetic

resources (J Ruane & A Sonnino, eds.). FAO. Rome, pp. 61-78.

Pasparakis G, Bouropoulos N. (2006) Swelling studies and in vitro release of verapamil from

calcium alginate and calcium alginate-chitosan beads. International Journal of

Pharmaceutics, 323:34-42.

Patel TR, Harding SE, Ebringerova A, Deszczynski M, Hromadkova Z, Togola A, Paulsen

BS, Morris GA, Rowe AJ. (2007) Weak self-association in a carbohydrate system.

Biophysical Journal, 93:741-749.

Paulet F, Engelmann F, Glaszmann JC. (1993) Cryopreservation of apices of in vitro plantlets

of sugarcane (Saccharum sp. Hybrids) using encapsulation-dehydration. Plant Cell

Reports, 12:525-529.

Pearce RS. (1988) Extracellular ice and cell shape in frost-stressed cereal leaves: a low-

temperature scanning-electron-microscopy study. Planta, 175:313-324.

Pegg DE. (1984) Red cell volume in glycerol/sodium chloride/water mixtures. Cryobiology,

2:234-239.

Pennycooke JC, Towill LE. (2000) Cryopreservation of shoot tips from in vitro plants of

sweet potato [Ipomoea batatas (L.) Lam.] by vitrification. Plant Cell Reports, 19:733-737.

Phunchindawan M, Hirata K, Sakai A, Miyamoto K. (1997) Cryopreservation of encapsulated

shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures. Plant

Cell Reports, 16:469-73.

Piccaglia R, Dellacecca V, Marotti M, Giovanelli E. (1993) Agronomic factors affecting the

yields and the essential oil composition of peppermint (Mentha x piperita L.). Acta

Horticulture, 344:29-40.

142


Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at

low temperatures. Nature, 164:666.

Pritchard HW, Dickie JB. (2003) Predicting Seed Longevity: the use and abuse of seed

viability equations. In: Seed Conservation, Turning Science into Practice. (RD Smith, JB

Dicke, SH Linington, HW Pritchard & RJ Probert, eds.). Royal Botanic Gardens. Kew, pp.

655-721.

Rabea EI, El Badawy MT, Stevens CV, Smagghe G, Steurbaut W. (2003) Chitosan as

antimicrobial agent: Applications and mode of action. Biomacromolecules, 4:1457-1465.

Rai MK, Asthana P, Singh SK, Jaiswal VS. (2009) The encapsulation technology in fruit

plants-A review. Biotechnology Advances, 27:671-79.

Rajasekaran K. (1996) Regeneration of plants from cryopreserved embryogenic cell

suspension and callus cultures of cotton (Gossypium hirsutum L.). Plant Cell Reports,

15:859-864.

Rajendran SK, Basu SK. (2009) Alginate-Chitosan Particulate System for Sustained Release

of Nimodipine. Tropical Journal of Pharmaceutical Research, 8:433-440.

Rall WF, Fahy GM. (1985) Ice-free cryopreservation of mouse embryos at -196 °C by

vitrification. Nature, 24:387-402.

Rall WF, Mazur P, Souzu H. (1978) Physical-Chemical Basis of the Protection of Slowly

Frozen Human Erythrocytes by Glycerol. Biophysical Journal, 23:101-120.

Ralston G. (1993) Introduction to Analytical Ultracentrifugation. Beckman Instruments.

Fullerton.

Ramløv H, Wharton DA, Wilson PW. (1996) Recrystallization in a Freezing Tolerant

Antarctic Nematode, Panagrolaimus davidi, and an Alpine Weta, Hemideina maori

(Orthoptera; Stenopelmatidae). Cryobiology, 33:607-613.

Rao PS, Suprasanna P, Ganapathi TR, Bapat VA. (1998) Synthetic seeds: concepts, methods

and application. In: Plant tissue culture and molecular biology. (PV Srivastava, ed.).

Narosa Publishing House. New Delhi, pp. 607-619.

143


Ravi-Kumar MNV. (2000) A review of chitin and chitosan applications. Reactivity and

Functionality of Polymers, 46:1-27.

Reddy MV, Arul J, Angers P, Couture L. (1999) Chitosan treatment of wheat seeds induces

resistance to Fusarium graminearun and improves seed quality. Journal of Agricultural

Food Chemistry, 47:1208-1216.

Reed BM. (1988) Cold acclimation as a method to improve survival of cryopreserved Rubus

meristems. CryoLetters, 9:166-171.

Reed BM. (2008) Plant cryopreservation: A practical guide. Springer. New York.

Reinhoud PJ. (1996) Cryopreservation of tobacco suspension cells by vitrification. Ph.D.

Thesis. Rijks University. Leiden.

Rinaudo M. (2006) Chitin and chitosan: Properties and applications. Progress in Polymer

Science, 31:603-632.

Robinson W. (1931) Free and bound water determinations by the heat of fusion of ice method.

Journal Biological Chemistry, 92:699-709.

Romanazzi G, Nigro F, Ippolito A, Di Venere D, Salerno M. (2002) Effects of pre-and

postharvest chitosan treatments to control storage grey mold of table grapes. Journal of

Food Science, 67:1862-1867.

Roos YH. (1995) Glass transition: related physicochemical changes in foods. Food

Technology, 49:97-102.

Roos YH, Karel M. (1991) Applying state diagrams to food processing and development.

Food Technology, 45:66-68.

Ruan SL, Xue QZ. (2002) Effects of chitosan coating on seed germination and salt-tolerance

of seedlings in hybrid rice (Oryza sativa L.). Acta Agronomica Sinica, 28:803-808.

Sacandé M, Buitink J, Hoekstra FA. (2000) A study of water relations in neem (Azadirachta

indica) seed that is characterized by complex storage behavior. Journal Experimental

Botany, 51:635-643.

144


Sakai A. (1997) Potentially valuable cryogenic procedures for cryopreservation of cultured

plant meristems. In: Conservation of Plant Genetic Resources in Vitro. (MK Lazdan & EC

Cocking, eds.). Science Publishers. New York, pp. 53-66.

Sakai A. (2000) Development of cryopreservation techniques. In: Cryopreservation of tropical

plant germplasm. (F Engelmann & H Takagi, eds.). International Plant Genetic Resources

Institute. Rome, pp. 1-7.

Sakai A. (2003) Proceedings of the International Workshop on Cryopreservation of Bio-

Genetic Resources. International Technical Cooperation Center, Rural Development

Administration. Suwon, pp. 3-18.

Sakai A. (2004) Plant cryopreservation. In: Life in the Frozen State (B Fuller, N Lane & EE

Benson, eds.). CRC Press. London, pp. 329-345.

Sakai A, Engelmann F. (2007) Vitrification, encapsulation-vitrification and droplet-

vitrification: a review. CryoLetters, 28:151-172.

Sakai A, Kobayashi S, Oiyama I. (1990) Cryopreservation of nucellar cell of navel orange

(Citrus sinensis Obs. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports, 9:30-33.

Sakai A, Kobayashi S, Oiyama I. (1991) Cryopreservation of nucellar cells of navel orange

(Citrus sinensis Osb.) by a simple freezing method. Plant Science, 74:243-248.

Sakai A, Matsumoto T, Hirai D, Niino T. (2000) Newly developed encapsulation�–dehydration

protocol for plant cryopreservation. CryoLetters, 21:53-62.

Sakai A, Ötsuka K, Yoshida S. (1968) Mechanism of survival in plant cells at super-low

temperatures by rapid cooling and rewarming. Cryobiology, 4:165-173.

Sakar D, Naik PS. (1998) Cryopreservation of shoot tips of tetraploid potato (Solanum

tuberosum L.) clones by vitrification. Annals of Botany, 82:455-461.

Salinas J. (2002) Molecular mechanisms of signal transduction in cold acclimation. In:

Frontiers in Molecular Biology. (BD Hames & DM Glover, eds.). Oxford University Press,

Oxford, pp. 116-139.

145


Sanchéz-Dominguéz D, Bautista-Baños S, Ocampo PC. (2007) Efecto del quitosano en el

desarrollo y morfologia de Alternaria alternata (FR.) Keissl. Anales de Biologia, 29:23-

32.

Sant R, Panis B, Taylor M, Tyagi A. (2008) Cryopreservation of shoot-tips by droplet

vitrification applicable to all taro (Colocasia esculenta var. esculenta) accessions. Plant

Cell, Tissues and Organ Culture, 92:107-111.

Saragusty J, Arav A. (2010) Current progress in oocyte and embryo cryopreservation by slow

freezing and vitrification. Reproduction, 141:1-19.

Schäfer-Menhur A, Schumacher HM, Mix-Wagner G. (1997) Cryopreservation of potato

cultivars - design of a method for routine application in genebanks. Acta Horticulturae,

447:477-482.

Schilcher H. (1988) Quality requirements and quality standards for medicinal, aromatic and

spices plants. Acta Horticulture, 249:33-44.

Schiller M, von der Heydt H, März F, Schmidt P. (2002) Quantification of sugars and organic

acids in hygroscopic pharmaceutical herbal dry extracts. Journal Chromatography A,

968:101-111.

Scottez C, Chevreau E, Godard N, Arnaud Y, Duron M, Dereuddre J. (1992)

Cryopreservation of cold-acclimated shoot tips of pear in vitro cultures after encapsulation-

dehydration. Cryobiology, 29:691-700.

Senaratna T. (1992) Artificial seeds. Biotechnology Advances, 10:379-392.

Snedeker WH, Gaunya WS. (1970) Dimethyl sulfoxide as a cryoprotective agent for freezing

bovine semen. Journal of Animal Science, 30:953-956.

Senula A, Keller JER, Sanduijav T, Yohannen T. (2007) Cryopreservation of cold-acclimated

mint (Mentha spp.) shoot tips using a simple vitrification protocol. CryoLetters, 28:1-12.

Shibli RA. (2000) Cryopreservation of black iris (Iris nigricans) somatic embryos by

encapsulation-dehydration. CryoLetters, 21:39-46.

146

http://www.sciencedirect.com/science/journal/00219673


Shibli RA, Shatnawi MA, Subaih WS, Ajlouni MM. (2006) In vitro conservation and

cryopreservation of plant genetic resources: a review. World Journal of Agricultural

Sciences, 2:372-382.

Shibli RA, Smith MAL, Shatnawi M. (1999) Pigment recovery from encapsulated-dehydrated

Vaccinium pahalae cryopreserved cells. Plant Cell, Tissue and Organ Culture, 55:119-123.

Simões CMO, Spitzer V. (2007) Óleos voláteis. In: Farmacognosia: da planta ao medicamento.

UFRGS. Porto Alegre, pp. 467-496.

Singh J. (1979) Freezing of protoplasts isolated from cold-hardened and non-hardened winter

rye. Plant Science Letters, 16:195-201.

Sinha VR, Kumria R. (2001) Polysaccharides in colon-specific drug delivery. International

Journal of Pharmaceutics, 224:19-38.

Slade L, Levine H. (1995) Glass Transitions and Water-Food Structure Interactions.

Advances in Food and Nutrition Research, 38:103-269.

Slade L, Levine H, Reid DS. (1991) Beyond water activity: recent advances based on an

alternative approach to the assessment of food quality and safety. Critical Reviews in Food

Science and Nutrition, 30:115-360.

Song YS, Adler D, Xu F, Kayaalp E, Nureddin A, Anchan RM, Demirci U. (2010)

Vitrification and levitation of a liquid droplet on liquid nitrogen. Proceedings of the

National Academic of Sciences of the United States, 107:4596-4600.

Sperling LH. (1986) Introduction to Physical Polymer Science. Interscience. New York, pp.

224-295.

Steponkus PL, Langis R, Fujikawa S. (1992) In: Advances in Low Temperature Biology. (PL

Steponkus, ed.). JAI Press. Hamptomill, pp. 1-16.

Steponkus PL, Uemura M, Webb MS. (1993) A contrast of the cryostability of the plasma

membrane of winter rye and spring oat-two species that widely differ in their freezing

tolerance and plasma membrane lipid composition. In: Advances in Low-temperature

Biology. (PL Steponkus, ed.), Vol. 2. JAI Press. London. pp. 211�–312.

147


Steponkus PL, Webb MS. (1992) Freeze-induced dehydration and membrane destabilization

in plants. Water and Life. Springer-Verlag. Berlin, pp. 338-362.

Sun WQ. (1999) State and Phase Transition Behaviors of Quercus rubra Seed Axes and

Cotyledonary Tissues: Relevance to the Desiccation Sensitivity and Cryopreservation of

Recalcitrant Seeds. Cryobiology, 38:372-385.

Sun T, Yao Q, Zhou D, Mao F. (2008) Antioxidant activity of N-carboxymethyl chitosan

oligosaccharides. Bioorganic & Medicinal Chemistry Letters, 18:5774-5776.

Sun T, Zhou D, Xie J, Mao F. (2006) Preparation of chitosan oligomers and their antioxidant

activity. Chemistry of Materials Science, 225:451-456.

Suzuki M, Ishikawa M, Okuda H, Noda K, Kishimoto T, Nakamura T, Ogiwara I, Shimura I,

Akihama T. (2006) Physiological changes in gentian axillary buds during two-step

preculturing with sucrose that conferred high levels of tolerance to desiccation and

cryopreservation. Annals of Botany, 97:1073-1081.

Swan TW, O�’Hare D, Gill RA, Lynch PT. (1999) Influence of preculture conditions on the

post-thaw recovery of suspension cultures of Jerusalem artichoke (Helianthus tuberosus

L.). CryoLetters, 20:325-336.

Swenson J, Teixeira J. (2010) The glass transition and relaxation behavior of bulk water and a

possible relation to confined water. Journal of Chemical Physics, 132:014508.

TA Instruments. Determination of Polymer Crystallinity by DSC. Thermal Analysis

Application Brief. No. TA123

http://www.tainstruments.com/main.aspx?n=2&id=181&main_id=338&siteid=11

(10/01/2013)

Tahtamouni RW, Shibli RA. (1999) Preservation at low temperature and cryopreservation in

wild pear (Pyrus syriaca). Advances in Horticultural Science, 13:156-160.

Takeda K, Murata K, Yamashita S. (1998) Thermodynamic investigation of glass transition in

binary polyalcohols. Journal of Non-Crystaline Solids, z213:273-279.

Teixeira AS, González-Benito ME, Molina-García AD. (2012) Tissue and cytoplasm

vitrification in cryopreservation monitored by low temperature scanning electron

148

http://www.tainstruments.com/main.aspx?n=2&id=181&main_id=338&siteid=11


microscopy (cryo-SEM). In: Current microscopy contributions to advances in science and

technology. (A Méndez-Vilas, ed.). Formatex. Badajoz, pp. 872-879.

Teixeira AS, Gonzalez-Benito ME, Molina-García AD. (2013) Glassy state and

cryopreservation of mint shoot tips. Biotechnology Progress, 29:707-717.

Terbojevich M, Muzzarelli RAA. (2000) Chitosan. In: Handbook of hydrocolloids. (G

Phillips & P Williams, eds.). Woodhead. Cambridge, pp. 367-378.

Thomashow MF. (1998) Role of Cold-Responsive Genes in Plant Freezing Tolerance. Plant


Touchell DH & Dixon KW. (1994) Cryopreservation for seedbanking of Australian species.

Annals of Botany, 74:541-546.

Towill LE. (1990) Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell

Reports, 9:178-180.

Towill LE. (1996) Vitrification as a method to cryopreserve shoot tips. In: Plant tissue culture

concepts and laboratory exercises. (RS Trigiano & DJ Gray, eds.). CRC Press. Boca Raton,

pp. 297-304.

Towill LE. (2002) Cryopreservation of Mentha (mint). In: Cryopreservation of Plant

Germplasm II. Springer. Heidelberg, pp. 151-163.

Towill LE, Bonnart R. (2003) Cracking in a vitrification solution during cooling or warming

does not affect growth of cryopreserved mint shoot tips. CryoLetters, 24:341-346.

Towill LE, Jarret RL. (1992) Cryopreservation of sweet potato (Ipomoea batatas (L.) Lam.)

shoot-tipsby vitrification. Plan Cell Reports, 11:175-178.

Ubbink J, Kruger J. (2006) Physical approaches for the delivery of active ingredients in foods.

Trends in Food Science and Technology, 17:244-254.

Uchendu EE, Reed BM. (2008) A comparative study of three cryopreservation protocols for

effective storage of in vitro-grown mint (Mentha spp.). CryoLetters, 29:181-188.

Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. (1999) Polymeric systems for

controlled drug release. Chemical Reviews, 99:3181-3189.

149


Uragami A. (1990) Cryopreservation by vitrification of cultured cells and somatic embryos

from mesophyll tissue of asparagus. Acta Horticulturae, 271:109-116.

Uragami A, Sakai A, Nagai M, Takahashi T. (1989) Survival of cultured cells and somatic

embryos of Asparagus officinalis cryopreserved by vitrification. Plant Cell Reports, 8:418-

421.

Umrath W. (1983) Calculation of the freeze-drying time for electron-microscopical

preparations. Mikroskopie, 40:9-34.

Velikov V, Borick S, Angell CA. (2001) The glass transition of water, based on

hyperquenching experiments. Science, 294:2335-2338.

Verdonck E, Schaap K, Thomas L. (1999) A discussion of the principles and applications of

modulated temperature DSC (MTDSC). International Journal of Pharmaceutics, 192:3-20.

Vertucci CW. (1989) The effects of low water contents on physiological activities of seeds.

Physiology Plantarum, 77:172-176.

Vertucci CW. (1990) Calorimetric studies of the state of water in seed tissues. Biophysical

Journal, 58:1463-1471.

Vold IMN. (2004) Periodate oxidised chitosans: Structure and solution properties. Ph.D.

Thesis. Norwegian University of Science and Technology. Trondheim.

Volk GM, Caspersen A, Walters C. (2007) Shoot tip structural and biophysical responses to

plant vitrification solution 2 (PVS2) exposure. Cryobiology, 55:358.

Volk GM, Harris JL, Rotindo KE. (2006) Survival of mint shoot tips after exposure to

cryoprotectant solution components. Cryobiology, 52:305-308.

Volk GM, Walters C. (2006) Plant vitrification solution 2 lowers water content and alters

freezing behavior in shoot tips during cryoprotection. Cryobiology, 52:48-61.

Vujovic T, Sylvestre I, Ruzic D, Engelmann F. (2011) Droplet-vitrification of apical shoot

tips of Rubus fruticosus L. and Prunus cerasifera Ehrh. Scientia Horticulturae, 130:222-

228.

Walstra P. (2002) Physical Chemistry of Foods. CRC Press. New York.

150


Walters C, Wheeler L, Stanwood PC. (2004) Longevity of cryogenically stored seeds.

Cryobiology, 48:229-244.

Wang N, Reed BM. (2003). Development, detection, and elimination of Verticillium dahliae in

mint shoot cultures. Hortscience, 38:67-70.

Wesley-Smith J, Vertucci CW, Berjak P, Pammenter NW, Crane J. (1992) Cryopreservation

of Desiccation-Sensitive Axes of Camellia sinensis in Relation to Dehydration, Freezing

Rate and the Thermal Properties of Tissue Water. Journal of Plant Physiology, 140:596-

604.

Wesley-Smith J, Walters C, Pammenter NW, Berjak P. (2001) Interactions among water

content, rapid (nonequilibrium) cooling to -196ºC, and survival of embryonic axes of

Aesculus hippocastanum L. seeds. CryoLetters, 42:196-206.

Wharton DA. (2011) Cold tolerance of New Zealand alpine insects. Journal of Insect


Wiggins PM. (1990) Role of water in some biological processes. Microbiology Reviews, 54:

432-449.

Wolf AV, Brown MG, Prentiss PG. (1985) Concentrative properties of aqueous solutions. In:

Handbook of Chemistry and Physics. (RC Weast, ed.) CRC Press. Boca Raton, pp. 219-

271.

Wolfe J, Bryant G, Koster KL. (2002) What is �‘unfreezable water�’, how unfreezable is it and

how much is there? CryoLetters. 23:157-166.

Woods EJ, Zieger MA, Gao DY, Critser JK. (1999) Equations for obtaining melting points for

the ternary system ethylene glycol/sodium chloride/water and their application to

cryopreservation. Cryobiology, 38:403-407.

Xie W, Xu P, Liu Q. (2011) Antioxidant activity of water-soluble chitosan derivatives.

Bioorganic & Medicinal Chemistry Letters, 11:1699-1701.

Yamada T, Sakai A, Matsumura T, Higuchi S. (1991) Cryopreservation of apical meristems

of white clover (Trifolium repens L.) by vitrification. Plant Science, 78:81-87.

151


Yavin S, Arav A. (2007) Measurement of essential physical properties of vitrification

solutions. Theriogenology, 67:81-89.

Yue Y, Angell CA. (2004) Clarifying the glass-transition behaviour of water by comparison

with hyperquenched inorganic glasses. Nature, 427:717-720.

Yue Y, Angell CA. (2005) Water behaviour: Glass transition in hyperquenched water?

(reply). Nature, 435:E1-E2.

Zame nik J, Bilav ík A, Faltus M. (2007) Importance, involvement and detection of glassy

state in plant meristematic tissue for cryopreservation. Cryobiology, 5:324-378.

Záme ník J, Faltus M, Bilav ík A, Kotková R. (2012) Comparison of Cryopreservation

Methods of Vegetatively Propagated Crops Based on Thermal Analysis. In: Current

Frontiers in Cryopreservation. (I Katkov, ed.). InTech. New York, pp. 335-358.

Zhou YG, Yang YD, Qi YG, Zhang ZM, Wang XJ, Hu XJ. (2002) Effects of chitosan on

some physiological activity in germinating seed of peanut. Journal Peanut Science, 31:22-

25.

152

11. ANEXOS/ANNEXES

153

11. ANNEXES

Annex I. COMPOSITION OF VITRIFICATION SOLUTIONS

Figure annex 1. Molecular structures of the components of the vitrification solutions.

11. ANEXOS/ANNEXES

154

Table 11.1. Molar composition of plant vitrification solutions. mol of each species in the PVS Chemical species

Molecular weight (g mol-1) PVS1 PVS2 PVS2 mod PVS3 PVS3 mod

Sorbitol 182.17 0.050 - - - - EG 62.07 0.209 0.242 0.266 - - DMSO 78.13 0.077 0.192 0.211 - 0.064 PG 76.09 0.171 - - - - Glycerol 92.09 - 0.326 0.411 0.5439 0.543 Sucrose 342.3 - 0.400 0.400 0.146 0.146 Water 18.02 3.557 2.236 1.737 1.587 1.337

Table 11.2. Water content on relation to total mass and molar relations for PVS.

water mass/total mass mol water/mol total mol water/mol solutes 0.609 0.875 7.018 0.354 0.659 1.928 0.270 0.574 1.349 0.222 0.697 2.303 0.187 0.640 1.776

11. ANEXOS/ANNEXES

155

Annex II. PUBLICATIONS

Tissue and cytoplasm vitrification in cryopreservation monitored by low temperature scanning electron microscopy (cryo-SEM) A.S. Teixeira1, M.E. González -Benito2 and A.D. Molina-García1 1ICTAN-CSIC, José Antonio Novais 10, Madrid, 28040, Spain 2Dpto. de Biología Vegetal, E.U.I.T. Agrícola, Universidad Politécnica de Madrid, Ciudad Universitaria 28040, Madrid,

Spain

Cryopreservation of cells and tissues frequently relies on inducing cytoplasm vitrification for storage with almost complete stoppage of both chemical reactions and physical processes and without ice crystal biological damage. The most frequently employed technique used for monitoring ice formation and vitrification in these systems is differential scanning calorimetry (DSC). This technique, which was used in this study to observe ice formation in cryoprotecting agent solutions and mint shoot tips, is fairly sensitive for detecting ice formation, but not so for directly observing glass transition. Besides, the possibility of coincident thermal phenomena obscuring the small glass transition signature in DSC and the lack of any spatial resolution, make valuable the information obtained from other sources. Low temperature scanning electron microscopy (cryo-SEM) is able to show ice crystals formed in the cooling process employed to introduce samples into the microscope, very similar to the cooling step of cryopreservation protocols. The images from samples that, after DSC evidence, have no ice crystals and are vitrified, appear completely unetched in cryo-SEM micrographs. Consequently, cryo-SEM is proposed as a suitable method to ascertain cells and tissues effective vitrification in cryopreservation.

Keywords glass transition; ice dynamics; DSC; mint tips

1. Introduction Cryopreservation comprises a family of procedures allowing the long-term storage of viable biological material at low temperature. Currently many different tissue and cell types (of human, animal, plant or microorganisms origin) are cryopreserved [1-3]. Low temperature storage is employed to avoid deterioration or viability loss occurring at room temperature. But the cooling and warming processes involved are also likely to irreversibly damage cells and tissues. Ice crystal formation is known to cause cell death and loss of viability of stored material [4], so the procedures applied are designed to avoid it. Ice formation requires an initial nucleation step, energetically disfavourable, that involves the reorganization of a large number of water molecules driven by stochastic Brownian movement. Only when nucleii have reached a certain size, ice crystal growth becomes energetically favourable, even at temperatures well below the equilibrium freezing point [5]. The protocols developed and the general strategy followed differs greatly, depending on the material type, tissue, and even concrete species. Nevertheless, most protocols aim at reducing the intracellular water content (either by air or osmotic dehydration, or extracted by the extracellular formation of ice), increasing at the same time the intracellular solute concentration. The result is an increase of the cytoplasmic microviscosity, slowing the water reorganization process required for ice nucleation. Viscosity, which is also increasing steeply as the system temperature decreases, can reach such high values that movement is virtually stopped. In this situation, denominated glassy or vitreous state, most chemical reactions and physical processes are considered to be detained, and ice formation is deemed as impossible [6, 7]. Plant cryopreservation is often performed by means of two step procedures in which first, the water content is reduced (increasing, so, both solute concentration and cytoplasm viscosity) and then temperature is quickly reduced, to cross the glassy state thermal border, the glass transition temperature, TG. Although there exist many other fluids with heat transfer and cryogenic properties more favourable than liquid nitrogen (LN), because cryopreservation is, after all, a set of standard preparative procedures, quick plunging into nitrogen is usually applied, as there exist a LN widespread supply system, at reasonable cost. Besides, the glass transition of many cryopreserved specimens often lies within the interval -120 to -90ºC, and the temperature of boiling LN (-196ºC) provides a large enough temperature difference to ensure a fast cooling drive, as well as a safe storage ambient, well below TG [5, 8]. Droplet-vitrification protocol is widely applied to different plant species. In it, in order to increase heat transfer rates, the tissue portion to be preserved is included into a small drop of vitrification solution (after a set of previous treatments, causing dehydration but also inducing natural defence mechanisms, useful to protect cells towards both dehydration and low temperature). Several drops of this solution (very concentrated in sugars and related low-molecular weight cryoprotecting compounds) are placed into a small strip of aluminium foil, which is then immersed directly, either into LN for cooling, or, at the recovery stage, into room-temperature or warm culture medium. This protocol is particularly successful when applied to mint shoot tips, and the reported viability reached is high [7, 9].

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

© 2012 FORMATEX 872

The transition between liquid and glassy state, not a proper phase transition, can be detected by monitoring a number of properties ultimately related to molecular mobility [10], such as volumetric or mechanical ones. In the context of cryopreservation, the glass transition is most frequently studied by means of differential scanning calorimetry (DSC). This technique allows to detect even small amounts of water freezing (due to its high phase change enthalpy) and also to directly observe glass transition as a step in the heat capacity (CP) baseline. DSC is, however, not a very sensitive technique for this purpose, as the CP change observed is often very small. Sensitive equipment, yielding a stable baseline, is required, and as the step span depends on the amount of vitrified material, in many occasions its observation is problematic [11]. Low temperature scanning electron microscopy (cryo-SEM) is a user-friendly microscopic technique, allowing an easy, quick and artefact free observation of biological materials. The main draw-back of electron microscopy applications to biological materials is their high water content and its incompatibility with the high vacuum required. Cryo-SEM allows the observation of these materials without tedious dehydration procedures, often causing loss or alteration of structural features in specimens. This technique is particularly useful for the observation of the effect of the cryopreservation processes, as the cooling into LN step can be considered equivalent to the final stage of the cryopreservation protocol. Contrast in this technique is low for high water content biological material, poor in high atomic weight atoms. So, an etching step is included, in which a temperature rise allows the partial sublimation of ice, creating depressions that are visible after gold covering. We are considering in this chapter the employment of cryo-SEM as a tool to visualize glassy state in cells and tissues submitted to cryopreservation processes. Mint shoot tips treated following the droplet-vitrification protocol were used as a test material, and the steps of the protocol with decreasing water content were compared to show examples of specimens with intracellular ice and vitrified.

2. Materials and Methods

2.1 Plant Vitrifying Solution 2

Plant Vitrifying Solution 2 (PVS2) was prepared as 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v dimethyl sulfoxide and 0.4 M sucrose in plant growth medium salts [6]. For Some DSC and Cryo-SEM experiments, PVS2 was diluted with milli-Q deionized water to 25%, 50% and 75%.

2.2 Plant material pre-culture and mint shoot tips extraction

A scheme of the cryopreservation protocol showing the stages in which the specimens were observed is shown in Fig 1. Shoot tips were extracted from Mentha x piperita in vitro plants, maintained by monthly subculture on MS medium [12] with 3% sucrose. Incubation took place at constant temperature (25°C) with a photoperiod of 16 h, and an irradiance of 50 µlmol m-2 s-1 from fluorescent tubes. Shoots were cut into one-node segments, transferred to fresh medium and incubated at alternating temperatures of 25°C (day) and -1°C (dark), always with 16 h photo- and thermoperiod, 50 µlmol m-2 s-1irradiance, provided by fluorescent tubes. After 3-weeks of culture under these conditions, shoot tips (1-2 mm) were isolated from the axillary buds.

2.3 Mint shoot tips dehydration steps

Shoot tips were pre-cultured overnight in a Petri dish with 2 mL of liquid MS medium containing 0.3 M sucrose, over filter paper, at 25°C (observation stage a). Thereafter, the explants were transferred to a Petri dish with 2 mL of loading

Figure 1. Scheme of the steps of the droplet-vitrification cryopreservation protocol employed, see text for more details on media and solutions employed.

a: preculture

Overnight, in MS medium with 0.3 M sucrose

b: loading c: dehydration

20 min incubation in 2M glycerol, 0.4 M

20 min incubation in PVS2 solution

LN



solution (2 M glycerol + 0.4 M sucrose), over filter paper, for 20 min, at room temperature (observation stage b). Finally, they were dehydrated in 2.0 mL PVS2, in a Petri dish, on filter paper, for 20 min at 0ºC (observation stage c).

2.4 Low temperature scanning electron microscopy Cryo-SEM observations were performed using a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold plate (Oxford, UK). Cryo-SEM allows sample observations without the need of prior chemical fixing or drying processes. Three shoot tips in the same stage of the cryopreservation protocol (see above) were fitted on a special bracket with their axes vertically aligned, and this piece was introduced in LN under low pressure, physically fixing tissues for microscopic observation. Then, the holder was introduced in the pre-chamber of the Cryotrans cold plate, at -180 ºC, and specimens were fractured perpendicularly to their axis, to obtain suitable observation surfaces. Finally, samples were inserted in the microscope, etched for three min at -90ºC, gold-covered, and observed at -150/-160ºC under secondary electron mode. Drops of PVS2 solution and 50% water dilution were deposited on the microscope sample holder and cooled in LN at reduced pressure. They were not fractured, but directly etched for three min at -90ºC, gold-covered, and observed at -150/-160ºC under secondary electron mode.

2.5 Differential scanning calorimetry

DSC experiments were performed with a TA-1000 instrument (TA Instruments, New Castle, DE, USA) using a scanning rate of 10ºC min-1. Five shoot tips in the same stage of the cryopreservation protocol were introduced in a DSC pan, which was sealed and weighed. 10 µl samples of PVS2 and water dilutions were also enclosed in DSC pans. Both sample types were submitted to cooling scans from room temperature to -85ºC, short (1 min) equilibration periods at this temperature, followed by warming scans, back to room temperature. Later, pans with tips were punctured and dried in an oven at 85ºC for 72 hours, when they showed constant weight. Thermograms were analysed using the standard procedures provided in the Universal Analysis Program (TA Instruments). Ice thawing thermal events (more precise than freezing events) were considered. The routine produced the temperature corresponding to the event onset and that to its peak, the former corresponding to the equilibrium freezing temperature. The process associated enthalpy, proportional to the event area, was also obtained.

3. Results and Discussion

3.1 Plant Vitrifying Solution 2 DSC studies PVS2 solution was employed as a model of a simple vitrifying system. Its composition was adjusted to enable tissue dehydration and protection towards low temperature effects [6]. Some of its components are membrane-penetrating, while some others are not. Its water content is 40,3 % and it is known to vitrify at -112ºC [13]. Figure 2 shows heating DSC thermograms obtained with pure PVS2 solution and several of its water dilutions. The pure PVS2 solution shows no ice formation events either at the cooling (not shown) or heating steps. The diluted mixtures show proof of ice formation. The samples of 25% and 50% PVS2 concentration show melting events corresponding to ice formed in the cooling step. The 75% PVS2 samples had the water mobility so reduced that ice was not formed in the cooling step. However, some ice was formed and quickly melted, during the heating step.

3.2 Plant vitrifying solution 2 cryo-SEM observation

Figure 3 shows the surface of a drop of PVS2 solution and its 50% water dilution deposited on the microscope sample holder without fracture. The clear lumps on the drop surface are volatile substances deposits originated by the lack of a clean surface generated by freeze-fracture. The pure PVS2 drop shows no darker regions that could be associated to ice crystal formation and later sublimation during the etching process. Meanwhile, the 50% PVS2 dilution shows dark holes interpreted as ice crystals formed in the cooling step and later evacuated by sublimation during etching. The pure PVS2 solution is known to be in glassy state at the -150ºC of the microscope stage [13]. It is shown to form no ice upon cooling, after the presented DSC results. Its 50% dilution however, is forming ice crystals, after these DSC measurements, and this is in good agreement with the microscopic observations, where the dark holes that can be observed after etching would correspond to the ice crystals formed. We conclude that pure, vitrified PVS2 solution undergoes no sublimation and no etching-enhanced contrast. Vitrified water has an amorphous structure [14,15]. Interactions among water molecules and other solution components are strong, though disordered, which may impair cooperative phenomena and may have an important role in phase change kinetics. The null or very slow sublimation of vitreous water has been previously reported [16, 17].



Specimens in the stages a (preculture), b (loading) and c (dehydrated) of the droplet-vitrification protocol were studied, after Fig. 1. Figure 4 shows their typical re-warming DSC thermograms. Table 1 shows the thermal parameters derived from these DSC experiments: Tf(onset) and Tf(peak), respectively, the onset temperature of the melting endotherm (corresponding to the equilibrium freezing temperature) and its peak temperature. The freezing enthalpy ( H) is also showed.

Table 1. Thermal parameters of the thawing events obtained from mint tips DSC thermograms.

Tf(onset) (ºC) Tf(peak) (ºC) H (J g-1)a -5.9 ± 1.1 -1.35 ± 0.34 292 ± 16 b -15.5 ± 0.1 -7.27 ± 0.21 188 ± 14 c - - -

3.3 Mint tips DSC studies Specimens both in steps a and b of the cryopreservation protocol undergo freezing (not shown) and melting events in the respective cooling and re-warming scans. Freezing events at cooling scans take place at the nucleation temperature, shifted to lower values by a considerable supercooling degree and are not appropriate to obtain the equilibrium freezing temperature, especially in a situation of high solute concentration and consequent low molecular mobility, favouring supercooling. Both Tf(onset) and Tf(peak) decrease from a to b, which is in good agreement with a significant increase in the solute concentration with the second incubation step. The amount of frozen water (proportional to the freezing enthalpy) is consequently reduced also, from a to b, as less water is available for ice formation when the solute concentration increases. Thermograms corresponding to specimens in step c show a flat baseline, with no freezing or thawing event in either cooling or warming DSC scans, spanning from room temperature -85 ºC (only warming scans from -50 to 15 ºC are shown.

3.4 Mint tips cryo-SEM studies Figure 5 shows typical cryo-SEM micrographs of mint shoot tips cooled in LN in the microscope cryo-unit, as described. Specimens in both a and b protocol steps showed a clearly visible tissular and cellular structure, with some organelles and vacuolar membrane elements present, among a dark ice crystal mosaic surrounded by a clearer supercooled cryo-concentrated solution matrix. Specimen a shows larger ice crystals while the alteration of cellular

Figure 2. Typical DSC thermograms corresponding to re-warming processes of PVS2 solution and its 25%, 50% and 75% water dilutions. Scanning rate was 10 ºC min-1.



structures by ice is more evident than in b. Meanwhile, specimen c shows a grey continuous mass masking all structural elements in the field, not affected by the etching procedure applied, and ascribed to vitrified solution.

The combined results of DSC and cryo-SEM would be in good agreement, implying that part of the water contained in the system for specimens on steps a and b becomes frozen when cooled under -85ºC (with less amount of ice formation in the latter) while specimens in step c would avoid ice formation, presumably getting vitrified in the cooling process, as they showed no ice trace, either microscopically or calorimetrically. This is also in good agreement with the literature data for viability as a result of applying the droplet-vitrification method to several systems, including mint apices [6]. DSC is the most frequently employed technique used for monitoring ice formation and glass transition in cryopreserved systems. While the high value of the phase change enthalpy for ice melting ensures the detection of even very small amounts of ice, glass transition has a much smaller reflection on DSC thermograms. Besides, glass

a b

Figure 3. Cryo-SEM micrographs of pure PVS2 solution (a) and 50% water dilution (b). The bar corresponds to 10 µm.

a

b c

-8 -6 -4 -2 0 2 4 6

-60 -50 -40 -30 -20 -10 0 10 20 Temperature (ºC)

Hea

t flo

w (

W g

-1 )

1

Figure 4. Typical DSC thermograms corresponding to re-warming processes of mint shoot tips in different stages of the droplet cryopreservation protocol: a, preculture; b, loading, and c, dehydration. Each experiment was performed with five shoot tips. Scanning rate was 10 ºC min-1. (See Materials and Methods and Figure 1 for more details).



transitions, showed in thermograms as a small baseline displacement, are much less energetic than freezing of thawing processes and are often difficult to be observed in low-mass samples, such as those here considered (total sample mass for each DSC experiment for c-step specimens, about 3.5 mg; total mass of water in these samples, about 2.5 mg). Besides, many other associated phenomena, causing a stronger distortion on the heat capacity baseline, can take place at the same time [11]. A common example is lipid melting which, being very wide processes can cover underlying glass transition taking place of the aqueous phase [18]. A further difficulty is the global character of the information obtained by calorimetric methods. Although many different types of cells and tissues could be present in cryopreserved specimens, the information on ice formation and/or vitrification given by DSC refers to an average over the whole specimen. The main interest lies, however, in the particular class of cells and tissues that may have been identified as essential for survival. In these cases, the possible use of a tool such as cryo-SEM could be very valuable. Tissues or cells with ice crystal marks or vitrified could be readily discriminated, no matter their size or water content. Other phenomena contributing to the energetic state of the sample, such as lipid melting or other unrelated phase transition, are not interfering, and besides, each particular type of cell can be identified and its glassy state directly checked. It must be noted that the cryo-SEM technique is directly reporting the lack of sublimated ice during etching [16, 17]. Ice formation is completely impaired in vitrified solutions, but in regions over TG but close to it (at least up to about 20ºC over TG), although ice has the physical possibility to be formed, its occurrence is very unlikely in a short time period, as the molecular mobility is already very low. So, the cryo-SEM methods would not allow discrimination between these two possible states, both of them not permitting ice formation, at least for short time scales.

4. Conclusions Low temperature scanning electron microscopy is useful to monitor ice formation in cells and tissues, in conditions very close to that of cryopreservation protocols. The information obtained from this technique is in perfect agreement with the ice formation and vitrification data obtained by differential scanning calorimetry. The combined used of these two techniques yielding complementary information is considered as very promising for further studies concerning the mechanism of both cryopreservation and glass formation and evolution.

Acknowledgements Work funded from project “CRYODYMINT” (AGL2010-21989-C02-02), Spanish “Plan Nacional I+D+i 2008-2011”. A.S.T. supported by the CSIC JAE-Pre program, partially funded from the European Social Fund. The expert technical work of F. Pinto is acknowledged.



a

b

c

Figure 5. Cryo-SEM micrographs of mint shoot tips in different steps of the droplet cryopreservation protocol: preculture, a; loading, b; dehydrated c. The bar corresponds to 10 µm.



References [1] Towill LE. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Rep. 1990;9:178-180. [2] Mazur P, Pinn IL, Kleinhans FW. The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at

that temperature. Cryobiology. 2007;54:223-233. [3] Nadarajan J, Mansor M, Krishnapillay B, Staines HJ, Benson E, Harding K. Applications of differential scanning calorimetry

in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds. Cryoletters. 2008;29: 95-110.

[4] Mazur P, Seki S, Pinn IL, Kleinhans FW, Edashige K. Extra- and intracellular ice formation in mouse oocytes. Cryobiology. 2005;51:29-53.

[5] Zamecnik J, Bilavcik A, Faltus M. Importance, involvement and detection of glassy state in plant meristematic tissue for cryopreservation. Cryobiology. 2007;55:324-378.

[6] Sakai A, Engelmann F. Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters. 2007;28:151-172.

[7] Senula A, Joachim Keller ER, Sanduijav T, Yohannen T. Cryopreservation of cold-acclimated mint (Mentha SPP.) shoot tips using a simple vitrification protocol. Cryoletters 2007;28:1-12.

[8] Reed BM (ed.). Plant Cryopreservation: A Practical Guide. New York, NY: Springer; 2008. [9] Volk GM, Walters C. Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during

cryoprotection. Cryobiology. 2006;52:48–61. [10] Kauzmann W. The nature of the glassy state and the behavior of liquids at low temperatures. Chem. Rev. 1948;43:219–256. [11] Verdonck E, Schaap K, Thomas, L. A discussion of the principles and applications of Modulated Temperature DSC

(MTDSC). Int. J. Pharm.1999;192:3–20. [12] Murashige T, Skoog F. A revised medium for rapid bioassays with tobacco tissue cultures. Physiol. Plantarum.1962;15:473-

497. [13] Teixeira AS, Faltus M, Záme ník J, Kotková R, González-Benito ME, Molina-García AD. Invariance of the Glass Transition

Temperature of Plant Vitrification Solutions with Cooling Rate. Cryobiology. 2012;65: in press. [14] Mishima O, Stanley HE. The relationship between liquid, supercooled and glassy water. Nature,1998; 396:329-

335. [15] Angell CA. Amorphous water. Annual review of physical chemistry,2004;55 (1):559-583. [16] Umrath W. Calculation of the freeze-drying time for electron-microscopical preparations. Mikroskopie. 1983;40:9-34. [17] Franks F. Metastable water at subzero temperatures. J. Microsc. Oxford. 1986;141(Part 3):243-249. [18] Teixeira AS, Ballesteros D, Molina-García AD, Walters C. Interactions between water and triacylglycerols may explain faster

aging rates in stored germplasm at low temperature. Cryobiology. 2012;65: in press.



Glassy State and Cryopreservation of Mint Shoot Tips

Aline S. TeixeiraICTAN, Instituto de Ciencia y Tecnolog!ıa de Alimentos y Nutrici!on, CSIC, Madrid 28040, Spain

M. Elena Gonz!alez-BenitoDpto. de Biolog!ıa Vegetal, E.U.I.T. Agr!ıcola, Universidad Polit!ecnica de Madrid, Ciudad Universitaria, Madrid 28040, Spain

Antonio D. Molina-Garc!ıaICTAN, Instituto de Ciencia y Tecnolog!ıa de Alimentos y Nutrici!on, CSIC, Madrid 28040, Spain

DOI 10.1002/btpr.1711Published online March 27, 2013 in Wiley Online Library (wileyonlinelibrary.com)

Vitrification refers to the physical process by which a liquid supercools to very low tem-peratures and finally solidifies into a metastable glass, without undergoing crystallization ata practical cooling rate. Thus, vitrification is an effective freeze-avoidance mechanism andliving tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly vis-cous and stops all chemical reactions that require molecular diffusion, its formation leads tometabolic inactivity and stability over time. To investigate glassy state in cryopreservedplant material, mint shoot tips were submitted to the different stages of a frequently usedcryopreservation protocol (droplet-vitrification) and evaluated for water content reductionand sucrose content, as determined by ion chromatography, frozen water fraction and glasstransitions occurrence by differential scanning calorimetry, and investigated by low-tempera-ture scanning electron microscopy, as a way to ascertain if their cellular content was vitri-fied. Results show how tissues at intermediate treatment steps develop ice crystals duringliquid nitrogen cooling, while specimens whose treatment was completed become vitrified,with no evidence of ice formation. The agreement between calorimetric and microscopicobservations was perfect. Besides finding a higher sucrose concentration in tissues at themore advanced protocol steps, this level was also higher in plants precultured at 25/21!Cthan in plants cultivated at 25!C. VC 2013 American Institute of Chemical Engineers Biotech-nol. Prog., 29:707–717, 2013Keywords: ice crystal, dehydration, droplet-vitrification, DSC, cryo-SEM

Introduction

Biologically material, most frequently that being used forplant propagation (i.e., germplasm), can be stored at lowtemperature for long periods. Plant germplasm, particularlyseeds, pollen, or reproductive tissues, is often kept at liquidnitrogen (LN) temperature to avoid deterioration.1 Cryopre-servation is currently an important tool for germplasm long-term storage, requiring minimal space and maintenance, andthe number of species and cultivars cryopreserved quicklyincreases.2 Low-temperature storage of living tissues andcells must avoid ice crystal formation (especially intracellu-lar) during the whole process (cooling, storage, and finalrewarming), as ice results lethal for cells, impairing speci-mens viability.3,4

Most techniques used include an initial step designed toreduce the tissues water content (with the limit of avoidingdehydration damage) and increase the cytoplasm internal vis-cosity. This step may include the incorporation of extrinsicviscosity enhancing substances (such as sugars, glycerol, orethylene glycol) and the induction of cold adaptation and

defense mechanisms by the plant cells. A second step con-sists of quick cooling by plunging into LN to avoid ice for-mation. The chances of ice crystallization are limited byboth a decrease in the amount and mobility of intracellularwater and an increase in cooling rates, hindering the rear-rangement of water molecules into ice, a necessary previousnucleation step. Cooling to LN temperature places the sys-tem well into the glassy state. Once vitrified, despite sometime-depending phenomena still taking place, relatively largemolecular reorganizations, as those required for ice forma-tion, are not possible and specimens can be stored for hypo-thetically indefinite periods, without ice being formed.5 Athird step consists of the rewarming process, which must beperformed quickly enough to avoid ice nucleation whilecrossing by the temperature region comprised between theglass transition temperature (TG) and the equilibrium freez-ing point (Tf).

6–8 Progressive dehydration and rapid coolinghave been found to avoid the formation and growth of lethalintracellular ice in plant cells.9

Many apparently independent physical properties undergolarge changes associated to the glass transition within similartemperature ranges. For example, the TG values determinedby calorimetry, thermal expansivity, or viscosity are oftennearly identical.10 This is a result of all these properties

Correspondence concerning this article should be addressed to A. S.Teixeira at [email protected]

VC 2013 American Institute of Chemical Engineers 707

being parallelly influenced by changes in molecular mobilityoccurring during the glass transition.

The initial works on mint cryopreservation by vitrificationincluded gradual dehydration using a mixture of ethyleneglycol, dimethyl sulfoxide (DMSO), and polyethylene gly-col.1 A successful method among those usually used forplant germplasm cryopreservation is that named droplet-vitri-fication.11 It is derived from the droplet-freezing technique12

developed for preserving shoot tips, which are placed indroplets of cryoprotective medium and cooled slowly in aprogrammable freezer. In the droplet-vitrification method,the specimen is initially incubated in solutions with increas-ing concentration of cryoprotecting and dehydrating agentsfor different periods. Then it is treated and included in asmall drop of cryoprotecting agents solution (containing highconcentrations of glycerol, ethylene glycol, DMSO, and su-crose13) and deposited on a small strip of aluminum foil.Finally, the strip with the droplets is quickly plunged intoLN and then stored in a cryovial in a LN container. Warm-ing is carried out similarly, plunging the strip plus dropletsinto warm liquid medium containing 1.2 M sucrose and, af-ter 20 min unloading, shoot tips are retrieved and placed onrecovery medium. The results of this method, in terms of vi-ability, have been good for a variety of systems.2,11

The main advantage of this technique is enabling veryhigh cooling and warming rates because of the small volumeof medium in which the explants are placed.2 Many proto-cols involve exposure of plant shoot apices to PVS2 at0!C.13,14 To prevent cellular damage, cryoprotectant solu-tions both dehydrate and penetrate cells to stabilize proteinsand membranes. However, the mode of action of each com-ponent may differ with cell type, species, temperature, andother solution components.15,16 A particular component ofthe cryoprotectant solutions used, DMSO, enhances chemicalpenetration into cells and, in spite of its own cytotoxicity—for which testing the treatment duration is essential—it hasbeen found to promote survival of cryoexposed cells.8,17,18

The interplay between exogenous and endogenous carbohy-drates plays an important role on plant resistance to stresses.For example, a two-step preculture treatment of gentian budswith increasing sucrose concentrations produced a raise ofabscisic acid, followed by an increase of endogenous sucroseconcentration, all of which resulting in a higher dehydrationtolerance.19 Various high-performance liquid chromatography(HPLC) techniques have been developed to facilitate carbohy-drate analyses in an effort to better understand their role inbiochemical processes. High-performance ion chromatogra-phy (IC) coupled with pulsed amperometric detection (PAD)is an efficient method to quantify carbohydrates in naturalsamples and food products.20–24

Differential scanning calorimetry (DSC) is a well-knowntechnique used for studying thermal properties of thermallydriven processes. It has been used for long in cryopreserva-tion studies, as it yields interesting information on freezingand vitrification processes.5,8,25–27 Water freezing latent heatis large, so calorimetry is a sensitive method to monitor iceformation or thawing, even in small quantities. Besides theamount of ice formed, after knowledge of the system’s watercontent, the ratio of frozen–unfrozen water may also beobtained. DSC also yields Tf, and its reduction speaks ofincreasing solute concentrations. TG is also acquired, if scansare performed at sufficiently low temperature, although DSCis not particularly sensitive for this purpose.

Scanning electron microscopy (SEM) is a powerful anduser-friendly microscopic technique, able to provide a wealthof structural information on a wide range of samples. Whencombined with a cryo-stage (cryo-SEM), it is especiallyinteresting to study the microstructure of biological systems,not altered in sample preparation.28 Low-pressure LN frozensamples preserve most structural relations present in theoriginal sample, with little or no artifacts. Cryo-SEM allowssample observation without need for prior chemical fixing ordrying. Ice crystals formed are small and not altering tissuestructures. Ice formed inside cells can be observed, afterfracture in the cryo-stage and a suitable etching process, asblack “void” regions surrounded by gray ridges formed byhighly cryo-concentrated solution either frozen as euthectic,unfrozen, or even vitrified.

In this work, these three powerful techniques, DSC, cryo-SEM, and HPLC, have been applied to follow the behaviortoward LN cooling of specimens of plant germplasm—mint(Menta 3 piperitha) shoot tips—in different steps of thedroplet-vitrification protocol. To date, no publication pro-vides data obtained during this cryopreservation protocol,but only of its final outcome. Actually, very little physicalinformation exists on actual cryopreserved or vitrified plantspecimens. Besides, the combined use of information result-ing from these physicochemical techniques allows us to bet-ter understand the phenomena taking place between thenormal and the vitrified states of plant tissues and back torestore its viability as germplasm. Interesting relationsamong sucrose and cryoprotectant content in cells and itsfreezing and vitrification behavior are found, as well as withthe other factors ruling over the physical control of ice for-mation: water content and cooling rate. The data presentedhere are expected to help to design improved cryopreserva-tion procedures, involving less damaging cryoprotectantsand/or suitable for problematic species.

Materials and Methods

Plant material preculture and shoot tips extraction

Shoot tips were extracted from in vitro shoots of Mentha3 piperita. In vitro plants were monthly subcultured on me-dium MS29 with 3% sucrose and incubated at constant tem-perature (25!C) with a photoperiod of 16 h and an irradianceof 50 mmol m22 s21 from fluorescent tubes. One-node seg-ments were obtained from these shoots, transferred to freshmedium, and incubated at alternating temperatures of 25!C(day) and 21!C (night), always with 16 h photoperiod andthermoperiod, 50 mmol m22 s21 irradiance, provided by fluo-rescent tubes (stage a, see Figure 1). After 3 weeks of cul-ture under these conditions, shoot tips (1–2 mm) wereexcised from axillary buds.

Incubation and dehydration of shoot tips

A protocol based on that of Senula et al.11 was followed.Five excised shoot tips were precultured overnight with 2mL of liquid MS medium containing 0.3 M sucrose at 25!Cover filter paper (stage b, see Figure 1). Thereafter, theexplants were transferred to a Petri dish with 2 mL of load-ing solution (2 M glycerol1 0.4 M sucrose) over filter paper,for 20 min, at room temperature (stage c). Finally, they wereosmotically dehydrated in 2 mL PVS2 (plant vitrification so-lution 2: 30% w/v glycerol, 15% w/v ethylene glycol, 15%w/v DMSO, and 0.4 M sucrose in plant growth medium13)

708 Biotechnol. Prog., 2013, Vol. 29, No. 3

in a Petri dish, on filter paper, for 30 min at 0!C (stage d).Immersion in LN was carried out in the cryo-SEM or insidethe aluminum pans of DSC, depending on the experiment. Aschematic of these steps can be seen in Figure 1.

Plant recovery and viability

Treated and control shoot tips were warmed from LN tem-perature by plunging into warm liquid medium with 1.2 Msucrose for 20 min. After the unloading of cryoprotectants,shoot tips were cultured on regrowth medium, composed ofsolid MS medium supplemented with 0.5 mg L21 BAP1 0.3M sucrose, cultivated in the dark for 24 h and finally placedinto the culture room at 22–25!C and 16 h illumination. Forcontrol samples (2LN), all steps were carried out, includingthe incubation in PVS2 for 30 min. After that the explantswere rinsed in 1.2 M sucrose solution for 20 min and thenplaced on the regrowth medium.

Survival and regrowth were calculated as rates over thetotal number of shoot tips used. Survival was defined includ-ing all forms of visible viability (evidence of green struc-tures or callus) observed 1–2 weeks after rewarming.Regrowth was defined as the formation of small plantlets, 4weeks after rewarming.

Low-temperature SEM

Low temperature SEM (cryo-SEM) observations were per-formed with a Zeiss DSN 960 scanning microscope equippedwith a Cryotrans CT-1500 cold plate (Oxford, UK). Threeshoot tips in the same stage of the cryopreservation protocolwere fitted on a special bracket with their axes verticallyaligned. The geometry of sample and sample holder requiredoptimization for both freezing and fracturing procedures:these steps require stable mechanical mounting of the sampleon a support. This piece was plunged into LN under lowpressure, physically fixing tissues for microscopic observa-tion. This cooling step was considered equivalent to thecooling process taking place in the actual cryopreservationprocess by LN immersion. The holder was then inserted inthe prechamber of the Cryotrans cold plate, at 2180!C, andspecimens were fractured perpendicularly to their axis toobtain a suitable observation surface (a cartoon of the tipsbefore introduction in the microscope and after fracture canbe seen in Figure 2). Later, samples were inserted in themicroscope and etching (partial ice sublimation induced toprovide contrast) was performed for 3 minutes at 290!C.Once etched and recooled, the sample was retrieved into thecryopreparation chamber and coated with high-purity Au,which acts as a conductive contact for electrical charge. The

etched and coated sample was reinstated into the cooledSEM stage and observed using an accelerating voltage of 15kV at 2150/2160!C.

Dry matter and water content determination

Six sets of five tips were used for determining the drymater content (dm) in shoot tips at the different stages of thecryopreservation protocol. It resulted from weighing afteroven drying ("85!C, 72 h) and was expressed as relative tothe total tip mass. The water content (Wc) of shoot tips couldbe calculated for stages a and b, directly as the differencebetween its total mass and dm. It was expressed as relativeto the total shoot tip mass (Wc(s)) or the dry mass (Wc(dm)).

Differential scanning calorimetry

DSC experiments were performed with a Mettler-ToledoDSC 30 instrument (Mettler-Toledo, Griefsen, Switzerland).Cooling was performed using the calorimeter control (10!Cmin21), or for higher rates, by quenching the samples in LN(see below). The response of shoot tips apices to each stepin the mint protocol was evaluated. Five shoot tips in thesame stage of the cryopreservation protocol were introducedin a DSC pan, which was sealed and weighed. Shoot tipshad been sequentially treated with MS medium, loading so-lution, and PVS2. Samples were submitted to a cooling scanfrom room temperature to 2150!C and a short 5 min equili-bration at this temperature, followed by a warming scan (at

Figure 1. Scheme of the steps of the droplet cryopreservation protocol used.

See text for more details on media and solutions used.

Figure 2. Cartoon showing the three mint tips on the micros-copy holder before insertion in the microscope (A)and after equatorial freeze fracture (B).

Biotechnol. Prog., 2013, Vol. 29, No. 3 709

10!C min21), back to room temperature. Calorimetric datawere collected from two replicates per treatment.

For the quenching method shoot tips, sealed into a DSCpan, were quickly plunged into LN. Pans were subsequentlycold-loaded into a prechilled DSC (2150!C) for analysis.

Later, pans were punctured and dried in an oven at 85!C,for 72 h, when they showed constant weight. Thermogramswere analyzed using the standard procedures provided in theMettler-Toledo STARe software. Ice thawing thermal events(more precise than freezing events) were used for extractionof Tf. The routine produced the temperature corresponding tothe event onset, Tf(onset), corresponding to the equilibriumfreezing temperature and that to the peak, Tf(peak).

The equilibrium freezing temperature can be related to thewater activity (aw) of the system to its osmolality (p) and toits solute content.30,31 For comparative purposes, a “sucroseequivalent” concentration (Suceq) and a “sodium chlorideequivalent” (NaCleq) were determined from Tf, assuming thatthe only solute present was sucrose or sodium chloride,respectively. Tf(peak) data were used for the determination ofthese parameters, as it was considered to better represent thebehavior of the whole phase change. Equation (1)30 was fol-lowed for aw determination:

ln aw 5DHf w# $Aw Tf2Tf%# $= TfTf%# $; (1)

where DHf(w) is the pure water freezing enthalpy (specific,per water gram), Aw is the water molecular weight, and Tf*is the equilibrium freezing point of pure water. The osmolal-ity was calculated using Eq. 231:

p5DTf=1:86; (2)

where DTf is the freezing point depression, which for waterat atmospheric pressure, being the pure substance freezingpoint 0!C, equals to Tf with a positive sign.

Suceq and NaCleq could not be calculated from Raoult’slaw, using analytical equations,32 and only valid for morediluted solutions. Experimental data from literature30,33 andcalorimetric determinations performed on concentrated su-crose solutions (data not shown) were used to relate thesehypothetic solute concentrations with the observed cryo-scopic temperature decrease.

The process-associated enthalpy, phase change enthalpyDHf, proportional to the event area, was also obtained. Itwas expressed as relative to either the total shoot tip mass(DHf(s)), its dry mass (DHf(dm)), or its water content (DHf(w))for allowing easier data comparison.

Water fractions: frozen water (Wf) and unfrozen water(Wu) were calculated by comparison of DHf and the purewater freezing enthalpy (DHf(w)5 333.4 J g21 at 0!C), to-gether with the total water contents previously determined,using Eq. (3):

Wf5 DHf=DHf w# $! "

;Wu5Wc2Wf : (3)

Specific enthalpy values (per sample gram) were alwaysused after the corresponding oven dry pan weighs. Enthalpyvalues measured at temperatures different from the corre-sponding phase change equilibrium temperature, 0!C, areunderevaluated, as a result of the difference between waterand ice heat capacities (Cpw and Cph, respectively). So, thevalue of DHf(w) was corrected using Eq. (4), before applyingEq. (3). Single freezing temperatures, Tf(peak), were used, andCpw and Cpi data were obtained from a data calculation

routine produced in this laboratory,34 after interpolationwhen required, and other sources.35,36

DHf corr5DHf2#Tf

0

CpWdT2#0

Tf

Cp jdT

$ %(4)

Frozen (Wf) and unfrozen (Wu) water contents wereexpressed as relative to the total shoot tip mass (Wf(s) andWu(s)), its dry mass (Wf(dm) and Wu(dm)), or its total watercontent, when available, (Wf(w) and Wu(w)).

The glass transition can be observed as a small step in theheat capacity thermogram baseline. The corresponding heatcapacity increment is very small and, in our experiments,only could be appreciated by using 30 shoot tips in the sameDSC pan.

For comparative purposes, calorimetric experiments wereperformed with samples of the cryoprotectant solutions usedin each stage of the droplet protocols (i.e., liquid MS me-dium with 0.3 M sucrose, loading solution, and PVS2).Experiments were performed in the same conditions thanthose carried out with shoot tip specimens.

Sucrose content assessment in shoot tips

Specimens were analyzed after each stage of the cryopre-servation procedure for its sucrose content. Five shoot tipswere weighed (average fresh weight 3.06 0.1 3 1023 g)and mortar-ground into a fine powder in LN. As grindingproceeded, the vessel was slowly warmed. Immediately, 1.5mL Milli-Q water was added and the mixture was filteredthrough a syringe-driven filter (Millex-GS, 0.22 lm).

The determination of sucrose was carried out by an IonChromatography 817 Bioscan (Metrohm, Herisau, Switzer-land) system equipped with a Metrosep Carb 1–250 columnand a PAD with a gold electrode. A three-step PAD protocolwas used with the following time intervals (ms) and poten-tials (mV): t1, 400/E1510.05 (detection); t2, 200/E2510.75 (cleaning); and t3, 400/E3520.15 (regenera-tion). Samples (1.5 mL) were injected using an autosampler(model, 838 Advanced Sample Processor, Metrohm, Herisau,Switzerland) and the flow rate through the column was 1 mLmin21 and the eluent was 100 mM NaOH.

Identification of sucrose was performed by comparisonwith the retention time of the pure standard sucrose (Merck)and the data were acquired using IC Net 2.3 software. Su-crose content was expressed as ppm and the data were themean of three replicates. Results (Suc) are expressed as ppmof the total tip mass.

Results and Discussion

Detection of glassy state and ice by cryo-SEM

Two different observation approaches were compared: sec-ondary electron and backscattered electron image. Basically,the same information was obtained with both; however, thecellular structures could be better appreciated using backscat-tered electrons, and this was used in the observations andmicrographs presented here.

Figure 3 shows the micrographs showing shoot tips cooledin LN in the microscope cryounit as described in the Materi-als and Methods section, in different moments of the dropletcryopreservation protocol: control (A); stage a, cold treat-ment (B); stage b, preculture (C, D); stage c, loading (E, F);and stage d, dehydration (G, H). Micrographs C, E, and G


show lower magnification images of the shoot tips axialplane. The concentric cell distribution in the equatoriallyfractured tips could be appreciated. There were no noticeabledifferences among central or more external cells, indicatingthat the fracture was close to the apical dome, so effectivehomogeneity for temperature and solute and water concentra-tions could be assumed for tip cells.

Ice crystals formed in the more concentrated solutionswere smaller because of the limitations to crystal growthcaused by the higher solution viscosity. On the other hand,conversion of water into ice during cooling increased theconcentration of solutes in the more concentrated regions.The etching procedure applied gave rise to a contrast in the

SEM micrograph, removing part of the ice formed by subli-mation and leaving the darker regions separated by sector or“ridges” of freeze-concentrated solution that can be appreci-ated in micrographs A–F. This behavior allowed visualiza-tion of individual crystals. Sublimation is only taking placeat an appreciable rate out of pure ice crystals, whereas waterin glassy state has a sublimation rate almost negligible.37,38

So, when solutions become vitrified, they cannot be etchedin this way. Micrographs G and H show a complete lack ofthe structural details revealed by etching.

Determination of preculture effect by cryo-SEM

Figures 3A,B show, respectively, typical cryo-SEM micro-graphs after 3 weeks of culture at 25!C (control apices) or at25/21!C. Widespread cellular disruption occurred in controlapices, as it can be observed in micrograph 3A, where icecan be seen as dark areas inside the cell’s cytoplasm andvacuole; meanwhile, clearer parts would correspond to thesupercooled cryoconcentrated solution matrix. Cold-treatedshoot tips (3B) showed the formation of smaller ice crystals,mostly in the vacuole space, which indicates a higher solutecytoplasmic concentration with respect to control apices. Theobserved size crystal reduction is understood because of anincrease in the amount of endogenous cryoprotecting agentsdeveloped by the plant during the cold hardening procedure,confirming the positive influence of tips cold hardeningbefore cryopreservation observed by other workers.11

Observed plant recovery and viability

Both survival and recovery reached a 96% value for speci-mens treated with the complete droplet cryopreservation pro-tocol (i.e., quenched in LN after stage d, dehydration inPVS2). Meanwhile, survival and recovery of previous stages,a to c, were 0% for all cases. The control samples (2LN)showed a 100% survival and recovery.

Analysis of the droplet-vitrification protocol stepsby cryo-SEM

Micrographs of specimens in the stages b (preculture) (C,D), c (loading) (E, F), and d (dehydration) (G, H) of thedroplet-vitrification protocol can be seen in Figure 3. Speci-mens in both b and c stages showed a clearly visible tissularand cellular structure, with some organelles and vacuolarmembrane elements present. Specimens in stage b showedlarger ice crystals and a higher degree of alteration of cellu-lar structures by ice than those in stage c. Tips treated withalternating temperatures (see previous section) had a similaraspect to those in step b (preculture). The ice crystal sizedifference could be due to concentration differences amongsamples, as the crystals formed in the more diluted solutioncan grow to a larger final size, whereas those occurring inconcentrated solutions, where molecular mobility isimpaired, would be smaller.

During the treatment in the loading solution, the cells ofspecimens in stage c of the protocol accumulated exogenousglycerol and other solutes of endogenous origin. These cellswere clearly partly plasmolyzed as a result of the osmoticdehydration imposed by the loading treatment, which wasrelated to the progressive acquisition of tolerance towardfreezing during the protocol.

PVS2, the cryopreservation solution used in the last step,affected cellular freezing properties.8 It has components that

Figure 3. Cryo-SEM micrographs of shoot tips cooled in LN inthe microscope cryounit (see Materials and Methods).

Control (A); stage a, cold treatment (B); stage b, preculture (Cand D); stage c, loading (E and F); and stage d, dehydration (Gand H). The bar corresponds to 10 lm (A, B, D, F, and H) or 50lm (C, E, and G).


are penetrating (dimethyl sulfoxide, glycerol, and ethyleneglycol) and nonpenetrating (sucrose) for the cellular mem-brane of most cell types.39 This complex solution served todehydrate shoot tips and to change the behavior of the waterremaining within shoot tips.

Micrographs of specimens in the stage d of the protocol,after osmotic dehydration in PVS2 solution, presumably witha lower water content and more concentrated in solutesinduced by permeation from the cryopreservation solution,showed a gray continuous, little detailed surface, indicatingvitrification (Figures 3G,H).

Changes in the dry mass and water content during thedroplet-vitrification protocol

The dry mass and water content of shoot tips, derivedfrom its weight difference after drying, are shown in Table1. For step a and b, where water was the only volatile sub-stance (overseeing very small quantities of organic com-pounds), Wc could be obtained by this procedure and ispresented in Table 1, expressed as related to the total samplemass Wc(s) or to the dry mass Wc(dm). The water content oftips undertook a small decrease with the first steps of thecryopreservation protocol, associated to sucrose incubationin stage b. Water content for stages c and d could not bederived from oven-drying data, as the amount of volatilesubstances apart from water was high. Nevertheless, it couldbe assumed that there was a large decrease in the water con-tent associated to step c, as inferred from the increase in dm,which was in good agreement with the reported microscopicobservations showing a smaller ice crystal size in these sam-ples and with the sucrose content data (see below).

Stage d, following the same arguments, would show thecomplex result of water osmotically driven out of the cell bythe strong potential of the very concentrated PVS2 solution,rich in the nonpenetrating sucrose, entrance of the penetratingagents DMSO, ethylene glycol, and glycerol, and water reten-tion inside the cell by the increased internal presence of thesecryoprotectors,8,39–42 notwithstanding the increasing presenceof endogenous cryoprotecting (and often water binding)agents, as a result of the plant response to the induced stress.

Measurement of ice heat of fusion using DSC

Figure 4 shows typical rewarming DSC thermograms forthe same specimens types (at stages a–d) previously cooled

at a rate of 10!C min21. Table 1 shows the thermal parame-ters derived from DSC experiments, Tf(onset) and Tf(peak),respectively, the onset temperature of the melting endotherm(corresponding to the equilibrium freezing temperature) andits peak temperature. The freezing enthalpy (DH) is alsoincluded. The information that both enthalpy and equilibriumfreezing temperature can provide is of the highest interest, asit yields information on the water content within cells and,in a context where it is difficult to know the detailed cyto-plasm composition and interacting effects of endogenous andexogenous solutes, to draw conclusions on the availability ofthis water, to form ice, to permit diffusion-driven processesand even to keep the stability of protein and membranes.Specimens both in stages b and c of the cryopreservationprotocol showed freezing (not shown) and melting events inthe respective cooling and rewarming scans.

The equilibrium freezing temperature is a good raw datato determine accurately solution characteristics, such aswater activity and frozen and unfrozen water.32 However,

Table 1. Compositional and Thermal Parameters of the Thawing Events Obtained from DSC Thermograms for Mint Shoot Tips Specimens atDifferent Stages (a–d) of the Droplet Cryopreservation Protocol

Cold Treatment(Stage a)

Preculture(Stage b)

Loading(Stage c)

Dehydration(Stage d)

dm (gdm gsample21) 0.1176 0.009 0.1516 0.009 0.3446 0.049 0.2526 0.013

Wc(s) (gwater gsample21) 0.8836 0.009 0.8496 0.009 – –

Wc(dm) (gwater gdm21) 7.566 0.65 5.646 0.39 – –

Tf(onset) (!C) 22.966 0.06 24.96 1.8 27.936 0.79 –

Tf(peak) (!C) 20.486 0.08 20.586 0.21 25.56 1.1 –

DHf(s) (J gsample21) 2416 16 209.856 0.12 93.06 7.2 –

DHf(dm) (J gdm21) 1,8006 650 1,3946 83 2741 60 –

DHf(w) (J gw21) 2726 14 247.16 2.5 – –

Wf(s) (gwater gsample21) 0.7246 0.47 0.6326 0.000 0.2906 0.023 0

Wf(dm) (gwater gdm21) 6.206 0.88 4.206 0.25 0.866 0.19 0

Wf(w) (gwater gwater21) 0.8206 0.045 0.7446 0.007 – 0

Wu(s) (gwater gsample21) 0.1596 0.038 0.2176 0.009 – –

Wu(dm) (gwater gdm21) 1.346 0.22 1.446 0.14 – –

Wu(w) (gwater gwater21) 0.1806 0.045 0.2566 0.007 – –

aw 0.99546 0.001 0.99446 0.002 0.94706 0.001 –

p (Osm) 0.266 0.04 0.316 0.12 2.976 0.60 –

Figure 4. Typical DSC thermograms corresponding torewarming processes of shoot tips in differentstages of the droplet cryopreservation protocol:cold treatment (stage a), preculture (stage b), load-ing (stage c), and dehydration (stage d).

Each experiment was performed with five shoot tips. Theinsert shows an expanded thermogram section which allowsthe glass transition, obtained with 30 tips, to be appreciated.Scanning rate was 10!C min21 (See Materials and Methodsand Figure 1 for more details). The ordinates scale of thethermograms baseline is arbitrary.


the precision yielded by DSC for melting temperatures, espe-cially in complex systems, such as natural tissues, is limited.The onset temperature is considered usually as the best esti-mation of the equilibrium freezing point. But, in complexsolutions, compartmentalized in different isolated pools, withmarkedly different composition and water contents (cyto-plasm, vacuoles, intercellular space, different cells, and tis-sues) the onset temperature would rather correspond to themelting of the most concentrated pool, which would thaw atthe lowest temperature.

We have considered that, for these sample types, the peaktemperature was giving a better estimation of the averagemelting of the specimens studied. Moreover, the DSC-meas-ured temperature was affected by the thermal differences (aswell as the compositional ones) within the sample, not negli-gible at a relatively fast scanning speed, as 10!C min21.

The temperatures determined were decreasing steeply withthe progress of the cryopreservation protocol, which spokeabout the increase in the “number” of solute molecule con-centration. The last stage showed no thawing event, andthere would be no frozen water at all in these conditions.From Tf, the aqueous solution parameters aw and p werederived and shown in Table 1.

The thawing enthalpy is, in practice, a much more accurateparameter than freezing enthalpy, depending less on kinetic,geometric, or compartmentalization issues. The phase changeenthalpy, which is presented in Table 1 as relative to eitherthe total sample mass, DHf(s), the dry mass, DHf(dm), or thetotal water content, DHf(w), decreased as the protocol pro-gresses. After correction for the different values of the purewater melting enthalpy at the actual phase change temperaturefor each of these processes, the amount of thawed water ineach process could be calculated by simple comparison withthis quantity. The resulting frozen water content, presented inTable 1 relative to either the total sample mass, Wf(s), the drymass, Wf(dm), or the total water content, Wf(w), again markedlydecreased as the protocol progressed. The last step could alsobe comprised in this trend, as Wf would equal to zero.

The unfrozen water fraction (derived from the frozenwater fraction) can help to visualize the changes undergoingin the cryopreserved tissues, and is also shown in Table 1relative to the total sample mass, Wu(s), the dry mass, Wu(dm),or the total water content, Wu(w). Traditionally, frozen waterwas understood and often named as free water, whereas theunfrozen fraction was considered as bound water.43 Thesedenominations are much criticized nowadays,44 as the differ-ences found in the actual binding of these two water frac-tions are very small, if any at all.45–50

For comparative purposes, Figure 5 shows typical calori-metric thermograms obtained during rewarming for the cryo-protective solutions used (at stages b–d), in the sameconditions than those applied in the experiments of Figure 4.DHf(s) for stage b (liquid MS medium with 0.3 M sucrose)was 2286 2 J g sample

21, whereas for stage c (loading solu-tion) it was 1386 2 J g sample

21. Stage d (PVS2) showed nomelting exotherm. DHf(w) for stage b amounted to 2506 3 Jgw

21. The Tf(onset) were 23.97!C6 0.2!C for stage b and24.68!C6 0.3!C for stage c.

Effects of quench-cooling on heat of fusion of ice

In Figure 6, thermograms of the rewarming DSC stage ofmint shoot tips cooled by quenching are compared with

those at a rate of 10!C min21 in specimens of step a. It canbe seen that, in the quench cooled sample, the thawing tem-perature decreased and the enthalpy increased slightly. Theseresults, although consistent in all repeats and for experimentsin different conditions (data not shown), were unexpected,and of difficult explanation. A possibility is that the fastcooling might trap solute molecules within the ice phase,which would later contribute to an average effective meltingat lower temperatures.51–53

Nevertheless, this behavior, although may be further fromequilibrium than those observed in the slow cooling, wouldcorrespond more closely to the real events taking place incryopreservation practice.

Detection of glass transition by DSC

Specimens at the last stage of the cryopreservation proto-col did not show any freezing event, in any case, either atthe cooling (not shown) or melting runs of DSC studies.Nevertheless, in many experiments, the step in the heatcapacity baseline showing the actual glass transition in DSC

Figure 5. Typical DSC thermograms corresponding torewarming processes of the cryopreservation solu-tions used in different stages of the droplet cryo-preservation protocol: preculture (stage b): liquidMS medium containing 0.3 M sucrose, loading(stage c): loading solution, and dehydration (staged): PVS2.

The insert shows an expanded thermogram section whichallows the glass transition to be appreciated. Scanning ratewas 10!C min21 (See Materials and Methods and Figure 1for more details). The ordinates scale of the thermogramsbaseline is arbitrary.

Figure 6. DSC scans showing warming at 10!C min21 forshoot tips treated at the preculture stage (b) of thedroplet cryopreservation protocol.

Cooling rate was 10!C min21 or quenching (direct immersionof the DSC pan in LN).


thermograms could not be appreciated. Cooling rates of10!C min21, controlled by the calorimeter itself, or exter-nally driven, by quick introduction into LN (quenching)were tested, but the glass transition could only be observed(for both cooling rates) for samples containing 30 shoot tips.Calorimetry is often claimed to be a rather insensitivemethod to observe the glass transition,54 and this is espe-cially applicable here, as the dehydrated tips have a verysmall weight (total average sample mass for each DSCexperiment—five tips—for d-stage specimens was "3.5 mgand the total mass of water in these samples was 2.5 mg).

As shown in the insert of Figure 5, PVS2 solution is alsoshowing a glass transition in these conditions, centered at2120!C.

Changes in sucrose content during the droplet-vitrificationprotocol

The actual composition of the cellular contents in cryopre-servation conditions is critical for the formation of ice or thevitrification of the system. Most studies rather equate thiscomposition (or the physical behavior of the cytoplasm) tothat of the cryopreservation solutions applied, which is notnecessarily correct. A simple extraction procedure and a gen-eral chromatographic method for small carbohydrate mole-cule quantification calibrated for sucrose were applied, as afirst approach to work out this composition. The sucrose con-tent data obtained are presented in Table 2, expressed asrelated to the total sample mass, Suc(s), or to the dry mass,Suc(dm), reflecting the increase in sucrose content within tis-sues, not only as a result of the actual increase of the contentof this compound but also of the decrease in that of water.

There was a strong increase in its level between stages band c (Table 2). Meanwhile, the sucrose incubation of thepreculture leading to stage b only implied a moderateincrease (about 10%) of the internal sucrose level; the load-ing with glycerol gave rise to a dramatic increase of thisconcentration (sevenfold). The stage d, dehydration, afterPVS2 treatment underwent a further increase, but muchmore moderate. This could be interpreted as a reflection ofthe actual cellular water content, and was in good agreementwith the data of water contents and its frozen and unfrozenfraction shown in Table 1.

Other sources for changes in sucrose levels, different fromthe alteration in the water content, were possible, such as theactual penetration of sucrose, which could be facilitated bythe presence of DMSO in the PVS2 solution, an agentknown for promoting the penetration of other solutes throughmembranes.2

Equivalent solute concentrations (isoosmotic) can beobtained from the freezing equilibrium temperatures obtainedby DSC.55 The observed decrease in this temperature can berelated by Raoult’s law to the molal concentration of the

solution. In the case of tissues, it would rather be an averageof the different solution pools in the different cell types andsubcellular compartments of the specimens studied. The ana-lytical equations are only valid for solutions much morediluted than those considered here, and their applicationwould overestimate the solvent concentration required toinduce the observed cryoscopic decrease. The Tf(peak)obtained was, so, compared with tabulated data sets, comple-mented when necessary with experimental DSC determina-tions for concentrated solutions (data not shown). Equivalentsucrose concentration, Suceq, and equivalent sodium chlorideconcentrations, SaCleq, were calculated and presented in Ta-ble 2. These coarse estimations could not describe the com-plex composition of cellular fluids. Nevertheless, as theobserved decreases in Tf were caused by the increased num-ber of molecules in solution, the amount of small molecularweight compounds (either dissociated as NaCl or not, as su-crose) would have a more determinant role than the presenceof larger macromolecules and other specific plant-generatedsubstances.

When comparing the chromatographic sucrose contentwith these isoosmotic solute estimated concentrations, itcould be seen that the total solute content of cellular com-partments must be much higher than the sucrose amountreported. Especially, a high increase in the estimated soluteconcentrations could be appreciated after incubation with theloading solution, may be reflecting the entrance of significantamounts of glycerol in cells, apart from the already com-mented dehydration. It must be noted, when comparing Ta-ble 2 parameters, that although the sucrose contentchromatographically determined refers to the total or drysample mass of the shoot tips, the estimated sucrose and so-dium chloride contents, as originated from the cryoscopicdecrease, refer to the solution mass (only considering solutesand solvent).

General discussion

The combined results of DSC and cryo-SEM are in goodagreement, implying that part of the water contained in thesystem for specimens on steps a and b becomes frozen whencooled in LN (with less amount of ice formation in the lat-ter), while specimens in step c would avoid ice formation,presumably getting vitrified in the cooling process, as theyshowed no ice trace, either microscopically or calorimetri-cally. This is also in good agreement with our data for via-bility and recovery and those of literature, resulting ofapplying the droplet method to several systems, includingmint apices.11,56–64

The different experimental approaches used agree in areduction of the cellular water content with the cryopreserva-tion protocol progress. The major dehydration would takeplace during the loading solution incubation (stage c, showingmuch lower water content than b). This agrees well with the

Table 2. Solute Content of Mint Shoot Tips at the Different Stages of the Droplet Cryopreservation Protocol

Cold Treatment(stage a)

Preculture(stage b)

Loading(stage c)

Dehydration(stage d)

Suc(s) (ppmtotal sample weight) 5,9006 600 6,5006 300 46,9006 700 53,0006 4,000Suc(dm) (g gdm

21) 0.0596 0.006 0.0446 0.002 0.1376 0.002 0.2096 0.015Suceq (ppm)* 8,800 10,800 470,000 –

NaCleq (ppm)* 8,000 10,000 91,000 –

*These concentrations, as derived from the freezing point depression, refer to the solution mass (mass of solutes plus mass of solvent), not takinginto consideration any nondissolved substances.


dry weight evolution, as well as the calorimetric frozen watercontent and the observed decrease of the equilibrium freezingtemperature. The decrease in the size of ice crystals observedby cryo-SEM and the increases in the sucrose contentobtained by ionic chromatography would confirm this result.

Regarding the last and most interesting stage d, there isless information available, as on one hand, the presence oflarge quantities of volatile substances precludes the measure-ment of a reliable water content by differential weighing,and on the other hand, there were no freezing eventsobserved that could yield information about water fractionsor cryoscopic decrease. Nevertheless, the sucrose contentdata could imply a further dehydration degree over stage c,unless a significant amount of the sucrose contained in PVS2solution could have penetrated cells facilitated by DMSO.The evidences of vitrification derived from the cryo-SEMobservations and the glass transition detected in DSC (aswell as the lack of freezing or thawing events at any scan-ning rate) justify a higher cytoplasm viscosity in stage dthan in stage c specimens, evidently not vitrified. This vis-cosity increase could be originated either by a further watercontent reduction or by the entrance in cells of the viscosityenhancing PVS2 components (or both).

The incubation with glycerol, a known penetratingagent,8,39–42 has been reported to promote water exit fromcells, driven by the osmotic gradient, but also a substitutionof part of this water by glycerol inside cells.39 The compari-son of total shoot tips specimen and dry mass (Table 1) withwater content data from other workers,8 whose mint shoottip protocol is not equivalent but similar enough, allows tocalculate that for stage c specimens, at a water content of"1.8 gwater gdm

21, and the amount of glycerol possibleincorporated to shoot tips would be small (about 0.1 gwatergdm

21). Although in this case most of the effect of the proto-col step would be, then, simple osmotic dehydration, thesame type of calculation applied to stage d yields, for awater content of "0.7 gwater gdm

21, an entrance of extrinsiccompounds corresponding to nearly 60% of the final speci-men mass (about 2.3 gwater gdm

21).

A general reflection on these data may acknowledge thenatural variability of the properties determined here. In spiteof the efforts to prepare equivalent start point materials andto treat them in a similar way, differences in size, permeabil-ity, microscopic scale damage, and all short of small compo-sitional and microstructural differences would exist in a waynot easy to monitor. Comparison with other similar experi-ments (data not shown), it can be observed that this variabil-ity would have a larger impact on the equilibrium freezingtemperature data, while the enthalpy and associated waterfractions would be more constant. And, an absolute observa-tion is that for all repeats, sets of experiments, and evencooling rates applied, the final stage of the cryopreservationprotocol is never producing calorimetrically detectable for-mation of ice. This is in good agreement with the viabilityresults found for this protocol,11 viability that is incompati-ble with the formation of ice.

The calorimetric properties (melting enthalpies and equilib-rium freezing and glass transition temperatures) of thecryoprotecting solutions are not too different of those of thespecimens treated with them. In addition to some differencesbeing evident, this calorimetric similarity cannot be takingas a proof of equal composition and/or behavior in otherrespects.

The probabilities of ice formation in specimens under TGand over TG but at temperatures not too elevated (roughly,no more than 20!C over TG) are similar, if a short frametime is considered, such as those corresponding to cooling orwarming processes in cryopreservation. But, this may be notso for longer time periods, such as those corresponding tostorage. Although storage is normally carried out below2150!C, well under these systems TG, it must be remem-bered that, when over TG, at long storage periods, ice forma-tion is always a real possibility.38,65

A further consideration can be derived from the lack ofice observations in DSC at the last stage of the cryopreserva-tion protocol, when the cooling is carried out at 10!C min21,a rather slow speed. This, generally observed for this sam-ples and this protocol (data not shown), would be indicativeof a possible excessive amount of cryoprotecting agents,although, for the biological material used here, we find thatthere is no appreciable damage, at least in terms of viabilityand recovery. Cooling at 10!C min21 would mean that thesamples are in the ice formation window (roughly, withoutconsidering the differences in ice formation probabilities fordifferent temperature regions, between 220 and 2110!C)for about 9–10 min. Quench cooling would mean a perma-nence in this ice formation possible temperature window foronly a few seconds (data not shown). Considering only theeffects of the cryopreservation protocol in the formation ofice (not valuating here the possible membrane or proteinprotection toward low temperature or dehydration, for exam-ple) a reduction in the concentration of the vitrification solu-tion used could produce specimens that may be ice-formation prone at low cooling rates but would not form iceat quenching rates, which are those actually used in practicalcryopreservation. This observation should be taken into con-sideration, especially when the effect of some of the cryo-protecting components of these solutions, such as DMSO, iscriticized for its possible mutagenic or cytotoxic effects.66

Conclusions

Specimens of mint shoot tips at the final step of the drop-let-vitrification cryopreservation protocol become vitrifiedand no ice is formed, which is associated to the lack of dam-age in the process and the resulting high viability. Mean-while, shoot tips at previous steps of the protocol show theformation of ice by both microscopic and calorimetric obser-vations, which is a possible cause of damage leading to theobserved low viability of these specimens. The agreementbetween both cryo-SEM and DSC techniques is good andthe potential of its associated use is clearly shown.

Acknowledgments

This work has been carried out thanks to project“CRYODYMINT” (AGL2010-21989-C02-02) of the SpanishMinistry of Science and Innovation. A.S. Teixeira was sup-ported by the CSIC, within the JAE-Pre program, partiallyfunded from the European Social Fund. The expert technicalwork of F. Pinto is also acknowledged.

Literature Cited

1. Towill LE. Cryopreservation of isolated mint shoot tips by vitri-fication. Plant Cell Rep. 1990;9:178–180.

2. Sakai A, Engelmann F. Vitrification, encapsulation-vitrificationand droplet-vitrification: a review. CryoLetters. 2007;28:151–172.


3. Muthusamy J, Staines HJ, Benson EE, Mansor M, KrishnapillayB. Investigating the use of fractional replication and taguchitechniques in cryopreservation: a case study using orthodoxseeds of a tropical rainforest tree species. Biodivers Conserv.2005;14:3169–3185.

4. Nadarajan J, Mansor M, Krishnapillay B, Staines HJ, Benson E,Harding K. Applications of differential scanning calorimetry indeveloping cryopreservation strategies for Parkia speciosa, atropical tree producing recalcitrant seeds. CryoLetters.2008;29:95–110.

5. Zamecnik J, Bilavcik A, Faltus M. Importance, involvement anddetection of glassy state in plant meristematic tissue for cryopre-servation. Cryobiology. 2007;5:324–378.

6. Niino T, Sakai A, Yakuwa H, Nojiri K. Cryopreservation of invitro-grown shoot tips of apple and pear by vitrification. PlantCell Tissue Organ Cult. 1992;28:261–266.

7. Reinhoud PJ. Cryopreservation of tobacco suspension cells byvitrification. Doctoral Paper, Rijks University, Leiden, TheNetherlands, 1996.

8. Volk GM, Walters C. Plant vitrification solution 2 lowers watercontent and alters freezing behavior in shoot tips during cryo-protection. Cryobiology. 2006;52:48–61.

9. Wesley-Smith J, Walters C, Pammenter NW, Berjak P. Interac-tions among water content, rapid (nonequilibrium) cooling to2196!C, and survival of embryonic axes of Aesculus hippocas-tanum L. seeds. CryoLetters. 2001;42:196–206.

10. Kauzmann W. The nature of the glassy state and the behaviorof liquids at low temperatures. Chem Rev. 1948;43:219–256.

11. Senula A, Joachim Keller ER, Sanduijav T, Yohannen T. Cryo-preservation of cold-acclimated mint (Mentha spp.) shoot tipsusing a simple vitrification protocol. CryoLetters. 2007;28:1–12.

12. Kartha KK, Leung NL, Mroginski LA. In vitro growth-responses and plant-regeneration from cryopreserved meristemsof cassava (Manihot esculenta Crantz), Z Pflanzenphysiol.1982;107:133–140.

13. Sakai A, Kobayashi S, Oiyama I. Cryopreservation of nucellarcell of navel orange (Citrus sinensis Obs. var. brasiliensisTanaka) by vitrification. Plant Cell Rep. 1990;9:30–33.

14. Reed BM. Plant Cryopreservation: A Practical Guide. NewYork: Springer; 2008.

15. Dereuddre J, Scottez C, Arnaud Y, Duron M. R!esistance d’apexcaulinaires de vitroplants de Poirier (Pyrus communis L. cvBeurr!e Hardy), enrob!es dans l’alginate, "a une d!eshydratationpuis "a une cong!elation dans l’azote liquide: effet d’un endur-cissement pr!ealable au froid. C R Acad Sci. 1990;10:317–323.

16. Jouve L, Hoffmann L, Hausman JF. Polyamine, carbohydrate,and proline content changes during salt stress exposure of aspen(Populus tremula L.): involvement of oxidation and osmoregula-tion metabolism. Plant Biol. 2004;6:74–80.

17. Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrifica-tion as an approach to cryopreservation. Cryobiology.1984;21:407–426.

18. Benson EE. An introduction to plant conservation biotechnol-ogy. In: Benson EE, editor. Plant Conservation Biotechnology.London: Taylor & Francis; 1999:3–10.

19. Suzuki M, Ishikawa M, Okuda H, Noda K, Kishimoto T, Naka-mura T, Ogiwara I, Shimura I, Akihama T. Physiologicalchanges in gentian axillary buds during two-step preculturingwith sucrose that conferred high levels of tolerance to desicca-tion and cryopreservation. Ann Bot. 2006;97:1073–1081.

20. Carpenter JF, Dawson PE. Quantification of dimethyl sulfoxidein solutions and tissues by high performance liquid chromatog-raphy. Cryobiology. 1991;28:210–215.

21. Cheng X, Kaplan LA. Simultaneous analyses of neutral carbo-hydrates and amino sugars in freshwaters with HPLC-PAD. JChromatogr Sci. 2003;41:434–438.

22. Lee YC. Carbohydrate analyses with high-performance anion-exchange chromatography. J Chromatogr A. 1996;720:137–149.

23. Masuda T, Kitahara K, Aikawa Y, Arai S. Determination of car-bohydrates by HPLC-ECD with a novel stationary phase pre-pared from polystyrene-based resin and tertiary amines. J AmChem Soc. 2001;17:895–898.

24. Schiller M, von der Heydt H, M#arz F, Schmidt P. Quantificationof sugars and organic acids in hygroscopic pharmaceuticalherbal dry extracts. J Chromatogr A. 2002;968:101–111.

25. Matsumoto T, Sakai A, Yamada K. Cryopreservation of in-vitrogrown apical meristems of wasabi (Wasabia japonica) by vitrifi-cation and subsequent high plant-regeneration. Plant Cell Rep.1994;13:442–446.

26. Yamada T, Sakai A, Matsumura T, Higuchi S. Cryopreservationof apical meristems of white clover (Trifolium repens L.) by vit-rification. Plant Sci. 1991;78:81–87.

27. Gonz!alez-Benito ME, Mendoza-Condori VH, Molina-Garc!ıaAD. Cryopreservation of in vitro shoot apices of Oxalis tuber-osa Mol. CryoLetters. 2007;28:23–32.

28. Craig S, Beaton CD. A simple cryo-SEM method for delicateplant tissues. J Microsc. 1996;182:102–105.

29. Murashige T, Skoog F. A revised medium for rapid bioassayswith tobacco tissue cultures. Physiol Plantarum. 1962;15:473–497.

30. Fullerton GD, Keener CR, Cameron IL. Correction for solute/solvent interaction extends accurate freezing point depressiontheory to high concentration range. J Biochem Biophys Methods.1994;29:217–235.

31. Elliott JAW, Prickett RC, Elmoazzen HY, Porter KR, McGannLE. A multisolute osmotic virial equation for solutions of inter-est in biology. J Phys Chem B. 2007;111:1775–1785.

32. Guignon B, Torrecilla JS, Otero L, Ramos AM, Molina-Garc!ıaAD, Sanz PD. The initial freezing temperature of foods at highpressure. Crit Rev Food Sci Nutr. 2008;48:328–340.

33. Wolf AV, Brown MG, Prentiss PG. Concentrative properties ofaqueous solutions. In: Weast RC, editor. Handbook of Chemis-try and Physics. Boca Raton: CRC Press; 1985:D-219–D-271.

34. Otero L, Molina-Garc!ıa AD, Sanz PD. Some interrelated ther-mophysical properties of liquid water and ice. I. A user-friendlymodelling review for food high-pressure processing. Crit RevFood Sci Nutr. 2002;42:339–352.

35. Angell CA, Shuppert J, Tucker JC. Anomalous properties ofsupercooled water. Heat capacity, expansivity, and proton mag-netic resonance chemical shift from 0 to – 38!. J Phys Chem.1973;77:3892–3899.

36. Angell CA, Ogunl M, Slchlna WJ. Heat capacity of water atextremes of supercooling and superheating. J Phys Chem.1982;86:998–1002.

37. Umrath W. Calculation of the freeze-drying time for electron-microscopical preparations. Mikroskopie. 1983;40:9–34.

38. Franks F. Metastable water at subzero temperatures. J Microsc.1986;141:243–249.

39. Benson EE. Cryopreservation theory. In: Reed BM, editor. PlantCryopreservation: A Practical Guide. New York: Springer;2008:15–32.

40. Sch#afer-Menhur A, Schumacher HM, Mix-Wagner G. Cryopre-servation of potato cultivars—design of a method for routineapplication in genebanks. Acta Hort. 1997;447:477–482.

41. Hub!alek Z. Protectants used in the cryopreservation of microor-ganisms. Cryobiology. 2003;46:205–229.

42. Panis B, Lombardi M. Status of cryopreservation technologiesin plants (food and forest crops). In: Ruane J, Sonnino A, edi-tors. The Role of Biotechnology in Exploring and ProtectingAgricultural Genetic Resources. Rome: FAO; 2006:61–78.

43. Robinson W. Free and bound water determinations by the heatof fusion of ice method. J Biol Chem. 1931;92:699–709.

44. Franks F. Unfrozen water - yes - unfreezable water - hardly -bound water - certainly not. CryoLetters. 1986;7:207.

45. Hills BP, Takacs SF, Belton PS. A new interpretation of protonNMR relaxation time measurements of water in food. FoodChem. 1990;37:95–111.

46. Wiggins PM. Role of water in some biological processes.Microbiol Rev. 1990;54:432–449.

47. Hills BP, Cano C, Belton PS. Proton NMR relaxation studies ofaqueous polysaccharide systems. Macromolecules. 1991;1:2944–2950.

48. Slade L, Levine H, Reid DS. Beyond water activity: recentadvances based on an alternative approach to the assessment offood quality and safety. Crit Rev Food Sci Nutr. 1991;30:115–360.


49. Hills BP, Tang H, Belton PS, Khaliq A, Harris RK. NMR Oxy-gen-17 studies of the state of water in a saturated sucrose solu-tion. J Mol Liq. 1998;75:45–59.

50. Wolfe J, Bryant G, Koster KL. What is ‘unfreezable water’,how unfreezable is it and how much is there? CryoLetters.2002;23:157–166.

51. Obbard R, Iliescu D, Cullen D, Chang J, Baker I. SEM/EDScomparison of polar and seasonal temperate ice. Microsc ResTech. 2003;62:49–61.

52. Ohnoa H, Igarashib M, Hondota T. Salt inclusions in polar icecore: location and chemical form of water-soluble impurities.Earth Planet Sci Lett. 2005;232:171–178.

53. Hashimoto T, Tasaki Y, Harada M, Okada T. Electrolyte-dopedice as a platform for atto- to femtoliter reactor enabling zepto-mol detection. Anal Chem. 2011;83:3950–3956.

54. Verdonck E, Schaap K, Thomas L. A discussion of the princi-ples and applications of modulated temperature DSC (MTDSC).Int J Pharm. 1999;192:3–20.

55. Buckley KA, Conway EJ, Ryan HC. Concerning the determina-tion of total intracellular concentrations by the cryoscopicmethod. J Physiol. 1958;143:236–245.

56. Keller ERJ, Dreiling M. Potato cryopreservation in Germany—using the droplet method for the establishment of a new largecollection. Acta Hort. 2003;623:193–200.

57. Halmagyi A, Deliu C, Coste A. Plant regrowth from potatoshoot tips cryopreserved by a combined vitrification-dropletmethod. CryoLetters. 2005;26:313–322.

58. Leunufna S, Keller ERJ. Cryopreservation of yams using vitrifi-cation modified by including droplet method: effects of coldacclimation and sucrose. CryoLetters. 2005;26:93–102.

59. Panis B, Piette B, Swennen R. Droplet vitrification of apicalmeristems: a cryopreservation protocol applicable to all Musa-ceae. Plant Sci. 2005;168:45–55.

60. Kim HH, Lee JK, Yoon JW, Ji JJ, Nam SS, Hwang HS, ChoEG, Engelmann F. Cryopreservation of garlic bulbil primordiaby the droplet-vitrification procedure. CryoLetters. 2006;27:143–153.

61. Kryszczuk A, Keller J, Gr#ube M, Zimnoch-Guzowska E. Cryo-preservation of potato (Solanum tuberosum L.) shoot tips usingvitrification and droplet method. Food Agric Environ.2006;4:196–200.

62. Sant R, Panis B, Taylor M, Tyagi A. Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasiaesculenta var. esculenta) accessions. Plant Cell Tissue OrganCult. 2008;92:107–111.

63. Halmagyi A, Deliu C, Isac V. Cryopreservation of Malus culti-vars: comparison of two droplet protocols. Sci Hortic.2010;124:387–392.

64. Vujovic T, Sylvestre I, Ruzic D, Engelmann F. Droplet-vitrifica-tion of apical shoot tips of Rubus fruticosus L. and Prunus cera-sifera Ehrh. Sci Hortic. 2011;130:222–228.

65. Angell CA. Supercooled water. In: Franks F, editor. Water—AComprehensive Treatise, Vol. 7. New York: Plenum Press;1982:215–338.

66. Harding K. Genetic integrity of cryopreserved plant cells: areview. CryoLetters. 2004;18:217–230.

Manuscript received Oct. 26, 2012, and revision received Jan. 11,2013.


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