Secondary somatic embryos (SSEs) of cocoa, a recalcitrant tropical, seed-producing species, were cryopreserved using a vitrification approach and Differential Scanning Calorimetry (DSC) was employed to optimise sucrose preculture and Plant Vitrification Solution 2 (PVS2) incubation. The objective of the study was to evaluate the influence of sucrose preculture and PVS2 dehydration on water content of SSE that will enable it to survive cryostorage. SSEs were precultured for 3 or 5 days on media containing 0.5 M or 0.75 M sucrose and cryoprotected in loading solution (2 M glycerol and 0.4 M sucrose in medium) for 20 min before they were dehydrated with cold PVS2 for 0 - 90 min. Thermal analysis revealed the occurrence of ice crystallization in the SSEs with the extent declining with increasing PVS2 exposure. Maximal survival of SSEs was promoted by preculture on 0.5 M sucrose medium and dehydration with PVS2 for 45 - 60 min, which was characterised by small ice crystallization. Exposure of SSEs beyond 60 min leads to excessive dehydration as characterized by no change in the thermograms. Based on these findings, preculture of SSEs on 0.5 M sucrose medium and dehydration with cold PVS2 for 60 min has been adopted for the successful cryopreservation of cocoa germplasm.
References
[1]
Maximova, S.N., Alemanno, L., Young, A., Ferriere, N., Traore, A. and Guiltinan, M.J. (2002) Efficiency, Genotypic Variability, and Cellular Origin of Primary and Secondary Somatic Embryogenesis of Theobroma cacao L. In Vitro Cellular & Developmental Biology-Plant, 33, 163-172. https://doi.org/10.1079/IVP2001257
[2]
Fang, J.-Y., Wetten, A. and Hadley, P. (2004) Cryopreservation of Cocoa (Theobroma cacao L.) Somatic Embryos for Long-Term Germplasm Storage. Plant Science, 166, 669-675. https://doi.org/10.1016/j.plantsci.2003.11.002
[3]
Charoensub, R., Phansiri, S., Yongmanitchai, W. and Sakai, A. (2003) Routine Cryopreservation of in Vitro-Grown Axillary Apices of Cassava (Manihot esculenta Crantz) by Vitrification: Importance of a Simple Mononodal Culture. Scientia Horticulturae, 98, 485-492. https://doi.org/10.1016/S0304-4238(03)00004-9
[4]
Drew, R., Azimi, M. and Ashmore, S. (2005) Development of Advanced Technologies for Germplasm Conservation of Tropical Species. Acta Horticulturae, 694, 97-100. https://doi.org/10.17660/ActaHortic.2005.694.12
[5]
Dumet, D., Engelmann, F., Chabrillange, N. and Duval, Y. (1993) Cryopreservation of Oil Palm (Elaeis guineensis Jacq.) Somatic Embryos Involving a Desiccation Step. Plant Cell Reports, 12, 352-355. https://doi.org/10.1007/BF00237434
[6]
Li, M., Zhao, X., Hong, S., Zhang, X., Li, P., Liu, J. and Xie, C. (2009) Cryopreservation of Plantlet Nodes of Diosocorea opposita Thunb. Using a Vitrification Method. CryoLetters, 30, 19-28.
[7]
Mandal, B.B., Chandel, K.P.S. and Dwivedi, S. (1996) Cryopreservation of Yam (Dioscorea spp.) Shoot Apices by Encapsulation-Dehydration. CryoLetters, 17, 165-174.
[8]
Takagi, H., Thinh, N.T. and Kyesmu, P.M. (1998) Cryopreservation of Vegetatively Propagated Tropical Crops by Vitrification. Acta Horticulturae, 461, 485-494. https://doi.org/10.17660/ActaHortic.1998.461.57
[9]
Wu, Y.-J., Huang, X.-L., Chen, Q.-Z., Li, X.-J. and Engelmann, F. (2007) Induction and Cryopreservation of Embryogenic Cultures from Nucelli and Immature Cotyledon Cuts of Mango (Mangifera indica L. var Zihua). Plant Cell Reports, 26, 161-168. https://doi.org/10.1007/s00299-006-0232-4
[10]
Volk, G.M. and Walters, C. (2006) Plant Vitrification Solution 2 Lowers Water Content and Alters Freezing Behaviour in Shoot Tips during Cryopreservation. Cryobiology, 52, 48-61. https://doi.org/10.1016/j.cryobiol.2005.09.004
[11]
Benson, E.E., Reed, B.M., Brennan, R.M., Clacher, K.A. and Ross, D.A. (1996) Use of Thermal Analysis in the Evaluation of Cryopreservation Protocols for Ribes nigrum L. Germplasm. CryoLetters, 17, 347-362.
[12]
Dereuddre, J. and Kaminski, M. (1992) Application of Thermal Analysis in the Cryopreservation of Plant Cells and Organs. Journal of Thermal Analysis, 38, 1965-1978. https://doi.org/10.1007/BF01979606
[13]
Dumet, D., Block, W., Worland, M.R., Reed, B.M. and Benson, E.E. (2000) The Use of Physical and Biochemical Studies to Elucidate and Reduce Cryopreservation-Induced Damage in Hydrated/Desiccated Germplasm. In: Engelmann, F. and Takagi, H., Eds., Cryopreservation of Tropical Plant Germplasm, Current Research Progress and Its Applications, JIRCAS, Tsukuba, 43-56.
[14]
Kim, H.-H., Yoon, J.-W., Kim, J.-B., Engelmann, F. and Cho, E.-G. (2005) Thermal Analysis of Garlic Shoot Tips during a Vitrification Procedure. CryoLetters, 26, 33-44.
[15]
Vertucci, C.W. and Stushnoff, C. (1992) The State of Water in Acclimating Vegetative Buds of Apple and Amelanchier and Its Relationship to Winter Hardiness. Physiologia Plantarum, 86, 503-511. https://doi.org/10.1111/j.1399-3054.1992.tb02162.x
[16]
Volk, G.M., Harris, J.L. and Rotindo, K.E. (2006) Survival of Mint Shoot Tips after Exposure to Cryoprotectant Solution Components. Cryobiology, 52, 305-308. https://doi.org/10.1016/j.cryobiol.2005.11.003
[17]
Nadarajan, J., Mansor, M., Krishnapillary, B., Staines, H.J., Benson, E.E. and Harding, K. (2008) Application of Differential Scanning Calorimetry in Developing Cryopreservation Strategies for Parkia speciosa, a Tropical Tree Producing Recalcitrant Seeds. CryoLetters, 29, 95-111.
[18]
Wesley-Smith, J., Vertucci, C.W., Berjak, P., Pammenter, N.W. and Crane, J. (1992) Cryopreservation of Desiccation-Sensitive Axes of Camelia sinensis in Relation to Dehydration, Freezing Rate and the Thermal Properties of Tissue Water. Journal of Plant Physiology, 140, 596-604. https://doi.org/10.1016/S0176-1617(11)80795-9
[19]
Wesley-Smith, J., Walters, C., Pammenter, N.W. and Berjak, P. (2001) Interactions of Water Content, Rapid (Non-Equilibrium) Cooling to −196 °C and Survival of Embryonic Axes of Aesculus hippocastanum Seeds. Cryobiology, 42, 196-206. https://doi.org/10.1006/cryo.2001.2323
[20]
Mazur, P. (2004) Principles of Cryobiology. In: Fuller, B.J., Lane, N. and Benson, E.E., Eds., Life in the Frozen State, CRC Press, Boca Raton, 3-65. https://doi.org/10.1201/9780203647073.ch1
[21]
Dumet, D., Block, W., Worland, M.R., Reed, B.M. and Benson, E.E. (2000) Profiling Cryopreservation Protocols for Ribes ciliatum Using Differential Scanning Calorimetry. CryoLetters, 21, 367-378.
[22]
Rall, W.F. (1987) Factors Affecting the Survival of Mouse Embryos Cryopreserved by Vitrification. Cryobiology, 24, 387-402. https://doi.org/10.1016/0011-2240(87)90042-3
[23]
Sakai, A., Kobayashi, S. and Oyiama, I. (1990) Cryopreservation of Nucellar Cells of Navel Orange (Citrus sinensis Osb. Var brasiliensis Tanka) by Vitrification. Plant Cell Reports, 9, 30-33. https://doi.org/10.1007/BF00232130
[24]
Vertucci, C.W., Berjak, P., Pammenter, N.W. and Crane, J. (1991) Cryopreservation of Embryonic Axes of an Homoiohydrous (Recalcitrant) Seed in Relation to Calorimetric Properties of Tissue Water. CryoLetters, 12, 339-350.
[25]
Kaczmarczyk, A., Zanke, C., Senula, A., Grübe, M. and Keller, E.R.J. (2011) Thermal Analyses by Differential Scanning Calorimetry for Cryopreservation of Potato Shoot Tips. I International Symposium on Cryopreservation in Horticultural Species, ISHS Acta Horticulturae, Vol. 908, 39-46. https://doi.org/10.17660/ActaHortic.2011.908.2
[26]
Gonzalez-Arnao, M.T., Panta, A., Roca, W.M., Escobar, R.H. and Engelmann, F. (2008) Development and Large Scale Application of Cryopreservation Techniques for Shoot and Somatic Embryo Cultures of Tropical Crops. Plant Cell Tissue Organ Culture, 92, 1-13. https://doi.org/10.1007/s11240-007-9303-7
[27]
Mazur, P. (1977) The role of Intracellular Freezing in the Death of Cells Cooled at Supraoptimal Rates. Cryobiology, 14, 251-272. https://doi.org/10.1016/0011-2240(77)90175-4
[28]
Adu-Gyamfi, R. and Wetten, A. (2012) Cryopreservation of Cocoa (Theobroma cacao L.) Somatic Embryos by Vitrification. CryoLetters, 33, 494-505.
[29]
Block, W. (2003) Water Status and Thermal Analysis of Alginate Beads Used in Cryopreservation of Plant Germplasm. Cryobiology, 47, 59-72. https://doi.org/10.1016/S0011-2240(03)00069-5
[30]
Mukherjee, P., Mandal, B.B., Bhat, K.V. and Biswas, A.K. (2009) Cryopreservation of Asian Dioscorea bulbifera and D. alata L. by Vitrification: Importance of Plant Growth Regulators. CryoLetters, 30, 100-111.
[31]
Reed, B.M. (2005) In Vitro Storage of Hops (Humulus L.) Germplasm. Acta Horticulturae, 668, 250-256. https://doi.org/10.17660/ActaHortic.2005.668.32
[32]
Crowe, J.H., Crowe, L.M., Carpenter, J.F. and Aurell Wistrom, C. (1987) Stabilization of Dry Phospholipid Bilayers and Proteins by Sugars. Biochemical Journal, 242, 1-10. https://doi.org/10.1042/bj2420001
[33]
Sánchez-Romero, C., Swennen, R. and Panis, B. (2009) Cryopreservation of Olive Embryogenic Cultures. CryoLetters, 30, 359-372.
[34]
Touchell, D.H., Turne, S.R., Bunn, E. and Dixon, K.W. (2002) Cryostorage of Somatic Tissues of Endangered Australian Species. In: Towill, L.E. and Bajaj, Y.P.S., Eds., Cryopreservation of Plant Germplasm II, Springer, Berlin Heidelberg, 357-372. https://doi.org/10.1007/978-3-662-04674-6_25
[35]
Fang, J.-Y., Sacandé, M. and Pritchard, H. (2009) Influence of Freezable/Non-Freezable Water and Sucrose on the Viability of Theobroma cacao Somatic Embryos Following Desiccation and Freezing. Plant Cell Reports, 28, 883-889. https://doi.org/10.1007/s00299-009-0691-5
[36]
Baudot, A. and Odagescu, V. (2004) Thermal Properties of Ethylene Glycol Aqueous Solutions. Cryobiology, 48, 283-294. https://doi.org/10.1016/j.cryobiol.2004.02.003
[37]
Sakai, A. (2000) Development of Cryopreservation Techniques. In: Engelmann, F. and Takagi, H., Eds., Cryopreservation of Tropical Plant Germplasm, Current Research Progress and Its Applications, JIRCAS, Tsukuba, 1-7.
[38]
Steponkus, P.L., Langis, R. and Fujikawa, S. (1992) Cryopreservation of Plant Tissue by Vitrification. JAI Press Ltd., Hampton Mill.
[39]
Fang, J.-Y. and Wetten, A. (2011) Importance of Structural Integrity of Somatic Embryos for Long-Term Cryopreservation of Cocoa (Theobroma cacao L.) Germplasm. African Journal of Agricultural Research, 6, 3954-3961.
