**6. Conclusion**

Many plant species have been successfully cryopreserved through the development of various cryopreservation methods. As a standard protocol, vitrification and droplet vitrification are widely applied. Shoot tips are the preferred material for cryostorage as they contain the meristem and an organised structure, with direct shoot development avoiding unstructured phases, which could lead to mutations. Preconditioning of plants (cold acclimation or sugar preculture) can have positive effects on survival and regeneration after cryopreservation, which could be due to increased membrane stability. Cryopreserved plants have been found to be genetically stable in most cases, but epigenetic changes have been detected, although most of the molecular analyses have only compared fractions of the genome.

Success in cryopreservation cannot be guaranteed for all plants, as some species are recalcitrant to tissue culture or the cryopreservation process. Fundamental studies looking at membrane composition, membrane damage and repair are likely to help to elucidate why some species are cryosensitive and how cryopreservation protocols can be improved for those species.

Current Issues in Plant Cryopreservation 431

Channuntapipat, C., Sedgley, M. & Collins, G. (2003). Changes in methylation and structure

Chen, X.-L., Li, J.-H., Xin, X., Zhang, Z.-E., Xin, P.-P. & Lu, X.-X. (2011). Cryopreservation of

Crowe, J.H., Crowe, L.M., Carpenter, J.F., Rudolph, A.S., Wistrom, C.A., Spargo, B.J. &

Dai, F., Huang, Y., Zhou, M. & Zhang G. (2009). The influence of cold acclimation on

Day, J.G., Harding, K.C., Nadarajan, J. & Benson, E.E. (2008). Cryopreservation,

Day, J.G. & Stacey, G.N. (2007). *Cryopreservation and Freeze-Drying Protocols*. Humana Press

Decruse, S.W., Seeni, S. & Pushpangadan, P. (1999). Cryopreservation of alginate coated

Dowdle, J., Ishikawa, T., Gatzek, S., Rolinski, S. & Smirnoff, N. (2007). Two genes in

Engelmann, F. (2004). Plant cryopreservation: Progress and prospects. *In Vitro Cellular &* 

Fabre, J. & Dereuddre, J. (1990). Encapsulation-dehydration: A new approach to

Fang, J.Y., Wetten, A. & Johnston, J. (2008). Headspace volatile markers for sensitivity of

Fleck, R.A., Benson, E.E., Bremner, D.H. & Day, J.G. (2000). Studies of free radical-mediated

Foubert, I., Dewettinck, K., Van de Walle, D., Dijkstra, A.J. & Quinn, P.J. (2007). Physical

Taylor & Francis Group, ISBN 0-8493-9688-3, LLC, Boca Raton, FL.

cocoa (*Theobroma cacao* L.) somatic embryos to cryopreservation. *Plant Cell Reports*

cryoinjury in the unicellular green alga *Euglena gracilis* using a non-destructive hydroxyl radical assay: a novel approach for developing protistan cryopreservation

properties: structural and physical characteristics, In: *The Lipid Handbook with CD-ROM*, Gunstone, F.D., Harwood J.L. & Dijkstra A.J. (Eds.), pp. 471-534, CRC Press,

cryopreservation of *Solanum* shoot-tips. *CryoLetters* 11:pp. 423-426.

organization. *Chemistry and Physics of Lipids* 14:pp. 236-246.

*Soc. Hort. Sci.* 128:pp. 890-897.

*Journal of Botany* 77:pp. 397-403

*Biophysica Acta* 947:pp. 367-84.

60327-374-9, Totowa, NJ.

27: pp. 453-461.

*Biologia Plantarum* 153:pp. 257-262.

Inc., ISBN 978-1-58829-377-0, Totowa, NJ.

regeneration. *CryoLetters* 20:pp. 243-250.

*Developmental Biology-Plant* 40:pp. 427-433.

strategies. *Free Radic Res* 32:pp. 157-70.

of DNA from almond tissues during in vitro culture and cryopreservation. *J. Amer.* 

in vitro-grown apical meristems of *Lilium* by droplet-vitrification. S*outh African* 

Anchordoguy, T.J. (1988). Interactions of sugars with membranes. *Biochimica et* 

antioxidant enzymes and antioxidants in sensitive and tolerant barley cultivars.

conservation of bioresources at ultra low temperatures. In: *Molecular Biomethods Handbook*. Walker J.M & Rapley R. (Eds.), pp. 917-947, Humana Press, ISBN 978-1-

shoot tips of in vitro grown *Holostemma annulare* (Roxb.) K. Schum, an endangered medicinal plant: Influence of preculture and DMSO treatment on survival and

*Arabidopsis thaliana* encoding GDP l galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. *The Plant Journal* 52:pp. 673-689. Dowgert, M.F. & Steponkus, P.L. (1984). Behavior of the plasma membrane of isolated protoplasts during a freeze-thaw cycle. *Plant Physiol* 75:pp. 1139-1151. Emmelot, P. & Vanhoeven, R.P. (1975). Phospholipid unsaturation and plasma membrane
