Abstract
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.
Highlights
Maximal survival of secondary somatic embryos (SSEs) was promoted by preculture on 0.5 M sucrose medium and dehydration with Plant Vitrification Solution 2 (PVS2) for 45 - 60 min, which was characterised by small ice crystallization
The objective of the study was to evaluate the influence of sucrose preculture and PVS2 dehydration on water content that allow somatic embryo to survive in liquid nitrogen
Embryos were treated with 0.5 ml loading solution (LS) (2 M glycerol and 0.4 M sucrose prepared in embryo development (ED) medium) for 20 min before they were dehydrated with ice-cold PVS2 (23) for 45 - 90 min
Summary
Cocoa is conventionally propagated using seeds which are considered recalcitrant and cannot be stored using conventional freezer-based storage protocols. The encapsulation-dehydration of somatic embryos is relatively labour-intensive and the asymmetrical position of some embryos at encapsulation can lead to non-uniform drying during their desiccation over silica gel. To address these limitations we adopted the vitrification approach which has proved successful in cryopreserving other tropical species [3]-[9]
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