SAFEGUARDING COCOA (THEOBROMA CACAOL) GERMPLASM BY CRYOPRESERVATION-THE VITRIFICATION APPROACH

Abstract

The need to conserve biodiversity of cocoa has become paramount due to the action of a range of diseases , pests and environmental hazard . s In 1983 bushfie r destroyed 60, 000 ha of cocoa plantation in Ghana and as of 1982 185.5 million trees have been cut down in Eastern region of Ghana alone due to Cacao Swollen Shoot Virus (CSSV) infection . In order to safeguard germplasm of cocoa there is the need to use in vitro-based techniques to support in situ conser. vation Successful cryopreservation of cocoa has already been demonstrated using an encapsulation-dehydration method but this is a labour intensive protocol requirng a relatively high level of technical skill and the objective of the current work is to explore the e fficacy of a vitrification-based approach. Floral-derived secondary somatc i embrys o (SSE) of cocoa genotype AMAZ 15 were utilised. In order to optimise the vitrification procedure the effect of preculturing SSEs on sucrose and dehydration with plant vitrification solution 2 (PVS2) was studied. SSEs were precultured on embryo development (ED) medium supplemented with either 0.5 or 0.75 M sucrose for 3 or 5 d and dehydrated with cold PVS2 for 45-105 min before storage in liquid nitrogen (LN). Preculturing the embryos on 0.5 M sucrose for 5d and dehydrating them in PVS2 for 60 min led to significantly higher post-cryo survival than any other treatment (74.5±6 .4. %) So as to minimise cryo-injury due to cation induced free radical formation , nutrient cation sources were removed from the ED solution and/or the recovery medium (ED), the former treatment resulting in a significant bene. fit The influence of size of cotyledonary stage SSEs on postcryo' survi al was studied for two size ranges. SSEs between 2-3 mm survived better than those between 4-5 mm. The protocol was effective across five other genotypes so far tested without further procedural variation . In order to accelerate bulking up of clones, embryos regenerated following cryopreserion vat were used as explant sources and the freezing process was not found to have any inhibitory effect on their embryogenic potential. In order to optimise the dehydration process Differentl ia Scanning Calorimetry (DSC) was used to examine SSEs during cooling and warming . Partial vitrification , as evidenced by glass transition and ice melting, was achieved when embryos were exposed to PVS2 for 45 and 60 min which correlated with higher post cryo sur. ival Application of somatic embryogenesis and cryopreservation to eradicate Cacao Swollen Shoot Virus (CSSV) was also studied . While somatic embryogenesis was able to reduce CSSV transmission in genotypes studied the assessment of any 'cryotherapy' ef fect was limited by the availability of post-cryo regenerants. Methylation sensitive amplified polymorphism screening shows that the 25°C maintained SSEs, cryopreserved SSEs and embryos further regenerated from the cryostored explants exhibited epigenetic changes . The epigenetic variation increased with increasing multiplication.