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Title: Safeguarding cocoa (Theobroma cacao L.) germplasm by cryopreservation : the vitrification approach
Author: Adu-Gyamfi, Raphael
ISNI:       0000 0004 2721 5693
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2011
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The need to conserve biodiversity of cocoa has become paramount due to the action of a range of diseases, pests and environmental hazards. In 1983 bushfire 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 conservation. Successful cryopreservation of cocoa has already been demonstrated using an encapsulation-dehydration method but this is a labour intensive protocol requiring a relatively high level of technical skill and the objective of the current work is to explore the efficacy of a vitrification-based approach. Floral-derived secondary somatic embryos (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 rin 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 benefit. The influence of size of cotyledonary stage SSEs on postcryo' survival 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 cryopreservation 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 Differential 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 survival. 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' effect was limited by the availability of post-cryo regenerants. Methylation sensitive amplified.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available