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Title: Virus elimination in cocoa (Theobroma cacoa L.) through somatic embryogenesis
Author: Edward, Rebicca
ISNI:       0000 0004 2717 8503
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2011
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Cacao swollen shoot virus (CSSV) is a major pathogen that has seriously constrained cocoa production in West Africa, particularly Ghana and Nigeria. To date, cordon sanitaire control measures in Ghana have failed to prevent the spread of the condition caused by the virus, Cacao swollen shoot virus disease (CSSVD). The aim of this study was to assess the efficacy of cocoa somatic embryogenesis to produce virus-free clonal propagation material both for replanting and to facilitate the safe international exchange of germplasm. CSSVD detection in the field is hampered by the sometimes slow appearance of visual symptoms and their complete absence in mild strains. Polymerase Chain Reaction (PCR)-based screening, a sensitive tool for microbial pathogen identification, is employed in this study because of its capacity for CSSV detection prior to the appearance of visual symptoms. Degenerate PCR primers were developed in order to improve the CSSV-strain dependence of earlier tests. Accordingly it was found that these primers (comprising a mixture of 48 variable redundant nucleotide bases) were capable of detecting 37 out of a putative 56 CSSV strains maintained at the Cocoa Research Institute of Ghana, four more than the sequence specific primers. For tissue culture studies, cocoa staminodes cultures were established from flowers of CSSV-infected cocoa genotypes CL 19/10 strain 1A and Amelonado Plant 2 to produce callus, primary and secondary somatic embryos, with genotype AMAZ 15 from the Intermediate Cocoa Quarantine Facility used as a virus-free control. In terms of primary somatic embryo production tested across five cocoa genotypes the Nestle protocol proved to be more efficient than the Penn State protocol, although the distinction was less clear with virus-infected donor trees. PCR- based CSSV detection proved that virus could be detected at callus, primary somatic embryos and secondary somatic embryo stages, although the frequency of CSSV was reduced, indicating that the progress of the virus was progressively impeded. Assessment on the presence of plasmodesmata within CSSV-infected cocoa tissues by using transmission electron microscopy was hampered with the presence of artefacts in all sections mounted on Formvar films. Plasmodesmata were not detected may be indicative of a low frequency of plasmodesmata and may also be due to the techniques and methods used. Trials were conducted to assess Nicotiana benthamiana plantlets as alternate CSSV indicators and while they did receive viral DNA this was at too Iowa frequency to make them a viable option for glasshouse- based viral screening. The overall findings of this work do, however, support the use of somatic embryogenesis as a means of improving CSSV-free clonal propagation of cocoa.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available