Towards optimization of parameters for regeneration and agrobacterium-mediated transformation of sweetpotato
Njagi, Irene Wangari
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Sweetpotato [Ipomoea batatas (L) lam] is one of the highest yielding crops in terms of production per unit area. The crop is very important to developing countries, which produce 95% of the total world production. In Kenya sweetpotato is an important food security and income-generating crop with an annual production of 750,000 tonnes/hectare (T/ha) on approx 75,000 ha of land. This gives an average root yield of about 10 T/ha compared to 50 T/ha observable experimentally. Both biotic and abiotic factors constrain sweetpotato production. In Kenya the biotic factor that most constraints sweetpotato production is viral diseases. Sweetpotato feathery mottle virus (SPFMV) is the most widespread of the viruses. When SPFMV interacts with the sweetpotato chlorotic stunt virus (SPCSV) they form the sweetpotato virus disease complex (SPVD). In susceptible varieties, yield loss caused by SPVD can be as high as 80%. Conventional breeding methods have had limited success in the control of SPVD. Therefore there is need for a biotechnological approach to creating virus resistance in sweetpotato. The approaches that have been undertaken to produce transgenic sweetpotato include expression of viral replicase genes, anti-sense RNAs and viral coat protein genes. In this study an Agrobacterium-mediated transformation and somatic regeneration protocol was adopted for two Kenyan sweetpotato varieties, KSP36 and CPT560. The effect of some plant growth regulators and two selective agents was tested. In transformation experiments, a coat protein gene of SPFMV was introduced into sweetpotato leaf and stem explants using Agrobacterium-mediated transformation. Transient GUS histochernical analysis was used as a control test in the transformation process. Transformed explants were regenerated by the method of somatic embryogenesis. For selection of transformed explants paramomycin was found to be effective at 25mg/l while kanamycin was effective at 20mg/I for KSP 36. For CPT 560 both paramomycin and kanamycin were effective at 25mg/l. The lower concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations proved better in regeneration as opposed to the higher 2,4-D concentrations. Zeatin/IAA (indole acetic acid) combination was more effective at embryo production as opposed to kinetin/2,4-D combination. Out of the 37 regenerants tested by PCR, 20 plants tested positive for the coat protein gene. Nine of these were of CPT560 variety whereas 11 were of KSP36. A Chisquare analysis of the PCR results showed that there was no significant difference between the transformation results of KSP36 and CPT560. This protocol can be recommended for other sweetpotato varieties.