Agrobacterium Tumefaciens-mediated transformation of three sweet potato varieties with xerophyta viscosa Peroxiredoxin 2 gene conferring drought stress tolerance
Kamwendo, Phillip Mike
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Sweet potato [Ipomoea batatas (L) Lam] is an important food crop and belongs to the family Convolvulaceae. It is a creeping herbaceous perennial vine that is cultivated as an annual crop in the world. Sweet potato is ranked fourth in terms of importance as staple food mostly in the developing world. Countries in Eastern and Southern Africa account for 76% of sweet potato production in Africa. Drought is one of the major abiotic constraints to production of many arable crops with yield losses estimated at 20% although values as high as 100% have been documented under severe conditions resulting into total crop failure. Although several strategies such as conventional breeding, crop introduction, hybridization have been employed in the past to improve drought tolerance, these efforts need to be complemented. Biotechnology offers alternative tools to the development of drought tolerant cultivars via techniques such as genetic engineering. In this study, three test cultivars of sweet potato namely KSP 36, PIPI and Zambezi were regenerated and transformed for drought tolerance via Agrobacterium tumefaciensmediated transformation system using a drought tolerant Xerophyta viscosa peroxiredoxin 2 gene (XvPrx2) isolated from "the resurrection" plant Xerophyta viscosa. The three cultivars were subjected to transformation experiments where Agrobacterium strain EHA101 bearing a standard plasmid was used to infect the transgene into the selected leaf and stem explants in a factorial design. It was established that any time interval from 30 to 60 minutes is sufficient for infection as there were no significant differences (p:0.15) in infection time regimes. Response to Agrobacterium infection, transformation and regeneration was genotype dependent. A total of 10 plantlets were regenerated from over 300 calli used giving regeneration frequencies (RF) of 0.02 for KSP 36, 0.04 for both Zambezi and PIPI. The survival rate of calli on selection was used to calculate the transformation frequencies (TF) which varied significantly (p:_0.001) among the three cultivars as follows KSP36 (0.26), PIPI (0.66) and Zambezi (0.75). Using 0.25mg/L zeatin and lmg/L Indole-3-acetic acid (IAA) only promoted root formation. but when kinetin (2mg/L) and IAA (l mg/L) were used shoot formation was promoted. Leaf explants recorded 87% calli formation whereas stems recorded less than 20%. There were significant differences in cultivar response to regeneration and transformation at p:_0.001 with Zambezi giving the best response. Seven putative transformants from Zambezi cultivar were identified to have been transformed through PCR giving a transformation frequency of 3.3% and transformation efficiency of 2.67%.