Genetic transformation of pigeon pea (cajanus cajan [L.] Millsp) varieties adapted to Eastern and Southern Africa
Changa, Taity Timothy
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Pigeonpea (Cajanus cajan (L.) Millsp) is an important grain legume. It is Kenya's third most important legume after common beans and cowpea. The potential yield of the crop in the region has not been realized due to insect infestation that causes losses of up to 65% in severe attacks and Fusarium wilt disease which is the most devastating disease in the region. This important crop has limited genetic variability within its centers of origin and cultivated lines which make it hard for improvement against disease and pests through conventional breeding. Modern biotechnology offers a window to overcome this hurdle. Through genetic engineering, genes from unrelated organisms carrying traits of interest can be transferred to the target organisms in order to improve both quality of the harvested grain as well a,, yield quantity. The objective of this study was to optimize a transformation protocol previously reported by Sharma and co-workers from ICRISAT in India using Agrobacterium tumefaciens-mediated transformation to engineer pigeonpea lines adapted to Eastern Africa using cotyledonary leaf explants. GUS reporter gene and neomycin phosphotransferase selectable marker gene (nptII) were used, nptlI was used to identify transgenic plants in kanamycin-containing medium. These genes were harboured in four Agrobacterium tumefciens strains; LBA 4404, EHA 105, C58C1 and AGLO. Four pigeonpea varieties were used in this research; short duration, ICPL 86012 and ICPL 87091, medium duration ICEAP 00554 and the long duration 00040. In this study, ICEAP 00040 germinated poorly and did not produce sufficient leaf explants for transformation while regenerated shoots of ICEAP 00554 did not develop beyond shoot elongation. Therefore, the two short duration varieties were used for transformation and their shoots regenerated on media supplemented with 125mg/1 of kanamycin. ICPL 86012 recorded a putative transformation frequency of 3% each with strains LBA 4404 and EIIA 105 and 2% with AGLO. ICPL 87091 achieved a 3% transformation frequency with strain EHA 105 that were confirmed with PCR amplification of the nptII and GUS genes. A large number of independent transformed plants can be generated usine leaf explants; this protocol can therefore be used to transform pigeonpea adapted to Eastern and Southern Africa to develop transgenic lines with genes of agronomic importance.