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Abstract
Selecting the explant genotypes is a crucial step in in-vitro culture and Agrobacterium-mediated transformation system due to its host range specificity. Immature embryos of five winter and three spring wheat (Triticum aestivum L.) cultivars were evaluated for tissue culture response involving three callus initiation media. Percentage of callus induction varied widely with the genotype and initiation medium used, with values ranging from 5.3% to 100%. Bob White (spring) and NE92458 (winter) were equal and most embryogenic; Pronghorn and 2137 (both winter) were the poorest. CM4C medium was found to be the best medium for initiating embryogenic callus among three media tested. A standard regeneration procedure was used. The genotypes with the highest regeneration efficiencies were Bob White, Fielder and NE92458, (1.8, 1.4 and 1.6 plants/explant, respectively).
An efficient and reproducible Agrobacterium tumefaciens-mediated system for wheat transformation was developed and genetically transformed wheat plants were produced using precultured immature embryos as the explant. The embryos were inoculated with a disarmed A. tumefaciens strain C58C1 carrying a binary vector pPTN115 coding for the β-glucuronidase gene (GUS), and a selectable marker, the neomycin phosphotransferase II gene (NPT II). A. tumefaciens cell density (OD660 = 0.6) for inoculation was found to be crucial. Two hours of inoculation period and co-cultivation for 48 h was favorable for the regeneration of transgenic plants. The transformation efficiency was comparable to that obtained by microprojectile bombardment method used routinely for wheat transformation. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants. Both marker genes, gus and nptII, were expressed and co-segragated in the T 1 progeny plants. In the majority of cases, the genes showed Mendelian segregation predicted for transgene insertion at a single locus.
