Regeneration via somatic embryogenesis from leaf basal segments and genetic transformation of bread and emmer wheat by particle bombardment

Abstract

Direct gene transformation methods such as microprojectile bombardment have been successfully employed for obtaining transgenics in cereals in general and wheat in particular. As success of any transformation strategy depends largely upon the regeneration capability of the target explant, the present investigation employs leaf basal segments to achieve high regeneration response via somatic embryogenesis. Basal segments of 5-day-old seedlings of T. aestivum var. CPAN1676 and T. dicoccum var. DDK1001 were cultured on callusing medium for 3 weeks at 26±1°C, discontinuous light followed by a culture period of 15 days at 21±1°C in continuous light. The calli were then transferred to auxin-free medium for regeneration in discontinuous light at 26±1°C. Regeneration via somatic embryogenesis was observed within 2 weeks in T. aestivum var. CPAN1676 and T. dicoccum var. DDK1001 (68 and 82%, respectively). This embryogenic calli were employed further to obtain hygromycin resistance by particle bombardment in T. aestivum and T. dicoccum. A transformation efficiency of 8.6, 7.5 and 4.9% was obtained in T. aestivum var. CPAN1676, PBW343 and T. dicoccum DDK1001, respectively. Presence of the transgene hptII (hygromycin) in T₀ plants was confirmed by Southern hybridization.