Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds

Abstract

A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l(-1) TDZ followed by three cycles of selection on medium with 0.5 mg l(-1) BA and increasing concentrations of hygromycin (20-40-60 mg l(-1)). Selected shoot clusters were transferred to medium with 0.5 mg l(-1) BA for proliferation and 0.2 mg l(-1) BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l(-1) NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor.