No abstract

The first successful attempt to produce stably transformed castor plants through direct gene transfer using particle gun (BioRad) is described. Decotyledonated embryos from mature seeds were germinated and the embryonic axis was induced to proliferate on Murashige and Skoog (MS) medium supplemented with 0.5 mg l(-1) thidiazuron (TDZ) and subjected to bombardment after 5-7 days of pre-incubation. The physical parameters for transient transformation were optimized using the UidA gene encoding beta-glucuronidase (GUS) as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker. Statistical analysis revealed that helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type and number of bombardments had significant influence on transformation efficiency, while the effect of genotype was non-significant. Of the different variables evaluated, embryonic axes from mature seeds, a target distance of 6.0 cm, helium pressure of 1,100 psi, 0.6 microm gold microcarriers, single time bombardment and with both pre- and post-osmoticum were found ideal. Selection of putative transformants was done on MS medium supplemented with 0.5 mg l(-1) BA and hygromycin (20, 40 and 60 mg l(-1)) for 3 cycles. The stable integration of the incorporated gene into castor genome was confirmed with PCR and Southern analysis of T0 and T1 plants. Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. The present results demonstrate the possibility of transformation of embryonic meristematic tissues of castor through particle delivery system.

No abstract

Castor (cv. DCS-9) has been transformed through Agrobacterium-mediated and particle gun bombardment methods using appropriate vectors containing the Bt chimeric gene cry1EC driven by enhanced 35S promoter. About 81 and 12 putative transformants were regenerated following selection on hygromycin and kanamycin, respectively. Southern analysis of DNA extracted from T(0) plants confirmed integration of the introduced gene in castor genome. The integration and inheritance of the introduced genes was demonstrated up to T(4) generation by PCR and Southern analysis. Southern analysis of two events having single and two copies showed the same pattern of integration in the subsequent generations. Insect feeding experiments conducted in the laboratory by releasing neonate larvae of castor semilooper and S. litura on leaf tissues excised from transgenic and control plants showed varying degrees of larval mortality and slow growth in larvae fed on transgenic leaf tissue. Field bioassays against Spodoptera litura and castor semilooper conducted for eight events in T(1)-T(4) generations under net confinement were more informative and events conferring resistance to the two major defoliators were identified.

No abstract