Agrobacterium-mediated transformation of Basmati rice and system development for transformation of wheat via multiple meristem bombardment

The research presented in this dissertation includes transformation of Basmatic Variety 370 of indica rice via the Agrobactrium system, confirmation of insect resistance of second-generation transgenic rice, and system development for transformation of wheat using the biolistic bombardment.; A lepidopteran/dipteran-specific synthetic gene, cry2A, from Bacillus thuringiensis (Bt) was sub-cloned under the control of the CaMV promoter. Agrobacterium-mediated transformation method was used to transfer two different Bt genes (cry1Ab and cry1Ac) in Basmatic rice. Conditions for transformation of rice were optimized using the β-glucoronidase (gus) reporter gene system. A total of 64 transgenic plants were produced from 15 hygromycin-resistant calli. Molecular analysis of R0 and R1 progeny plants confirmed integration, transcription and translation of all transgenes with a 100% cointegration of linked genes. The expression of Cry1Ab and Cry1Ac was estimated up to 0.1% of total soluble proteins. Inheritance of the introduced genes to R1 progeny was found to be in agreement with Mendelian segregation in most of the transgenic lines. Further, Real-Time PCR was used to confirm the transgene copy numbers. Since it is illegal to bring rice pests from outside of the U.S., European corn borer (Ostrinia nubilalis) was used to bioassay non-transgenic and transgenic rice plants. Excised-leaves of plants at tillering stage, two-week-old whole-plants and mature whole-plants were used in feeding assays. Significant (P < 0.0001) differences compared to non-transgenic plants were observed in all bioassys conducted.; Efforts were also made to develop a less genotype dependent in vitro regeneration system as a pre-step toward genetic transformation of wheat (Triteum aestivum L.). Among different concentrations of phytohormones, 2 and 4 mg/l of BA (8.8 and 17.7 μM) in combination with 0.5 mg/l 2,4-D (2.26 μM) produced the best results that were significantly (P = 0.05) different from other hormone combinations. The system developed here is capable of producing multiple shoot clumps and whole plants in four different wheat genotypes tested. Scanning electron microscopy of cultures showed a proliferating budding state that gave rise to adventitious shoots, as well as, somatic embryos on further multiplication. These shoot clumps regenerated normally and produced fertile plants containing viable seeds. This in vitro regeneration system was tested for its usefulness for the production of transgenic wheat using bioslistic bombardment (PDS-1000/He) system. Different bombardment parameters were studied for transient expression of gus. Among parameters used, 0.6–0.9 μm tungsten particles, 13 cm distance of the target tissue and point of bombardment and pressure of 1100 and 1550 psi gave maximum transient expression of gus (i.e. 43.5 to 45%). Gold particles were also used in comparisons with tungsten particles but did not yield significant (P = 0.05) increase in transient expression of gus.