Genetic transformation of kenaf (Hibiscus cannabinus L.) (Agrobacterium tumefaciens)

This study was conducted to investigate genetic transformation of kenaf via Agrobacterium tumefaciens (A.A.) and/or biolistics (PDS-1000/He; gene gun). Initially, standard shoot regeneration media for kenaf leaf explants were optimized for cultivars Everglades 41 and Tainung 2. Factors affecting both A.t. and biolistics-mediated transformations were investigated. Vectors utilized for optimizations contained both chimaeric nptII and gus genes. Post-transformation, leaf explants were selected on media containing geneticin. Optimized parameters for A.t. transformation (using cointegrate vectors) based on GUS transient expression assayed histochemically included preculture and inoculation times of 2 days and 10 min, respectively. Addition of acetosyringone to bacterial and coculture media could enhance the transformation frequency, but this was dependent on the plasmid construct utilized. Additional wounding with tungsten particles using the gene gun decreased the transformation frequency.; Optimized parameters for biolistics included 900 psi helium pressure, 1/4 inch (6 mm) gap and 7.5 cm target distance. Greatest numbers of GUS histochemical spots were observed with leaf explants precultured for 7 days, then shot once with 2 μg DNA adhered onto M10 (0.7 μm) tungsten particles. Transient and stable transformation frequencies (based on number of explants surviving on selection media after 6 months) for both gene transfer methods were cultivar and seed clonal line (seedling lines maintained as independent clones) dependent.; Greater numbers of T₀ transgenic kenaf plants were produced from biolistics (14 shoots from 8 independent explants) compared to A.t. (4 shoots from 2 independent explants). Initially, evidence of transformation was provided by polymerase chain reaction (PCR) and then confirmed by PCR-based Southern and T₁ progeny analyses.; A chimaeric insect resistance gene [cryIA(c )] was introduced via A.t. and biolistics. Three transgenic plants from each gene transfer method [3 independent explants (A.t.); 2 independent explants (biolistics)] were confirmed for the presence of the cryIA (c) gene using PCR, PCR-based Southern and T ₁ progeny analyses. Fall armyworm larvae were susceptible to leaves from one transgenic plant obtained via A.t. in feeding trials, however, another plant obtained via biolistics did not affect larval feeding or growth.; A chimaeric bar vector (pLMBAR; conferring resistance to the herbicide Ignite) was constructed. Plasmid pLMBAR was used to bombard kenaf leaf explants. The presence of the bar gene was confirmed in kenaf shoots and leaf clumps using PCR.