Abstract:We report here the suitable explant with high efficiency of transformation and the positive effects of timentin over other antibiotics like carbenicillin and cefotaxime on the elimination of Agrobacterium tumefaciens during the genetic transformation of popular indica rice (Oryza sativa L.). The tissues assayed were embryogenic calli, embryos with endosperm contamination, intact seeds, leaf blades, leaf bases and coleoptiles. The frequency of transient β-glucuronidase (GUS) expression as revealed by histochemical assay was 90% for embryogenic calli, which was the highest among the explants used. On the basis of disc-diffusion assay, the maximum zone of inhibition (29 mm) at 250 mg/l was obtained for timentin. In tissue culture conditions the frequency of Agrobacterium recurrence after 20 days of infection was minimum (2.3%) at 200 mg/l of timentin. At 250 mg/l of timentin there was no Agrobacterium growth, besides, there were no negative effects on the callus growth unlike other antibiotics, hence it was selected as the optimum concentration for high frequency callus proliferation and regeneration. The effect of the parameters evaluated was determined by the callus proliferation during selection, reduction in browning, transient GUS expression and stable transformation efficiency (23.3%). The resultant plants were stable transformants as confirmed by a molecular analysis of the gus and hpt genes. The developed transformation protocol will be very helpful for the information on indica rice cultivars in general and on IR 64 in particular.
A novel synthetic cry2AX1 gene was codon optimized and a sequence encoding cotton rbcS1b transit peptide was fused upstream of coding sequence. The fusion cry2AX1 gene, driven by maize ubiquitin1 promoter was cloned in a pUH plant transformation vector. Agrobacterium mediated transformation was carried out with pUH-ctp-2AX1 construct using leaf discs of tobacco as model plant. Screening by PCR revealed presence of cry2AX1 gene in all nine putative transformants and expression of cry2AX1 protein in PCR positive T 0 tobacco plants ranged from 1.5 to 10.0 ng/g. The detached leaf bit bioassay of tobacco transformants with Helicoverpa armigera showed 30 per cent mortality even at lower level of cry2AX1 expression. The results indicated a newly developed construct was functionally expressed in tobacco plant.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.