DEVELOPMENT OF EFFICIENT TRANSFORMATION SYSTEMS TO ENHANCE INSECT RESISTANCE IN RICE (Oryza sativa L.)
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Date
2007
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Indira Gandhi Krishi Vishwavidyalaya, Raipur
Abstract
Rice is the world‟s important food crop, serving as a major staple food for about 3 billion people. The pests particularly stem borers causes million dollars worth of loss annually. Development of resistant varieties plays a pivotal role in management of insect pests such as yellow stem borer (Scripophaga incertulus), stripped stem borer (Chilo supperessalis) and leaf folder (Cnaphalocrocis medinalis). Despite of the fact that more than 30,000 rice accessions have been screened for resistance till date, the sufficient level of resistance was not found in rice collection (Wu et al., 1999). The only viable option to control borers and leaf folder insects of rice are either through the use of chemical insecticides or use of genetic engineering to introduce genes of alien origin conferring resistance to these pests. Genetic engineering using Bt gene(s) is an attractive alternative of developing rice genotypes resistant to stem borers. Most of the rice genotypes have been transformed using tissue culture based techniques, Agrobacterium- mediated and Biolistic approaches. However, some technological limitations like these methods of transformation are genotype dependent, time consuming and recalcitrant nature of indica rice to in-vitro regeneration with very low transformation efficiency has limited the routine use of this technology.
The present study was therefore conceived with the objectives to assess the various factors affecting tissue culture response and subsequently the tissue culture based transformation systems viz., Agrobacterium- mediated and Particle gun mediated DNA delivery and to develop insect resistant indica rice plants using cryIAc and mVIP genes. For developing high efficiency callusing and plant regeneration systems for two rice varieties, Swarna and Mahsuri, suitable concentrations of carbohydrate maltose, a gelling agent gel rite and plant growth
regulators were identified and a high efficiency in vitro regeneration protocol from seed derived calli were developed for indica rice varieties under study. Partial desiccation of embryogenic calli (18-24 hours) prior to regeneration has significantly increased regeneration efficiency by 23.15% in Swarna and 33.55% in Mahsuri.
The cryIAc gene was successfully introduced into rice variety Swarna using Particle gun mediated transformation system for developing insect resistant rice plants. Twenty of 58 putative transformants of rice variety Swarna were found positive for cryIAc gene, whereas, no transformants were found positive for mVIP gene. Later, the integration of cryIAc gene was confirmed initially by PCR and then by southern analysis.
To develop a high efficiency non- tissue culture based in planta transformation system for indica rice, various factors for Agrobacterium based floral dip method were examined using a reporter gene, GUS/INT driven by CaMV35S promoter. Our study suggested that female floral part mainly egg cells of emasculated rice flowers are the target of T- DNA transfer in floral dip method. The parameters viz., density of Agrobacterium inoculum, dipping time with gentle shaking, removing of excess Agrobacterium culture adhered on the surface and pollen density to allow double fertilization and seed development were standardized for indica rice. The high transformation efficiency of 22.95% was recorded using the procedure developed in this study. This opens up avenues for developing rice transgenic rapidly using less sophisticated, easy and by less technically skilled personals. Development of genotype independent non-tissue culture based high efficiency transformation procedure for indica rice will also enhance the gene validation research in rice.
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Keywords
rice, genetic processes, planting, genes, developmental stages, bacteria, transgenics, application methods, dna, regeneration