Attempts for genetic transformation of cotton through ternary transformation system in Agrobacterium mediated transformation- a strategy for improvement of gene transfer method

Cotton tissue in culture is a recalcitrant plant system. It was thus necessary to make available of a suitable protocol, efficient enough to cause Agrobacterium mediated transformation. An efficient gene transfer system should have the capacity to result high transformation incidence. The transformation frequency in any plant species is influenced by T- DNA transfer efficiency by the virulent Agrobacterium tumifaciens strain. A critical step in the development of robust Agrobacterium mediated transformation system in recalcitrant cotton is the establishment of optimal conditions for efficient T-DNA delivery into target tissue and recovery of transgenic plants. A dramatic increase in efficiency of T-DNA delivery was achieved by constitutive expression of additional vir genes, a mutant of virG, virGN54D in resident pCAMBIA vector in Agrobacterium strain LBA4404. Taking advantage of this phenomenon, we designed a strategy for ternary transformation system of a plant transformation by providing an additional plasmid containing virGN54D along with standard binary system of Agrobacterium mediated transformation. To start with, the influence of ternary system of transformation was tested by monitoring the expression of gus A gene, as marker. Analysis of stably transformed cell lines showed that, although the T-DNA transfer frequency is greatly enhanced and incidence of higher level of transformation in case of ternary system in comparison to binary system of transformation was observed. Agrobacterium mediated transformation of cotton via somatic embryogenic mode of plant regeneration remains by far the most dependable method of choice. This is due to the reliability and efficiency by which transgenic plants are recovered. Independent transformants showed stable expression of the transformed gus A gene and its seed transmission to the next filial generation.