Transformation of Non- Agrobacterium (Rhizobia) Species by Binary Vector: Identification of a Tool for Plant Genetic Engineering

Abstract: The aim of this project is to identify new Rhizobia species exhibiting transformation efficiency of binary vectors, which work as better genetic engineering tool than Agrobacterium species. Agrobacterium was widely considered to be the only bacterial genus capable of transferring genes into the genomes of plants. Other plant-associated bacteria, including Sinorhizobium, Rhizobium, and Mesorhizobium, can be modified to mediate gene transfer into tobacco and Arabidopsis plants and, in the case of Sinorhizobium meliloti, into rice plants. Rhizobia could become a new resource for crop improvement and therefore an alternative to the patented Agrobacterium- mediated technology Rhizobia are phylogenetically diverse; currently they are classified into five genera of a-proteobacteria (Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, and Mesorhizobium) and at least two genera of ß-proteobacteria (Burkholderia and Ralstonia). In addition to forming symbiotic nodules on legumes, rhizobial strains are members ofsoil or rhizophere communities or occur as endophytes, e.g. in rice. Two rizobial strains which have been isolated from root nodules of the aquatic legumes Aeschnomene fluminensis (IRBG271) and Sesbania aculeate (IRBG74) were previously found to promote rice growth. The close genetic relationship between Agrobacterium and rhizobia led to test the widely used freeze-thaw method for Agrobacterium transformation on rhizobia. Until recently, Agrobacterium was considered to be the only bacterium used for gene transformation into plant genomes. This project explores the possibility of using rhizobia as a vehicle for gene transfer into plants. Rhizobium transformants carrying binary vectors can be engineered to carry genes of interest (nitrogen fixation gene, toxic gene to provide compatibility from other strain) offering opportunity to modulate the rhizobium plant interaction in a novel way. Along with the rate of transformation, this study could also be helpful in assessing the quality of transformation, which in turn could be helpful in determining the competency of these modified strains with particular plant types. As a result a new study focusing on the essence of symbiotic relationships between both species could be carried out. The aim of the present project is to carry out comparative study to check the efficiency for transformation in different non-agronomic strains (rhizobia) like Mesorhizobium loti and Rhizobium leguminosaum. Plasmids (Binary vectors) to be used for transformation is pGA482. As a result a new study focusing on the essence of symbiotic relationships between both species could be carried out.