Plant & Animal Genomes XIII Conference Plant & Animal Genomes XIII ConferenceJanuary 15-19, 2005
Town & Country Convention Center
San Diego, CA
Rajesh PN* , Fred Muehlbauer , Kevin McPhee
Agrobacterium tumefaciens-mediated transformation is the most preferred of all available transformation methods for grain legume species. Our objective was to establish stable transformation of large genomic inserts using a suitable Agrobacterium tumefaciens (A. t) strain that will have long term application in functional analyses of disease resistance genes in chickpea. Here we report stability analysis of seven large inserts ranging in size from 20 to 100 kb and transformation of the BAC clone, 15(O)o9 which is 46kb in size. Our results show that genomic fragments up to 100kb transferred by triparental mating were stably maintained in A.t. strain Agl0. Clone identity was confirmed by fingerprinting. 15(O)o9 contains 8 genes as determined by blast analysis and 36 predicted genes by genscan analysis. Since this particular large insert was linked to ascochyta blight resistance and also gene rich, we transformed 15(O)o9 into a chickpea line for complementation analysis. The explants were screened on selection media for 23 weeks. To identify the presence of the inserts in the potential transformant by PCR, we used the markers spanning this large insert as well as primers designed from the binary construct. Our results were positive for all markers in the To transformants. Work is underway to determine the copy number and the inheritance of this particular fragment in subsequent generations.