|P.N., Rajesh - WASHINGTON STATE UNIV|
Submitted to: International Chickpea and Pigeonpea Newsletter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 1, 2007
Publication Date: November 1, 2007
Citation: P.N., R., Muehlbauer, F.J., Mcphee, K.E. 2007. Stability analysis of chickpea large genomic DNA inserts in Agrobacterium.. International Chickpea and Pigeonpea Newsletter.online Interpretive Summary: Genetic transformation using Agrobacterium tumefaciens is a valuable tool for gene discovery and in the study of gene funcition. The ablility to study genes in their native state including their flanking regulatory sequences requires the manipulation of very large DNA segments. Through the use of Bacterial Artificial Chromosome libraries it is possible to study such large segments, but the stability of these segments in Agrobacterium tumefaciens must be verified prior to intiation of transformation experiments. This study evalutated the stablity of seven large DNA segments ranging from 20 - 100 kb in length in two A. tumefaciens strain Agl0. All these fragments were successfully recovered from strain Agl0; however, individual colonies were found with some deletions. Results from this study demonstrate the necessity and importance of selecting an appropriate and suitable A. tumefaciens strain and that verifying the stability of the DNA segment in the suitable strain prior to initiating transformation experiments.
Technical Abstract: Agrobacterium tumefaciens-mediated transformation of large DNA inserts directly into plants facilitates the transfer of gene clusters and flanking regulatory elements. It is recommended that the integrity of large genomic fragments in Agrobacterium be verified prior to plant transformation. In this study, the stability of large genomic DNA inserts in A.t. was assessed using seven fragments ranging in size from 20 to 100 kb obtained from a chickpea Bacterial Artificial Chromosome (BAC) library constructed using the pCLDO4541 (V41) binary vector. Six inserts were associated with ascochyta blight resistance and one was linked to fusarium wilt race 3 resistance in chickpea. These BAC clones were transformed into A. tumefaciens strain Agl0 using triparental mating. Stability of the clones in A. tumefaciens was assessed by transforming the BAC clones back into E. coli - ElectroMAXTM DH10BTM strain and evaluated using Pulsed Field Gel Electrophoresis. All fragments up to 100kb in size were stably transformed into Agl0 by triparental mating and recovered intact. Clone identity was confirmed by fingerprinting. Our results show that genomic fragments up to 100kb transferred by triparental mating were stably maintained in A. tumefaciens strain Agl0. However, despite the presence of intact plasmids, evidence of deletions from individual colonies was also observed in Agl0 which emphasizes the need to verify the presence and integrity of the plasmid being transferred.