Transformation Fava Beans by Agrobacterium using Chitinase, Glucanase and CryIA (b) genes
In this study two plasmid vectors that are appropriate for plant transformation were made by preparation of gene cassettes for β-1, 3-glucanase from barley, chitinase from bean and cryIA (b) from Bacillus thuringiensis (BT). Each of these genes were cloned under the control of the CaMV35S promoter and the Nos terminator in pBI121 binary vector. pBI-Chi and pBI-Glu and recombinant plasmid vectors were constructed via cloning of chitinase , β-1,3- glucanase and cryIA (b) genes, respectively, instead of the gus gene in T-DNA region of pBI121 vector. Construction of pBI-ChiGlu recombinant plasmid vector was performed by means of cloning both of the complete chitinase and glucanase gene cassettes in pBI121 vector, with the intention of production synergistic effects against fungal infection.pBI-ChiBt recombinant plasmid vector containing both of the complete chitinase and Bt gene cassettes was also constructed in order to contemporaneous plants resistance to pest pathogens and fungal in a single transformation event. pBI-ChiGlu and pBI-ChiBt that have been introduced into the A. tumefaciens strain LBA4404 that was subsequently used for transformation. Results indicate that embryogenic calli are well appropriate as objective material for Agrobacterium tumefaciens-mediated transformation in Faba bean.Seventeen well established shoots were transferred to new MLS medium including suitable antibiotics.Finally six independent transgenic plants were successfully rooted on kanamycin-containing selection media and then transferred to soil after 20 days .Four plants out of six putative transgenic plants displaied to contain the end part of the chit transgene and nos terminator.The corresponding piece, 700 bp of the chit gene, was amplified using specific primer.These putative transgenic plants were also be measured for the presence of the bgn13.1 and cryIA (b) genes by PCR using specific primers.Two pieces with expected sizes (1221 bp and 640 bp) for bgn13.1 and cryIA (b) genes subsequently was amplified from three the transgenic plants and other three plants had not these fragments.A group of virG primers was used to discover Agrobacterium pollution that might have threw off the selection. Expression of chit , and β-1,3-glucanase genes at the transcriptional level in the transformed plants were proven by means of RT-PCR but expression of cryIA(b)was not proven.The expected dimension of the amplified cDNA pieces were identificated in the transformed plants,that confirming the permanent annexation and expression of T-DNA in the genome of kanamycin-screened plants.
Agrobacterium ,β-1,3-glucanase; BT; Chitinase; Cloning; Recombinant Plasmid
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Published by INSIGHT - Indonesian Society for Knowledge and Human Development