Optimization of Nisin Gene Transfer by Agrobacterium in Carrot Plant

Nisin is a natural antibacterial that is used as a preservative in foods and drugs. The production of transgenic carrots containing recombinant nisin protein was investigated by Agrobacterium, LBA4404 strain and pBI121 plasmid. PBI121 plasmid was contains NPTII reporter genes with promoter CaMV35S and terminator NOS, respectively. In order to prepare recombinant plasmid, nisin gene first was identified . After codon optimization, were cloned between bamHI and SacI in pBI121 vector. Root, leaf, stem and nods explants of carrot were inoculation with agrobacterium suspension containing inoculum with different concentrations and were placed for different times of co-cultivation and they were also cultured in callus induction medium containing kanamycin and cefotaxime. The calli that were able to grow on the selective medium were selected and were transferred to the regeneration medium . Callus induction was observed in 40% of the explants. The produced embryos, after germination were transferred to the regeneration medium. The highest percentage of kanamycin resistant plants (45%) were related to the use of stem explants and bacterium concentrations of 0.6 to 0.8 and different co-cultivation times of 1 and 2 days and concentration of 250 mg/L cefotaxime. In order to ensure no contamination of plants to Agrobacteria and confirm the accuracy of gene transfer, polymerase chain reaction was performed using specific primers of vir gene and the accuracy of gene transfer was confirmed. Polymerase chain reaction was showed that 60% of hypothetical transgenic plants had at least one copy of nptII and nisin genes in their genomes.