Abstract
In this article, we describe a highly efficient and reproducible method of Agrobacterium-mediated transformation protocol for several gene transformations (cry1Ab, rolB, and PhMV) in tomatoes. Tomato fruit is rich in minerals, essential amino acids, sugar, vitamin B, iron, phosphorus, and dietary fiber. Tomato Crop production has some major problem which includes early ripening, reduction in fruit shelf life and devastating diseases that caused by viruses, bacteria, fungi, and nematodes, etc. The study aims to use the Agrobacterium-mediated transformation method to improve the tomato shelf life and make tomato plants resistant to such types of diseases caused by bacteria, viruses, and fungi, etc. Tomato was transformed via Agrobacterium tumefaciens strain LBA4404 harbouring Cry1Ab synthetic gene for toxin proteins. And same strain was inserted with another CP of PhMV that cloned into the binary vector pBI 121 for resistance to PhMV infection, and strain GV3101 harbouring the binary vector pICBV19 for reducing the expression of vis1 gene. Tomato seed culture on MS media after sterilization with 75% ethanol and distilled water. Plantlet root formation was on different kanamycin MS mediums. Constricted plasmid vectors with a gene if interest allowed to be transferred into Agrobacterium tumefaciens using the freeze-thaw method. The engineered strain suspension was centrifuged and re-suspended with MS media. Tomato seeds were obtained, then 0.5 cm per cotyledon, and put into the Agrobacterium suspension and co-cultivated and incubated and a transgenic plant, analysis was carried out using PCR, real-time Reanalysis, Northern blot analysis, and statistical analysis. For tomato varieties, the protocol developed showed very high transformation efficiency. The optimized transformation process is simple, effective and no need for tobacco, Petunia, tomato suspension feeder layer, or acetosyringone as chemical transduction.
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