Transformation of the Cry1Ac and bar genes to forage pea (pisum arvense l.) via agrobacterium tumefaciens

The objective of this study was to develop a genetic transformation protocol for forage pea (Pisum arvense L.) using the bacterium Agrobacterium tumefaciens. The scope of this study included experiments on three main areas: the regeneration of adventitious shoots, the optimisation of gene transfer, and the PCR-based confirmation of transgenic candidate plants that are resistant to insect pests. In the first stage of the study, the adventitious shoot regeneration capacities of cotyledonary node and root node explants of the Ateş genotype of forage pea were assessed on Murashige and Skoog (MS) media, with the explants being supplemented with various concentrations of plant growth regulators. Six different combinations were tested, supported by 6-Benzyl Amino Purine (BAP) and Naphthalene Acetic Acid (NAA) hormones. The following concentrations of hormones were applied: 1.00, 2.00, 3.00, 4.00, 5.00, and 6.00 mg l⁻¹ BAP + 0.2 mg l⁻¹ NAA. Based on the regeneration results, the highest direct shoot regeneration for both cotyledonary node and root node explants was achieved using the medium with 3 mg l⁻¹ BAP + 0.2 mg l⁻¹ NAA. To induce rooting of shoots that successfully completed regeneration, MS medium supplemented with 1.00 mg l⁻¹ Indole-3-Acetic Acid (IAA) was used, and effective root formation was observed in this medium. The appropriate herbicide doses for the selection of explants treated with Agrobacterium were determined by testing MS media containing four different concentrations (2.00, 4.00, 6.00, and 8.00 mg l-1) of phosphinothricin. Consequently, a concentration of 2.00 mg l-1 PPT was determined to be the optimal setting for the effective selection of transformants in the designated selection medium. To optimise genetic transformation, the 35S-cry1Ac-NosT gene cassette was used alongside the NPT-II selectable marker gene, which was cloned into Agrobacterium for transformation. vectors, while the Bar gene was introduced using the LBA PGG Bar cassette. A total of 175 transgenic candidate plants carrying the Cry1Ac and Bar genes were obtained at the end of the transformation studies. PCR analysis revealed transformation frequencies of 36.66% and 17.65%, respectively, for the Cry1Ac and Bar genes. This study demonstrates that an effective genetic transformation system can be established in forage peas and similar crops to develop resistance against insect pests.