Evaluation of Factors Influencing In Vitro Regeneration and Transformation Protocols to Produce Salinity Tolerant Tomato (Solanum lycopersicum L.)

Introduction: Tomato (Solanum lycopersicum L.) is one of the essential vegetables worldwide, for consumption and mitigating malnutrition. Genetic transformation conceivably overcomes its yield challenges due to salinity, a crucial constraint for the economical use of ۳۰% of cultivable lands in the coastal region of Bangladesh. Therefore, a robust and reproducible protocol has been established for in vitro regeneration and transformation to develop transgenic salt-tolerant tomato plants.Materials and Methods: During micropropagation, cotyledonary leaf explants were excised and cultured on MS media containing different combinations and concentrations of plant growth regulators. In transformation, the pre-cultured explants were inoculated and co-cultivated with Agrobacterium. Then they were transferred to the antibiotics-supplemented media to achieve salt-tolerant putative transformed plants. The transformation was confirmed by β-glucuronidase (GUS) assay and PCR for the antiporter gene.Results: Maximum regeneration response was achieved from the explants abaxially positioned at a ۱.۵ cm distance apart. BARI Tomato ۱۴ and BINA Tomato ۳ showed the highest shoot regeneration response (%) on MS media supplemented with ۲ mg/L BAP and with ۰.۱ mg/L IAA for BARI Tomato ۲ and BARI Tomato ۱۵. Bacterial culture of OD۶۰۰ ۰.۶۸ for ۳۰ min and a Co-cultivation period of ۴۸ h resulted in the highest transformation frequency (۴۷%) in Agrobacterium-mediated transformation with pBI۱۲۱ in BARI Tomato ۳. The highest regeneration frequency (۲۰.۵%) was obtained in transformation with pH۷WG۲_OsNHX۱_۱.۶.Conclusions: The optimized procedure is simple, efficient, and can be used for micro-propagation and the production of tolerant varieties to increase yield in saline areas. 

Keywords:

Nutritional Crop , Tissue culture , Agrobacterium-mediated Transformation , Salt Tolerance , Phytohormones , Explants Spacing