Optimization for Agrobacterium-Mediated Transformation of Reporter Gene in Tomato

Abstract

In tropical and subtropical regio tomato (Solanum lycopersicum) has economical vegetable crop and extensively cultivated. The current study purpose is to optimize the conditions for in vitro germination of various tomato seeds cultivars and develop an efficient Agrobacterium-mediated transformation procedure using the GUS reporter gene. Half-strength MS medium supplemented with 2 mg/L NAA and 2 mg/L BAP were used to culture seeds, which shows reduced time required for germination. Four different cultivars of tomato using hypocotyl and cotyledonary leaf as explants and subjected to transformation using Agrobacterium tumefaciens strain EHA101 with vector pZY102 holding the GUS gene. For infection bacterial culture with OD₆₆₀ of 0.3–0.4 was used with acetosyringone for 15 minutes. Co cultivation of explants was done at 22°C in the dark for 2 days, after pre selection of 7 days followed by histochemical GUS assay at 37°C. The results of present study showed that optimized conditions for germination significantly increased seed emergence and both explant types showed successful transformation with difference among cultivars. The optimized infection conditions and presence of acetosyringone enhanced T-DNA delivery and expression of GUS. Higher efficiency of transformation was observed under controlled infection parameter and co-cultivation. This study reveals that type of explant and conditions for culture plays an important role in improving transformation efficiency in tomato. It is concluded that a protocol which is reliable and efficient for Agrobacterium-mediated transformation has been developed; still, to achieve higher rate of transformation and stable expression of gene continuous optimization is required.