Efficient Regeneration and Agrobacterium-Mediated Transformation Method For Cultivated and Wild Tomato

Agrobacterium -based stable transformation is an imperative tool for functional genomics studies and crop improvement which is needed to maximize the benefits of emerging technologies like CRISPR/Cas9. In the present study, we report a highly efficient protocol capable of transforming different tomato cultivars. The three cultivated varieties of Solanum lycopersicum , Pusa Ruby, Arka Vikas, and Pusa early dwarf, and one wild species Solanum peruvianum were used. The CRISPR-based vectors pDIRECT and pMOD were used to prepare constructs for the transformation. We have compared effect of different types of explants, age of explants, different strains of A. tumefaciens , different cell densities of A. tumefaciens used for co-cultivation, and different temperatures of co-cultivation on regeneration and transformation. Among the growth hormones, zeatin (2 mg/l) and IAA (0.1 mg/l) were used as a source of cytokinin and auxin for regeneration. Enhanced rooting was observed with 0.1 mg/l IAA in the rooting medium. Among the cultivated varieties, S. lycopersicum cv. Arka Vikas showed maximum regeneration efficiency of 54.8% and wild species S. peruvianum displayed 65% efficiency. Pusa early dwarf showed the lowest regeneration efficiency of 17%; however, enough plantlets of this recalcitrant cultivar have been recovered proving the utility of the present method. More than 80% of the regenerated plantlets yielded PCR-positive transformed plants for all the varieties and species tested in the study. Copy number analysis using a novel qPCR method was also performed for transgenic plants. Here, we describe a protocol with minute details and care to be taken for efficient tomato transformation, with a single standard protocol capable of transforming any tomato variety.