[Springer] Efficient Regeneration and Agrobacterium-Mediated Transformation Method For Cultivated and Wild Tomato Copy

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.