Modification of plant regeneration medium decreases the time for recovery of Solanum lycopersicum cultivar M82 stable transgenic lines

Abstract

Abstract Background: Tomato (Solanum lycopersicum) has rapidly become a valuable model as a result of the availability of a high quality reference genome, extensive genetic resources, and efficient gene transfer methodology. A high-throughput method to obtain transgenic lines sooner than standard methods has the potential to greatly advance gene function studies. Results: The goal of this study was to optimize our current gene transfer (transformation) method for tomato by investigating medium components that would result in a decreased time for recovery of transgenic lines. The methodology reported here is based on infection of 6-day-old cotyledon explants from Solanum lycopersicum cultivar M82 in vitro-grown seedlings with Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBI121. This vector contains the β-glucuronidase reporter gene and the neomycin phosphotransferase II selectable marker gene that confers resistance to kanamycin. Modification of our standard plant regeneration medium by the addition of indole-3-acetic acid (IAA) at concentrations of either 0.05 mg/l or 0.1 mg/l greatly decreased the time for recovery of transgenic lines by 6 weeks as compared to our standard medium that contains zeatin alone. Moreover, addition of 1 mg/l IAA to the root induction medium resulted in faster root development. We observed 50% and 54% transformation efficiency when plant regeneration medium containing 0.05 mg/l and 0.1 mg/l IAA, respectively, was used for transformation experiments. Transgenic lines recovered with the optimized method were similar in development to our standard method. No negative effects were observed as a result of the addition of IAA to the medium. Conclusions: The results showed that addition of IAA at concentrations of either 0.05 or 0.1 mg/l to the plant regeneration medium reduced the total time for recovery of stable transgenic lines by 6 weeks. The ability to recover transgenic lines in a shorter time results in higher throughput for the introduction of gene constructs and has the potential to decrease the time needed to complete investigations of gene function. The ability to recover transgenic lines for each gene construct in a shorter time saves labor and resource expenses because culture maintenance is also reduced.