Modification of plant regeneration medium decreases the time for recovery of Solanum 1 lycopersicum cultivar M 82 stable transgenic lines 2 3

11 Background: Tomato (Solanum lycopersicum) has rapidly become a valuable model as a result 12 of the availability of a high quality reference genome, extensive genetic resources, and efficient 13 gene transfer methodology. A high-throughput method to obtain transgenic lines sooner than 14 standard methods has the potential to greatly advance gene function studies. 15 16 Results: The goal of this study was to optimize our current gene transfer (transformation) 17 method for tomato by investigating medium components that would result in a decreased time 18 for recovery of transgenic lines. The methodology reported here is based on infection of 6-day19 old cotyledon explants from Solanum lycopersicum cultivar M82 in vitro-grown seedlings with 20 Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBI121. This vector 21 contains the β-glucuronidase reporter gene and the neomycin phosphotransferase II selectable 22 marker gene that confers resistance to kanamycin. Modification of our standard plant 23 regeneration medium by the addition of indole-3-acetic acid (IAA) at concentrations of either 24 0.05 mg/l or 0.1 mg/l greatly decreased the time for recovery of transgenic lines by 6 weeks as 25 compared to our standard medium that contains zeatin alone. Moreover, addition of 1 mg/l 26 IAA to the root induction medium resulted in faster root development. We observed 50% and 27 54% transformation efficiency when plant regeneration medium containing 0.05 mg/l and 0.1 28 mg/l IAA, respectively, was used for transformation experiments. Transgenic lines recovered 29 with the optimized method were similar in development to our standard method. No negative 30 effects were observed as a result of the addition of IAA to the medium. 31 32 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/046839 doi: bioRxiv preprint first posted online Apr. 2, 2016;