Efficient production of transgenic potato (S. tuberosum L. ssp. andigena) plants via Agrobacterium tumefaciens-mediated transformation

Abstract

Potato is an important target crop for biotechnological applications and is a valuable model system for studying signaling processes. Efficient transformation is critical for rapid genetic analyses. The production of transgenic potato shoots within 4 weeks from the time of initial inoculation of leaf explants by Agrobacterium tumefaciens has been established with the Solanum tuberosum subspecies andigena. Vigorous stock plants, the precise, uniform wounding of the midrib on the leaf explant, and the composition of the regeneration media play key roles in the development of this efficient shoot regeneration protocol. To produce callus from leaf explants in 7 days, basal medium was supplemented with optimized concentrations of benzyl-aminopurine and napthalene acetic acid. Incubation on basal medium supplemented with a combination of zeatin riboside, napthalene acetic acid, and gibberellic acid induced shoot formation from callus after 28 days of incubation. In this improved protocol, the commonly used pre-culture of explants with nutrient medium was eliminated and the Agrobacterium inoculation medium was not supplemented with any phytohormones. Induction of roots from putative transformed shoots was achieved in hormone-free basal medium supplemented with kanamycin. Normal, healthy root formation was observed within 5 days and 91% of the selected shoots rooted on kanamycin. By using RT-PCR analysis with gene specific primers, all rooted shoots out of 20 selected from five different lines exhibited expression of the full-length StBEL5 transgene driven by the CaMV 35S promoter. The protocol described here is simple, efficient, and produces transgenic shoots in just 4 weeks after inoculation with Agrobacterium.