Genetic transformation of Solanum chrysotrichum by Agrobacterium tumefaciens and the production of antifungal saponins

Abstract

Solanum chrysotrichum (Solanaceae) synthesizes a family of six antifungal spirostanol saponins designated as SC-1 to SC-6. The production of saponins by wild-type plants is variable depending on the environmental conditions. In order to develop an in vitro system for the sustained production of these saponins, transformed cell suspension cultures of S. chrysotrichum were established from nodal explants of 3-mo-old plantlets by infecting with the Agrobacterium tumefaciens strain C58/pBI12. From these cultures, kanamycin-resistant and phytohormone-independent cell suspension line C58 5.1.1 was obtained. PCR and Southern blot analyses were used to confirm the integration of the wild-type T-DNA into the plant genome. Batch cultures of the C58 5.1.1 cell line were grown in phytohormone-free MS liquid medium for 25 d. First-order growth kinetics and the production of the antifungal saponins (SC-2, SC-3, and SC-4) were determined by dry weight and quantified by HPLC, respectively, from the cells as well as the culture medium. Based on the cell biomass, the specific growth rate was 0.09 d−1 and the yield of SC-2 reached 5.5% of dry weight, representing 40 times higher amount than that produced in plant leaves. SC-3 was recovered with a maximum yield of 0.9% of dry weight, whereas SC-4 was accumulated at 1.1% of dry weight. Saponins SC-2 and SC-3 were also excreted into the culture medium in low concentrations.