The addition of an organosilicone surfactant to Agrobacterium suspensions enables efficient transient transformation of in vitro grapevine leaf tissue at ambient pressure

Abstract

Transient Agrobacterium-mediated transformation of plant tissue has become a standard technique for rapid in vivo analysis of gene expression and function. In grapevine, the efficacy of transient leaf transformation is limited by the ability of bacterial suspensions to penetrate into the tissue. Current protocols therefore use the temporary application of a vacuum or site-specific syringe infiltration to improve transformation efficiencies. We show that supplementing Agrobacterium suspensions with a commercially available organosilicone surfactant (Pulse® penetrant) elevates transformation efficiency at ambient pressure. The transformation efficiency of leaf tissue of in vitro grown Vitis vinifera ‘Sauvignon blanc’ plantlets submerged in Agrobacterium suspension was increased 65-fold by the addition of Pulse® penetrant at low concentration (0.03 % v/v). A quick and precise determination of transformation efficiency was achieved by measuring red pigmentation of cells transiently transformed with the transcriptional activator of anthocyanin biosynthesis, VvMYBA1. A variable increase in transformation efficiency was also observed in eight commercial wine grape varieties and one rootstock variety. Pulse® penetrant can therefore be used to achieve transient transformation of grapevine by simply dipping in vitro leaf material into bacterial suspension culture.