A fast, simple, high efficient and one-step generation of composite cucumber plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation

Abstract

Agrobacterium rhizogenes-mediated transformation is widely used in different species with various purposes. The development of composite plants (wild-type shoot with transgenic roots) has been a milestone for functional characterization of genes. Previously, composite plants were generated by two steps from inducing of hairy roots to growing in the growth medium. Hairy roots were induced in an induction medium and the growth of composite plants generated were in another different growth medium. The composite plants produced was subject to transplanting. Here, we describe an improved and optimized protocol for generation of composite plant achieved by one-step in cucumber, which has not been reported previously in living plants. Incubation of explants post inoculation to induce transgenic roots and the growth of rooted explants were in the same medium. The primary root of 5-day-old seedling was excised and the slant cut of residual hypocotyl with 1 cm length was inoculated with A. rhizogenes harboring the desired gene construct followed by directly planted into a pot with wet sterile vermiculite. More than 90% of the infected seedlings can produce positive transgenic root. In addition, we further used the one-step transformation protocol to analyze the function of Arabidopsis YAO promoter. The result indicated that pYAO::GUS was highly conserved expression in whole root and high activity in the root tips. Therefore, a fast, expedient, high efficient, and one-step transformation method of composite cucumber produced is established, which is suitable for promoter functional analysis and other root-related events.