Optimization of transient GUS expression of Agrobacterium-mediated transformation in Dierama erectum Hilliard using sonication and Agrobacterium

Abstract

Efficient Agrobacterium-mediated transformation was established via sonication of embryonic shoot apical meristems (ESAMs) of Dierama erectum Hilliard. Effects of explant types, co-cultivation time, acetosyringone concentration, Agrobacterium concentration and different gene delivery methods were evaluated for higher efficiency of genetic transformation in D. erectum. An explant type (ESAMs), concentration of Agrobacterium inoculum (OD600 of 1.6) and acetosyringone (50mgL−1), and co-cultivation duration (3days) were optimized for efficient genetic transformation of D. erectum. The transformation efficiency varied with explant types (from 0 to 60%), concentrations of bacteria (10 to 55%) and acetosyringone (50 to 90%) and period of co-cultivation (30 to 70%). The transformation efficiency was best with ESAMs explants compared with callus clusters. The gene delivery method via sonication-assisted Agrobacterium-mediated transformation (SAAT) provided higher transformation efficiency (40%) GUS expression compared with agrobacterial monolayer and agrobacterial suspension which gave less than 5% transformation efficiency. The putative transgenic plants which histochemically expressed GUS, were confirmed further with PCR and 35.3% of the plants were GUS positive. Stable integration of the transgene was not demonstrated hence the GUS expression observed was regarded as transient. This newly developed transformation system may facilitate improvement of D. erectum characteristics and other related geophytes.