Optimization of Agrobacterium-Mediated Genetic Transformation and Regeneration of Transgenic Plants in Indian Cultivar of Barley (Hordeum vulgare L. cv. BL 2)

Abstract

An efficient Agrobacterium-mediated genetic transformation protocol has been developed for Indian winter barley. Several parameters affecting Agrobacterium-mediated gene delivery were investigated and optimized. These include use of acetosyringone, surfactants in the infection medium, antinecrotic treatment of explants and heat and centrifugation pre-treatment. Addition of acetosyringone in the infection and co-cultivation media was advantageous and resulted in increase in transformation efficiency from 3.7 to 11.1 %. Addition of surfactants (Tween-20/Pluronic acid F 68) at 0.1 % (v/v) had also resulted in improved gene delivery. Pre-treatment of immature embryos with antinecrotic mixture had significant positive effect on percent callus survival and transformation efficiency. Temperature pre-treatment of immature embryos prior to infection resulted in an increase in the number of GUS positive calli with optimum temperature of 40 °C for 3 min. However, centrifugation of immature embryos had a negative effect on barley transformation. Regenerated transgenic plants obtained after 8 to 10 weeks of selection were further rooted for 2 weeks. The plantlets established in green house condition showed normal growth. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of regenerated transformants, respectively. Molecular analysis of transgenic plants containing II gene (nptII) demonstrated the efficacy of optimised protocol for Agrobacterium-mediated genetic transformation in Indian cultivar of barley for the first time.