Efficient in vitro plant regeneration and generation of transgenic plants in barley (Hordeum vulgare L.) using particle bombardment

Abstract

Plant regeneration from immature embryos of 15 Indian barley genotypes (Hordeum vulgare L.) was examined. Highest percent callus induction and number of regenerated plantlets were obtained in cultivars BL 2 (94.4 %; 12.1), RD 2668 (92.6 %; 9.1) and RD 2552 (90.8 %; 7.8). The highly responding cultivar BL 2 was selected for further development of transformation protocol. The plasmid DNA isolated from pCAMBIA1381 harbouring hptII gene as selectable marker and gusA gene as reporter was used. Particle bombardment was used for transformation of immature embryos and regeneration of transgenic plants in Indian barley genotype for the first time. Transformation experiments were carried out using different parameters and optimum conditions for DNA delivery was standardized. The transient expression of gusA gene was investigated as a preliminary test of optimum DNA delivery and for selecting the most appropriate bombardment parameters. The optimum conditions were: gold microparticles (diameter 1.0 μm) shot with 1,100 psi rupture disc pressure. The 3 cm distance between rupture disk and macrocarrier and 9 cm target tissue distance yielded high transient GUS expression. The immature embryos were bombarded twice to increase area for efficient gene delivery. Osmotic medium optimization with 0.4 M sorbitol and preculture of immature embryos for 5 days prior to bombardment resulted into efficient gene transfer in barley. Selection of transformed tissue was performed after 7 days resting step on selection medium containing 50 mg l−1 hygromycin. After two more selection steps, green shoots were rooted on MSB5 medium with 50 mg l−1 hygromycin. PCR analysis using primers specific for hptII and gusA genes and Southern blot analysis with hptII probes confirmed the stable integration of transgene in barley genome. Molecular analysis of T1 generation plantlets revealed the amplification of selectable marker hptII gene in the progeny.