Particle bombardment mediated transformation of barley
Particle bombardment transformation was conducted using the Particle Delivery system PDS-1000/He (Bio-Rad) as per the protocol described by Harwood and Smedly, 2009. For this the entire fragment representing selection, reporter and TaHSFA6b cassette was isolated by digesting the pANIC6B:TaHSFA6b plasmid by Eco RV andPme I restriction enzymes (Supplementary Figure S1). One day after isolation, 25–35 immature embryos were placed keeping scutellum side up, on callus induction media containing 0.4M mannitol in the centre of each plate. Embryos were kept on the osmoticum medium for 4-6 hours before the bombardment and post bombardment the plates were kept at 8oC overnight. Next day, embryos were removed from the osmoticum plates and transferred to callus induction media containing 50 mg/L hygromycin for selection and kept at 22°C in the dark. After 14 days calli derived from transformed embryos were sub-cultured on fresh callus induction plates with 50 mg/L hygromycin. After four weeks, these calli were further, transferred to regeneration media (MS supplemented with 0.5mg/l 6-BAP) with reduced hygromycin concentration to 25 mg/L, and placed at 22°C in 16:8 light and dark. Well-developed plantlets with proper roots were carefully removed from the plates and transferred to Magenta boxes without any growth regulator further for 4-6 weeks. Well rooted plantlets were transferred to the pots with 1:1 mixture of farm soil and soilrite (Kel Perlite, India) for hardening and kept in green house.
After 5 days post bombardment and growth on callus induction media, histochemical assay for Gus activity was performed by dipping the transformed calli in Gus buffer solution for 24 hours at 37 °C to check transformation (Jefferson et al,1987).