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).