ESTABLISHMENT OF REGENERATION AND TRANSFORMATION OF Brassica napus

Abstract

Production of genetically enhanced canola varieties with desirable new traits requires the establishment of efficient regeneration and transformation protocols. In the current work, experiments were conducted using two commercial cultivars Sarow-4 and Bactool, hypocotyl explants and Agrobacterium strain LBA4404 (harboring pISV2678 plasmid, which contains both gus and bar genes as reporter and selectable marker, respectively). Six days old hypocotyls segment were co-cultivated on callus induction medium (CIM) containing MS basal salt mixture and supplemented with 1mg/l 2,4-D for 3 days, Agrobacterium inoculation was conducted and cultures were incubated for 3 days before being placed on selection medium for a further 11 days. Two weeks old cultures were transferred to shoot induction RG3 medium supplemented with 4 mg/l BAP, 0.2 mg/l NAA, 5 mg/l AgNo3, 500 mg cefotaxime and 3 mg/l bialaphos. Shoots were rooted on rooting R4 medium containing 0.3 mg/l IBA. Regenerated plantlets were successfully established in soil and plants were allowed to produce seeds. T1 seeds were used for evaluation of transformation frequency using histochemical GUS assay, leaf painting, PCR and RT-PCR. Our results indicated higher transformation frequency in Sarow-4 cultivar when compared to Bactool.