Agrobacterium-mediated genetic transformation of safflower (Carthamus tinctorius L.)

Reproducible and highly efficient protocols for shoot regeneration and genetic transformation mediated by Agrobacterium have been established for safflower (Carthamus tinctorius L.). Agrobacterium tumefaciens strain LBA 4404 with gus reporter gene and hygromycin (hpt gene) as plant selection marker was used as the plant transformation vector. Genetic transformation experiments were carried out to evaluate the efficacy of various parameters such as genotype, seedling age, co-cultivation period, bacterial titer, enzymatic pre-treatment of target tissues, use of compounds that induce vir-gene enhancer, acetosyringone (AS), explant type and explant injury to enhance transformation efficiency. Transformation frequency was high when root and hypocotyl explants of 8-day-old seedlings of safflower cv. HUS-305 were co-cultivated with bacterial cell density of 0.5 OD600 during a period of 2 days followed by selection regime of 10–15–15 mg/l hygromycin. The frequency of rooting of the primary transformants was low (18.0%) when compared with the regenerated shoots (70.0%), and seven shoots survived on transfer to soil. The putative transformants were confirmed by β-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR), reverse-transcriptase PCR (RT-PCR) and Southern blot analysis. With the optimized transformation protocol, putative transformed shoots were obtained with frequency of 51.0% within 8–10 weeks of culture initiation.