High frequency regeneration and Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica)

An efficient protocol for high frequency regeneration and Agrobacterium tumefacies mediated transformation of Broccoli (Brassica oleracea var. italica) has been developed. Hypocotyl and leaf segment explants were transferred on to MS medium supplemented with different concentrations and combinations of IAA (0-0.57 mu M) and Kinetin/BAP/Zeatin (8.9-22.2 mu M). Hypocotyl explants showed the highest regeneration potential. Maximum number of shoots (16 per explant) were obtained on MS medium supplemented with 0.57 mu M IAA and 22.2 mu M BAP. The concentrations of extracellular calcium, nitrate and ammonium nitrogen in the medium had a marked effect on regeneration. The optimal media for maximum shoot proliferation (30 per explant) contained 3 mM CaCl2, 1900 mg l(-1) KNO3, 825 mg l(-1) (NH4)(2)SO4, 22.2 mu M BAP and 0.57 mu M IAA. Rhizogenesis from isolated shoots was obtained on B-5 medium supplemented with 2.85 mu M IAA. The high frequency regeneration system served as an excellent tool to establish an efficient transformation method of broccoli. Hypocotyl explants were co-cultivated with the Agrobactetium tumefaciens strain LBA 4404, containing a binary vector with the neomycin phosphotransferase (nptii) gene and a P-glucuronidase (gus) gene, each under the control of a separate CaMV35S promoter. Primary selection of transformed explants was performed on high kanamycin (100 mg l(-1)) containing medium for 10 days and regeneration was achieved on the optimised regeneration medium containing lower concentration (50 mg l(-1)) of kanamycin. The frequency of transgenic plants was calculated on the basis of GUS activity detected by histochemical X-gluc test. The effect of pre-culture and growth of bacterial culture was studied. Explants precultured for 2-days prior to inoculation with A. tumefaciens resulted in improved transformation frequency. Bacterial cultures grown to the optical density of 2.0 resulted in the highest transformation rate (60%). Evidence for integration of nptii gene was obtained by the PCR. All transgenic plants grew normally. The present study demonstrates the improved high frequency regeneration and transformation of B. oleracea var. italica.