High Frequency Regeneration and Agrobacterium tumefa- ciens-mediated Transformation of Broccoli (Brassica oleracea var. italica)

摘要

An efficient protocol for high frequency regeneration and Agrobacterium tumefades mediated transformation of Broccoli (Brassica oleracea var, italica) has been developed. Hypocotyl and leaf segment ex-plants 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 supplementedwith 0.57 mu M IAA and 22.2 muM 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 CaCl_2,1900 mg~(-1) KNO)3, 825 mg l~(-1) (NH_4)_2SO_4, 22.2 mu M BAP and 0.57 mu M IAA. Rhizogenesis from isolated shoots was obtained on 65 medium supplemented with 2.85 |J.M IAA. The high frequency regeneration system served as an excellent tool to establishan efficient transformation method of broccoli, Hypocotyl ex-plants were co-cultivated with the Agrobactenum tume-faciem strain LBA 4404, containing a binary vector with the neomycin phosphotransferase (nptii) gene and a beta-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 his-tochemical X-gluc test. The effect of pie-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.