Agrobacterium tumefaciens-mediated transformation of cabbage with betaine aldehyde dehydrogenase gene.

Abstract

Cabbage(Brassica oleracea var.capitata) is one of the most popular and widely cultivated vegetable crops in the world. In this paper,BADH(betaine aldehyde dehydrogenase) gene derived from spinach was transformed into the genome of cabbage line 03079 mediated by Agrobacterium tumefaciens. To establish the ideal transformation platform,the main factors which affect the transformation efficiency were optimized through the orthogonal design of L9(34) ,including the type of infection medium,concentration of acetosyringone,the period of infection time and co-culture time. The results indicated that No. 9 was the best treatment combination,i.e,AA liquid medium was the optimal infection medium for the cabbage transformation,and co-culturing for 2 days after infection(20 min) would be favorable for the transformation. If supplemented with 200 μmol L-1 acetosyringone in infection medium,the transformation efficiency would be improved and the transformed plant regenaration ratio reached at 54.26%. Based on this optimal transformtion system,we obtained many transgenic plants,and PCR analysis using BADH gene primers and Southern blot analysis indicated that the BADH gene had been integrated into genome of cabbage. The BADH enzymes activity of transgenic plants were tested after treated with NaCl,drought-tolerance and PEG stress. The results showed that the average values of the activity of BADH enzymes,which varied from 2.1 U to 3.6 U per mg in transgenic plants,were 1 to 3 times higher than that of un-transgenic plants,and there was a significant difference between transgenic and un-transgenic plants using Duncan′s Multiple Range Test. Furthermore,the average value of relative electronic conductivity(varying from 16.2% to 32.6%) of transgenic plants were significantly lower than that of un-transgenic plants,which indicated that the protection ability of membrane penetration was enhanced when BADH activity increasing and the stress resistance of the transgenic plants was improved by introduction BADH gene into cabbage. At the same time,most of the transgenic cabbage plants were vigrously growing under drought 、salt and polyethylene glycol(PEG) stresses. The average height increasing percentage of transgenic plants were significantly higher than that of un-transgenic plants. Those results showed that the traits of drought or salt tolerance of transgenic cabbage plants were improved after transformed with BADH gene,and our transgenic plants would be used as fundamental germplasm for stress-tolerant breeding of cabbage.