Study On Highly Efficient Transformation System Of Brassica Rapa Mediated By Agrobacterium

With the accomplishment of the whole genome sequencing, Brassica rapa has become a modelspecies for functional genomics study, for which a highly efficient genetic transformation platform ofB.rapa is more and more required. However, B.rapa is a tough species in genetic transformation,whichhas seriously influenced gene excavation and function study in Brassica rapa.To establish highefficiency transformation system will offer important values for theoretical and practical studies.From eleven cultivars, we selected six cultivars with high regeneration rate, includingNanjingaijiaohuang(N1),TianjingNo.2(N2),HualiangNo.5(N3),Shiyuehong(N4),Huangyangxiaobaicai(N5) and Hanyangqingxiaobaicai(N11). The factors affecting the transformation frequency,such as theconcentration of Kanamycin, seedling age, the pH of co-culture medium, the concentration of AgNO3and hormone in differentiation medium, were studied.The plasmid vector PBI121-Cry1Aa weretransferred into N1,N2,N4and N11cultivar mediated by Arobacterium, through the established highlyefficient genetic transformation system.The major results were obtained as follows:1.The regeneration frequences of N1,N2,N3,N4,N5and N11were higher among eleven cultivars. Theoptimal concentration of Kanamycin was25mg/L for the hypocotyl of N1、N2、N4and N11;The optimalconcentration of Kanamycin was20mg/L for the hypocotyl of N3and N5.2.An efficient transformation mediated by Agrobacterium was established. The best differentiationmedium was Zeatin2.0mg/L+IAA0.1mg/L+AgNO30mg/L; the optimal pH of co-culture medium was5.8.3. Transgenic plants with high insect resistance were obained: The expression vector PBI121-Cry1Aawas constructed. The expression vector PBI121-Cry1Aa was transformed into N1、N2、N4and N11withthe established Agrobacterium-mediated method, and the frequency of transformation were1.65%、0.52%、0.13%and3.65%,respectively. PCR showed that61transgenic plants were positive among theobtained63transgenic plants from N11cultivar. About96.8%of them turned out to be transgenic.TheT1plants of transgenic N11cultivar were verified and the Cry1Aa gene was integrated into the genomeDNA of Hanyangqingxiaobaicai according to analysis of PCR and Southern blotting hybridization.Real-time PCR showed that Cry1Aa gene was normally transcribed in the T1plants. According to theassay of insect-resistance in vitro,the mean larval mortality of Plutella xylostella and cabbage caterpillarwere siginificantly lower（P<0.05）than the control group, showing significant insect-resistance.4. SAD-RNAi transformants were obained: in order to develop transgenic plants of high stearate acid(C18:0) content, a SAD-RNAi vector was constructed and transformed into the hypocotyls of B.rapa c.v.Hanyangqinbaicai.15transgenic plants were obtained with the transformation frequency of1.81%.