Transformation Of GAFP Gene Into Brassica Napus Without Selectable Marker And Resistance Identification

Plant tissue culture has great potential for epigenetic variation. DNA methylation is important to genome activity. HPLC-assisted quantification was carried out to assess the DNA methylation level of explants during tissue culture of Brassica napus L.(cv. Yangyou9). The methylation levels were detected in hypocotyls cultured in media with different hormone concentrations. Dissected tissue cultured with0.1mg/L2,4-D and1.0mg/L6-BA presented the lowest methylation level and highest induction rate of callus (90.78%), respectively. Different time points of cultured explants also showed significant methylation variations. Explants cultured after6and21days exhibited methylation ratios of4.33%and8.07%, respectively. The methylation ratio increased to38.7%after30days of cultivation, which indicated that the methylation level of hypocotyls changed during tissue culture. Moreover, the methylation level in callus was the highest during rapeseed regeneration, followed by regenerated plantlets and hypocotyls. These results indicate that the function of hormones and differentiation of callus are relevant to the methylation levels during tissue culture.In this study, hypocotyls of Brassica napu L. were used as the explants, and the GAFP were transformed to the rapeseed mediated by Agrubacterium. During selection, we got69To plants with resistance,7were determinated as the transgenic plants by the PCR analysis, the positive ratio of transformation was10.14%. After selfing, we also can detecte the positive fragment from the T1plants by PCR analysis which indicated that the the GAFP gene had been integrated into the genome, and4were the GAFP transgenic plants without selection marks. In order to get the physiological index of desease resistance in T1plants, the detached leaves of seedlings were vaccinated by Sclerotinia mycelium, the results showed that:compared with the control, the transgenic plants had a higher content level of MDA; the enzyme activities of SOD, POD, CAT and PAL were enhanced significantly, while there were no significant changes of the level of Pro. The above results suggested that the desease resistance of T1transgenic plants showed a significant improvement as compared with the wild-type plants. In this study, through the dual carrier, we first reported to get the transgenic rapeseeds with important agronomic traits while no selection marks.