玉米3种非组培转基因方法转化外源bar基因研究

Comparison of Three Non-tissue Culture Plant Transformation Approaches

本试验用3种非组培型转基因方法,即花粉介导、子房注射、萌动种胚法在玉米上转化bar基因,经大田筛选及PCR和PCRSouthern检测,证明均可获得转化植株。还分析了3种方法的转化机理,并通过转化率与操作简便程度的比较,认为花粉介导优于萌动种胚法,二者又优于子房注射法。

At present, high velocity microprojectile and Agrobacterium tumeficiens infection are two main methods to transform cereal such as wheat and maize. The former is not only expensive, but also with low transgenic and high infertile frequency, and the later also takes immature embryo as the object, spending much time in tissue culture, and is with low transgenic frequency too. Although acetosyringone has been introduced into Agrobacterium tumeficiens infection, the transgenic frequency is still not high. At the same time, all these transgenic experiments can only be done in instrumented laboratory by few scientists. In this study, three non-tissue culture methods were used to transform foreign bar gene into maize. T1 seeds were identified by Basta resistance, PCR amplification and Southern blotting, T2 genetic rate and phenotype, in order to select a simple highly effective transgenic approach. The three non-tissue culture plant transformation methods are introduced as follows： （1） Pollen mediation： in the morning of flowering stage, pollen was collected and suspended into 10% sucrose solution with appropriate amount of plasmid, then treated 10 times with ultrasonic （400 W 10 s, interval 5 s）, finally the filaments were smeared with the pollen solution bagged and marked. （2） Co-cultivation of germinated embryos with Agrobacterium tumefaciens： first, mature seeds were submerged into warm water for 4 h, then the germinated embryos were wounded a little at their growing point and put into Agrobacteriun tumefaciens solution with acetosyringone on shaker for 24 h, then sown in pot for 3 d and irrigated with 0.2% PPT （phosphinothricin） solution, finally the seedlings were transplanted in the field. （3） Ovary injection： when selfing line began flowering, the ears were bagged silking, and artificially selfed. 20 d later after selfing, the ears were opened and sterilized with alcohol cotton, then injected plasmid solution to embryos one by one with microinjector, finally rebagged loosely and marked for harvesting seeds. There were remarkable difference between treatment and control in field screening with Basta （Fig.2, Fig.3 and Fig.4） ： The PPT injury trace area on control leaves was bigger and stricter than that on treated leaves, even though there was a little injury on treated leaves. The control leaves were so badly withered, curled and died. The PCR products from DNA samples treated with three ways showed the target bands （Fig,5）, and Southern-blotting of 5 samples （Fig.6） was positive. Table 2 showed that the PCR positive rate of germinated embryos was highest among 3 methods, and that of pollen mediation was higher than that of ovary injection; meanwhile, 2 statistic （Table 3） showed that the most of PCR positive ratios was fit in with Mendel＇ s law. In phenotypes of T1 and T2 among the 3 methods, there were no distortion from both pollen mediation and germinated embryos, but different types of distortions, such as no tassel and bunch ear plants （Fig. 7, Fig. 8） from ovary injection. In addition, the grain number per ear from ovary injection was about 5.57 % of normal seed number, while 6 % from pollen mediation, as to germinated embryo treatment, once a transgenic seed was obtained, it would bring up a healthy TO ear. So germinated embryo is superior to other two ways. The mechanisms of the three methods were also discussed in the paper. Finally, it has been proven that transgenic plants can be obtained through all these methods. Concerning the standpoint of transformation rate and easiness of manipulation, the best one is pollen mediation, co-cultivation of germinated embryos with Agrobacterium tumefaciens is the second, and all of them are better than ovary injection.