摘要
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 mark
出处
《作物学报》
CAS
CSCD
北大核心
2005年第12期1648-1653,共6页
Acta Agronomica Sinica
基金
国家自然科学基金(30340033)
山西省科委攻关项目(981001)
仲恺农业技术学院项目(G2360218)资助。
关键词
非组培转化
花粉介导
子房注射
萌动种胚法
Non-tissue culture transformation method
Pollen mediation
Co-cultivation of germinated embryos with Agrobacterium turnefaciens
Ovary injection
