Wheat Transformation by Pollen-Tube Pathway and High Effective Screening System
Huang Yihong1* Liu Chunguang2 Ma Hongxiang1 Liu Genqi2 Zhou Miaoping1 Sun Yongru2 Yu Guihong1 Lu Weizhong11 Institute of Agrobiological Genetics and Physiology, Jiangsu Academy of Agricultural Sciences, Nanjing, 2100142 Institute of Genetics and Developnental Biology, Chinese Academy of Sciences, Beijing, 100101
A screening method can obtain quickly and effectively transgenic wheat plants from a great deal of progenies by pollen-tube pathway transformation is reported in this experiment. The plasmid vector pBI35S-gafp -NP1-bar containing selective marker gene bar?disease- resistance genes GAFP (Gastrodia Antifugal protein) and NP-1(Rabbit Defensin) genes, and the acceptors is wheat varieties Alondra and Yangmai158. The screening to the transformation seeds is separated as 3 steps: at first, sowing densely T0 seeds in a plastic box, and when the seedlings are in 3-leaves stage, spraying 120mg/L Basta solution on these seedlings to screen them. After 10 days, the most of them turn yellow and died, and only 1% Basta-resistance seedlings survive. Then, the PCR analysis with gafp primer to the T0-plants shows that 62.5% Basta-resistance plants are positive. Finally, germinating T1-seeds derive from PCR positive T0-plants, we take mixture samples according T0-lines and extract their DNA. PCR analysis is performed with gafp and bar genes primers again, and the PCR result is still positive. It confirms that the gafp and bar genes have integrated into the wheat, and inherited stability from T0-plants to T1-plants,and shows that the screening method is effective and reliable.