| Maize (Zea mays L.)is an important cereal, forage and economic crop in the world today. It has very important significance for establishment of efficient, stable genetic transformation system of maize germplasm improvements and good receptor and plantlet regeneration system is the prerequisite of genetic transformation. In this study taking the immature embryos of the inbred lines A188, Zong31. B73and Mo17as explants, the influence factors of maize immature embryo tissue culture system and Agrobacterium mediated transformation system were studied in details and at last insect resistance gene bt-slm have been transformed into maize lines. The main conclusions are as follows:1. The establishment of immature embryo tissue culture system. In this study, immature embryos of maize inbred lines A188, Zong31and Mol7were proved suitable for transformation because of the higher embryogenic callus induction rate and better plantlet regeneration ability. NB medium was more appropriate for callus induction of the A188, Zong31and Mol7, and the MB was more suitable for callus induction of B73. The tested materials not only have the highest rate of callus, and more for the embryogenic callus only when the immature embryos between1.0-2.0mm after pollination of9-12days was used, and2.0-3.0mg/L2,4-D was added into induction medium. During the induction period, AgNO3adopted by adding/free in turn in sub-culture medium was helpful for improving embryogenic callus induction rate, of which optimal concentration was10.0mg/L.0.25mg/L s-3307on inhibition of germ had reached extremely significant level and no significant impact on embryogenic callus induction rate.2,4-D concentrations in the third subculture of embryogenic callus reduced had stimulative effect on the callus embryogenic maintenance and micro bud formation.50.0g/L sucrose and0.5mg/L6-BA were optimal for shoot differentiation. The inbred line had the highest rate of root differentiation in0.6mg/L IBA.2. On the basis of the former study, the effects of Agrobacterium twnefaciens mediated maize transformation of various factors were optimized, genetic transformation system for maize inbred lines of immature embryo of inbred lines A188, Zong31, Mol7were established. The results showed as follow:1.0-2.0mm immature embryo was most suitable for the transformation receptor. When an infection solution and co-cultivation medium was added with100μmol/L AS, resistant callus induction rate was the highest compared to other combinations. Suitable bacterium concentration of OD600=0.5, and immersed for10min, co-cultured for3days, and the immature embryo were soaked in infection solution for2-4h before infection were conducive to the increase of T-DNA transfer efficiency. Using this transformation system, through the PCR initial detection, a total of32plants of insect resistant transgenic plants received, of which are16plants of A188,4plants of Zong31or Mo17, B73×A1885plants, Zong31×A1882plants, Mol7x A1881plant. Rate of PCR positive plants was0.588-4.160%. Bt-slm accounted for the percentage of soluble protein checking by ELISA up to0.030%and was11.795times higher than the control group. This initial establishment of Agrobacterium-mediated genetic transformation of maize system should facilitate the foundation for further useful value of the gene into corn inbred lines. |
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