Production of insect resistant, transgenic turfgrass plants with an insecticidal protein gene.

在我们的项目中，我们获得了以下研究成果。利用改进的愈伤组织诱导培养基获得了较高的胚性愈伤组织诱导频率，表明草坪草植物在离体条件下建立了高效的细胞-植株再生体系。2.在电穿孔中，修饰缓冲成分和电条件提高了基因向原生质体的转移效率。3.在粒子轰击中，调整几种机械条件可以提高基因向完整培养细胞的转移效率。4.通过电穿孔获得了带有标记基因的转基因植株，并通过pcr - southern分析证实了该转化。分析了不同类型的转基因启动子在转化子间基因表达水平的差异。5.在不同类型的bt基因中，我们发现CryIA(c)产生的蛋白对草坪草最严重的害虫——Jananese lawn grass cutworm和Bluegrass webworm的杀虫活性最强。6.将CryIA(c)基因（野生型）引入草坪草。采用蓝草网虫生物测定法检测转基因植株的抗虫性。然而，没有一种转基因植物表现出对这种昆虫的抗性。7.为了提高CryIA（c)基因的杀虫活性，我们打算1)制备一个具有更强启动子的CryIA(c)基因，2）修改CryIA(c)基因编码区的序列。我们希望利用具有较强启动子的基因或修饰基因，可以生产出具有较强抗虫性的转基因草坪草植物。

In our project, we obtained the following research results.1.A higher frequency of embryogenic callus induction from seeds was obtained using a revised callus induction medium, indicating the establishment of an efficient cell-to-plant regeneration system of turfgrass plants in vitro. 2.In electroporation, modification of buffer components and electric conditions improved gene transfer efficiency to protoplasts. 3.In particle bombardment, adjustment of several mechanical conditions improved gene transfer efficiency to intact culture cells. 4.Transgenic plants with a marker gene were obtained by electroporation, and the transformation was confirmed by PCR-southrn analysis. Difference in gene expression level between transformants with different types of promoters of transgenes was analyzed. 5.Among various types of bt genes, we found that the CryIA(c) produced the protein which gave the strongest insecticidal activity to Jananese lawn grass cutworm and Bluegrass webworm, the severest harmful insects of turfgrass. 6.The CryIA(c) gene (wild-type) was introduced into turgrass plants. Insect resistance of the transgenic plants was examined by bioassay with Bluegrass webworm. However, none of the transgenic plants showed resistance to the insect. 7.To enhance the insecticidal activity of the CryIA(c) gene, we intend 1)to produce a CryIA(c) gene with a stronger promoter, and 2)to modify the sequence of the coding region of the gene. We hope that the use of the gene with a stronger promoter or the modified gene may lead to the production of transgenic turfgrass plants with substantial insect resistance.