Study On The Wheat Transformation With Leaf Senescence-Inhibition Gene P_SAG12-IPT

In an agricultural setting, the use of variety to increase crop production is an economic and simple way. At present, leaf presenility is a relatively serious problem for some wheat varieties (or pedigrees) in China. It may limit yield potential and influence variety extension. Because of the environment influence, traditional breeding method that is based on phenotype is not efficient in crop improvement. In recent years, plant transformtion technology has become a potential tool in this field. With leaf senescence - inhibition gene PsAGi2 - IPT that was found by Dr. Gan and Amasino transformed tobacco and rice, the transgenic plants showed significant to delay leaf senescence and to increase seed setting and yield. The growth period and the form of transgenic plants were same as control. In this study, transgenic wheat plants of XiNongl376 with PsAGia - IPT gene were obtained by pollen tube pathway and Agrobacterium - mediated transformation of seeds. The transgene integration, expression and inheritance were studied by means of target character and molecular biology method. An Agrobacterium - mediated transformation system for wheat in normal and selection of transgenic wheat progeny with Kanamycin were investigated. The main results showed as follows:l.One transgenic wheat plant with PSAGU - IPT gene from XiNongl376 and XiNong2611was obtained by Agrobacterium - mediated transformation of seeds. By PCR amplification, GUS histochemical and Southern blot hybridization analysis, PSAGIZ - IPT gene was confirmed to integrate into the wheat genome. PCR analysis of TI progeny proved that their segregation pattern fell into Mendel model 3:1 and inherited steadily. Morphology observation and the analysis of leaf cytokinin content, chlorophyll content, leaf senescence development and agronomical character showed that the difference of transgenic wheat and control was little. The result indicted that PSAOII - IPT gene might not express(gene silencing) or express partly in transgenic wheat plant2.A few resistant-callus from immature embryo calli of XiNongl376 and JingHua 1 were obtained by Agrobacterium - mediated transformation. GUS histochemical assay proved that PSAGU - IPT gene was transferred into immature embryo calli of XiNongl376 and transformed in it. However, transgenic plant regeneration from that resistant-callus could not be produced. The selection and regeneration effects to the wheat immature embryo calli with different concentration Hygromycin and different regeneration mediumwere studied. The results showed that the optimal concentration of Hygromycin was HOmg/L and the ideal regeneration medium was MST with lAAl.Omg/L and KTl.Omg/L. XiNongl376 wheat variety was more suitable for Agrobacteriwn-mediated transformation than JingHua 1.S.Five transgenic wheat plant with PSAGII-IPT gene from XiNongl376 were obtained by pollen tube pathway-mediated transformtion with the plasmid of lOOng/ul, 400ng/ul and 700ng/ul. By PCR amplification, GUS histochemical, Dot and Southern blot hybridization analysis, the target gene with specific promoter was confirmed to integrate into the wheat genome. The transformation efficiency of 700ng/ul concentration plasmid was high. Morphology observation and the analysis of leaf cytokinin content, chlorophyll content, leaf senescence development and agronomical character showed that the leaf senescence of the two transgenic wheat was obviously delayed. The results indicted that PsAGi2-IPT gene might specifically express in the senescent leaf of the two transgenic wheat and not express, partly express or not specifically express in other plants. PCR analysis of TI progeny proved that mostly transgenic plants could inherit steadily and their segregation pattern fell into Mendel model 3:1. Some line did not segregate normally. In the late growth period, part of TI progeny showed significant feature to delay leaf senescence and the problem of leaf presenility was improved. According to this paper, the use of PSAGII-IPT gene to improve wheat varieties that had the defect of l...