Study On Transfoemation Of Wheat With KN2and PYL5Gene By Biolistic Particle

Wheat is one of the major food crops for human. Wheat production plays a decisive rolein the world food security. The planting area of wheat mainly distributed in the arid andsemi-arid areas. Drought stress is the most harmful abiotic stress on wheat production and isone of the important factors limiting wheat growth, crop yield and quality. Therefore, it hasimportant significance to cultivate new wheat varieties with drought resistance for alleviatingthe world food security problem. In addition, with the extension of planting area of weakwinter wheat, the freezing damage has become one of the main natural disasters limitingwheat growth and yield increase. Many new varieties with high quality have been developed,using conventional breeding methods and promote the wheat production, but it still can notmeet the needs of wheat production. With the continuous development of biologicaltechnology and theory, transgenic breeding techniques have gradually become an effectivecomplement of conventional breeding methods. Therefor, it has important theoreticalsignificance and practical value to improve wheat cold resistance and drought resistance bytransgenic breeding techniques.The elite wheat varieties planted in HuangHuai areas in recent years, including Xinong889, Xinong2000, and Xiaoyan22, were selected as materials in this study. A superoxidedismutase gene (KN2) from antarctic fish and ABA receptor protein gene (PYL5) fromArabidopsis were transformed respectively into immature callus by biolistic particle. KN2isdriven by corn ubiquitin promoter and PYL5was driven by inducible promoter Rd29A. Thepurpose of this study is to obtain transgenic wheat with the target genes. The results will laythe foundation for studying the function of these two genes in stress environments and createnew wheat variety with drought resistance and cold resistance. This study has achieved thefollowing results:(1) Comparison of tissue culture of the immature embryos from three wheat varietiesThere are no obvious differences on the frequency of immature embryos callus inductionamong these three wheat varieties, which are all above97percent. But the quality of callushas a little difference: Xinong2000> Xiaoyan22> Xinong889. Based on the number ofregenerated plantlets, the differentiation capacities of three wheat varieties have some difference: Xiaoyan22> Xinong889> Xinong2000.(2) Transformation of wheat with KN2gene by biolistic particle and molecular identificationThe over expressed vector pUBI::KN2, which contained the superoxide dismutase geneKN2controlled by the ubiquitin promoters, was introduced into three wheat varieties bybiolistic particle. Immature embryos callus of Xiaoyan22were divided into two sections: onesection was transformed only with the target gene KN2, the other section wereco-transformated with KN2and Bar into the receptor. Xinong889and Xinong2000wereonly introduced the target gene KN2.900immature embryos callus of Xinong2000werebombarded by biolistic particle, and10regenerated plants were obtained. But none of thetransgenic positive plants was identified.1440immature embryos callus of Xinong889werebombarded by biolistic particle, and22regenerated plants were obtained. One out of22wasidentified to be transgenic positive plants by PCR analysis in the T0plants. Thetransformation frequency was0.07%.1800immature embryos callus of Xiaoyan22werebombarded with KN2gene by biolistic particle, and50regenerated plants were obtained. Oneout of50was identified to be transgenic positive plants by PCR analysis. The transformationfrequency was0.06%.1680immature embryos callus of Xiaoyan22were bombarded withKN2and Bar gene by biolistic particle, and17regenerated plants were obtained. Four out of17were identified to be transgenic positive plants with KN2gene. The transformationfrequency was0.24%. Six out of17were identified to be transgenic positive plants with bargene. The transformation frequency was0.36%. The co-transformation frequency was0.24%.The seed2positive plants which only introduce KN2gene were harvested and9T1generationplants were got, but none of the transgenic positive plants was identified by PCR analysis.The seeds of4positive plants which co-transformed with KN2and Bar were harvested and5plants of T1generation were got, out of which2were identified with the gene KN2.(3) Transformation of wheat with PYL5gene by biolistic particle and molecular identificationThe ABA receptor protein gene (PYL5) driven by inducible promoter Rd29A wasintroduced into wheat variety Xinong889by biolistic particle.1500immature embryos calluswere bombarded, and23regenerated plants were obtained. But none of the transgenicpositive plants was identified.1800immature embryos callus were bombarded with PYL5andbar gene by biolistic particle, and9regenerated plants were obtained, two out of which wereidentified to be transgenic positive plants with PYL5gene. The transformation frequency was0.11%. Five out of9were identified to be transgenic positive plants with bar gene. Thetransformation frequency was0.28%and the co-transformation frequency was0.11%.