Genetic Transformation By Transferring Transcription Factor DREB4A And W17Genes Into Wheat(Triticum Aestivum L.) And Related Research

Drought, high salt and low temperature are the main abiotic stresses affecting wheatgrowth and development. A number of researches have indicated that DREB and ERFtranscription factors play a significant role in improving plant stress resistance. DREB4A is akind of DREB transcription factor genes and its expression product can enhance the resistanceof plants to abiotic stresses such as drought, high salt and low temperature while W17belongsto ERF gene family and its overexpression can boost plant resistance to bacteria and fungus.Genetic engineering technique has provided a new path for plant stress-resistance breeding, ofwhich particle bombardment with immature embryos as receptor materials is the mostcommonly used method. This study optimized the transformation system mediated by genegun in wheat and transformed DREB4A and W17genes into wheat using this method. Aa aresult, new transgened wheat materials were created, which was essential for furtherdeveloping stress-resistant wheat varieties. Meanwhile, wheat mature embryo in vitro culturewas studied, which laid the foundation for establishment of genetic transformation systemwith mature embryos as receptors.Main research results are as follows:1. The result of GUS transient expression demonstrated that bombardment distance, goldparticle amount and plasmid DNA amount are the main factors affecting the particlebombardment transformation efficiency in wheat immature embryos. Transient expressionefficiency of GUS gene achieved the highest level when bombardment distance was6cm,which was66.7%, and was13%higher than that of9cm. Besides, transient expressionefficiency of GUS gene increased with increasing gold particle amount and the efficiency wasthe highest when the amount was120μg per bombardment, which was70%. The efficiencywas between66.7%and70%when the amount was0.5-1.5per bombardment. Study alsorevealed that particle bombardment had a great influence on callus regeneration of immatureembryo and the regeneration rate decreased25.8%on average.2. Linear DREB4A gene and Bar gene were cotransformed into receptor material Jimai19by particle bombardment and48regenerated resistant plants were obtained. The result of PCRdetection showed that positive plants of DREB4A and Bar genes were12and21respectively and transformation rate were0.2%and0.38%respectively. Cotransformed plants were10andcotransformation rate was0.18%.3. Linear W17gene and Bar gene were transformed into receptor material Xinchun9byparticle bombardment and64regenerated resistant plants were obtained. The result of PCRdetection showed that positive plants of W17and Bar genes were7and7respectively andtransformation rate were0.49%and0.49%respectively. Cotransformed plants were5andcotransformation rate was0.35%.4. The plasmid of W17RNAi was transformed into receptor material Xinchun9byparticle bombardment and115regenerated resistant plants were obtained. The result of PCRdetection showed that positive plants were10and transformation rate were0.42%.5. Comparison study of callus induction and regeneration rates among20wheat varietiesrevealed that callus induction rate was generally high in tested materials, which was between66.7%and100%while the differentiation rate was3.6%－35.8％. However, the differencewas significant among genotypes, of which the green plantlet differentiation rates of Fanmai8,Zhoumai27and Xingmai6were higher(over30%). Callus differentiation rate was higherusing cross cutting method in wheat mature embryos in vitro culture, which was54.5%－73.3％.Callus induction and regeneration rates were boosted when2-4mg/L Dicamba was addedwhile callus differentiation rate was higher using differential medium without hormone orwith2mg/L6-BA or KT, which were38.5％,45.8％and37％respectively.