Establishment Of Agrobacterium-Mediated Transformation System Using Wheat Immature Embryo Callus

Genetic transformation technology greatly contributes to functional genomics research and molecular design breeding.Hexaploid wheat（Triticum aestivum L.）,as one of the most important crops,the genetic transformation technology of which is far behind other crops,that restricts the functional genome research and breeding application of wheat.Therefor,it is urgent needed to establish efficient and convenient wheat genetic transformation system.Recently,wheat genetic transformation methods has made a breakthrough today.However,most of wheat transformation system uses wheat immature embryos as explants based on the transformation system purchased from Japan Tobacco Company,which has strict restrictions on sampling time,culture conditions and transformation receptor genotype.Using wheat immature embryo callus for genetic transformation can reduce amount of immature embryo,we can produce plenty of callus through subgeneration for genetic transformation,which ease immature embryo materials restrictions imposed by season and growth conditions.We can also increase the efficiency of genetic transformation through the optimization of callus status.In order to establish wheat genetic transformation system with independent intellectual property rights,this study proposed by improve the quality of wheat callus,the optimization of genetic transformation system,the development of wheat genetic transformation receptor,improved methods of medium hormone,set up a regeneration system and establishment of Agrobacterium-mediated transformation system using wheat immature embryo callus,which provides important technical support for wheat genetic transformation.It also laid the foundation for further promoting functional genome research and breeding application of wheat.Specific research results are as follows:（1）Exploration of efficient callus induction and regeneration system using wheat callusThe key factors such as embryo extraction time,germ removal method,medium type,and hormones were explored and optimized for callus induction using wheat Fielder and KN199immature embryos as explants.In this study,we found that the quality of induced calluses were the best by using the 14-day immature embryos after flowering,and we also found that excision of germ before inoculation induction medium was more conducive for callus induction:compared with germination after inoculation,callus induction efficiency was 77.00%and 68.00%for Fielder and KN199,which increased by 7.00%and 6.00%,respectively.Addition of 20 g L^-1 mannitol to the callus induction medium could greatly improve the induction efficiency of Fielder and KN199 embryonic callus to 88.00%and 75.00%.Addition of 0.5 mg L^-1 thidiazuron（TDZ）to the differentiation medium could significantly improve the regeneration rate of callus:the regeneration rate of callus in Fielder and KN199 could achive to 83.33%and 67.78%,respectively.（2）Agrobacterium-mediated transformation using wheat immature embryo callusBased on the establishment of the wheat immature embryo callus regeneration system,we further explored the effects of the time of callus preculture,Agrobacterium strain,screening antibiotics and other factors,establishment of Agrobacterium-mediated transformation system using wheat immature embryo callus in this study.we found that 3 weeks was the optimal pre-culture time of wheat embryonic callus,and the transient expression efficiency of GFP fluorescence mediated by Agrobacterium was 8.50%,and the calluses produced by immature embryos could be used for genetic transformation within 2 subgenerations.Compared with other Agrobacterium strains,C58C1 could improve the transient expression efficiency of GFP fluorescence in callus by 12.00%,and phosphinothricin screening could increase the regeneration rate of green buds after callus infection to 43.00%.Using the optimized genetic transformation system above,21.67%regeneration rate and 4.16%positivity transgenic rate coud be obtained,the efficiency of genetic transformation of immature embryo callus of wheat was greatly improved than previous studies.（3）Screening for the genotypes with high tissue culture ability from natural varietiesBy using the optimized culture conditions,we screened 16 wheat varieties mainly planted all over the country,and found that Yangmai 16 and Mianyang 15 had higher regeneration efficiency,which provide key clues for expanding the wheat genetic transformation acceptors.These results laid a good foundation for the establishment of an efficient transformation system based on immature embryo callus of wheat.