The Optimization Of Regeneration System From Wheat Mature Embryo And The Construction Of Plant Expression Vector PBin–HARDY

In the process of crop’s growth and development, drought and salt stress whichwere often the main type of adversity stress factor, were the key factors of affectingcrop yield and quality.For saving resource and promoting the sustainable developmentof agriculture economy, cultivating new crop varieties with high resistance had veryimportant significance.The genotype of explants、the composition of mineral and the selection of plantauxin had great influence on vitro regeneration and tissue culture.In the process oftissue culture of wheat mature embryo、the appropriate choice of mature embryogenotype、adding moderate concentration of Ca2+and2,4-D were the key factors onthe efficient regeneration system of wheat mature embryo. Furthermore, it would lay afoundation to improve the efficiency of wheat mature embryo’s regeneration systemand genetic transformation.HARDY gene belonging to the AP2/EREBP transcription factors was the memberof AP2/ERF-like transcription factor family.Under drought stress, HARDY gene hadbeen induced to generate regulatory protein which could enhance the droughtresistance of plant and improve the plant water use efficiency.Then it could improvethe drought-resistant and water-saving capacity of crops.The experiment aimed to optimize the system of the wheat mature embryoregeneration and to construct the plant expression vector, the results were as follows:(I) optimizing the regeneration system of the wheat mature embryo1. Compared with the embryonic callus induction rate and its differentiation ofZhongmai175、CB037、Ganchun24、the Bobwhite、Ganchun20、Yangmai15, theresults showed that：in the process of wheat mature embryo regeneration system,plant regeneration rates of Zhongmai175、CB037、GanChun24were higher,whichwere good receptors of genetically modified materials.2. In the process of wheat mature embryo culture,adding3.5~5.0mmol/L Ca2+to the medium of Adi and embryonic induction culture medium,the higher rate ofgreen bud was induced;adding2.0~5.0mg/L2,4-D to the medium above,it couldeffectively increase the induction rate and differentiation rate of wheat mature embryo callus.3. On the process of wheat mature embryo culture,changes of endogenoushormone IAA and ABA were the key for embryonic callus induction,the results wereshowed as below,0~7d,the content of IAA and ABA declined sharply;7~35d,IAA andABA were showing a trend of first rising, then downward.The content of IAA andABA were reaching its peak on the day of21and28, respectively.(II) HARDY successfully cloned and done the biological analysis of its protein;constructing a prokaryotic expression vector of pET28a (+)-HRD which was tested byIPTG in E.coli BL21(DE3);constructing plant expression vector pBin-HRD whichwould be prepared for the research of wheat genetic breeding of HARDY.