Selection Of High Frequency Regenerration Genotype In Wheat And Optimization Of Transformation System Of Biolistics

Although biolistic genetic transformation system of Wheat is relatively mature, itstransformation rate is still lower influenced by a variety of factors. It is important factors thatregeneration frequency of donor material and screening methods of resistant callus andregenerated plants affect the conversion efficiency. Thus, the way by selecting the highfrequency regeneration of genotype, optimizing medium and screening methods improves theconversion efficiency and screening results. Furthermore, it could optimize the biolisticgenetic transformation system of wheat and promote the breeding process of wheat.In order to filter out high frequency regeneration genotype provide receptors for thegenetic transformation of wheat,20wheat genotypes immature embryo in vitro culture in thisexperiment studied the differences between the different genotypes and callus induction andregeneration. Secondly, the immature embryo callus induction medium was carried out atdifferent levels of ammonium nitrate, intended to improve training effectiveness and improvethe regeneration frequency. Finally, the Gus gene and Bar gene were cotransformed intowheat callus by biolistic, and comparison of callus culture and differentiation through thedifferent options for Bialaphos concentration and screening times, establishs appropriatescreening procedures to improve screening efficiency, at the same time, it is the foundationfor optimizing the transformation system of biolistic. The following conclusions of thisexperiment:(1) Comparison study of the results that20wheat genotypes immature embryos culturedin vitro founds callus induction and plant regeneration rates were significantly differentbetween genotype. Callus induction rate was higher overall level, with an average of84%,between68.75%and100%; Embryogenic callus induction rate average of58.7%;Redifferentiation overall lower rate, with an average of34.77%, the largest up to76.23%, thesmallest up to8.38%. Among them, Xinong1376, Lunxuan987, Shi4185and CB037withhigh embryonic callus induction rate and green plant regeneration rate is suitable for wheattransformation receptor genotype.(2) The constant total amount of nitrogen in the immature embryo callus inductionmedium, the proportion of ammonium nitrate and potassium nitrateincreased to7:10, willincrease the callus and embryogenic callus induction and regeneration rate, compared with the control, were increased respectively by10.8%,271%and8.32%. When nitrate is constant, sothat the total nitrogen by adding ammonium nitrogen increased10mM, increased the callusand embryogenic callus induction and plant regeneration rates with increased up12.38%,173%and50%.(3) The result of Gus gene transient expression showed that Gus transient expression isthe highest adding0.5mg/L of Bialaphos in callus induction medium, there is no significantlydifferent between adding1.0mg/L of Bialaphos and the control (no added Bialaphos).However, regeneration rate of resistant plants and the PCR results of Gus gene showed thehighest conversion rate reached4.14%, after culture in this way induction mediumsupplemented with1mg/L for2weeks, subculture induction medium added2mg/L train oneweek, respectively differentiation medium add4mg/L,6mg/L screened twice.