Optimization Of Agrobacterium-mediated Transformation System Of Wheat Callus And Function Verification Of Salt Tolerant Gene From Halogeton Glomeratus In Arabidopsis Thaliana

Wheat?Tritieum aestivum L.?is an important global food crop and plays an important role in ensuring world food security.Due to the aggravation of land salinization worldwide,the yield and quality decline of wheat are becoming increasingly prominent.At the same time,the limitations of conventional breeding methods in salt tolerant breeding of wheat are becoming increasingly apparent.Therefore,it has a broad application prospect that using biotechnology to introduce salt tolerant genes into wheat and improve its salt tolerance.High frequency wheat regeneration system is a necessary condition for bioengineering operation.Halogeton glomeratus is a strong salt tolerant riparian plant,which is mainly grown in arid saline land in Northwest China.It is also an excellent gene pool for salt tolerance research.In this study,wheat mature embryos were used as explants to construct a stable and efficient wheat regeneration system,and the salt tolerant gene was transformed into wheat callus.Meanwhile,salt tolerance candidate genes cloned from Halogeton glomeratus were used for functional verification in Arabidopsis thaliana.The main results are as follows:1.The best method of pretreatment of callus by mature embryo of wheat was to soak the seeds after sterilizing the seeds for 12h,and then peeled the whole embryo and destroyed the germ growth point.The embryo was also cut into four parts by cross cutting and inoculated on the inducible medium.This method not only can maked callus induction rate higher,but also prevented embryos from developing into seedlings directly.2.We chose different hormone ratios to optimize the growth system of wheat callus,and finally determined the appropriate proportion of callus induction and differentiation hormone:2,4-D of 2.5mg/L was beneficial to callus induction.The complete induction system was MS medium+CH?0.5 g/L?+Pro?0.5 g/L?+2,4-D?2.5 mg/L?+sucrose?15 g/L?+maltose?15 g/L?+agar?4 g/L?,pH=5.6⁶.0;The differentiation system was MS+KT?3.0 mg/L?+6-BA?2.0 mg/L?+sucrose?15 g/L?+maltose?15 g/L?+agar?4 g/L?,pH=5.6⁶.0.3.The suitable conditions for genetic transformation were determined that the concentration of bacteria was OD₆₀₀=0.6,which was good for the condition of callus infection,and the survival rate was relatively high.When cef concentration was600mg/L,the bacteriostasis effect of Agrobacterium tumefaciens was better.It is also found that acetyl syringone and ultrasonic wave can play supplementary roles in genetic transformation.4.In this part,we used the T₂ generation of Arabidopsis thaliana which had transformed the salt tolerant genes,to explore the effect of transformed genes on salt tolerance from germination rate,root growth condition,phenotype and physiological change,respectively.The result as follows:Transgenic Arabidopsis thaliana increased the germination rate than control,and had little difference under 30 mmol/L NaCl stress and 120 mmol/L NaCl stress stress.The difference was significant in60⁹0mmol/L NaCl stress.The length and number of root of transgenic Arabidopsis thalianas were different from those of the wild type.With the increase of stress concentration,both the wild type and the transgenic lines showed a shorter main root and the trend of the lateral root increase.The change amplitude of the transgenic lines was relatively small and stable.The transgenic lines showed obvious salt tolerance compared with the wild type under salt stress,and showed stronger salt tolerance under different stress concentrations.The chlorophyll content of Arabidopsis thaliana decreased significantly after salt stress,and the decline of wild type was the largest,which showed that the photosynthetic system of the transgenic lines were destroyed more.The MDA content and relative conductivity reflected that the lipid membranes of transgenic Arabidopsis thaliana were less damaged,which shows a stronger salt tolerance than the wild type.