Optimization Of Regeneration Culture System From Shoot Tip Of Wheat And Transformation Of CL5227 Gene Into Wheat

In this study,we use three wheat varieties Ganchun20,Ganchun24,Zhongmai175 as the experimental materials,and research the callus culture,plant regeneration of shoot tip.In order to research the factors such as genotype,growt h days of shoot tip and the location of segment,medium type,plant hormones(2,4-D,6-BA and KT)concentration that influence callus induction,differentiation and plant regeneration.The callus culture and p lant regeneration system of shoot tip of Zhongmai 175 were established and optimization.We have finished genetic transformation on callus of shoot tip,and researched the effect of various factors on agrobacterium transformation.Agrobacterium mediated transformation system was established on Zhongmai175,and transferred the Cl5227 gene into wheat successfully.The main results were as follows:(1)According to study on callus culture and plant regeneration of shoot tip on Zhongmai 175,the results showed that the effects of different genotypes on callus induction and differentiation of shoot tips were very significant;We selected the Zhongmai 175 which has good regeneration ability for the next experiment;The first segment of shoot tip for 3 days was the best part of callus culture;The best medium for callus induction was medium: MS basic component +2,4-D 2mg/L+CH 0.5g/L+ Pro 0.5g/L+Gln 0.25g/L+ Sucrose 30g/L+ Agar 6g/L,p H=5.8;Concentration of 2,4-D significantly affected the callus induction rate of shoot tip.With the increase of the concentration of 2,4-D,the induction rate increased first and then decreased,and the concentration of 2mg/L was the most suitable for callus induction of 2,4-D;The best medium for callus differentiation was medium: MS basic component +2,4-D 2mg/L+CH 0.5g/L+Pro 0.5g/L+Gln 0.25g/L+Sucrose 30g/L+Agar 6g/L+KT 2mg/L+6-BA 2mg/L,p H=5.8.When the concentration of 6-BA and KT reached to 2mg/L in the differentiation medium,the differentiation rate reached the maximum.(2)According to study the agrobacterium mediated transformation system on Zhongmai175.The results showed that,the time of co culture,the concentration of bacilli and the time of infection had great effect on the transformation system.The survival rate of callus showed a decreasing trend with the extension of infection time.30 min was the most suitable infection time,after 40 min of infection time,some of callus appeared death.Differentiation rate reached the maximum when t he time was 30 min and OD600=0.6;In this study,we researched the effects of co culture time on transformation of shoot tip callus,differentiation rate reached the maximum when the time of co cultured for 3 days.The Days of co culture were more than 4 days,the callus appeared brown and even died,differentiation rate decreased significantly.The optimum time of co culture was 3 days;Cef was added into the induction screening medium and differentiation screening medium to restrain the growth of agrobacterium.The result showed that 500 mg/L Cef could effectively restrain the growth of agrobacterium,and had little effect on the growth of shoot tip callus.In order to select the resistant callus,we used after 21 days induced screened callus,the concentration of Kan was 5mg/l,then the differentiation rate reach the highest value,which could be used to select resistant callus.(3)We have got two resistant regenerated wheat plants after resistance screening.But just only one resistant regenerated plant were positive after the PCR.