Establishment Of Agrobacterium Mediated Genetic Transformation System Of Dongnongdongmai1 Mature Embryo

‘Dongnongdongmai1’ is the only winter wheat which has been confirmed can live through the winter in the alpine region of Heilongjiang Province at present, Agrobacterium mediated genetic transformation technology which has an important significance in genetic improvement of wheat. The mature embryos of ‘Dongnongdongmai1’ was used as explants to explore the influence of callus induced、differentiation and plant regeneration, including the influence factors of medium, inoculation method, the additive concentration combination, the concentration of 2,4-D、6-BA and KT. Preliminary established the tissue culture and plant regeneration system from mature embryos of winter wheat ‘Dongnongdongmai1’. Making use of Agrobacterium-mediated transform the callus from mature embryos on the basis of this system and studied the factors that influence the efficiency of genetic transformation, preliminary established Agrobacterium mediated transformation system of the winter wheat mature embryos.The results as follows:(1)Through culture and plant regeneration of mature embryos callus test on winter wheat Dongnongdongmai1, the results showed that different inoculation methods significantly effect on mature embryo callus differentiation rate, embryo cut slightly endosperm>broken embryo method>complete embryo. Orthogonal test of additives obtained the best additive hormone ratio was 100 mg·L-1 glutamine+ 0.1 mg·L-1 NAA+0.3 g·L-1 inositol+1 g·L-1 proline+0.5 mg·L-1 ABA. The concentration of 2,4-D significantly affected the differentiation and plant regeneration of mature embryos, with the increasing of 2,4-D concentration, the induction rate was increased at first and then decreased,the differentiation rate and plant regeneration rate decreased. The 6-BA of differentiation medium was extremely significant effect on plant regeneration, reaches the maximum at 4 mg·L-1; the differentiation of KT on callus is higher than that of 6-BA, but significantly lower than the effect of 6-BA on plant regeneration. The system of the winter wheat callus differentiation and plant regeneration rate can reach 93.33% and 66.64%.(2) The type of strains、co-culture mode、preculture time、the concentration of bacteria、time of infection and co-culture had a great impact on the efficiency of genetic transformation. Strains EHA105 infection was more apparent than LBA4404; trained on callus processing which stable GUS expression rate was about 2.8 times higher than medium;the GUS expression rate was risingwith the increasing of pre-culture time;the GUS expression rate showed a trend of rise after the fall of first with the increasing of bacterial concentration and infection time, and reach the maximum when the OD value of 0.8 and infection time of 3h; with the increasing of co-culture time, the stable GUS expression rate showed the tendency of gradually reduce.Through orthogonal test obtained the optimal transformation method was pre-cultivate 18 d, bacteria concentration OD value was 0.8, 1h of infection time, co-culture 5d, GUS stable expression rate reached 83.33%, establish the Agrobacterium mediated genetic transformation system of mature embryos.(3) Based on the of Agrobacterium mediated genetic transformation system, strains whichusing the GUS gene and hygromycin resistance selection transform of the mature embryos of‘Dongnongdongmai1’,obtained the resistance wheat regeneration plants,extracted the transgenicplants genome DNA,then the PCR test was positive, the transformation efficiency is 2.86%,laid asolid foundation for Agrobacterium mediated genetic transformation.