| Tall fescue(Festuca arundinacea Schreb.) is an excellent worldwide cool-season turfgrass,with high resistance to stress and strong adaptive ability. In recent years,tall fescue used is increasing rapidly in China.However,water requirement of tall fescue is very high in summer,and it and been regarded as one of the main factors that restrict the spread of tall fescue.So,improving drought tolerance is an important object in breeding tall fescue.The embryo callus from tall fescues were transformed with OsWAX2-1 (AK070469,homologous with Arabidopsis WAX2 gene) mediated by Agrobacterium.The results of PCR assay and sequencing indicated that target gene has been transferred into tall fescue.The main results are as follows.1.High efficient regeneration system of tall rescue were establishedThe sterilization method for tall fescue mature seeds:Dipping in 70%alcohol for 5 min plus in 5.25%sodium hypochlorite solution for 15 min,or dipping in 70%alcohol for 5 min plus in 10%Benzalkonium Chloride Solution for 1 min can reach the lowest contamination rate with the highest seed germination rate.The high efficient callus induction medium is MS+0.5g·L-1 casein+5mg·L-1 2,4-D+0.02mg·L-1KT,the induction rate was 86.1%.The high efficient callus differentiation medium is MS+0.5mg·L-1casein+0.6mg·L-1TDZ+0.5mg·L-1NAA,the differentiation rate was 91.9%.2.Establishment of tall fescue transformation systemFactors influencing Agrobacterium-mediated tall fescue transformation were investigated,the optimal conditions for transformation were 3 days pre-culture time,concentration(OD600) of Agrobacterium solution was 0.8,pH value of Agrobacterium solution was 5.2,immersed by Agrobacterium for 30min,co-cultivated with Agrobacterium for 3 day,transfer the infected callus to selection medium after 10 days refresh culture.3.Obtained transgenic tall fescueIt was confirmed that the OsWAX2-1 were integrated into the tall fescue genome by PCR analysis and sequence analysis.
|
PDF Full Text Request