Study On Agrobacterium Tumefaciens-Mediated Transformation Of Strawberry With RdreB1BI Gene Of Rice

Strawberry(Fragaria ananassa Duch),perennial herbaceous plant in the Rosaceae family,which is a major berry crop around the world.However,safe hibernation and deficiency of cold tolerance varieties are outstanding problems.In this case,biotechnology which developed speedly is an alternative efficient strategy to solve the problems. RdreB1BI gene is important transcription factors which related to plant development and physiology.It was investigated that RdreB1BI transcription factors are able to improve tolerance to drought,high-salt and cold stresses in Arabidopsis.But there is no research in transformation of strawberry with RdreB1BI gene of rice at present.So in this study, RdreB1BI was used as purpose gene,the transformation of strawberry by Agrobacterium-mediated was studied.The experiment research results were as follows:1.The ideal adventitious shoot regeneration system of strawberry 'Toyonoka','Xuemi' and 'Guinugan' by leaf and petiole explants were established In this study.The results showed that the highest adventitious shoot regeneration rate of Toyonoka's leaf disc was obtained on MS medium supplemented with 2.0mg/L TDZ,0.2 mg/L IBA and 0.1mg/L 2,4-D.The highest adventitious shoot regeneration rate of Toyonoka's petiole was obtained on MS+B5 medium supplemented with 2.0mg/L TDZ,0.2mg/L IBA and 0.1mg/L 2,4-D. The highest adventitious shoot regeneration rate of Xuemi's leaf disc was obtained on MS medium supplemented with 1.5mg/L TDZ and 0.2mg/L IBA.The highest adventitious shoot regeneration rate of Xuemi's petiole was obtained on MS+B5 medium supplemented with 2.0 mg/L TDZ,0.2mg/L IBA.The highest adventitious shoot regeneration rate of Guinugan's leaf disc was obtained on MS medium supplemented with 2.0mg/L BA and 0.1mg/L IBA.The highest adventitious shoot regeneration rate of Guinugan's petiole was obtained on MS medium supplemented with 2.0 mg/L BA and 0.2mg/L IBA.For 'Toyonoka' and 'Xuemi' Dark treatment for two weeks could increase the adventitious shoot regeneration.But for 'Guinugan' Dark treatment for one week could increase the adventitious shoot regeneration.The ideal leaf ages was 10 to 30 days of 'Toyonoka' and 'Guinugan' leaf.The ideal leaf ages was 10 to 20 days of 'Xuemi' leaf.The ideal leaf ages was 10 to 20 days of the three varieties petiole.There was no effect of leaf burl on adventitious shoot regeneration of strawberry.2.The region of rd29A gene promoter was amplified by PCR technique.Sequence analysis showed that the fragment contained the main regulation motifs.Two a-ntisense expression vectors called pYH4215-rd29A and pYF7713-Rdre were obtained.3.By optimizing the transformation system of strawberry,a high-efficient transformation system was established.The bacterial concentration of OD600=0.4 and 10 minutes was the best condition.The available concentration of Cb was 200mg/L to eliminate Agrobacterium tumefaciens.The corresponding concentration of Kan was 20mg/L to screen.Co-culture for 3 days.The optimum Co-culture medium was MS+TDZ(2.0mg/L)+IBA(0.2mg/L),the optimum medium for elongation was MS+6-BA(0.5mg/L)+NAA(0.1mg/L)+Kan(20mg/L)+Cb(200mg/L),the optimum root inducing medium was MS+IBA(0.1mg/L)+Kan(20mg/L)+Cb(200mg/L).4.The transgenic plants contained RdreB1BI were obtained.The result of PCR and RT-PCR showed that the target gene was successfully integrated into the genome of strawberry.The concentration of praline indicated cold resistance of transformation strawberry had improved.