Studies On Optimization Of Regeneration From Mature Conyledons Of Prunus Mume And Genetic Transformation Of PmMYBs Genes

Mei flower(Prunus mume Sieb.et Zucc.),belonging to Prunus Genus,Rosaceae,is the deciduous wooded plants with important ornamental value in landscape application.It is originated in warm humid climate conditions and is difficult to live throught the winter in the North,Northeast and Northwest of China.The new germplasm with targeted characters can be quickly got through the method of genetic transformation,but a high regeneration system is an important prerequisite for genetic transformation.In this research,using the mature embryo cotyledons of ’Fenpi Gongfen’x’Xue Mei’as material,on the basis of predecessors’ works,the basic media,the combinations of hormones and culture conditions were optimized.Meanwhile,the immature embryo cotyledons and mature embryo cotyledons of Mei flowers were taken as the receptor,and the PmMYBs was transformted into Mei flower by agrobacterium.The main results were as follows:(1)Regeneration system of mature embryo cotyledons was optimized based on predecessors’ research about cotyledons regeneration of Prunus,through the screening of basic culture medium and comparing the effect of BA,TDZ and IBA on plantlet regeration.The result showed that mature embryo cotyledons of Mei flower could induce adventious shoots regeneration when cultured in the 1/2 MS medium supplemented with 1.0 mg/L TDZ,0.5 mg/L BA and 1.0 mg/L IBA;the highest induction rate of mature embryo cotyledons could reach 38.02%when the medium supplemented with CH at 500 mg/L;through comparing the regeneration rate and growing conditions,the highest regeneration rate was obtained when mature embryo cotyledons are cultured at dark for 12 days and then transferred to light condition with normal growth.The shoots with 3cm length were transferred into 1/2 MS+BA 3.0 mg/L which could induce callus on the stem base,then were soaked into the 200 mg/L IBA for 20s,and are cultured in QL medium to develop roots.The roots appeared after about 45 days cultivation.(2)Agrobacterium-mediated transformations of cotyledons of immature and mature embryosImmature embryo cotyledons were cultured in the 1/2 MS medium supplemented with TDZ at 0.2 mg/L,BA at 2.0 mg/L,IBA at 1.0 mg/L and mature embryo cotyledons were cultured in the 1/2 MS medium supplemented with TDZ at 1.0 mg/L,BA at 0.5 mg/L,IBA at 1.0 mg/L,CH at 100 mg/L for 3 days pre-culture at the dark,then infected 15 minutes and co-cultured for 3 days in the medium supplemented with AS at 100μmol/L at the dark.After that,selective culture was conducted with Km at 20 mg/L and Cef at 300 mg/L,and the immature and mature embryo cotyledons delay selection time was 7d and 10d,respectively.Fifteen resistance plantlets from immature embryo cotyledons of Prunus mume transformted with PmMYBs were produced,of which ten are positive through the PCR detection.Twenty-five resistance plantlets from mature embryo cotyledons of Prunus mume transformted with PmMYBs were produced,of which,fifteen ones are positive via the PCR detection.