Construction Of Agrobacterium Tumefaciens Mediated Transient Transformation System For Prunus Xueluoensis

Prunus xueluoensis belongs to the Rosaceae genus Prunus（Subg.Cerasus）,and is a unique Prunus germplasm resource in China.It is distributed in Xueluozhai in Hubei,Dawei Mountain in Hunan,Lushan and Jinggangshan in Jiangxi,and Emeishan in Sichuan.It grows in mountain shrubs or Pinus massoniana pine forests at an altitude of 1,100 to 1,500 m.P.xueluoensis is a unique ornamental value compared with other species of the same genus because of its short juvenile phase,early flowering period,a large number of flowers,light color,long flowering period and low tree shape,and it is an excellent material for making bonsai and planting innovative research.Due to the small number of wild individuals of P.xueluoensis,the seed emergence rate is low under natural conditions,and the regeneration system is lacking,which limits its promotion and application.Therefore,there is an urgent need to establish its efficient tissue culture system and genetic transformation system to provide a theoretical basis for the conservation of P.xueluoensis resources,germplasm innovation and molecular improvement.In this study,the effect of different hormone ratios on the tissue culture of P.xueluoensis was investigated,and the best medium for tissue culture was selected to establish a stable in vitro regeneration system of P.xueluoensis.Meanwhile,the effects of different Kan concentrations,Cef concentration,pre-culture,bacterial solution concentration,infestation time and co-culture time on the genetic transformation of P.xueluoensis were studied,and the molecular biology of the resistant plants was identified,and the genetic transformation system of P.xueluoensis was initially constructed.The main research results are as follows:（1）P.xueluoensis was used as the experimental material to establish its efficient regeneration system.The best disinfection method for germination of P.xueluoensis embryos was a combination of 75%alcohol soaking for 5 min and 10%Na Cl O soaking for 25 min,and the average germination rate could reach 84.44%.The optimal medium formulation for P.xueluoensis embryo germination culture was MS+200 mg/L GA3+4 mg/L 6-BA,and the average germination rate could reach94.44%.The optimal medium formulation for the induction culture of P.xueluoensis was MS+2.00mg/L 6-BA+1.00 mg/L NAA+200 mg/L VC,and the average induction rate could reach 76.66%.The optimal medium formulation for adventitious shoot differentiation culture of P.xueluoensis was MS+1.00 mg/L 6-BA+0.10 mg/L NAA+200 mg/L VC,and the average induction rate could reach75.92%.The optimal medium formulation for the rooting culture of P.xueluoensis terminal buds was 3/4MS+0.50 mg/L NAA,and the average rooting rate could reach 74.08%.（2）Establishment of genetic transformation system of P.xueluoensis stem segments using Agrobacterium-mediated method.According to the growth of the explants,the concentration of Kan was chosen to be 30 mg/L during the screening culture,which could effectively screen the resistant plants.250 mg/L of Cef was chosen to effectively inhibit the growth of Agrobacterium and at the same time,it had less effect on the growth of the explants.Based on establishing the regeneration system of P.xueluoensis,we investigated the effects of four aspects:pre-culture time,bacterial solution concentration,infestation time and co-culture time on the infestation rate of Agrobacterium,and established the Agrobacterium-mediated genetic transformation system of P.xueluoensis.The order of influence of the four factors on the transformation rate was:concentration of bacterial solution>pre-culture time>infestation time>co-culture time.The optimal genetic transformation conditions for P.xueluoensis stem segments were:pre-culture for 2 d,bacterial solution concentration OD₆₀₀=0.6,infestation for 30 min,and total incubation time of 3 d.The highest genetic transformation efficiency of P.xueluoensis was achieved under this condition,and five transgenic positive plants were identified by PCR assay and GFP fluorescence detection,and the positivity rate could reach 10.42%.In summary,this study established a regeneration system using P.xueluoensis with axillary bud stem segments as explants.On this basis,the effects of various factors on the genetic transformation efficiency of P.xueluoensis were explored,and the preliminary establishment of the Agrobacterium-mediated genetic transformation system of P.xueluoensis stem segments laid the theoretical foundation and technical support for obtaining high-quality germplasm of P.xueluoensis and transgenic research.