Establishmentof Regeneration Andgenetic Transformation System Of Oncidium And Transformation Of Linalool Synthase Gene

Oncidium（Oncidium.spp）is a popular tropical orchidesin Orcidium for its beautiful color and form.However,the fragrant clutivars are very rare in this plant,and the transgenic technology can provide a new way to solve this problem quickly.In this study,Oncidium’Lemon green’was used as the material to establish and optimize an efficient regeneration system.and agrobacterium-mediated transformation method was used to establish a transgenic system using GUS gene as an indicator.Finally,a Lilis gene was introduced into‘Lemon green’by agrobacterium-mediated transformation method for a new fragrant cultivar.The main research results were as follows:（1）The establishment and optimization of the transgenic system of Oncidium.The Oncidium’Lemon Green’sterile seedlings were used as materials for the protocorm induction,proliferation,differentiation,rooting and transplanting.Then the induced protocorm were use as materials to compare the effects of protocorm diameter,different hormone ratios,mannitol and other factors on the growth of protocorm.Finally the best growth conditions were confirmed.The results showed that NAA was the most important factor,and the treatment 5（1/2 MS with 0.5 mg/LNAA,0.5 mg/L6-BA and 0.3 mg/LTDZ）had the highest induction rate of 48%.The different content of activated carbon with different carbon source received significantly different protocorm proliferation rate.Suppliments of different natural compounds for the plantlets rooting was significantly better than that of the control,and the coconut water was the best one with nearly 4 roots per plantlet.The survival rate of the seedlings can reach 100%after transplanting.The optimal induction medium for protocorm was 1/2MS+0.5 mg/L NAA+0.5 mg/L 6-BA+0.3 mg/L TDZ.The optimal multiplication medium was 1/2 MS+4.0 mg/L 6-BA+0.4mg/L NAA+1.0 g/L activated carbon+20 g/L trehalose.The optimal rooting medium was1/2 MS+0.8 mg/L 6-BA+0.2 mg/L NAA+10%coconut water+30 g/L sucrose.The optimal growth conditions of the receptors were 1/2 MS+0.2 or 0.6 mg/L 6-BA+0.4 mg/L NAA mg/L combined with the slice size of 1.5mm protocorm.The best content and time of mannitol pretreatmen was 0.1⁰.25mol/Land 1h.（2）Establishment of genetic transformation system:The resistance sensitivity test of antibiotic concentration gradient to receptor growth was designed to obtain the best screening conditions,The best content of cefomycin was 60mg/L for the bacteriostatic effect of agrobacterium.The best content of kanamycin was 50mg/L for the protocorm selection.Before infection,the optimal concentration of hyperosmotic treatment for protocorm was 0.15mol/L.The best infection time was 1h,and the best OD₆₀₀00 value of the bacterial solution was 0.4.The best infection time was 30 or 50 min and the best co-culture time was 2 days.The beat sterilizing time was 20min.The best content of of AS during infection was 100μmol/L.The instantaneous expression rate of GUS could reached to64.7%.The Lslis-pBI121 plant expression vector was successfully constructed,and a total of 1800 procorms were transformed by the optimized transgenic system.After bacteriostatic screening,543 survived,with a survival rate of 30.2%,Resistance screening was continued,and 9 of them survived,with a survival rate of about 0.5%.The protocorms of transgenic Oncidium with resistance were screened.