Establishment Of Transformation System And Transfer Of Cold Induced Gene To Lavandula Angustifolia Mediated By Agrobacterium

Lavandula angustifolia cv.Munstead,one of the major natural aromatic plants,is a perennial subshrub plant of the Labiatae family native to Mediterranean areas.Due to the difficult overwintering of Lavandula angustifolia in China,its planting zone and economic values are restricted.Therefore,it is urgent to use transgenic engineering technique to improve the cold tolerance of Lavandula angustifolia.And the quality and output of its essential oils could be improved at the same time.In this study,transgenic Lavandula plant containing cold induced gene XCH were obtained through Agrobacterium-mediated transformation,based on the optimization of regeneration and transformation system.It was hoped that the cold tolerance and essential oil production of Lavandula angustifolia cv. Munstead could be improved.Useful information was provided for the widely quality improvement of Lavandula by transgenic technique.The major results are presented bellow:1.Culture mediums for different explants in vitro were optimized through experiment and several influencing factors were studied.Result showed that the optimal medium for direct regeneration of stem segment was MS +NAA 0.5mg/L+6-BA 2.0 mg/L.The optimal mediums for callus induction and regeneration and shoot differentiation were MS+2,4-D 0.1 mg/L+6-BA 0.5 mg/L and MS +NAA 0.5 mg/L+6-BA 2.0 mg/L,respectively. Relatively thickset regeneration shoots in shoot clusters originated from different explants were selected and subcultured on shoot regeneration medium MS +NAA 2.0 mg/L+6-BA 0.5 mg/L for about 30 days.Then they were cultured on rooting medium 1/2MS+IAA 0.5 mg/L+6-BA 0.1 mg/L for root formation.2.According to the study result of Cef sterilization concentration,Hyg screening pressure and the influencing factors of transformation efficiency,the transformation systems of Lavandula angustifolia mediated by Agrobacterium tumefaciens were preliminary setup using leaf induced callus and stem segment.The optimum condition for callus genetic transformation was 2 weeks' regeneration on MS+2.4-D0.1 mg/L+6-BA0.5 mg/L,2～3mm² in size,OD₆₀₀0.5,10 min infection time,2 d co-cultivation,10 d restoration,200 mg/L Cef,10 mg/L Hyg for 4 weeks and then 30 mg/L for about 4 weeks to select resistant callus,10 mg/L Hyg for shoot differentiation and regeneration,5 mg/L for rooting.The optimum condition for stem segment was 3 d pre-culture on MS+NAA 0.5 mg/L+6-BA 2.0mg/L,OD₆₀₀0.5,15 min infection time,3 d co-cultivation,7 d restoration,200 mg/L Cef,10 mg/L Hyg for shoot selection and 5 mg/L Hyg for rooting.GFP transient expression rates of the two transformation systems were high and were both above 90%.After selection for 3 weeks,the frequency of resistant callus was 37.7% and that of resistant stem segment was 15.6%.However,1 transgenic plant was obtained only in callus regeneration system.3.DNA of the transgenic plant was amplified by PCR,result of electrophoresis showed that the same line as positive control XCH(643 bp)was obtained.It was proved in molecular level that the cold induced gene was integrated into Lavandula angustifolia genome.And the transformation rate was 1.03%.