2.2.11烟草悬浮系BY-2的转化1）农杆菌一次过夜摇培，二次摇培稀释至OD6000.25继续培养6小时。2）取继代三或四天的4mL（8mL）BY2细胞，加入20μM（终浓度，有的文献为200μM）乙酰丁香酮。3）悬浮细胞加入200μL二摇农杆菌于10cm培养皿28℃暗培养两天。4）加有羧苄青霉素（500mgL-1/500μgmL-1）培养基50mL洗涤细胞，室温离心1000rpm，4min。洗涤1-2次。5）细胞沉淀在半固体培养基上铺板（羧苄青霉素500μgmL-1，潮霉素20μgmL-1），28℃暗培养两天。6）3-4周可见愈伤长出，用荧光显微镜检查阳性愈伤，继代一次。7）挑取悬浮细胞于20mL培养基摇培，有悬浮细胞出现后稀释至50mL摇培。至转化后的悬浮系生长速度和野生型一致，每周继代一次。Preparation:-SetuptobaccoBY-2cells.Threedaysbeforetransformation,startnewsubculture.Youwillneed5mlofcultureforeverytransformationShakeat25°C.-MakeMSliquidmedium(about80mlforeachtransformation).-Prepareagarplates:MSmedium+0.8%phytagarplus500μg/mlcarbenicillin(1persample)MSagar+500μg/mlcarbenicillin+50μg/mlkanamycin(upto3persample)-Agrobacteriumstraincontainingbinaryvector.Thedaybeforedoingthetransformation,inoculatealargesinglecolonyofeachAgrobacteriumstraininto3mlLBplusantibioticsinatesttube.(FormostpBINvectorsinLBA4404,youwilluse25μg/mlstreptomycinand50μg/mlkanamycin).Growinrollerdrumat28°CovernighCocultivation:1.MeasureOD600ofeachAgrobacteriumstrain.DilutetoOD600=1.0inLB.2.Foreachtransformation,aliquot5mlBY-2cellsintoa100mmPetridish.Besuretoincludeacontrol(nobacteria).3.Add5lacetosyringonetoBY-2cells(stockis20mMinethanol).Swirlgentlytomix.4.AddAgrobacteriacarryingthebinaryvector.Foreachvector,setuptwotransformationsusingtwodifferentamountsofAgrobacteria,i.e.,25land100l.Swirltomix.5.WrapplateswithParafilmandincubateat25°Cfortwodays.Washing(2daysaftersettingupcocultivation):7.Usingawidebore10mlpipette,transfertheBY-2cellsfromeachplateintoa15mlcentrifugetube.Rinsetheplatewithanadditional5-7mlMS,andaddtothecentrifugetube.8.Letthecellssettleinthecentrifugetube.9.Carefullyremovethesupernatantbyaspiration.10.PourMSmediumintothetubewiththecellsto14-15mlfinalvolume.Mixbygentleinversion.11.PelletcellsasinStep.8.12.Repeatsteps9-11twomoretimesusingMSmedium.13.RepeatSteps9-11usingMSmediumplus500gpermlcarbenicillin.14.AddMS/carbenicillinto12ml.Mixbygentleinversion.Platingofcellso