PlantCellRep(2010)29:371–381DOI10.1007/s00299-010-0828-6ORIGINALPAPERChloroplasttransformationofrapeseed(Brassicanapus)byparticlebombardmentofcotyledonsLinCheng•He-PingLi•BoQu•TaoHuang•Jin-XingTu•Ting-DongFu•Yu-CaiLiaoReceived:19November2009/Revised:8January2010/Accepted:3February2010/Publishedonline:24February2010ÓSpringer-Verlag2010AbstractAprotocolforchloroplasttransformationofanchloroplastgenomeusingtwoprimersdesignedaccordingeliterapeseedcultivar(BrassicanapusL.)wasdevelopedtoArabidopsishomologs.TheaadAgenewasusedasabasedonoptimizedconditionsforcallusinductionandselectionmarkerregulatedbytheribosome-bindingsiteregenerationfromcotyledonarytissues.ComparisonofsixfromthebacteriophageT7gene10L,thetobacco16SdifferentmediawiththreeelitecultivarsshowedthatB5rRNApromoterandthepsbAterminator.Afterbombard-mediumplus3mg/lAgNO3supplementedwith0.6mg/lment,cotyledonarysegmentswereculturedforcallusfor-2,4-dichlorophenoxyaceticacidand0.2mg/l6-furfury-mationonmediacontaining10mg/lspectinomycinandlaminopurinewasoptimalforcallusformationandmain-regenerationwascarriedoutonmediumwith20mg/ltenancewithoutdifferentiation,whilethemediumsuitablespectinomycin.HeteroplasmicplastidtransformantswereforregenerationwasB5mediumsupplementedwith1mg/lisolated.Anoverallefficiencyforthechloroplasttrans-6-benzylaminopurine,1mg/l6-furfurylaminopurineandformationwasonetransplastomicplantperfourbom-0.5mg/la-naphthaleneaceticacid.Arapeseed-specificbardedplates.Southernblotanalysesdemonstratedproperchloroplasttransformationvectorwasconstructedwiththeintegrationofthetargetsequenceintotherapeseedchlo-trnIandtrnAsequencesamplifiedfromtherapeseedroplastgenomeviahomologousrecombination.TheexpressionoftheaadAgenewasconfirmedbyNorthernblotanalysis.AnalysisofT1transplastomicplantsrevealedthatthetransgenesintegratedintothechloroplastwereCommunicatedbyH.Jones.inheritablewitharatioofabout8%.TheseresultssuggestthatrapeseedmaybeasuitablecropforchloroplastL.ChengH.-P.LiB.QuT.HuangY.-