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1、水稻悬浮细胞培养的方法Theflasksweresetonagyratoryshakeragitatedat120rpmunderfluorescentlightof200-300luxat27°C.CellgrowthintheR-2mediumwassuperiortothatobtainedinB5,Heller,Murashige-SkoogandWhitemedia.Plant&CellPhysiol.14:1113-1121(1973)Experimentalcellresearch50,151-15
2、8(1968)MolecgenGenet161,67一76(1978)Ajaponicaricevariety,Nackdong,wasusedfortransformationbytheAgrobacteriumcocultivationmethodasdescribedpreviouslywiththefollowingmodifications[14].CalliwereinducedfromthescutellumofmatureseedsonanN6mediumcontaining2mg/l2,4-D.
3、AnA.tumefaciensstrainLBA4404carryingthepGA1647plasmidwasgrownfor3daysinanABliquidmediumsupplementedwith30mg/lhygromycinBand3mg/ltetracycline.Three-week-oldcalliwerecocultivatedwithAgrobacteriumona2N6-Asmediumsupplementedwith1mMbetainefor2–3daysindarknessat25C
4、.Thecocultivatedcalliwerewashedwithsterilewatercontaining100mg/lcefotaxime,andincubatedonanN6mediumcontaining40mg/lhygromycinBand250mg/lcefotaximefor3weeks.Activelygrowingcalliweretransferredontoaregenerationmedium,MSmediasupplementedwith0.1mg/lNAA,2mg/lkinet
5、in,2%sorbitol,1.6%phytagar(Gibco),50mg/lhygromycinB,and250mg/lcefotaxime.After2–3weeksundercontinuouslight(40molm?2s?1),plantletswerepottedandgrowninagrowthchamberwith10hlightperday.PlantMolecularBiology39:35–44,1999.NBBasicN6majorsaltsandironsource(Chu1975),
6、B5minorsaltsandvitamins(Gamborgetal.1968),30g/lsucroseNBNBBasic+2mg/l2,4-dichlorophenoxyaceticacid(2,4-D),500mg/lproline,500mg/lglutamine,300mg/lcaseinhydrolysate,2.6g/lPhytagel,pH5.8(Lietal.1993)R2BasicR2majorandminorsalts,vitaminsandironsource(Ohiraetal.197
7、3),2.5mg/l2,4-DCCLR2Basic+10g/lglucose,100mMacetosyringone,pH5.2liquidco-culturemediumCCSCCL+7g/lagaroseR2SR2Basic+30g/lsucrose,50mg/lhygromycin,400mg/lcefotaxime,100mg/lvancomycine,7g/lagarose,pH6.0NBSNBBasic+2.5mg/l2,4-D,500mg/lproline,500mg/lglutamine,300m
8、g/lcaseinhydrolysate,50mg/lhygromycin,400mg/lcefotaxime,100mg/lvancomycine,万古霉素7g/lagarose,pH6.0PRAGNBBasic+2mg/lBAP,1mg/lNAA,5mg/lABA,500mg/lproline,500mg/lglutamine,300mg/lcaseinhydroly
