发过多篇nature实验室的Protocols_Page[bioworlde.com].pdfVIP免费

1ProtocolICulturingofPlantCellSuspensionCulturesPurpose:Plantcellscanbegrownundersterileconditionsassuspensionorcallusculturesandgiventheappropriatehormonalsupplementstothegrowthmediacanbeinducedtoregenerateintoanentireplant.Thisphenomenonisuniquetoplantsandistermedtotipotency.Cellcultures(plant,animal,microbial,etc.)alsoofferadvantagesforcertaintypesofexperimentalmanipulationsbecausethecultureconditionsareeasilymanipulated.Thisprotocoldetailsproceduresnecessaryforthemaintenanceofplantcellsuspensioncultures.1.PreparationofMurashigeandSkoognutrientmedia-1liter4.3gmMSsalts(containsessentialsalts,nitrogen,potassium,phosphorus,etc.)2mlGamborg'svitamins5ml0.2mg/ml2,4-dichlorophenoxyaceticacid(hormone)30gmsucrosedissolvemixturein800mldistilledorpurifiedwateradjustpHto5.4-5.6bringvolumeto1literdispenseintoErlenmeyerflasks(50ml/250mlflask,100ml/500,200ml/1l)autoclave20min.Forplates,add8gmagarbeforeautoclaving2.Wipedownlaminarflowhoodwith70%ethanol3.Flametopsofflasks4.Dilutecellcultureswithfreshmediaifnecessary5.Dispenseculturesintoflaskscontainingfreshmedia(25mlcultureinto50mlfreshmedia6.Shakecellsatapproximately100rpm(providesaeration)7.Subculturethecultureswhenevertheculturesappeartodepletemediasource.2ProtocolIIInductionofPlantDefenseResponsesPurpose:Attemptstoinvestigateplantdefenseresponsesarecomplicatedbytwofactors.One,theinteractionisgenerallylimitedtoaveryfewcellsandthus,biologicalmaterialtoexamineislimiting.Two,itisoftendifficulttodiscernifaresponseisderivedfromtheplantorthepathogenwheninvestigatinganintactsystem.Fortunately,plantdefenseresponsestomicrobialpathogenscanbestimulatedbychallengingplantcellcultureswithcellsurfacecomponentsorextracellularproteinsoffungalpathogens.Thepathogenderivedfactorsthatcaninduceplantdefenseresponsesarereferredtoaselicitors(theyelicitaresponse).Thefollowingprotocoldescribesaninductiontreatmentoftobaccocellsuspensionculturestocellulase,afungalextra-cellularenzymeisolatedfromTrichodermaviride.1.PreparationofCellulase.Dissolve1mgofT...