(7.10)--TRANSIENT GENE EXPRESSION IN ARA细胞生物学细胞生物学.pdf

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TRANSIENTGENEEXPRESSIONINARABIDOPSIS

PROTOPLASTS

WenbinWeiKeithLindsey

(LeicesterBiocentre,UniversityofLeicester.)

*WehaveusedthismethodofPEG-mediateddirectgenetransferinto

Arabidopsisleafprotoplastsforanalysingthetransientgeneexpression

ofgusAreportergeneconstructs.Theprotocolworkswellforecotype

C24.Wehavenottriedothers.

1.PLANTMATERIAL

*Useaerialpartsof3-4week-oldseedlingsgerminatedfromsurface-

sterilisedseedandmaintainedasepticallyonhalfstrengthMS10.

2.SOLUTIONS

(i)HALFSTRENGTHMS10

HalfstrengthMurashigeandSkoog(1962)Medium,e.g.,Sigma

10gl-1sucrose

8gl-1Difcoagar

pH5.8,adjustedwith0.1MKOH,autoclave

(ii)ENZYMESOLUTION

(Damm,B.Willmitzer,L.1991.Arabidopsisprotoplasttransformationand

regeneration.In:K.Lindsey(ed.)PlantTissueCultureManual,A7.Kluwer

AcademicPublishers,Dordrecht.pp1-17.)

1%(w/v)CellulaseOnozukaR-10

0.25%(w/v)MacerozymeR-10

8mMCaCl2.2H20

0.5Mmannitol

pH5.5,adjustedwith0.5MKOH,filtersterilise

(iii)W5WASHSOLUTION

154mMNaCl

125mMCaCl2.2H20

5mMKCl

5mMglucose

0.5Mmannitol

pH5.8,adjustwith0.1MKOH,autoclave

(iv)SUCROSE

21%(w/v),autoclaved

(v)MANNITOL/MgSOLUTION

15mMMgCl2

0.1%MES

0.4Mmannitol

pH5.6,adjustwith0.1MKOH,autoclave

(vi)PEGSOLUTION

40%(w/v)polyethyleneglycol(PEG)6000inasolutionof:

0.4Mmannitolplus

0.1MCa(NO3)2.4H20

pH8.0,adjustwith0.1MKOH

*pHtakes2-3htostabilise

(vii)PROTOPLASTCULTUREMEDIUM

MurashigeandSkoog(1962)medium,e.g.,fromSigma)

file://localhost/Users/lreiser/Desktop/comguide%20Folder/chap_2_tissue_culture/10_tra