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GrainGenes Reference Report: TAG-86-721

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Reference	TAG-86-721
Title	Factors affecting transient gene expression in protoplasts isolated from very slowly growing embryogenic callus cultures of wheat (Triticum aestivum L.
Journal	Theoretical and Applied Genetics
Year	1993
Volume	86
Pages	721-730
Author	Zaghmout O Trolinder N
Abstract	Protoplasts isolated from embryogenic ('Mustang' and 'Chinese Spring') and non-embryogenic ('Mit') calli of wheat (Triticum aestivum L.) genotypes transiently expressed beta-glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm- and 250 microF for 2 s using 50 microgram/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed.
Keyword	[ Hide all but 1 of 24 ] 35s-promoter adh1-promoter alcohol-dehydrogenase beta-glucuronidase callus cauliflower-mosaic-caulimovirus electroporation embryogenesis enzyme activity gene expression genetic transformation histoenzymology leader-sequence nucleotide sequence plasmid-vectors promoters protoplast recombinant-dna regenerative-ability reporter genes tobacco-mosaic-tobamovirus transgenic-plants triticum aestivum vitis-vinifera

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