An approach towards genetically engineered cell fate mapping in maize using the Lc gene as a visible marker: transactivation capacity of Lc vectors in differentiated maize cells and microinjection of Lc vectors into somatic embryos and shoot apical meristems.
To establish a system for genetically engineered cell fate mapping, different vectors carrying the Lc gene, a member of the R gene family, were delivered into embryonic and meristematic cells of maize by the microinjection technique. Vectors in which the Lc cDNA is driven either by a constitutive promoter (CaMV 35S), with or without the Adh1 intron 1 of maize, or a tissue-specific promoter (phosphoenolpyruvate carboxylase, PEPC) as well as self-replicating wheat dwarf virus (WDV) vectors carrying a Lc-expression-cassette, have been tested. The ability of these vectors to transactivate was evaluated in mesophyll-derived protoplasts of the maize genotype appropriate for these microinjection experiments. The expression product of the introduced Lc gene can substitute for mutated R and B loci, resulting in anthocyanin production. Analogous results were obtained by microinjection into organized tissues, where transactivation of anthocyanin biosynthesis resulted in pigmented sectors in somatic embryos (B79) and in the leaves of plants regenerated from the cultivated shoot apical meristems (K55, r-g, b). The tissue-specific appearance of pigmented sectors in leaves, using the mesophyll-specific PEPC promoter suggests the possibility of using this approach for layer-specific cell fate studies. The presence of the introduced plasmids in leaves showing red sectors 20-30 days after injection was proven by PCR analysis.
Lusardi
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MC
Institute of Plant Sciences, Swiss Federal Institute of Technology, Zürich.
Neuhaus-Url
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Potrykus
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Neuhaus
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D001483:Base Sequence; D002454:Cell Differentiation; D002478:Cells, Cultured; D017931:DNA Primers; D005818:Genetic Engineering; D005819:Genetic Markers; D005822:Genetic Vectors; D008845:Microinjections; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics*; D011523:Protoplasts; D012639:Seeds / Q000166:cytology; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D003313:Zea mays / Q000166:cytology* / Q000196:embryology / Q000235:genetics
D017931:DNA Primers; D005819:Genetic Markers; D010940:Plant Proteins; D014157:Transcription Factors; C083017:R-S protein, Zea mays