Abstract
A visual marker genetically linked to a trait of interest would enable the unequivocal identification of transgenic seed containing the trait and ensure that genetically modified material could be easily differentiated from non-transgenic grain. Genes from maize that regulate the anthocyanin biosynthetic pathway, and are therefore responsible for directing the pattern of purple pigmentation, can be used to generate transgenic plants with unique, easily recognizable phenotypes. In the present study, a seed-specific maize globulin promoter was used to drive the expression of two transcription factor genes (Bp and C1) regulating the anthocyanin biosynthetic pathway in embryo and aleurone tissues resulting in maize seed with a distinct purple pigmentation. The following report describes the construction of expression vectors containing these two genes and an herbicide resistance selectable marker gene, the generation and characterization of transgenic cultures, the induction of pigmentation in somatic embryos, the regeneration of transgenic plants with pigmented seed and the co-segregation of integrated DNA with seed pigmentation and herbicide resistance. The co-segregation of seed pigmentation and herbicide resistance confirms the feasibility of using the expression of these genes in a ‘molecular stack’ for visual transgene identification.
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References
Belanger FC, Kriz AL (1989) Molecular characterization of the major maize embryo globulin encoded by the Glb1 gene. Plant Physiol 91:636–643
Bodeau JP, Walbot V (1992) Regulated transcription of the maize Bronze-2 promoter in electroporated protoplasts requires the C1 and R gene products. Mol Gen Genet 233:379–387
Bower R, Elliott AR, Potier BAM, Birch RG (1996) High efficiency, microprojectile-mediated co-transformation of sugarcane, using visible or selectable markers. Mol Bree 2:239–249
Chawla HS, Cass LA, Simmonds JA (1999). Expression of anthocyanin pigmentation in wheat tissues transformed with anthocyanin regulatory genes. Curr Sci 76(10):1365–1370
Coe EH, Neuffer MG, Hoisington DA (1988) The genetics of corn. In Sprague GF, Dudley J (eds) Corn and corn improvement. American Agronomy Society, pp 81–258
Cowen NM, Armstrong K, Smith KA (2004) Use of regulatory sequences in transgenic plants. United States Patent Application 20040158887
DePicker A, Stachel S, Dhaese P, Zambryski P, Hoodman HM (1982) Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet 1:561–573
Dooner HK, Roobins TP, Jorgensen RA (1991) Genetic and developmental control of anthocyanin biosynthesis. Ann Rev Genet 25:173–199
Goff SA, Klein TM, Roth BA, Fromm ME, Cone KC, Radicella JP, Chandler VL (1990). Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. EMBO J 9(8):2517–2522
Grotewold E, Chamberlin M, Snook M, Siame B, Butler L, Swenson J, Maddock S, St. Clair G, Bowen B (1998) Engineering secondary metabolism in maize cells by ectopic expression of transcription factors. Plant Cell 10:721–740
Grotewold E, Sainz MB, Tagliani L, Hernandez JM, Bowen B, Candler VL (2000) Identification of the residues in the Myb domain of maize C1 that specify the interaction with the bHLH cofactor R. Proc Natl Acad Sci USA 97:13579–13584
Hall G Jr, Allen GC, Loer DS, Thompson WF (1991) Nuclear scaffold attachment regions in higher plants. Proc Natl Acad Sci USA 88:9320–9324
Irani NG, Grotewold E (2005) Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells. BMC Plant Biol 5:7
Irani NG, Hernandez JM, Grotewold E (2003) Regulation of anthocyanin pigmentation. Rec Adv Phytochem 38:59–78
Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750
Klein TM, Roth BA, Fromm ME (1989) Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles. Proc Nat Acad Sci USA 86:6681–6685
Kwiatkowski RW, Lyamichev V, de Arruda M, Neri B (1999) Clinical, genetic and pharmacogenetic applications of the Invader assay. Mol Diagn 4(4):353–364
Linsmaier EM, Skoog F (1965) Organic growth factor requirements for tobacco tissue cultures. Physiol Plant 18:100–127
Ludwig SR, Bowen B, Beach L, Wessler SR (1990) A regulatory gene as a novel visible marker for maize transformation. Science 247:449–450
Ludwig SR, Habera LF, Dellaporta SL, Wessler SR (1989) Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci USA 86:7092–7096
McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Oldenburg MC, Siebert M (2000) New CleavaseR fragment length polymorphism method improves the mutation detection assay. BioTechniques 28:351–357
Paz-Ares J, Ghosal D, Wienand U, Peterson PA, Saedler H (1987) The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products with structural similarities to transcriptional activators. EMBO J 6:3553–3558
Paz-Ares J, Wienand U, Peterson PA, Saedler H (1986) Molecular cloning of the c locus of Zea mays: a locus regulating the anthocyanin pathway. EMBO J 5:829–833
Piazza P, Procissi A, Jenkins GI, Tonelli C (2002) Members of the c1/pl1 regulatory gene family mediate the response of maize aleurone and mesocotyl to different light qualities and cytokinins. Plant Physiol 128:1077–1086
Radicella JP, Brown D, Tolar LA, Chandler VL (1992) Allelic diversity of the maize B regulatory gene: different leader and promoter sequences of two B alleles determine distinct tissue specificities of anthocyanin production. Genes Dev 6:2152–2164
Radicella JP, Turks D, Chandler VL (1991) Cloning and nucleotide sequence of a cDNA encoding B-Peru, a regulatory protein of the anthocyanin pathway in maize. Plant Mol Biol 17:127–130
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: laboratory manuals. Cold Spring Harbor Lab press, Cold Spring Harbor, New York
Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204
Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493
Wohlleben W, Arnold W, Broer L, Hillemann D, Strauch E, Puehler A (1988) Nucleotide sequence of the phosphinothricin N-acetyletransferase gene from Streptomyces viridochromogenes Tue494 and its expression in Nicotiana tabacum. Gene 70:25–37
Zar JH (1974) Biostatistical analysis. Prentice-Hall, Inc., Englewood Cliffs, NJ, pp. 41–53
Acknowledgements
The authors would like to recognize Don Merlo for providing the Bp and C1 gene vectors and Beth Rubin–Wilson for the maize globulin-1 promoter. Special thanks to Krisi Curlee and Katy Kirkwood–Rodriguez for their care of the transgenic plants and the harvesting and sorting of transgenic seeds, Andy Worden and Charles Cai for molecular analysis and Nicole Hopkins and Lisa Schulenberg for their expert advice on Agrobacterium transformation of maize. Lastly, Jean Roberts and Beth Rubin–Wilson provided invaluable input during the planning of this project and a critical review of the manuscript.
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Shen, L.Y., Petolino, J.F. Pigmented Maize Seed via Tissue-specific Expression of Anthocyanin Pathway Gene Transcription Factors. Mol Breeding 18, 57–67 (2006). https://doi.org/10.1007/s11032-006-9018-1
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DOI: https://doi.org/10.1007/s11032-006-9018-1