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Use of an anthocyanin production phenotype as a visible selection marker system in transgenic tobacco plant

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Abstract

To develop a potentially alternative method for the selection of transgenic plants instead of antibiotic and herbicide resistance, anthocyanin pigmentation phenotype was examined to provide a visible selection marker system. Two regulatory genes of the anthocyanin biosynthetic pathway, the R2R3 MYB mPAP1 gene from Arabidopsis and the basic helix loop helix B-Peru gene from maize, were amplified by RT-PCR and then individually cloned into a plant expression vector. The requirement of these two genes for anthocyanin pigmentation was pre-confirmed via an in vivo assay using tobacco agro-infiltration. The mPAP1 and B-Peru vectors were further stably co-transformed into tobacco plants using Agrobacterium tumefaciens strain LBA4404. Tobacco plants harboring both genes could be readily selected through the manifestation of a red color due to anthocyanin accumulation in the whole plant body. The T1 segregants showed red or green phenotypes depending on the genotype. The need for both the mPAP1 and B-Peru genes for a red color phenotype due to anthocyanin pigmentation was further confirmed by genotyping of the T1 generation by genomic PCR analysis and an in vivo assay using agro-infiltration. From these results, we conclude that co-transformation with two individual vectors harboring a critical anthocyanin transcriptional factor has potential utility as an alternative visible selectable marker system for transgenic progeny selection in plants.

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References

  • Baerson SR, Rodriguez DJ, Tran M, Feng YM, Biest NA, Dill GM (2002) Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol 129:1265–1275

    Article  PubMed  CAS  Google Scholar 

  • Bernasconi P, Woodworth AR, Rosen BA, Subramanian MV, Siehl DL (1995) A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase. J Biol Chem 270:17381–17385

    Article  PubMed  CAS  Google Scholar 

  • Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383–2394

    PubMed  CAS  Google Scholar 

  • Bovy A, de Vos R, Kemper M, Schijlen E, Almenar Pertejo M, Muir S, Collins G, Robinson S, Verhoeyen M, Hughes S, Santos-Buelga C, van Tuinen A (2002) High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. Plant Cell 14:2509–2526

    Article  PubMed  CAS  Google Scholar 

  • Bradley JM, Davies KM, Deroles SC, Bloor SJ, Lewis DH (1998) The maize Lc regulatory gene up-regulates the flavonoid biosynthetic pathway of Petunia. Plant J 13:381–392

    Article  CAS  Google Scholar 

  • Choi MS, Yoon IS, Rhee Y, Choi SK, Lim SH, Won SY, Lee YH, Choi HS, Lee SC, Kim KH, Lomonossoff G, Sohn SH (2008) The effect of cucumber mosaic virus 2b protein to transient expression and transgene silencing mediated by agroinfiltration. Plant Pathol J 24:296–304

    Article  CAS  Google Scholar 

  • Daley M, Knauf V, Summerfelt K, Turner J (1998) Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Rep 17:489–496

    Google Scholar 

  • Höfgen R, Willmizer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877

    Article  PubMed  Google Scholar 

  • Huang S, Gilbertson LA, Adams TH, Malloy KP, Reisenbigler EK, Birr DH, Snyder MW, Zhang Q, Luethy MH (2004) Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors. Transgenic Res 13:451–461

    Article  PubMed  CAS  Google Scholar 

  • Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  PubMed  CAS  Google Scholar 

  • Kim JS, Chung TY, King GJ, Jin M, Yang TJ, Jin YM, Kim HI, Park BS (2006) A sequence-tagged linkage map of Brassica rapa. Genetics 174:29–39

    Article  PubMed  CAS  Google Scholar 

  • Kim CY, Ahn YO, Kim SH, Kim YH, Lee HS, Catanach AS, Jacobs JME, Conner AJ, Kwak SS (2010) The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system. Physiol Plant 139:229–240

    Article  PubMed  CAS  Google Scholar 

  • Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174

    Article  PubMed  CAS  Google Scholar 

  • Kortstee AJ, Khan SA, Helderman C, Trindade LM, Wu Y, Visser RGF, Allan A, Schouten HJ, Jacobsen E (2011) Anthocyanin production as a potential visual selection marker during plant transformation. Transgenic Res 20:1253–1264

    Article  PubMed  CAS  Google Scholar 

  • Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079

    Google Scholar 

  • Lim SH, Woo HJ, Lee SM, Jin YM, Cho HS (2007) d-Amino acid oxidase (DAO) gene as a novel selection marker for plant transformation. Korean J Plant Biotechnol 34:31–36

    Google Scholar 

  • Lloyd AM, Walbot V, Davis RW (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258:1773–1775

    Article  PubMed  CAS  Google Scholar 

  • Luo K, Zheng X, Chen Y, Xiao Y, Zhao D, McAvoy R, Pei Y, Li Y (2006) The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation. Plant Cell Rep 25:403–409

    Article  PubMed  CAS  Google Scholar 

  • Lutz KA, Svab Z, Maliga P (2006) Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system. Nat Protoc 1:900–910

    Article  PubMed  CAS  Google Scholar 

  • Mentewab A, Stewart CN (2005) Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants. Nat Biotechnol 23:1177–1180

    Article  PubMed  CAS  Google Scholar 

  • Park JM, Lee YK, Kang BK, Chung WI (2004) Co-transformation using a negative selectable gene for the production of selectable marker gene-free transgenic plants. Theor Appl Genet 109:1562–1567

    Article  PubMed  CAS  Google Scholar 

  • Quattrocchio F, Wing JF, Leppen HTC, Mol JNM, Koes RE (1993) Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell 5:1497–1512

    PubMed  CAS  Google Scholar 

  • Rommens CM, Humara JM, Ye J, Yan H, Richael C, Zhang J, Perry R, Swords K (2004) Crop improvement through modification of the plant’s own genome. Plant Physiol 135:421–431

    Article  PubMed  CAS  Google Scholar 

  • Schmülling T, Schell J (1993) Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras. Plant Mol Biol 21:705–708

    Google Scholar 

  • Sreekala C, Wu J, Gu K, Wang D, Tian D, Yin Z (2005) Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system. Plant Cell Rep 24:86–94

    Article  PubMed  CAS  Google Scholar 

  • Sugita K, Kasahara T, Matsunaga E, Ebinuma H (2000) A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency. Plant J 22:461–469

    Article  PubMed  CAS  Google Scholar 

  • Vetten N, Wolters AM, Raemakers K, van der Meer I, Stege R, Heeres E, Heeres P, Visser R (2003) A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nat Biotechnol 21:439–442

    Article  PubMed  Google Scholar 

  • Vionnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci USA 96:14147–14152

    Article  Google Scholar 

  • Wang J, Oard JH (2003) Rice ubiquitin promoters: deletion analysis and potential usefulness in plant transformation systems. Plant Cell Rep 22:129–134

    Article  PubMed  CAS  Google Scholar 

  • Wei HR, Wang ML, Moore PH, Albert HH (2003) Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. Plant Physiol 160:1241–1251

    Article  CAS  Google Scholar 

  • Zubko E, Scutt C, Meyer P (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 18:442–445

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a fund from the National Academy of Agricultural Science (PJ006834) and a grant from the Next-Generation BioGreen 21 Program (PJ008184), Rural Development Administration, Republic of Korea.

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Authors and Affiliations

  1. National Academy of Agricultural Science, Rural Development Administration, Suwon, 441-707, Republic of Korea

    Sun-Hyung Lim, Seong-Han Sohn, Dong-Hern Kim, Jae Kwang Kim, Jong-Yeol Lee, Young-Mi Kim & Sun-Hwa Ha

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  1. Sun-Hyung Lim
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  2. Seong-Han Sohn
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  3. Dong-Hern Kim
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  4. Jae Kwang Kim
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  5. Jong-Yeol Lee
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  7. Sun-Hwa Ha
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Correspondence to Sun-Hwa Ha.

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Lim, SH., Sohn, SH., Kim, DH. et al. Use of an anthocyanin production phenotype as a visible selection marker system in transgenic tobacco plant. Plant Biotechnol Rep 6, 203–211 (2012). https://doi.org/10.1007/s11816-012-0215-6

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  • DOI: https://doi.org/10.1007/s11816-012-0215-6

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