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The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes

  • Genetic Transformation and Hybridization
  • Published:
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Abstract

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. ‘Baltica’, respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

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Abbreviations

GA:

Gibberellic acid

gfp :

Green fluorescent protein gene

GFP:

Green fluorescent protein

gus :

β-Glucuronidase gene

GUS:

β-Glucuronidase

luc :

Luciferase gene

MS:

Murashige and Skoog (1962)

NAA:

Naphthaleneacetic acid

nptII:

Neomycin phosphotransferase gene

PCR:

Polymerase chain reaction

RT-PCR reverse:

Transcriptase-polymerase chain reaction

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Acknowledgements

A German Academic Exchange Service (DAAD) Scholarship and a German–Romanian Bilateral Project are acknowledged for supporting part of this research (ER-T). The EU Erasmus–Socrates programme provided partial support for CD. Dr. G. Hahne and Dr. J. Molinier kindly provided pHB2892.

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Authors and Affiliations

  1. Babes-Bolyai University, Plant Genetic Engineering Group, 400006, Cluj-Napoca, Romania

    Elena Rakosy-Tican, Cristian M. Aurori & Adriana Aurori

  2. School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK

    Camelia Dijkstra & Michael R. Davey

  3. Federal Centre for Breeding Research on Cultivated Plants, Institute of Agricultural Crops, Groß Lüsewitz, Germany

    Ramona Thieme

Authors
  1. Elena Rakosy-Tican
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  2. Cristian M. Aurori
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  3. Camelia Dijkstra
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  4. Ramona Thieme
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  5. Adriana Aurori
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  6. Michael R. Davey
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Correspondence to Elena Rakosy-Tican.

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Communicated by L. Peña

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Rakosy-Tican, E., Aurori, C.M., Dijkstra, C. et al. The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes. Plant Cell Rep 26, 661–671 (2007). https://doi.org/10.1007/s00299-006-0273-8

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