Plant Cell Reports

, Volume 26, Issue 5, pp 661–671

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

  • Elena Rakosy-Tican
  • Cristian M. Aurori
  • Camelia Dijkstra
  • Ramona Thieme
  • Adriana Aurori
  • Michael R. Davey
Genetic Transformation and Hybridization

DOI: 10.1007/s00299-006-0273-8

Cite this article as:
Rakosy-Tican, E., Aurori, C.M., Dijkstra, C. et al. Plant Cell Rep (2007) 26: 661. doi:10.1007/s00299-006-0273-8

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.

Keywords

Agrobacterium tumefaciens-mediated transformationChimerasGreen fluorescent protein reporter geneNeomycin phophotransferase marker geneTransgene silencing

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

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Elena Rakosy-Tican
    • 1
  • Cristian M. Aurori
    • 1
  • Camelia Dijkstra
    • 2
  • Ramona Thieme
    • 3
  • Adriana Aurori
    • 1
  • Michael R. Davey
    • 2
  1. 1.Babes-Bolyai University, Plant Genetic Engineering GroupCluj-NapocaRomania
  2. 2.School of BiosciencesUniversity of NottinghamLoughboroughUK
  3. 3.Federal Centre for Breeding Research on Cultivated PlantsInstitute of Agricultural CropsGroß LüsewitzGermany