Plant Molecular Biology

, Volume 62, Issue 1–2, pp 71–82 | Cite as

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

  • Wilmer Cuellar
  • Amélie Gaudin
  • Dennis Solórzano
  • Armando Casas
  • Luis Ñopo
  • Prakash Chudalayandi
  • Giuliana Medrano
  • Jan Kreuze
  • Marc Ghislain
Original Paper

Abstract

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophilamelanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.

Keywords

Cre-loxP recombination system Gene excision Selectable marker Solanum tuberosum Transgenic potato 

Abbreviations

CaMV 35S

Cauliflower mosaic virus 35S

cDNA

Complementary DNA

cox

Cytochrome oxidase

Ct

Cycle threshold

CTAB

Cetyl trimethyl ammonium bromide

EDTA

Ethylenediaminetetraacetic acid

hsp70

Heat-shock protein 70

LB

Luria-Bertani

loxP

Locus of X-ing over

MCS

Multiple cloning site

MS

Murashige and Skoog

M-MuLV

Moloney murine leukemia virus

NaCl

Sodium clhoride

nptII

Neomycin phosphotransferase II

NOS

Nopaline syntase

T-DNA

Transferred DNA

TBE

Tris-borate-ethylenediaminetetraacetic acid

pCIP

Plasmid International Potato Center

PCR

Polymerase chain reaction

RT-qPCR

Real time-quantitative polymerase chain reaction

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Wilmer Cuellar
    • 1
    • 2
    • 4
  • Amélie Gaudin
    • 1
  • Dennis Solórzano
    • 1
    • 2
  • Armando Casas
    • 1
    • 5
  • Luis Ñopo
    • 1
    • 6
  • Prakash Chudalayandi
    • 1
    • 7
  • Giuliana Medrano
    • 1
  • Jan Kreuze
    • 1
    • 3
  • Marc Ghislain
    • 1
  1. 1.Applied Biotechnology Laboratory, Germplasm enhancement and Crop Improvement DivisionInternational Potato Center CIPLimaPeru
  2. 2.Universidad Nacional Frederico VillarrealPueblo LibrePeru
  3. 3.Department of Plant Biology and Forest GeneticsSwedish University of Agricultural Sciences (SLU)UppsalaSweden
  4. 4.Department of Applied BiologyUniversity of HelsinkiHelsinkiFinland
  5. 5.Plant Science Initiative, E209 Beadle CenterUniversity of Nebraska-LincolnLincolnUSA
  6. 6.Arkansas Biosciences InstituteArkansas State UniversityJonesboroUSA
  7. 7.Department of Cell Biology and PhysiologyWashington UniversitySt LouisUSA

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