Skip to main content
SpringerLink
Log in

A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either β-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

35S :

Cauliflower mosaic virus 35S promoter

Als:

Acetolactate synthase

app1 :

An Arabidopsis embryo-specific promoter

Cre:

Cre recombinase

Gat:

Glyphosate acetyltransferase

Gus:

β-Glucuronidase

Hpt:

Hygromycin β-phosphotransferase

loxP :

Cre recombinase recognition site of phage P1

nos3 :

Nopaline synthase transcription terminator

pinii :

Potato proteinase inhibitor II protein transcription terminator

scp1 :

A synthetic plant constitutive promoter

T0 plants:

The first generation of transgenic soybean plants regenerated from tissue culture

References

  • Bayley CC, Morgan M, Dale EC, Ow DW (1992) Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site specific recombination system. Plant Mol Biol 18:353–361

    Article  PubMed  CAS  Google Scholar 

  • Bowen BA, Bruce WB, Lu G, Sims LE, Tagliani LA (2003) Synthetic promoters. United States Patent No. 6555673 B1

  • Castle LA, Siehl DL, Gorton R, Patten PA, Chen YH, Bertain S, Cho H-J, Duck N, Wong J, Liu D, Lassner MW (2004) Discovery and directed evolution of a glyphosate tolerance gene. Science 304:1151–1154

    Article  PubMed  CAS  Google Scholar 

  • Cuellar W, Gaudin A, Solórzano D, Casas A, Ñopo L, Chudalayandi P, Medrano G, Kreuze J, Ghislain M (2006) Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato. Plant Mol Biol 62:71–82

    Article  PubMed  CAS  Google Scholar 

  • Dale EC, Ow DW (1990) Intra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene 91:79–85

    Article  PubMed  CAS  Google Scholar 

  • Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88:10558–10562

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • de Frammond A, Back E, Chilton W, Kayes L, Chilton M-D (1986) Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation. Mol Gen Genet 202:125–131

    Article  Google Scholar 

  • Falco SC, Li Z (2000) S-adenosyl-l-methionine synthetase promoter and its use in expression of transgenic genes in plants. US patent publication WO 00/37662

  • Finer JJ, McMullen MD (1991) Transformation of soybean via particle bombardment of embryogenic suspension culture tissue. Vitro Cell Dev Biol-Plant 27:175–182

    Article  Google Scholar 

  • Gleave AP, Miltra DS, Mudge SR, Morris BAM (1999) Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol Biol 40:223–235

    Article  PubMed  CAS  Google Scholar 

  • Hoa TTC, Bong BB, Huq E, Hodge TK (2002) Cre/lox site-specific recombination controls the excision of a transgene from the rice genome. Theor Appl Genet 104:518–525

    Article  PubMed  CAS  Google Scholar 

  • Hoff T, Schnorr KM, Mundy J (2001) A recombinase-mediated transcriptional induction system in transgenic plants. Plant Mol Biol 45:41–49

    Article  PubMed  CAS  Google Scholar 

  • Jia H, Pang Y, Chen X, Fang R (2006) Removal of the selectable marker gene from transgenic tobacco plants by expression of Cre recombinase from a Tobacco Mosaic Virus vector through agroinfection. Trangenic Res 15:375–384

    Article  CAS  Google Scholar 

  • Kilby NJ, Davies GJ, Snaith MR (1995) FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis. Plant J 8:637–652

    Article  PubMed  CAS  Google Scholar 

  • Klimaszewska K, Rutledge RG, Seguin A (2005) Genetic transformation of conifers utilizing somatic embryogenesis. Methods Mol Biol (Clifton, NJ) 286:151–164

    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 

  • Kopertekh L, Jüttner J, Schiemann J (2004) Site-specific recombination induced in transgenic plants by PVX virus vector expressing bacteriophage P1 recombinase. Plant Sci 166:485–492

    Article  CAS  Google Scholar 

  • Kopertekh L, Schiemann J (2005) Agroinfiltration as a tool for transient expression of cre recombinase in vivo. Trans Res 14:793–798

    Article  CAS  Google Scholar 

  • Little EL, Magbanua ZV, Parrott WA (2000) A protocol for repetitive somatic embryogenesis from mature peanut epicotyls. Plant Cell Rep 19:351–357

    Article  CAS  Google Scholar 

  • Li Z, Thomas TL (1998) PEI1, an embryo-specific zinc finger protein gene required for heart-stage embryo formation in Arabidopsis. Plant Cell 10:283–398

    Google Scholar 

  • Lu HJ, Zhou XR, Gong ZX, Upadhyaya NM (2001) Generation of selectable marker-free transgenic rice using double right border (DRB) binary vectors. Aust J Plant Physiol 28:241–248

    Article  CAS  Google Scholar 

  • Luo H, Lyznik LA, Gidoni D, Hodges TK (2000) FLP-mediated recombination for use in hybrid plant production. Plant J 23:423–430

    Article  PubMed  CAS  Google Scholar 

  • Lutz KA, Bosacchi MH, Maliga P (2006) Plastid marker-gene excision by transiently expressed CRE recombinase. Plant J 45:447–456

    Article  PubMed  CAS  Google Scholar 

  • Maeser S, Kahmann R (1991) The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts. Mol Gen Genet 230:170–176

    Article  PubMed  CAS  Google Scholar 

  • Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: Applications, alternatives and biosafety. J Biotechnol 107:193–232

    Article  PubMed  CAS  Google Scholar 

  • Onouchi H, Yokoi K, Machida C, Matsuzaki H, Oshima Y, Matsuoka K, Nakamura K, Machida Y (1991) Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res 19:6373–6378

    Article  PubMed  CAS  Google Scholar 

  • Ow DW (2002) Recombinase-directed plant transformation for the post-genome era. Plant Mol Biol 48:183–200

    Article  PubMed  CAS  Google Scholar 

  • Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a transgene from the plant genome. Mol Gen Genet 234:49–59

    PubMed  CAS  Google Scholar 

  • Sairam R, Chennareddy S, Parani M, Zhang S, Al-Abed D, Abou-Alaiw W, Goldman S (2005) OBPC Symposium: Maize 2004 & beyond—plant regeneration, gene discovery, and genetic engineering of plants for crop improvement. Vitro Cell Dev Biol-Plant 41:411–423

    Article  CAS  Google Scholar 

  • Sakhanokho HF, Ozias-Akins P, May OL, Chee PW (2004) Induction of somatic embryogenesis and plant regeneration in selected Georgia and Pee Dee cotton lines. Crop Sci 44:2199–2205

    Article  Google Scholar 

  • Samoylov VM, Tucker DM, Thibaud-Nissen F, Parrott WA (1998) A liquid-medium-based protocol for rapid regeneration from embryogenic soybean cultures. Plant Cell Rep 18:49–54

    Article  CAS  Google Scholar 

  • Sreekala C, Wu L, 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 

  • Srivastava V, Anderson OD, Ow DW (1999) Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proc Natl Acad Sci USA 96:11,117–111, 121

    Google Scholar 

  • Stewart CN Jr, Adang MJ, All JN, Boerma HR, Cardineau G, Tucker D, Parrott WA (1996) Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cryIAc gene. Plant Physiol 112:121–129

    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 

  • Wang Y, Chen B, Hu Y, Li J, Li Z (2005) Inducible excision of selectable marker gene from transgenic plants by the Cre/lox site-specific recombination system. Trans Res 14:605–614

    Article  CAS  Google Scholar 

  • Xing A, Zhang Z, Sato S, Staswick P, Clemente T (2000) The use of the two T-DNA binary system to derive marker-free transgenic soybeans. Vitro Cell Dev Biol-Plant 36:456–463

    Article  CAS  Google Scholar 

  • Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003) Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 107:1157–1168

    Article  PubMed  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 

  • Zuo J, Niu QW, Moller SG, Chua NH (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnol 19:157–161

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. Lucy Liu from Pioneer Hi-Bred Int’l, Johnston, Iowa, USA for providing the anti-Gat antibody serum and E. coli expressed Gat protein; to Dr. Barbara J. Mazur from DuPont Agriculture & Nutrition, Wilmington, Delaware, USA for reviewing the manuscript.

Author information

Authors and Affiliations

  1. DuPont Agriculture & Nutrition, Experimental Station, E353-002C, Route 141 & Henry Clay Road, Wilmington, DE, 19880, USA

    Zhongsen Li, Aiqiu Xing, Bryan P. Moon, Susan A. Burgoyne, Anthony D. Guida, Huiling Liang, Catharina Lee, Cheryl S. Caster, Joanne E. Barton, Theodore M. Klein & Saverio C. Falco

Authors
  1. Zhongsen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aiqiu Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bryan P. Moon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Susan A. Burgoyne
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anthony D. Guida
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Huiling Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Catharina Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Cheryl S. Caster
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Joanne E. Barton
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Theodore M. Klein
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Saverio C. Falco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhongsen Li.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, Z., Xing, A., Moon, B.P. et al. A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants. Plant Mol Biol 65, 329–341 (2007). https://doi.org/10.1007/s11103-007-9223-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-007-9223-2

Keywords

Search

Navigation