Plant Cell Reports

, Volume 32, Issue 10, pp 1601–1613 | Cite as

Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection

  • Xiuping ZouEmail author
  • Aihong Peng
  • Lanzhen Xu
  • Xiaofeng Liu
  • Tiangang Lei
  • Lixiao Yao
  • Yongrui HeEmail author
  • Shanchun ChenEmail author
Original Paper


Key message

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus.


The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.


Citrus Genetic transformation ipt Marker-free Cre/loxP 



We are grateful to Prof Xiaochun Zhao (Citrus Research Institute, Chinese Academy of Agricultural Sciences) for his critical reading of the manuscript. This work was supported by grants from the National Natural Sciences Foundation of China (31272150, to X. Zou), the Ministry of Agriculture ‘Introduce International Advanced Agriculture Science and Technology’ (‘948’ project, to R. He), Program for Changjiang Scholars and Innovative Research Team in University (IRT0976, to S. Chen), and Natural Science Foundation Project of CQ (CSTC, to A. Peng).

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

299_2013_1470_MOESM1_ESM.docx (536 kb)
Supplementary material 1 (DOCX 535 kb)


  1. Atkins D, Young M, Uzzell S, Kelly L, Fillatti J, Gerlach WL (1995) The expression of antisense and ribozyme genes targeting citrus exocortis viroid in transgenic plants. J Gen Virol 76:1781–1790PubMedCrossRefGoogle Scholar
  2. Ballester A, Cervera M, Peña L (2007) Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination. Plant Cell Rep 26:39–45PubMedCrossRefGoogle Scholar
  3. Ballester A, Cervera M, Peña L (2008) Evaluation of selection strategies alternative to nptII in genetic transformation of citrus. Plant Cell Rep 27:1005–1015PubMedCrossRefGoogle Scholar
  4. Ballester A, Cervera M, Peña L (2010) Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants. Plant Cell Tiss Org Cult 102:329–336CrossRefGoogle Scholar
  5. Boscariol R, Almeida W, Derbyshire M, Mourão Filho F, Mendes B (2003) The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Rep 22:122–128PubMedCrossRefGoogle Scholar
  6. Boscariol RL, Monteiro M, Takahashi EK, Chabregas SM, Vieira MLC, Vieira LGE, Pereira LFP, de AA Mourão Filho F, Cardoso SC, Christiano RSC (2006) Attacin A Gene from Tricloplusia ni Reduces Susceptibility to Xanthomonas axonopodis pv. citri in Transgenic Citrus sinensis ‘Hamlin’. J Am Soc Hortic Sci 131:530–536Google Scholar
  7. Cervera M, Juârez J, Navarro A, Pina JA, Durân-Vila N, Navarro L, Peña L (1998a) Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage. Transgenic Res 7:51–59CrossRefGoogle Scholar
  8. Cervera M, Pina J, Juârez J, Navarro L, Peña L (1998b) Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep 18:271–278CrossRefGoogle Scholar
  9. Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88:10558–10562PubMedCrossRefGoogle Scholar
  10. Daniell H (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol 20:581–586PubMedGoogle Scholar
  11. Daniell H, Kumar S, Dufourmantel N (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends Biotechnol 23:238–245PubMedCrossRefGoogle Scholar
  12. Domínguez A, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2000) Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Rep 19:427–433CrossRefGoogle Scholar
  13. Domínguez A, de Mendoza AH, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2002a) Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic Mexican lime (Citrus aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein gene. Mol Breed 10:1–10CrossRefGoogle Scholar
  14. Domínguez A, Fagoaga C, Navarro L, Moreno P, Peña L (2002b) Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes. Mol Genet Genomics 267:544–556PubMedCrossRefGoogle Scholar
  15. Domínguez A, Cervera M, Pérez RM, Romero J, Fagoaga C, Cubero J, López MM, Juárez JA, Navarro L, Peña L (2004) Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol Breed 14:171–183CrossRefGoogle Scholar
  16. Ebinuma H, Sugita K, Matsunaga E, Yamakado M (1997) Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci USA 94:2117–2121PubMedCrossRefGoogle Scholar
  17. Endo S, Sugita K, Sakai M, Tanaka H, Ebinuma H (2002) Single-step transformation for generating marker-ree transgenic rice using the ipt-type MAT vector system. Plant J 30:115–122PubMedCrossRefGoogle Scholar
  18. Fagoaga C, López C, De Mendoza AH, Moreno P, Navarro L, Flores R, Peña L (2006) Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime. Plant Mol Biol 60:153–165PubMedCrossRefGoogle Scholar
  19. Febres VJ, Lee RF, Moore GA (2008) Transgenic resistance to Citrus tristeza virus in grapefruit. Plant Cell Rep 27:93–104PubMedCrossRefGoogle Scholar
  20. Febres V, Fisher L, Khalaf A, Moore GA (2011) Citrus transformation: challenges and prospects. Genetic transformation by intech. pp 101–122Google Scholar
  21. Fladung M, Becker D (2011) Targeted integration and removal of transgenes in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) using site-specific recombination systems. Plant Biol 13:223–223CrossRefGoogle Scholar
  22. Gambino G, Gribaudo I (2012) Genetic transformation of fruit trees: current status and remaining challenges. Transgenic Res 21:1163–1181PubMedCrossRefGoogle Scholar
  23. Ghorbel R, Domínguez A, Navarro L, Peña L (2000) High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus. Tree Physiol 20:1183–1189PubMedCrossRefGoogle Scholar
  24. Guo W, Duan Y, Olivares-Fuster O, Wu Z, Arias CR, Burns JK, Grosser JW (2005) Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene. Plant Cell Rep 24:482–486PubMedCrossRefGoogle Scholar
  25. Hare PD, Chua N-H (2002) Excision of selectable marker genes from transgenic plants. Nature Biotechnol 20:575–580Google Scholar
  26. He Y, Chen S, Peng A, Zou X, Xu L, Lei T (2011) Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds. Sci Hortic 128:99–107CrossRefGoogle Scholar
  27. Höfgen R, Willmitzer L (1990) Biochemical and genetic analysis of different patatin isoforms expressed in various organs of potato (Solanum tuberosum). Plant Sci 66:221–230CrossRefGoogle Scholar
  28. Khan RS, Ntui VO, Chin DP, Nakamura I, Mii M (2011) Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker. Plant Cell Rep 30:587–597PubMedCrossRefGoogle Scholar
  29. López C, Cervera M, Fagoaga C, Moreno P, Navarro L, Flores R, Peña L (2010) Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime. Mol Plant Pathol 11:33–41PubMedCrossRefGoogle Scholar
  30. López-Noguera S, Petri C, Burgos L (2009) Combining a regeneration-promoting ipt gene and site-specific recombination allows a more efficient apricot transformation and the elimination of marker genes. Plant Cell Rep 28:1781–1790PubMedCrossRefGoogle Scholar
  31. Luke Mankin S, Thompson WF (2001) New green fluorescent protein genes for plant transformation: intron-containing, ER-localized, and soluble-modified. Plant Mol Biol Rep 19:13–26CrossRefGoogle Scholar
  32. Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Stewart CN Jr, McAvoy R, Jiang X, Wu Y (2007) ‘GM-gene-deletor’: fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–374PubMedCrossRefGoogle Scholar
  33. Luo K, Sun M, Deng W, Xu S (2008) Excision of selectable marker gene from transgenic tobacco using the GM-gene-deletor system regulated by a heat-inducible promoter. Biotechnol Lett 30:1295–1302PubMedCrossRefGoogle Scholar
  34. Malnoy M, Boresjza-Wysocka EE, Norelli JL, Flaishman MA, Gidoni D, Aldwinckle HS (2010) Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene. Tree genet genomes 6:423–433CrossRefGoogle Scholar
  35. Mlynárová L, Nap J (2003) A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco. Transgenic Res 12:45–57PubMedCrossRefGoogle Scholar
  36. Mlynárová L, Conner AJ, Nap JP (2006) Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes. Plant Biotechnol J 4:445–452PubMedCrossRefGoogle Scholar
  37. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  38. Ow DW (2002) Recombinase-directed plant transformation for the post-genomic era. Plant Mol Biol 48:183–200PubMedCrossRefGoogle Scholar
  39. Peña L, Martín-Trillo M, Juárez J, Pina JA, Navarro L, Martínez-Zapater JM (2001) Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nat Biotechnol 19:263–267PubMedCrossRefGoogle Scholar
  40. Petri C, Hily JM, Vann C, Dardick C, Scorza R (2011) A high-throughput transformation system allows the regeneration of marker-free plum plants (Prunus domestica). Ann Appl Biol 159:302–315CrossRefGoogle Scholar
  41. Petri C, López-Noguera S, Wang H, García-Almodóvar C, Alburquerque N, Burgos L (2012) A chemical-inducible Cre-LoxP system allows for elimination of selection marker genes in transgenic apricot. Plant Cell Tiss Org Cult 110:337–346CrossRefGoogle Scholar
  42. Privalle LS, Wright M, Reed J, Hansen G, Dawson J, Dunder EM, Chang YF, Powell ML, Meghji M (2000) Phosphomannose isomerase—a novel system for plant selection. In: The biosafety of genetically modified organisms. International biosafety symposium Advisory Committee, pp 171–178Google Scholar
  43. Ramessar K, Peremarti A, Gómez-Galera S, Naqvi S, Moralejo M, Munoz P, Capell T, Christou P (2007) Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics. Transgenic Res 16:261–280PubMedCrossRefGoogle Scholar
  44. Ruiz O, Daniell H (2005) Engineering cytoplasmic male sterility via the chloroplast genome by expression of β-ketothiolase. Plant Physiol 138:1232PubMedCrossRefGoogle Scholar
  45. Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual (2001) 3rd edn. Cold Spring Harbor. NY Cold Spring Harbor LaboratoryGoogle Scholar
  46. Stoykova P, Stoeva-Popova P (2011) PMI (manA) as a nonantibiotic selectable marker gene in plant biotechnology. Plant Cell Tiss Org Cult 105:141–148CrossRefGoogle Scholar
  47. Sugita K, Matsunaga E, Ebinuma H (1999) Effective selection system for generating marker-free transgenic plants independent of sexual crossing. Plant Cell Rep 18:941–947CrossRefGoogle Scholar
  48. Tuteja N, Verma S, Sahoo RK, Raveendar S, Reddy IBL (2012) Recent advances in development of marker free transgenic plants: regulation and biosafety concern. J Biosci 37:162–197CrossRefGoogle Scholar
  49. Van Ex F, Verweire D, Claeys M, Depicker A, Angenon G (2009) Evaluation of seven promoters to achieve germline directed Cre-lox recombination in Arabidopsis thaliana. Plant Cell Rep 28:1509–1520PubMedCrossRefGoogle Scholar
  50. Vanblaere T, Szankowski I, Schaart J, Schouten H, Flachowsky H, Broggini GA, Gessler C (2011) The development of a cisgenic apple plant. J Biotechnol 154:304–311PubMedCrossRefGoogle Scholar
  51. Vervliet G, Holsters M, Teuchy H, Van Montagu M, Schell J (1975) Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains. J Gen Virol 26:33–48PubMedCrossRefGoogle Scholar
  52. Verweire D, Verleyen K, De Buck S, Claeys M, Angenon G (2007) Marker-free transgenic plants through genetically programmed auto-excision. Plant Physiol 145:1220–1231PubMedCrossRefGoogle Scholar
  53. Wong WS, Li GG, Ning W, Xu ZF, Hsiao W, Zhang LY, Li N (2001) Repression of chilling-induced ACC accumulation in transgenic citrus by over-production of antisense 1-aminocyclopropane-1-carboxylate synthase RNA. Plant Sci 161:969–977CrossRefGoogle Scholar
  54. Zelasco S, Ressegotti V, Confalonieri M, Carbonera D, Calligari P, Bonadei M, Bisoffi S, Yamada K, Balestrazzi A (2007) Evaluation of MAT-vector system in white poplar (Populus alba L.) and production of ipt marker-free transgenic plants by ‘single-step transformation’. Plant Cell Tiss Org Cult 91:61–72CrossRefGoogle Scholar
  55. Zou X, Li D, Luo X, Luo K, Pei Y (2008) An improved procedure for Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata L. Raf.) via indirect organogenesis. In Vitro Cell Dev Pl 44:169–177CrossRefGoogle Scholar
  56. Zuo J, Niu Q-W, Møller SG, Chua N-H (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nature biotechnol 19:157–161CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Citrus Research InstituteChinese Academy of Agricultural SciencesChongqingPeople’s Republic of China
  2. 2.National Center for Citrus Variety ImprovementChongqingPeople’s Republic of China

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