Skip to main content
Log in

Generation of selectable marker-free transgenic eggplant resistant to Alternaria solani using the R/RS site-specific recombination system

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

Marker-free transgenic eggplants, exhibiting enhanced resistance to Alternaria solani , can be generated on plant growth regulators (PGRs)- and antibiotic-free MS medium employing the multi-auto-transformation (MAT) vector, pMAT21 - wasabi defensin , wherein isopentenyl transferase ( ipt ) gene is used as a positive selection marker.

Abstract

Use of the selection marker genes conferring antibiotic or herbicide resistance in transgenic plants has been considered a serious problem for environment and the public. Multi-auto-transformation (MAT) vector system has been one of the tools to excise the selection marker gene and produce marker-free transgenic plants. Ipt gene was used as a selection marker gene. Wasabi defensin gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), was used as a gene of interest. Wasabi defensin gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledon explants of eggplant were cultured on PGRs- and antibiotic-free MS medium. Extreme shooty phenotype/ipt shoots were produced by the explants infected with the pMAT21-wasabi defensin (WD). The same PGRs- and antibiotic-free MS medium was used in subcultures of the ipt shoots. Subsequently, morphologically normal shoots emerged from the Ipt shoots. Molecular analyses of genomic DNA from transgenic plants confirmed the integration of the WD gene and excision of the selection marker (ipt gene). Expression of the WD gene was confirmed by RT-PCR and Northern blot analyses. In vitro whole plant and detached leaf assay of the marker-free transgenic plants exhibited enhanced resistance against Alternaria solani.

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

Access this article

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
Fig. 7

Similar content being viewed by others

References

  • Akama K, Puchta H, Hohn B (1995) Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker. Plant Cell Rep 14:450–454

    Article  CAS  PubMed  Google Scholar 

  • Araki H, Jearnpipatkula A, Tatsumi H, Sakurai T, Ushino K, Muta T, Oshima Y (1987) Molecular and functional organization of yeast plasmid pSR1. J Mol Biol 182:191–203

    Article  Google Scholar 

  • Arpaia S, Mennella G, Onofaro V, Perri E, Sunseri F, Rotino GL (1997) Production of transgenic eggplant (Solanum melongena L.) resistant to Colorado potato beetle (Leptinotarsa decemlineata Say). Theor Appl Genet 95:329–334

    Article  CAS  Google Scholar 

  • Ballester R, Cervera M, Peña L (2007) Efficient production of transgenic citrus plants using isopentenyltransferase positive selection and removal of the marker gene by site-specific recombination. Plant Cell Rep 26:39–45

    Article  CAS  PubMed  Google Scholar 

  • 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 Organ Cult 102:329–336

    Article  CAS  Google Scholar 

  • Bevan MW, Flavell RB, Chilton MD (1983) A chimeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304:184–187

    Article  CAS  Google Scholar 

  • Broekaert WF, Cammue BPA, De Bolle MFC, Thevissen K, de Samblanx GW, Osborn RW (1997) Antimicrobial peptides from plants. Crit Rev Plant Sci 16:297–323

    Article  CAS  Google Scholar 

  • Cammue BPA, De Bolle MFC, Schoofs HME, Terras FRG, Thevissen K, Osborn RW, Rees SB, Broekaert WF (1994) Gene encoded antimicrobial peptides from plants. In: Boman HG, Marsh J, Goode JA (eds) Antimicrobial peptides, Ciba Foundation Symposium 186. John Wiley & Sons, New York, pp 91–106

    Google Scholar 

  • Darbani B, Elimanifar A, Stewart CN, Camargo WN (2007) Methods to produce marker-free transgenic plants. Biotechnol J 2:83–90

    Article  CAS  PubMed  Google Scholar 

  • Ebinuma H, Komamine A (2001) MAT (multi-auto-transformation) vector system. The oncogenes of Agrobacterium as positive markers for regeneration and selection of marker-free transgenic plants. In Vitro Cell Dev Biol Plant 37:103–113

    Article  CAS  Google Scholar 

  • 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 99:2117–2121

    Article  Google Scholar 

  • Endo S, Kasahara T, Sugita K, Ebinuma H (2002) A new GSTMAT vector containing both ipt and iaaM/H genes can produce marker-free transgenic tobacco plants with higher frequency. Plant Cell Rep 20:923–928

    Article  CAS  Google Scholar 

  • Gressel J (1992) Indiscriminate use of selectable markers: sowing wild oats? TIBTECH 10:382

    Article  Google Scholar 

  • Hare P, Chua N-H (2002) Excision of selectable marker genes from transgenic plants. Nat Biotechnol 20:575–580

    CAS  PubMed  Google Scholar 

  • Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species—opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56

    Article  CAS  PubMed  Google Scholar 

  • Hewelt A, Prinsen E, Schell J, Van Onckelen H, Schmuülling T (1994) Promoter tagging with a promoterless ipt gene leads to cytokinin-induced phenotypic variability in transgenic tobacco plants: implications of gene dosage effects. Plant J 6:879–891

    Article  CAS  PubMed  Google Scholar 

  • Hill R, Sendashonga C (2006) Conservation biology, genetically modified organisms, and the biosafety protocol. Conserv Biol 20:1620–1625

    Article  PubMed  Google Scholar 

  • Hohn B, Levy A, Putcha H (2001) Elimination of selection markers from transgenic plants. Curr Opin Biotechnol 12:139–143

    Article  CAS  PubMed  Google Scholar 

  • Jaiwal PK, Sahoo L, Singh ND, Singh RP (2002) Strategies to deal with the concern about marker genes in transgenic plants: some environment friendly approaches. Curr Sci 83:128–136

    CAS  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Article  CAS  Google Scholar 

  • Kanzaki H, Nirasawa S, Saitoh H, Ito M, Nishihara M, Terauchi R, Nakamura I (2002) Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor Appl Genet 105:809–814

    Article  CAS  PubMed  Google Scholar 

  • Khan RS, Chin DP, Nakamura I, Mii M (2006a) Production of marker-free Nierembergia caerulea using MAT vector system. Plant Cell Rep 25:914–919

    Article  CAS  PubMed  Google Scholar 

  • Khan RS, Nishihara M, Yamamura S, Nakamura I, Mii M (2006b) Transgenic potatoes expressing wasabi defensin peptide confer partial resistance to gray mold (Botrytis cinerea). Plant Biotechnol 23:179–183

    Article  Google Scholar 

  • Khan RS, Nakamura I, Mii M (2010) Production and selection of marker-free transgenic plants of Petunia hybrida using site-specific recombination. Biol Plantarum 54:265–271

    Article  CAS  Google Scholar 

  • Khan RS, Ntui VO, Chin DP, Nakamura I, Mii M (2011a) Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker. Plant Cell Rep 30:587–597

    Article  CAS  PubMed  Google Scholar 

  • Khan RS, Nakamura I, Mii M (2011b) Development of disease-resistant marker-free tomato by R/RS site-specific recombination. Plant Cell Rep 30:1041–1053

    Article  CAS  PubMed  Google Scholar 

  • Kiba A, Saitoh H, Nishihara M, Omiya K, Yamamura S (2003) C-terminal domain of a hevein-like protein from Wasabia japnica has potent antimicrobial activity. Plant Cell Physiol 44:296–303

    Article  CAS  PubMed  Google Scholar 

  • Kumar PA, Mandaokar A, Sreenivasu K, Chakrabarti SK, Bisaria S, Sharma SR, Kaur S, Sharma RP (1998) Insect-resistant transgenic brinjal plants. Mol Breed 4:33–37

    Article  CAS  Google Scholar 

  • Lu L, Wu X, Yin X, Morrand J, Chen X, Folk WR, Zhang ZJ (2009) Development of marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] using standard binary vectors with bar as a selectable marker. Plant Cell Tiss Organ Cult 99:97–108

    Article  CAS  Google Scholar 

  • Matsunaga E, Sugita K, Ebinuma H (2002) Asexual production of selectable-marker free transgenic woody plants, vegetatively propagated species. Mol Breed 10:95–106

    Article  CAS  Google Scholar 

  • Medford JI, Horgan R, EI-Sawi Z, Klee HJ (1989) Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. Plant Cell 1:403–413

    CAS  PubMed Central  PubMed  Google Scholar 

  • Moreno M, Segura A, Garcia-Olmedo F (1994) Pseudothionin-St1, a potato peptide active against potato pathogens. Eur J Biochem 223:135–139

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Ntui VO, Thirukkumaran G, Azadi P, Khan RS, Nakamura I, Mii M (2010) Stable integration and expression of wasabi defensin gene in ‘‘Egusi’’ melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot. Plant Cell Rep 29:943–954

    Article  CAS  PubMed  Google Scholar 

  • Pedras MSC, Sorensen JI, Okanga FI, Zaharia I (1999) Wasalexins A and B, new phytoalexins from wasabi: isolation and synthesis, and antifungal activity. Med Chem Lett 9:3015–3020

    Article  CAS  Google Scholar 

  • Rogers OS, Bendich JA (1988) Extraction of DNA from plant tissues. In: Gelvin SB, Schiliperoort RA, Verma DPS (eds) Plant molecular biology manual, vol A6. Kluwer Academic Publishers, Dordrecht, pp 1–10

    Google Scholar 

  • Rotino GL, Gleddie S (1990) Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vectors. Plant Cell Rep 9:26–29

    Article  CAS  PubMed  Google Scholar 

  • Roy SD, Saxena M, Bhomkar PS, Pooggin M, Hohn T, Sarin NB (2008) Generation of marker-free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters. Plant Cell Tiss Organ Cult 95:1–11

    Article  CAS  Google Scholar 

  • Saelim L, Phansiri S, Suksangpanomrung M, Netrphan S, Narangajavana J (2009) Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants. Plant Cell Rep 28:445–455

    Article  CAS  PubMed  Google Scholar 

  • Saitoh H, Kiba A, Nishihara M, Yamamura S, Suzuki K, Terauchi R (2001) Production of antimicrobial defensin in Nicotiana benthamiana with a potato virus X vector. Mol Plant Microbe Int 14:111–115

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Scaramelli L, Balestrazzi A, Bonadei M, Piano E, Carbonera D, Confalonieri M (2009) Production of transgenic barrel medic (Medicago truncatula Gaernt.) using the ipt-type MAT vector system and impairment of recombinase-mediated excision events. Plant Cell Rep 28:197–211

    Article  CAS  PubMed  Google Scholar 

  • Sjahril R, Chin DP, Khan RS, Yamamura S, Nakamura I, Amemiya Y, Mii M (2006) Transgenic Phalaenopsis plants with resistance to Erwinia caratovora produced by introducing wasabi defensin gene using Agrobacterium method. Plant Biotechnol 23:191–194

    Article  CAS  Google Scholar 

  • Smigocki AC (1991) Cytokinin content and tissue distribution in plants transformed by a reconstructed isopentenyl transferase gene. Plant Mol Biol 16:105–115

    Article  CAS  PubMed  Google Scholar 

  • Sugita K, Matsunaga E, Ebinuma H (1999) Effective selection system for generating marker-free transgenic plants independent of sexual crossing. Plant Cell Rep 18:941–947

    Article  CAS  Google Scholar 

  • Sugita K, Matsunaga E, Kasahara T, Ebinuma H (2000) Transgene stacking in plants in the absence of sexual crossing. Mol Breed 6:529–536

    Article  CAS  Google Scholar 

  • Terras FRG, Eggernont K, Kovaleva V et al (1995) Small cysteine-rich antifungal proteins from radish: their role in host defense. Plant Cell 7:573–588

    CAS  PubMed Central  PubMed  Google Scholar 

  • Thirukkumaran G, Khan RS, Chin DP, Nakamura I, Mii M (2009) Isopentenyltransferase gene expression offers the positive selection of marker-free transgenic plant of Kalanchoe Blossfeldiana. Plant Cell Tissue Organ Cult 97:237–242

    Article  CAS  Google Scholar 

  • Thomma B, Eggermont K, Penninckx I, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci USA 95:15107–15111

    Article  CAS  PubMed  Google Scholar 

  • Vetten N, Wolters AM, Raemkers K, Meer I, Stege R, Heeres E, Heeres P, Visser R (2003) A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nat Biotechnol 21:4339–4442

    Google Scholar 

  • Weigel D, Glazebrook J (2006) Transformation of Agrobacterium Using the Freeze–Thaw Method. Cold Spring Harb Protoc. doi:10.1101/pdb.prot4666

  • Yoder JI, Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. Biotechnology 12:263–267

    Article  CAS  Google Scholar 

  • 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 Tissue Org Cult 91:61–72

    Article  CAS  Google Scholar 

  • Zhang X, Wang J, Letham DS, McKinney SA, Higgins TJV (1995) The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene. Planta 196:84–94

    Article  CAS  Google Scholar 

  • Zhang Y, Liu H, Li B, Zhang JT, Li Y, Zhang H (2009) Generation of selectable marker-free transgenic tomato resistant to drought, cold and oxidative stress using the Cre/loxP DNA excision system. Transgenic Res 18:607–619

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are very grateful to the Pulp and Paper Research group, Nippon Paper Industries, Tokyo, who kindly provided the MAT vector constructs. We are also thankful to the Egyptian Ministry of Higher Education for their financial support.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Raham Sher Khan.

Additional information

Communicated by H. Ebinuma.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Darwish, N.A., Khan, R.S., Ntui, V.O. et al. Generation of selectable marker-free transgenic eggplant resistant to Alternaria solani using the R/RS site-specific recombination system. Plant Cell Rep 33, 411–421 (2014). https://doi.org/10.1007/s00299-013-1541-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-013-1541-z

Keywords

Navigation