Plant Cell Reports

, Volume 29, Issue 9, pp 943–954 | Cite as

Stable integration and expression of wasabi defensin gene in “Egusi” melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot

  • Valentine Otang Ntui
  • Gunaratnam Thirukkumaran
  • Pejman Azadi
  • Raham Sher Khan
  • Ikuo Nakamura
  • Masahiro MiiEmail author
Original Paper


Production of “Egusi” melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.


Colocynthis citrullus Cotyledon Genetic transformation Antifungal resistance Wasabi defensin 









Murashige and Skoog


Neomycin phosphotransferase


Polymerase chain reaction


Wasabi defensin


  1. Ajibola OO, Eniyemon SE, Fasina OO, Adeeko KA (1990) Mechanical expression of oil from melon seeds. J Agric Eng Res 45:45–53CrossRefGoogle Scholar
  2. Ali GS, Reddy ASN (2000) Inhibition of fungal and bacterial plant pathogens by synthetic peptides: in vitro growth inhibition, interaction between peptides and inhibition of disease progression. Mol Plant Microbe Interact 13:847–885CrossRefPubMedGoogle Scholar
  3. Blondelle SE, Houghten RA (1991) Hemolytic and antimicrobial activities of twenty-four individual omission analogues of melitin. Biochemistry 30:4671–4678CrossRefPubMedGoogle Scholar
  4. Bohlmann H (1994) The role of thionins in plant protection. Crit Rev Plant Sci 13:1–16CrossRefGoogle Scholar
  5. Broekaert WF, Terras FRG, Cammue BPA, Osborn RW (1995) Plant defensins: novel antimicrobial properties as components of the host defense system. Plant Physiol 108:1353–1358CrossRefPubMedGoogle Scholar
  6. Commue BPA, De Bolle MFC, Terras FRG, Proost P, Van Damme J, Rees SB, Vanderleyden J, Broekeart WF (1992) Analysis of two novel classes of antifungal proteins from radish (Raphanus sativa L.) seeds. J Biol Chem 267:2228–2233Google Scholar
  7. Durrant WE, Dong X (2004) Systemic acquired resistance. Annu Rev Phytopathol 42:185–209CrossRefPubMedGoogle Scholar
  8. Ekundayo CA, Idzi E (1990) Mycoflora and nutritional value of shelled melon seeds (Citrullus vulgaris Schrad.) in Nigeria. Plant Food Hum Nutr 40:215–222CrossRefGoogle Scholar
  9. Elomaa P, Helariutta Y, Griesbach RJ, Kotilainen M, Seppanen P, Terri T (1995) Transgene inactivation in Petunia hybrida is influenced by the properties of the foreign gene. Mol Gen Genet 248:649–656CrossRefPubMedGoogle Scholar
  10. Evans KJ, Nyquist WE, Latin RX (1992) A model based on temperature and leaf wetness duration for establishment of Alternaria leaf blight of muskmelon. Phytopathology 82:890–895CrossRefGoogle Scholar
  11. Finnegan J, McElroy D (1994) Transgene inactivation: plants fight back. Bio/Technology 12:883–888CrossRefGoogle Scholar
  12. Florack DEA, Stiekema EJ (1994) Thionins: properties, possible biological roles and mechanisms of action. Plant Mol Biol 26:25–37CrossRefPubMedGoogle Scholar
  13. Gaba V, Zelcer A, Gal-On A (2004) Cucurbit biotechnology-the importance of virus resistance. In Vitro Cell Dev Biol Plant 40:346–358CrossRefGoogle Scholar
  14. Giwa SO, Chuah LA, Adam NM (2009) Optimization of the production of biodiesel from ‘Egusi’ melon (Colocynthis citrullus L.) oil using response surface methodology. International Advance of Technology Congress (ATC), PWTC, Malaysia, Article No. 028, November 3–5, 2009Google Scholar
  15. Grant M, Lamb C (2006) Systemic immunity. Curr Opin Plant Biol 9:414–420CrossRefPubMedGoogle Scholar
  16. Helmerhorts EJ, Reijnders IM, van’t Hof W, Veerman EC, Nieuw Amerongen AV (1999) A critical comparison of the haemolytic and fungicidal activities of cationic antimicrobial peptides. FEBS Lett 449:105–110CrossRefGoogle Scholar
  17. Jach G, Gornhardt B, Mundy J, Logemann J, Pinsdorf E, Leeh R, Schell J, Mass C (1995) Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8:97–109CrossRefPubMedGoogle Scholar
  18. Kanzaki H, Nirasawa S, Saitoh H, Ito M, Nishihara M, Terauchi R, Nakamura I (2002) Over expression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor Appl Genet 105:809–814CrossRefPubMedGoogle Scholar
  19. Khan RS, Nishihara M, Yamamura S, Nakamura I, Mii M (2006) Transgenic potatoes expressing wasabi defensin peptide confer partial resistance to gray mold (Botrytis cinerea). Plant Biotechnol 23:179–183Google Scholar
  20. Krapp A, Hofmann B, Schafer E, Stitt M (1993) Regulation of the expression of rbcS and other photosynthetic genes by carbohydrates: a mechanism for the ‘sink’ regulation of photosynthesis. Plant J 3:817–828CrossRefGoogle Scholar
  21. Krishnamoorthy V, Kumar N, Angappan K, Soorianathasundaram K (2004) Evaluation of new banana hybrids against black leaf streak disease. InfoMusa 13:25–26Google Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  23. Matzke M, Matzke AJM, Scheid OM (1994) Inactivation of repeated genes: DNA-DNA interaction? In: Paszkowaski J (ed) Homologous recombination and gene silencing in plants. Kluwer Academic Publishers, Dordrecht, pp 271–307Google Scholar
  24. Mohamed YF, DaFalla GA, Omara SK (1994) Races of Fusarium oxysporum f.sp melonis causing wilt of melons in central Sudan. Cucurbit Genet Coop 17:66–68Google Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plantarum 15:473–497CrossRefGoogle Scholar
  26. Ntui VO, Thirukkumaran G, Iioka S, Mii M (2009) Efficient plant regeneration via organogenesis in melon (Colocynthis citrullus L.). Sci Hortic 119:397–402CrossRefGoogle Scholar
  27. Ogawa Y, Mii M (2007) Screening for high active β-lactam antibiotics against Agrobacterium tumefaciens. Arch Microbiol 181:331–336CrossRefGoogle Scholar
  28. Oyolu C (1977) A quantitative and qualitative study of seed type in ‘Egusi’ (Colocynthis citrullus L.). Trop Sci 19:55–62Google Scholar
  29. Peach C, Velten J (1991) Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17:49–60CrossRefPubMedGoogle Scholar
  30. Purseglove JW (1991) Tropical crops (Dicotyledons). Longman, New YorkGoogle Scholar
  31. Risser G, Banihashemi Z, Davis DW (1976) A proposed nomenclature of Fusarium oxysporum f.sp. melonis races and resistance genes in Cucumis melo. Phytopathology 66:1105–1106CrossRefGoogle Scholar
  32. 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 1–10Google Scholar
  33. Saitoh H, Kiba A, Nishihara M, Yamamura S, Suzuki K, Terauchi R (2001) Production of antimicrobial defensin in Nicotiana benthamiana with a potato virus × vector. Mol Plant Microbe Interact 14:111–115CrossRefPubMedGoogle Scholar
  34. Sanford JC, Johnson SA (1985) The concept of parasite-derived resistance: deriving resistance genes from the parasites own genome. J Theor Biol 115:395–405CrossRefGoogle Scholar
  35. Schippers RR (2000) African indigenous vegetables: an overview of the cultivated species. Natural Resources Institute/ACP-EU Technical Centre for Agricultural and Rural Cooperation, Chatham, UKGoogle Scholar
  36. Schippers RR (2004) Cucurbitaceae. In: Légumes africains indigènes: présentation des espèces cultivées. Margraf Publishers/CTA, Wuerzburg, Allemagne, pp 113–182Google Scholar
  37. Sijen T, Wellink JB, van Kammen A (1996) RNA-mediated virus resistance: role of repeated transgenes and delineation of targeted regions. Plant Cell 8:2277–2294CrossRefPubMedGoogle Scholar
  38. 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 23:191–194Google Scholar
  39. Staub JE, Grumet R (1993) Selection for multiple disease resistance reduces cucumber yield potential. Euphytica 67:205–213CrossRefGoogle Scholar
  40. Thevissen K, Terras FRG, Broekaert WF (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microb 65:5451–5458Google Scholar
  41. van der Vossen HAM, Denton OA, El Tahir IM (2004) Citrullus lanatus. In: Grubben GJH, Denton OA (eds) Plant resources of tropical Africa. 2. Vegetables. CTA, Wageningen, The Netherlands; Backhuys Publishers, Leiden, The Netherlands, pp 185–191Google Scholar
  42. Van Wees SC, Van der Ent S, Pieterse CM (2008) Plant immune response triggered by beneficial microbes. Curr Opin Plant Biol 11:443–448CrossRefPubMedGoogle Scholar
  43. Vodouhe RS, Achigan-Dako GE, Adjakidje V (2001) Observation de la diversité génétique des Egusi collectées au Bénin et au Togo. In: Agbo BP et al (eds) Actes 2 de l’atelier scientifique sud et centre, 12–13 décembre 2001, Institut National des Recherches Agricoles du Bénin. INRAB, Niaouli, Bénin, pp 53–61Google Scholar
  44. Wu HW, Yu TA, Raja JAJ, Wang HC, Yeh SD (2009) Generation of transgenic oriental melon resistant to Zucchini yellow mosaic virus by an improved cotyledon-cutting method. Plant Cell Rep 28:1053–1064CrossRefPubMedGoogle Scholar
  45. Yevtushenko DP, Romero R, Forward BS, Hancock RE, Kay WW, Misra S (2005) Pathogen-induced expression of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco. J Exp Bot 56:1685–1695CrossRefPubMedGoogle Scholar
  46. Zitter AM (1999) Fusarium wilt of melon, a world wide problem in temperate and Tropical regions. Acta Hortic 492:157–162Google Scholar
  47. Zuniga TL, Zitter TA (1995) Field screening of melon varieties and lines for multiple race resistance to Fusarium oxysporum f. sp melonis. Cucurbit Genet Coop 18:43Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Valentine Otang Ntui
    • 1
  • Gunaratnam Thirukkumaran
    • 1
  • Pejman Azadi
    • 1
  • Raham Sher Khan
    • 1
  • Ikuo Nakamura
    • 1
  • Masahiro Mii
    • 1
    Email author
  1. 1.Laboratory of Plant Cell Technology, Graduate School of HorticultureChiba UniversityMatsudoJapan

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