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Levels of phytoene and β-carotene in transgenic honeydew melon (Cucumis melo L. inodorus)

  • Yan Ren
  • Haejeen Bang
  • Eun Jin Lee
  • Jean Gould
  • Keerti S. Rathore
  • Bhimanagouda S. Patil
  • Kevin M. CrosbyEmail author
Original Paper

Abstract

Using a previously reported optimized Agrobacterium tumefaciens-mediated transformation protocol, a watermelon phytoene synthase-C (PSY-C) gene was introduced into the elite honeydew breeding line ‘150’. Putative transformants were selected on kanamycin-containing medium (from 150 to 50 mg·l−1) and presence of the transgene was confirmed using polymerase chain reaction (PCR). Moreover, Southern blot analysis confirmed integration of the transgene and revealed presence of one to two copies of the PSY-C transgene in the different transgenic lines. In addition, reverse transcription (RT)-PCR analysis revealed transcript levels of the transgene in different tissues of these transgenic lines. Using flow cytometric analysis, it was found that all T0 transgenic plants were tetraploid. Moreover, altered phenotypes (color change) were observed for rind tissues of transgenic lines. Based on high performance liquid chromatography (HPLC), β-carotene content and phytoene accumulation in fruit of transgenic lines were 32-fold and 11 μg·g−1 FW higher than the levels found in the control plants.

Keywords

Honeydew (Cucumis melo L. inodorusPhytoene synthase Polygalacturonase Phytoene β-Carotene Tetraploid Flesh color 

Abbreviations

PCR

Polymerase chain reaction

HPLC

High performance liquid chromatography

PG

Polygalacturonase

PSY

Phytoene synthase

T0

Regenerated transgenic plant from the original inoculated explant

T1

The progeny of the original T0 transgenic plants

Notes

Acknowledgments

This research was supported by the USDA-CSREES (2008-34402-19195, 2009-34402-19831) and USDA-NIFA (2010-34402-20875), “Designing Foods for Health” grants, through the Vegetable and Fruit Improvement Center, Texas AgriLife Research. We wish to thank Drs. Jim Giovannoni and Li Li at Cornell University for providing binary vectors and technical advice. We also appreciate Dr. Yong Hun Chi’s help in providing E. coli for gene construction and technical advice.

Supplementary material

11240_2012_269_MOESM1_ESM.doc (8.5 mb)
Supplementary material 1 (DOC 8726 kb)

References

  1. Aggelis A, John I, Grierson D (1997) Analysis of physiological and molecular changes in melon (Cucumis melo L.) varieties with different rates of ripening. J Exp Bot 48:769–778CrossRefGoogle Scholar
  2. Aluru M, Xu Y, Guo R, Wang Z, Li S, White W, Wang K, Rodermel S (2008) Generation of transgenic maize with enhanced provitamin A content. J Exp Bot 59:3551–3562PubMedCrossRefGoogle Scholar
  3. Ayub R, Guis M, Amor MB, Gillot L, Roustan JP, Latché A, Bouzayen M, Pech JC (1996) Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nat Biotechnol 14:862–866PubMedCrossRefGoogle Scholar
  4. Bang H, Kim S, Leskovar DI, Davis AR, King SR (2006) Duplication of the phytoene synthase gene in the carotenoid biosynthetic pathway of watermelon. HortScience 41:1007 (abstract)Google Scholar
  5. Bird CR, Smith CJS, Ray JA, Moureau P, Bevan MW, Bird AS, Huges S, Morris PC, Grierson D, Schuch W (1988) The tomato polygalacturonase gene and ripening-specific expression in transgenic plants. Plant Mol Biol 11:651–662CrossRefGoogle Scholar
  6. Clendennen S, Kellogg JA, Wolf KA, Matsumura W, Peters S, Vanwinkle JE, Copes B, Pieper M, Kramer MG (1999) Genetic engineering of cantaloupe to reduce ethylene biosynthesis and control ripening. In: Kanellis A, Chang C, Klee H, Bleecker AB, Pech JC, Grierson D (eds) Biology and biotechnology of the plant hormone ethylene, vol II. Kluwer, Dordrecht, pp 371–379CrossRefGoogle Scholar
  7. Clough GH, Hamm PB (1995) Coat protein transgenic resistance to watermelon mosaic and zucchini yellows mosaic-virus in squash and cantaloupe. Plant Dis 79:1107–1109CrossRefGoogle Scholar
  8. Cunningham FX Jr, Gantt E (1998) Genes and enzymes of carotenoid biosynthesis. Annu Rev Plant Physio Plant Mol Biol 49:557–583CrossRefGoogle Scholar
  9. Curuk S, Ananthakrishnan G, Singer S, Xia X, Elman C, Nestel D, Cetiner S, Gaba V (2003) Regeneration in vitro from the hypocotyl of Cucumis species produces almost exclusively diploid shoots, and does not require light. HortScience 38:105–109Google Scholar
  10. Dhooghe E, van Laere K, Eeckhaut T, Leus L, van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro. Plant Cell Tiss Organ Cult 104:359–373CrossRefGoogle Scholar
  11. Diretto G, Al-Babili S, Tavazza R, Papacchioli V, Beyer P, Giuliano G (2007) Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway. PLoS ONE 2:e350PubMedCrossRefGoogle Scholar
  12. Ducreux LJM, Morris WL, Hedley PE, Shepherd T, Davies HV, Millam S, Taylor MA (2005) Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein. J Exp Bot 56:81–89PubMedGoogle Scholar
  13. Ezura H, Hiroshi A, Keiko Y, Oosawa K (1992) Highly frequent appearance of tetraploidy in regenerated plants, a universal phenomemon, in tissue cultures of melon (Cucumis melo L.). Plant Sci 85:209–213CrossRefGoogle Scholar
  14. Ezura H, Hitomi A, Higashi K, Sato T, Kubota M (1997a) Introduction of ACC synthase antisense gene to muskmelon (Cucumis melo L. var. reticulatus). In: Abak K, Büyükalaca S (eds) First international symposium on cucurbits, Adana, Turkey, AbstractGoogle Scholar
  15. Ezura H, Kikuta I, Oosawa K (1997b) Long-term ploidy stability of shoot primordium cultures and produced plants of melon. Plant Cell Tiss Organ Cult 48:31–35CrossRefGoogle Scholar
  16. Fan J, He Q, Wang X, Yu X (2007) Antisense acid invertase (anti-MAI1) gene alters soluble sugar composition and size in transgenic muskmelon fruits. Acta Hort Sinica 34:677–682Google Scholar
  17. Fang G, Grumet R (1993) Genetic-engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene. Mol Plant-Microbe Interact 6:358–367PubMedCrossRefGoogle Scholar
  18. Fraser PD, Bramley PM (2004) The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res 43:228–265PubMedCrossRefGoogle Scholar
  19. Fraser PD, Römer S, Kiano JW, Shipton CA, Mills PB, Drake R, Schuch W, Bramley PM (2001) Elevation of carotenoids in tomato by genetic manipulation. J Sci Food and Agri 81:822–827CrossRefGoogle Scholar
  20. Fraser PD, Römer S, Shipton CA, Mills PB, Kiano JW, Misawa N, Drake RG, Schuch W, Bramley PM (2002) Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner. Proc Natl Acad Sci USA 99:1092–1097PubMedCrossRefGoogle Scholar
  21. Fray RG, Grierson D (1993) Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression. Plant Mol Biol 22:589–602PubMedCrossRefGoogle Scholar
  22. Fuchs M, McFerson JR, Tricoli DM, McMaster JR, Deng RZ, Boeshore ML, Reynolds JF, Russell PF, Quemada HD, Gonsalves D (1997) Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus, zucchini yellow mosaic virus, and watermelon mosaic virus-2 is resistant to these three viruses in the field. Mol Breed 3:279–290CrossRefGoogle Scholar
  23. Gallagher CE, Matthews PD, Li F, Wurtzel ET (2004) Gene duplication in the carotenoid biosynthetic pathway preceded evolution of the grasses (Poaceae). Plant Physiol 135:1776–1783PubMedCrossRefGoogle Scholar
  24. Gonsalves C, Xue B, Yepes M, Fuchs M, Ling KS, Namba S, Chee P, Slightom JL, Gonsalves D (1994) Transferring cucumber mosaic virus-white leaf strain coat protein gene into Cucumis melo L. and evaluating transgenic plants for protection against infections. J Amer Soc Hort Sci 119:345–355Google Scholar
  25. Guis M, Botondi R, BenAmor M, Ayub R, Bouzayen M, Pech JC, Latche A (1997) Ripening-associated biochemical traits of cantaloupe charentais melons expressing an antisense ACC oxidase transgene. J Am Soc Horticult Sci 122:748–751Google Scholar
  26. Guis M, Amor MB, Latché A, Pech JC, Roustan JP (2000) A reliable system for the transformation of cantaloupe charentais melon (Cucumis melo L. var. cantalupensis) leading to a majority of diploid regenerants. Scientia Hort 84:91–99CrossRefGoogle Scholar
  27. Hao J, Niu Y, Yang B, Gao F, Zhang L, Wang J, Hasi A (2011) Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway. Biotechnol Lett 33:55–61PubMedCrossRefGoogle Scholar
  28. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plant. Transgenic Res 2:208–218CrossRefGoogle Scholar
  29. Karvouni Z, John I, Taylor JE, Watson CF, Turner AJ, Grierson D (1995) Isolation and characterization of a melon cDNA clone encoding phytoene synthase. Plant Mol Bio 27:1153–1162CrossRefGoogle Scholar
  30. Lau JM, Cooper NG, Robinson DL, Korban SS (2009) Sequence and in silico characterization of the tomato polygalacturonase (PG) promoter and terminator regions. Plant Mol Biol Rep 27:250–256CrossRefGoogle Scholar
  31. Lema-Rumińska J (2011) Flow cytometric analysis of somatic embryos, shoots, and calli of the cactus Copiapoa tenuissima Ritt. forma monstruosa. Plant Cell Tiss Organ Cult 106:531–535CrossRefGoogle Scholar
  32. Li Z, Yao L, Yang Y, Li A (2006) Transgenic approach to improve quality traits of melon fruit. Sci Hort 108:268–277CrossRefGoogle Scholar
  33. Li F, Vallabhaneni R, Wurtzel ET (2008) PSY3, a new member of the phytoene synthase gene family conserved in the Poaceae and regulator of abiotic stress-induced root carotenogenesis. Plant Physiol 146:333–1345CrossRefGoogle Scholar
  34. Montgomery J, Pollard V, Deikman J, Fischer RL (1993) Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp. Plant Cell 5:1049–1062PubMedGoogle Scholar
  35. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  36. Nicholass FJ, Smith CJS, Schuch W, Bird CR, Grierson D (1995) High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions. Plant Mol Biol 28:423–435PubMedCrossRefGoogle Scholar
  37. Nuňez-Palenius HG, Cantliffe DJ, Huber DJ, Ciardi J, Klee HJ (2006) Transformation of a muskmelon ‘Galia’ hybrid parental line (Cucumis melo L. var. reticulatus Ser.) with an antisense ACC oxidase gene. Plant Cell Rep 25:198–205PubMedCrossRefGoogle Scholar
  38. Nuňez-Palenius HG, Grumet R, Lester G, Cantliffe D (2008) Melon fruits: genetic diversity, physiology, and biotechnology features. Crit Rev Biotechnol 28:13–55PubMedCrossRefGoogle Scholar
  39. Ochatt SJ, Patat-Ochatt EM, Moessner A (2011) Ploidy level determination within the context of in vitro breeding. Plant Cell Tiss Organ Cult 104:329–341CrossRefGoogle Scholar
  40. Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E, Adams JL, Silverstone AL, Drake R (2005) Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nat Biotechnol 23:482–487PubMedCrossRefGoogle Scholar
  41. Qin X, Coku A, Inoue K, Tian L (2011) Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.). Planta 234:737–748PubMedCrossRefGoogle Scholar
  42. Ren Y, Bang H, Curtis IS, Gould G, Patil BS, Crosby KM (2012) Agrobacterium-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (Cucumis melo L.). Plant Cell Tiss Organ Cult 108:147–158CrossRefGoogle Scholar
  43. Shellie KC (2001) Reduced ethylene concentration and postharvest quality of transgenic netted melon (Cucumis melo L.) expressing S-adenosylmethionine hydrolase. HortScience 36:467Google Scholar
  44. Shewmaker CK, Sheehy JA, Daley M, Colburn S, Ke DY (1999) Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. Plant J 20:401–412PubMedCrossRefGoogle Scholar
  45. Silva JA, da Costa TS, Lucchetta L, Marini LJ, Zanuzo MR, Nora L, Nora FR, Twyman RM, Rombaldi CV (2004) Characterization of ripening behavior in transgenic melons expressing an antisense 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene from apple. Postharvest Biol Technol 32:263–268CrossRefGoogle Scholar
  46. Skroch PW, Nienhuis J (1995) Qualitative and quantitative characterization of RAPD variation among snap bean genotypes. Theor Appl Genet 91:1078–1085Google Scholar
  47. Tian H, Ma L, Zhao C, Hao H, Gong B, Yu X, Wang X (2010) Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development. Biochem and Biophys Res Commun 393:365–370CrossRefGoogle Scholar
  48. Wu H, Yu T, Raja JAJ, Wang H, Yeh SD (2009) Generation of transgenic oriental melon resistant to zucchini yellow mosaic virus by an improved cotyledon-cutting method. Plant Cell Rep 28:1053–1064PubMedCrossRefGoogle Scholar
  49. Yalçın-Mendi NY, Ipek M, Serbest-Kobaner S, Curuk S, Aka Kacar Y, Cetiner S, Gaba V, Grumet R (2004) Agrobacterium-mediated transformation of Kirkagac 637 a recalcitrant melon (Cucumis melo) cultivar with ZYMV coat protein encoding gene. Eur J Hort Sci 69:258–262Google Scholar
  50. Yalçın-Mendi NY, Sari N, Akyildiz A, Solmaz I, Ünek C, Ozkaya O, Serçe S (2010) Determination of gene escape and fruit quality characteristics in transgenic melon. Turk J Agric Sci 34:135–143Google Scholar
  51. Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Sci 287:303–305CrossRefGoogle Scholar
  52. Yoshioka K, Hanada K, Nakazaki Y, Minobe Y, Yakuwa T, Oosawa K (1992) Successful transfer of the cucumber mosaic virus coat protein gene to Cucumis melo L. Jpn J Breed 42:277–285Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Yan Ren
    • 1
  • Haejeen Bang
    • 1
  • Eun Jin Lee
    • 1
  • Jean Gould
    • 1
    • 2
  • Keerti S. Rathore
    • 3
  • Bhimanagouda S. Patil
    • 1
  • Kevin M. Crosby
    • 1
    Email author
  1. 1.Vegetable and Fruit Improvement Center, Department of Horticultural SciencesTexas A&M UniversityCollege StationUSA
  2. 2.Department of Ecosystem Science and ManagementTexas A&M UniversityCollege StationUSA
  3. 3.Department of Soil and Crop SciencesTexas A&M UniversityCollege StationUSA

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