Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Adventitious regeneration in vitro occurs across a wide spectrum of squash (Cucurbita pepo) genotypes

  • 278 Accesses

  • 22 Citations


Cucurbita pepo L. (squash, pumpkin) is a highly polymorphic vegetable species of major importance. Our study characterized a spectrum of C. pepo germplasm for the ability to regenerate in vitro by direct organogenesis from cotyledon explants. Cultivars tested included both cultivated subspecies, texana and pepo, and nearly all of their respective cultivar-groups. Direct shoot regeneration occurred in all accessions, and was generally high (56–94%), with a single exception of 22% (‘Bolognese’). There was no significant difference between the percentage regeneration of the two subspecies. Shoot regeneration per responding explant was uniform (1.2–1.6 shoots per explant). Only ‘True French’ produced statistically more shoots (3.9 per explant) than other accessions. The morphology of regeneration varied. Most cultivars produced long shoots, often fasciated, amid a few small buds. Some subspecies pepo cultivars (Beirut, Yugoslavia 7, Ma’yan and True French) produced short, massive, hollow shoots, sometimes accompanied by shoots that were more normal. Two subspecies texana cultivars (Creamy Straightneck and Small Bicolor) produced single (sometimes double) shoots without other buds. The production of chimeric (mixoploid) regenerants varied and there was a tendency to regenerate chimeric plants from the widest-fruited accessions (i.e. lowest length-to-width ratio) in each subspecies. Subspecies pepo Pumpkin Group ‘Tondo di Nizza’ showed significantly greater production of chimeric regenerants. In comparison with the great range of variation observed in fruit shape, the variation of in vitro responses (mostly less than 2-fold in regeneration and shoot production) was less than expected.

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


  1. Adelberg J (1998) Regeneration and frequency of tetraploid variants of Cucumis metuliferus are affected by explant induction on semi-solid medium versus the liquid/membrane system Plant Cell Rep. 17: 225–229

  2. Adelberg J, Rhodes B, Skorupska H, Bridges W (1994) Explant origin affects the frequency of tetraploid plants from tissue culture of melon HortScience 29: 689–692

  3. Ananthakrishnan G, Xia X, Elman C, Singer S, Paris H, Gal-On A, Gaba V (2003) Shoot production in squash (Cucurbita pepo) by in vitro organogenesis Plant Cell Rep. 21: 739–746

  4. Chee PP (1991) Somatic embryogenesis and plant regeneration of squash Cucurbita pepo L. cv. YC 60Plant Cell Rep. 9: 620–622

  5. Chee PP (1992) Initiation and maturation of somatic embryos of squash (Cucurbita pepo) HortScience 27: 59–60

  6. Colijn-Hooymans CM, Hakkert JC, Jansen J, Custers JMB (1994) Competence for regeneration of cucumber cotyledons is restricted to specific developmental stages Plant Cell Tissue Organ Cult. 39: 211–217

  7. Compton ME, Veilleux RE (1991) Variation for genetic recombination among tomato plants regenerated from three tissue culture systems Genome 34: 810–817

  8. Çürük 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: 105–109

  9. Çürük S, Çetiner S, Elman C, Xia X, Wang Y, Yeheskel A, Zilberstein L, Perl-Treves R., Watad AA, Gaba V (2005) Transformation of recalcitrant melon (Cucumis melo L.) cultivars is facilitated by wounding with carborundum Eng. Life Sci. 6: 169–177

  10. De Laat AMM, Blaas J (1984) Flow cytometric characterization and sorting of plant chromosomes Theor. Appl. Genet. 67: 463–467

  11. Esau K (1953) Plant Anatomy Wiley, New York

  12. Ezura H, Amagi H, Yoshioka K, Oosawa K (1992) Highly frequent appearance of tetraploidy in regenerated plants, an universal phenomenon in tissue cultured melon (Cucumis melo L.)Plant Sci. 85: 209–213

  13. FAOSTAT (2005) World agricultural data. (Date of last successful access 10 January 2006)

  14. Ferriol M, Pico B, Nuez F (2003) Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers Theor. Appl. Genet. 107: 271–282

  15. Gaba V, Elman C, Perl-Treves R & Gray DJ (1996) A theoretical investigation of the genetic variability in the ability of Agrobacterium to transform Cucumis melo L. In: Gomez-Guillamon ML, Soria C, Cuartero J, Tores JA & Fernandez-Munoz R (eds), Cucurbits Towards 2000. Proc. 6th Eucarpia meeting on Cucurbit Genetics and Breeding (pp. 172–178), Estacion Experimental La Mayora. Malaga, Spain

  16. Gaba V, Schlarman E, Elman C, Sagee O, Watad AA, Gray DJ (1999) In vitro studies on the anatomy and morphology of bud regeneration in melon cotyledons In Vitro Cell. Dev. Biol. Plant 35: 1–7

  17. Gaba V, Zelcer A, Gal-On A (2004) Cucurbit biotechnology – the importance of virus resistance In Vitro Cell. Dev. Biol. Plant 40: 346–358

  18. Galbraith DW, Harkins KR, Knapp S (1991) Systemic endopolyploidy in Arabidopsis thaliana Plant Physiol. 96: 985–989

  19. Gilissen LJW, van Staveren MJ, Creemers-Molenaar J, Verhoeven HA (1993) Development of polysomaty in seedlings and plants of Cucumis sativus LPlant Sci. 91: 171–179

  20. Gonsalves C, Xue B, Gonsalves D (1995) Somatic embryogenesis and regeneration from cotyledon explants of six squash cultivars HortScience 30: 1295–1297

  21. Gray DJ, McColley DW, Compton ME (1993) High-frequency somatic embryogenesis from quiescent seed cotyledons of Cucumis melo cultivars J. Am. Soc. Hort. Sci. 118: 425–432

  22. Guis M, Ben Amor M, Latche A, Peche 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 regenerantsSci. Hort. 84: 91–99

  23. Han JS, Oh DG, Mok IG, Park HG, Kim CK (2004) Efficient plant regeneration from cotyledon explants of bottle gourd (Lagenaria siceraria Standl.)Plant Cell Rep. 23: 291–296

  24. Jeffrey C (2001) Cucurbitaceae In: P. Hanelt, Inst. Plant Genet., Crop Res.(eds), Mansfeld’s Encyclopedia of Agricultural and Horticultural Crops. Springer, Berlin, pp. 1510–1557

  25. Jelaska S (1972) Embryoid formation by fragments of cotyledons and hypocotyls in Cucurbita pepo Planta 103: 278–280

  26. Jelaska S (1974) Embryogenesis and organogenesis in pumpkin explants Physiol. Plant. 31: 257–261

  27. Jelaska S (1986) Cucurbits In: Bajaj YPS (eds), Biotechnology in Agriculture and Forestry 2. Crops I Springer-Verlag, Berlin, pp. 371–386

  28. Kintzios S, Taravira N (1997) Effect of genotype and light intensity on somatic embryogeneisis and regeneration in melon (Cucumis melo L.) Plant Breed. 116: 359–362

  29. Kintzios S, Sereti E, Bluchos P, Drossopoulos JB, Kitsaki CK, Liopa-Tsakalidis A (2002) Growth regulator pretreatment improves somatic embryogenesis from leaves of squash (Cucurbita pepo L.) and melon (Cucumis melo L.) Plant Cell Rep. 21: 1–8

  30. Kudo N, Kimura Y (2001) Patterns of endopolyploidy during seedling development in cabbage (Brassica oleracea L.) Ann. Bot. 87: 275–281

  31. Oridate T, Atsumi H, Ito S, Araki H (1992) Genetic differences in somatic embryogenesis from seeds in melon (Cucumis melo L.)Plant Cell Tissue Organ Cult. 29: 27–30

  32. Orts MC, Garcia-Sogo B, Roche MV, Roig LA, Moreno V (1987) Morphogenetic response of calli from primary explants of diverse cultivars of melon HortScience 22: 666

  33. Lee YK, Chung WI, Ezura H (2003) Efficient plant regeneration via organogenesis in winter squash (Cucurbita maxima Duch.) Plant Sci. 164: 413–418

  34. Molina R.V., Nuez F. 1995 Characterization and classification of different genotypes in a population of Cucumis melo based on their ability to regenerate shoots from leaf explants Plant Cell Tissue Organ Cult. 43: 249–257

  35. Morris PC & Altmann T (1994) Tissue culture and transformation. In: Somerville CR & Meyerowitz EM (eds) Arabidopsis (pp. 173–222). Cold Spring Harbor Laboratory Press

  36. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant. 15: 473–498

  37. Paris HS (1986) A proposed subspecific classification for Cucurbita pepo Phytologia 61: 133–138

  38. Paris HS (2000) History of the cultivar-groups of Cucurbita pepo Hort. Rev. 25: 71–170

  39. Paris HS, Nerson H (2003) Seed dimensions in the subspecies and cultivar-groups of Cucurbita pepo Genet. Res. Crop Evol. 50: 615–625

  40. Paris HS, Yonash N, Portnoy V, Mozes-Daube N, Tzuri G, Katzir N (2003) Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers Theor. Appl. Genet. 106: 971–978

  41. Punja ZK, Abbas N, Saramento GG, Tang FA (1990) Regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii, C. melo, and C. metuliferous from explants through somatic embryogenesis and organogenesisPlant Cell Tissue Organ Cult. 21: 93–102

  42. Sinnott EW (1939) A developmental analysis of the relation between cell size and fruit size in cucurbits Am. J. Bot. 26: 179–189

  43. Sinnott EW (1960) Plant Morphogenesis McGraw-Hill, New York, Toronto, London

  44. Smulders MJM, Rus-Kortekaas W, Gilissen LJW (1994) Development of polysomaty during differentiation in diploid and tetraploid tomato (Lycopersicon esculentum) plants Plant Sci. 97: 53–60

  45. Sokal RR, Rohlf FJ (1981) Biometry 2. W.H. Freeman, New York

  46. Sugimoto-Shirasu K, Roberts K (2003) “Big it up”: endoreduplication and cell-size control in plants Curr. Opin. Plant Biol. 6: 544–553

  47. Tricoli DM, Carney KJ, Russel PF, McMaster JR, Groff DW, Hadden KC, Himmel PT, Hubbard JP, Boeshore ML, Reynolds JF, Quemada HD (1995) Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and/or zucchini yellow mosaic virus Biotechnology 13:1458–1465

  48. Urbanek A, Zechmann B, Muller M (2004) Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations Plant Cell Tissue Organ Cult. 79: 329–340

  49. Yang M, Loh CS (2004) Systemic endopolyploidy in Spathoglottis plicata (Orchidaceae) development BMC Cell Biol.5:33

  50. Winkelmann T, Kaviani K, Serek M (2005) Development of a shoot regeneration protocol for genetic transformation in Pelargonium zonale and Pelargonium peltatum hybrids Plant Cell Tissue Organ Cult. 80: 33–42

Download references


Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 513/05. This work was partially supported by Research Grant No. US2541-95R from BARD, The United States-Israel Binational Agricultural Research and Development Fund to V.G., K. Kathiravan received a BOYSCAST Fellowship from the Ministry of Science and Technology, Government of India.

Author information

Correspondence to Victor Gaba.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kathiravan, K., Vengedesan, G., Singer, S. et al. Adventitious regeneration in vitro occurs across a wide spectrum of squash (Cucurbita pepo) genotypes. Plant Cell Tiss Organ Cult 85, 285–295 (2006).

Download citation


  • chimeric regenerant
  • direct regeneration
  • multiple genotypes
  • ploidy
  • subspecies