Applications of Chromosome Doubling in Haploid Squashes and Pumpkins (Cucurbita spp.)

Abstract

Squashes and pumpkins are important vegetable species belonging to the genus Cucurbita of the family Cucurbitaceae. The genus Cucurbita (2n=40) comprises five cultivated species, the most economically important of which are Cucurbita pepo L., C. maxima Duch., and C. moschata Duch. The doubled haploidization technique, which allows the rapid and efficient obtaining of pure lines in variety breeding programs in many plant species, is also successfully applied in the Cucurbita genus. The irradiated pollen technique yields successful results in all three squash species in obtaining haploid plants. Successful applications have been made in projects conducted at our company to obtain haploid plants in squash. However, it is not possible to propose a practical protocol for chromosome doubling in squash and obtaining doubled haploid (DH) plants from haploid plants. Although the literature contains successful applications and reports of pure-line seeds, the doubling step remains a key challenge in the production of pure lines in squash. For this purpose, studies were conducted on chromosome doubling in haploid squash plants under in vitro and in vivo conditions. Concentration and duration of colchicine applications were tested, and stomatal examinations revealed diploid plants. The most effective method was determined to be a 3-hour application period using a 0.5% colchicine dose used in acclimated young haploid seedlings at the 3-4 leaf stage. Plants grown from this treatment were self-pollinated to obtain pure-line seeds. However, optimization studies on chromosome doubling in squash varieties are still needed.

Keywords

• Squashes and pumpkins
• haploid
• Cucurbita pepo
• C. moschata
• C. maxima
• colchicine

References

1. Abbey, L. (2016). Cucurbits physiological stages of growth. In M. Pessarakli (Ed.), Handbook of cucurbits: Growth, cultural practices, and physiology (pp. 151-170). CRC Press.
2. Ananthakrishnan, G., Xia, X., Amutha, S., Singer, S., Muruganantham, M., Yablonsky, S., Fischer, E., and Gaba, V. (2007). Ultrasonic treatment stimulates multiple shoot regeneration and explant enlargement in recalcitrant squash cotyledon explants in vitro. Plant Cell Reports, 26(3), 267-276. https://doi.org/10.1007/s00299- 006-0235-1
3. Anonymous. (2025a). FAOSTAT: Squash and pumpkin production quantity (tons) Food and Agriculture Organization [Data set]. United Nations. https:// www.fao.org/faostat/en/#data/QCL (Accessed: 07.11.2025).
4. Anonymous. (2025b). Fresh fruit and vegetable export data of Türkiye. Türkiye yaş sebze-meyve ihracat verileri. İstanbul Ticaret Borsası (İTB). https://www.itb.org.tr (Accessed: 11 November 2025).
5. Aslam, M., Farid, B., Khakwani, K., Maqbool, M. A., and Zou, H. (2017). In vivo maternal haploid seed production and chromosome doubling with different anti-microtubular agents in maize. International Journal of Agricultural and Biology, 19(1), 114-120. https://doi. org/10.17957/IJAB/15.0251
6. Baktemur, G., Yücel, N. K., Taşkın, H., Çömlekçioğlu, S., and Büyükalaca, S. (2014). Effects of different genotypes and gamma ray doses on haploidization using irradiated pollen technique in squash. Turkish Journal of Biology, 38(3), 318-327. https://doi.org/10.3906/biy-1309-5
7. Balkaya, A., and Karaağaç, O. (2006). Vegetable genetic resources of Turkey. Journal of Vegetable Science, 11(4), 81-102. https://doi. org/10.1300/J484v11n04_08
8. Bat, H., Altındağ, F. N., Yiğit, M. A., Ellialtıoğlu, Ş. Ş., and Çömlekçioğlu, N. (2021). Ploidy estimation in pepper and eggplant via stomata characteristics. International Journal of Agriculture Forestry and Life Sciences, 5(2), 139-146. https://izlik.org/JA25KP23EU

9. Caglar, G., and Abak, K. (1997). In vitro colchicine application of haploid cucumber plants. Cucurbit Genetics Cooperative Report, 20, 21-23. https:// www.ars.usda.gov/ARSUserFiles/60800500/ CGC/CGC%2020%20(1997).pdf#page=33 Chinnathambi, V., Panwar, S., Singh, K. P., Namita,
10. Lekshmy, S., and Mallick, N. (2023). Studies on in vitro chromosome doubling of haploid derived through androgenesis in marigold. Indian Journal of Horticulture, 80(4), 333-338. https:// doi.org/10.58993/ijh/2023.80.4.5
11. Dewitte, W., and Murray, J. A. (2003). The plant cell cycle. Annual Review of Plant Biology, 54(1), 235-264. https://doi.org/10.1146/annurev. arplant.54.031902.134836
12. Dhooghe, E., Van Laere, K., Eeckhaut, T., Leus, L., and Van Huylenbroeck, J. (2011). Mitotic chromosome doubling of plant tissues in vitro. Plant Cell, Tissue and Organ Culture, 104(3), 359-373. https://doi.org/10.1007/s11240-010- 9786-5
13. Doğan, O., Kara, Z., Yazar, K., and Ekinci, H. (2023). Diploit ve tetraploit asma genotiplerinde poliploidi doğrulama yöntemlerinin testi. Bahçe, 52(Özel Sayı 1), 10-17. (In Turkish)
14. Ebrahimzadeh, H., Soltanloo, H., Shariatpanahi, M. E., Eskandari, A., and Ramezanpour, S. S. (2018). Improved chromosome doubling of parthenogenetic haploid plants of cucumber (Cucumis sativus L.) using colchicine, trifluralin, and oryzalin. Plant Cell, Tissue and Organ Culture (PCTOC), 135(3), 407-417. https://doi. org/10.1007/s11240-018-1473-y
15. Ellialtıoğlu, Ş., Sarı, N., and Abak, K. (2002). Haploid bitki üretimi. M. Babaoğlu, E. Gürel, and S. Özcan (Ed.), Bitki Biyoteknolojisi I içinde (ss. 137-189). Selçuk Üniversitesi Vakfı Yayınları.
16. Ermolaev, A., Fomicheva, M., and Domblides, E. (2026). Advances in polyploid breeding of Cucurbitaceae crops: From polyploidy research to triploid seedless hybrid breeding. Crops, 6(1), 5. https://doi.org/10.3390/crops6010005
17. Ferriol, M., and Picó, B. (2008). Pumpkin and winter squash. In J. Prohens and F. Nuez (Eds.), Vegetables I: Asteraceae, Brassicaceae, Chenopodicaceae, and Cucurbitaceae (pp. 317- 349). Springer. https://doi.org/10.1007/978-0- 387-30443-4_10
18. Fomicheva, M., Kozar, E., and Domblides, E. (2025). Carrot (Daucus carota L.) haploid embryo genome doubling with colchicine and trifluralin. Horticulturae, 11(5), 505. https://doi.org/10.3390/ horticulturae11050505
19. Fomicheva, M., Kulakov, Y., Alyokhina, K., and Domblides, E. (2024). Spontaneous and chemically induced genome doubling and polyploidization in vegetable crops. Horticulturae, 10(6), 551. https:// doi.org/10.3390/horticulturae10060551 Gałązka, J., and Niemirowicz-Szczytt, K. (2013).
20. Review of research on haploid production in cucumber and other cucurbits. Folia Horticulturae, 25(1), 67-78. https://doi. org/10.2478/fhort-2013-0008
21. Harlan, J. R. (1951). Anatomy of gene centers. The American Naturalist, 85(821), 97-103. https:// doi.org/10.1086/281657
22. Hooghvorst, I., and Nogués, S. (2020). Opportunities and challenges in doubled haploids and haploid inducer-mediated genome-editing systems in cucurbits. Agronomy, 10(9), 1441. https://doi. org/10.3390/agronomy10091441
23. Kathiravan, K., Vengedesan, G., Singer, S., Steinitz, B., Paris, H. S., and Gaba, V. (2006). Adventitious regeneration in vitro occurs across a wide spectrum of squash (Cucurbita pepo) genotypes. Plant Cell, Tissue and Organ Culture, 85(3), 285- 295. https://doi.org/10.1007/s11240-006-9079-1
24. Kesici, S., Abak, K., and Tunar, M. (2000). Yazlık kabaklarda erkencilikte ve verimde heterozis etkisi. In III. Sebze Tarımı Sempozyumu (pp. 167- 172). Isparta, Türkiye.
25. Klíma, M., Shmeit, Y. H., Kopecký, P., Vítámvás, P., Kosová, K., Prášil, I. T. ve FernándezCusimamani, E. (2024). Impact of selected antimitotic substances on doubled haploid and polyploid regeneration in microspore cultures of swede (Brassica napus ssp. napobrassica (L.) Hanelt). Czech Journal of Genetics and Plant Breeding, 60(2), 79-85. https://doi. org/10.17221/84/2023-CJGPB
26. Kurtar, E. S. (2018). The effects of anti-mitotic agents on dihaploidization and fertility in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) androgenic haploids. Acta Scientiarum Polonorum Hortorum Cultus, 17(5), 3-14. https://doi.org/10.24326/ asphc.2018.5.1
27. Kurtar, E. S. and Seymen, M. (2021). Gynogenesis in Cucurbita species. J. M. Seguí-Simarro (Ed.), Doubled haploid technology: Volume 3: Emerging tools, cucurbits, trees, other species içinde (s. 123-133). Humana Press. https://doi. org/10.1007/978-1-0716-1335-1_9
28. Kurtar, E. S., Ellialtıoğlu, Ş. Ş. and Metin, D. (2024). Doku kültürü tekniklerinin hızlandırılmış ıslah amaçlı olarak sebze tohumculuğunda kullanımı. A. Balkaya ve L. Arın (Ed.), Sebze tohum üretimi ve teknolojisinde güncel ve yenilikçi uygulamalar (pp. 12–30). IKSAD Publishing House. https:// doi.org/10.5281/zenodo.13822756 (In Turkish)
29. Kurtar, E. S., Seymen, M. ve Kal, Ü. (2020). An overview of doubled haploid plant production in Cucurbita species. Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi, 30(3), 510-520. https:// doi.org/10.29133/yyutbd.741087
30. Lim, W. and Earle, E. D. (2009). Enhanced recovery of doubled haploid lines from parthenogenetic plants of melon (Cucumis melo L.). Plant Cell, Tissue and Organ Culture, 98(3), 351-356. https://doi.org/10.1007/s11240-009-9563-5
31. Marangoz, B. N., Çömlekçioğlu, N., Gürsoy, E., Altuntaş, B. and Ellialtıoğlu, Ş. Ş. (2025). Effects of colchicine applications on embryo yield and spontaneous chromosome doubling in pepper (Capsicum annuum L.) anther culture. Manas Journal of Agriculture, Veterinary and Life Sciences, 15(1), 76-90. https://doi.org/10.53518/ mjavl.1583740
32. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473-497.
33. Nikolova, V. and Niemirowicz-Szczytt, K. (1996). Diploidization of cucumber (Cucumis sativus L.) haploids by colchicine treatment. Acta Societatis Botanicorum Poloniae, 65(3-4), 311-317. https:// doi.org/10.5586/asbp.1996.048
34. Paris, H. S. (1989). Historical records, origins, and development of the edible cultivar groups of Cucurbita pepo (Cucurbitaceae). Economic Botany, 43(4), 423-443. https://doi.org/10.1007/ BF02935916
35. Robinson, R. W., and Decker-Walters, D. S. (1997). Cucurbits. CAB International. Wallingford, UK. 226 p. (Crop Production Science in Horticulture n.6).
36. Sari, N., and Abak, K. (1996). Effect of colchicine treatment with different doses and periods on in vitro chromosome doubling in haploid watermelon. Turkish Journal of Agriculture and Forestry, 20(6), 555-559.
37. Sauton, A., and Dumas de Vaulx, R. (1987). Production of haploid plants in melon (Cucumis melo L.) as a result of gynogenesis induced by irradiated pollen. Agronomie, 7(2), 141-147. https://doi. org/10.1051/agro:19870209
38. Singh, B., Yun, S., Gil, Y., and Park, M. H. (2025). The role of colchicine in plant breeding. International Journal of Molecular Sciences, 26(14), 6743. https://doi.org/10.3390/ijms26146743
39. Smith, B. D. (1997). The initial domestication of Cucurbita pepo in the Americas 10,000 years ago. Science, 276(5314), 932-934. https://doi. org/10.1126/science.276.5314.932
40. Smith, B. D. (2005). Reassessing Coxcatlan cave and the early history of domesticated plants in Mesoamerica. Proceedings of the National Academy of Sciences, 102(27), 9438-9445. https://doi.org/10.1073/pnas.0502847102
41. Solmaz, I., Sari, N., Gursoy, I., and Kasapoğlu, S. (2011). Comparison of in vivo and in vitro colchicine application for production of dihaploid ‘Kirkagac’ and ‘Yuva Hasanbey’ melons. African Journal of Biotechnology, 10(70), 15717-15724.
42. Tepe, Ş., Ellialtıoğlu, Ş., Yenice, N., and Tıpırdamaz, R. (2002). In vitro kolhisin uygulaması ile poliploid nane (Mentha longifolia L.) bitkilerinin elde edilmesi. Akdeniz Üniversitesi Ziraat Fakültesi Dergisi, 15(2), 63-69. (In Turkish)
43. Vaughn, K. C. (2006). The abnormal cell plates formed after microtubule disrupter herbicide treatment are enriched in callose. Pesticide Biochemistry and Physiology, 84(2), 63-71. https://doi. org/10.1016/j.pestbp.2005.03.006
44. Vural, G. E., Arı, E., Zengin, S., and Ellialtıoğlu, S. S. (2019). Development of androgenesis studies on eggplant (Solanum melongena L.) in Turkey from past to present. In Sustainable Crop Production (pp. 67-90). IntechOpen. https://doi.org/10.5772/ intechopen.88299
45. Whitaker, T. W., and Davis, G. N. (1962). Cucurbits: Botany, cultivation, and utilization. Interscience Publishers. New York, 250p.
46. Yapıcı, B., Gürsoy, E., Yıldız, B. N., İpek, E., Kavak, S., Kurtar, E. S., Gülşen, O., and Metin, D. (2023). Külleme ve ZYMV dayanımlı nitelikli yazlık kabak (Cucurbita pepo L.) hatlarında ışınlanmış polen tekniği ile dihaploidizasyon. 3rd International Antalya Scientific Research and Innovative Studies Congress (13-14 Şubat 2023, Antalya, Türkiye), s. 773-788. (In Turkish)
47. Yetişir, H., and Sarı, N. (2003). A new method for haploid muskmelon (Cucumis melo L.) dihaploidization. Scientia Horticulturae, 98(3), 277-283. https://doi.org/10.1016/S0304- 4238(02)00226-1
48. Yiğit, M., Tunalı, H., Çardak, T. Z., Altındağ, F. N., Şeker, E., Doğangüzel, E., and Ellialtıoğlu, Ş. Ş. (2023,). Parthenogenetic embryo induction in different zucchini genotypes by irradiated pollen technique [Abstract]. 6th International Agricultural Congress, p: 55. 31 August-4 September 2023
49. Yuan, S., Su, Y., Liu, Y., Li, Z., Fang, Z., Yang, L., Zhuang, M., Zhang, Y., Lv, H., and Sun, P. (2015). Chromosome doubling of microspore-derived plants from cabbage (Brassica oleracea var. capitata L.) and broccoli (Brassica oleracea var. italica L.). Frontiers in Plant Science, 6, Article 1118. https://doi.org/10.3389/fpls.2015.01118