Doubled Haploid Production in Cucurbita pepo L. Through Ovary Culture

Gynogenesis offers a promising route for doubled haploid (DH) production in Cucurbita, yet efficient protocols remain scarce. This study established a reproducible ovary culture system for Cucurbita pepo and evaluated zeatin riboside (ZR) as an alternative cytokinin. Ovaries collected at anthesis and one day before were cultured to screen nine media with different cytokinin–auxin combinations. Subsequently, four optimized ZR-based formulations were evaluated. Both floral stages showed morphogenic activity, but embryo formation occurred almost exclusively in pre-anthesis ovaries. Among ZR treatments, E6.1 (1 mg·L−1 ZR + 3 mg·L−1 NAA, 30 g·L−1 sucrose) achieved the highest embryogenic output (approximately 97 embryos per 100 explants), while high-sucrose media (120 g·L−1) induced abundant swollen ovules but poor conversion, suggesting that excessive osmotic pressure promotes morphogenesis but hampers embryogenic transition. In total, 415 embryos were obtained, and 52 regenerants were analyzed by flow cytometry, confirming haploid, diploid, and mixoploid plants and evidencing spontaneous chromosome doubling during in vitro development. A categorical A–D scoring system enabled early prediction of embryogenic potential. This represents the first successful application of ZR in cucurbit gynogenesis and highlights its value as a biologically compatible cytokinin for DH production. The findings open new avenues for testing ZR-based formulations in other Cucurbita species under different auxin and sucrose regimes.