Research on the creation and application of a doubled haploid for distant hybrid pumpkin

Distant hybridization is an important method for pumpkin germplasm innovation that can effectively expand the genetic range of germplasm resources. However, reproductive isolation hinders the application of this technology. This study explored the feasibility and advantages of gynogenesis through unfertilized ovary culture of the first filial generation of Cucurbita maxima Duch. and Cucurbita moschata Duch. First, the factors influencing the survival of unfertilized ovaries of interspecific hybrid pumpkins were screened. All four donors obtained regenerated plants. D2 (Jingxinzhen 2) had the highest rates of embryo-like structure (ELS) induction and plant induction, which were 52.99% and 35.24%, respectively. D4 (regenerated plants from ovary culture) had the highest plant regeneration rate, which was 68.24%. The average ELS induction rate of donors in M5 (1.0 mg·L⁻¹ 6-BA+0.04 mg·L⁻¹ TDZ) medium was the highest at 24.87%, which is more suitable for distant hybrid pumpkin ovary culture. Second, the ploidy identification results showed that there were haploid (n), diploid (2n), tetraploid (4n), hexaploid (6n), and octoploid (8n) plants among the regenerated plants (R1s). Furthermore, the simple sequence repeat (SSR) molecular marker results showed that 61.94% of the R1s were homozygous, and 38.06% were heterozygous. Third, sequence alignment and qRT-PCR analysis results indicate that the AtBBM homologous gene CmaCh01G000370.1 plays an important role in unfertilized ovary culture. Finally, field observations demonstrated that R1 combined characteristics of both C. maxima and C. moschata, and its successive generations of inbred lines consistently retained these traits without segregation. Four hybrid combinations between R1 and C. maxima as well as C. moschata yielded fertile seeds. This study established a stable and efficient culture system for distant hybrid pumpkin unfertilized ovaries, providing an innovative approach to solve the barrier of distant hybridization in Cucurbita.