Cucurbitaceae crops can obtain haploids through the induction of parthenogenesis via pollen irradiation (Kurtar et al.2010; Asadi et al.2021; Sari et al.2021). Haploids can also be induced through in vitro culturing of the nuclear tissues of haploids (Sari et al.2021; d'Hooghvorst et al.2021). Thick-skinned melons can induce parthenogenesis by irradiating pollen to induce haploids, followed by seed germination to obtain haploids (Glowacka et al.2009; Abe et al.2020). However, this method has low efficiency and many uncontrollable factors. Currently, there is a lack of hormone formulations and regeneration systems for thin-skinned melons, and there is no stable method for obtaining haploids.
The activity test of ovules at various developmental stages of thin-skinned melons indicates that during stages VI and VII, ovules exhibit high activity, which can increase the likelihood of nucellar degeneration within the ovule (Fig. 2A,B). Treatments such as high and low temperatures, as well as osmotic pressure, can enhance tissue activity. Fluctuating low-temperature treatment can induce anther development and significantly increase the yield of pollen-induced callus in Miscanthus (Al-Mayahi et al.2018). Low temperatures can eliminate the negative effects of high-molecular-weight compounds (HMCs) and significantly increase the genetic transformation rate of wheat (Zenser et al.2001). Dark storage of date palms at 5°C for 45 days results in a significant decrease in phenolic substances and enhances bud differentiation ability (Dai et al.2007). The effect of different temperatures on melon ovules shows that they are sensitive to temperature. Treatment at 4°C for 1 day can significantly increase activity, and the longer the treatment time, the lower the activity (Fig. 2F). Inducing callus formation is crucial for plant regeneration. Dark conditions can reduce sediment deposition, thin the cell wall, facilitate hormone entry into the cell, and maintain high levels of auxin and cytokinin (Ran et al.2023; Song et al.2023). Thin-skinned melon ovules can form callus tissue after dark culture for 1–2 days, and the longer the dark culturing, the slower the photomorphogenesis (Fig. 4A,B). Plant growth regulators (PGRs) are one of the key factors in culturing plant tissues. Plant tissues exhibit different sensitivities to different PGRs. For example, high concentrations of 2,4-D have a toxic effect on stem segments of watercress, while NAA can induce the formation of adventitious roots in stem segments (Lillo et al.2008; Liu et al.2022). ZT and BR can induce the formation of regenerated seedlings from ovules of thin-skinned melons, while KT30 and MLT can induce the formation of embryoids from ovules of thin-skinned melons. Increasing the proportion of TDZ, KT30, and ZT can induce callus formation from thin-skinned melon ovules. Increasing the proportion of IAA, NAA, and 2,4-D can induce callus development and the formation of regenerated plants (Fig. 3). Light and sucrose are important influencing factors for inducing the differentiation of callus buds. Continuous light-dark cycles can enhance callus growth rate (Ran et al.2023). Long photoperiod induces chloroplast development in callus tissue and significantly upregulates nitrogen assimilation-related genes, which are closely related to the formation of green cells and chloroplasts (Song et al.2023). Green cells activate the glucose-TOR signaling pathway, affecting the development of bud primordium or meristematic tissues and promoting bud differentiation (Xiong et al.2013; Ye et al.2022)). Long-term weak light conditions can induce callus tissue of thin-skinned melon ovules to rapidly expand on CIM. Subsequent increases in light intensity and PM sucrose content can induce the formation of green cells and bud differentiation, supporting this conclusion (Fig. 4A,B).
In conclusion, an efficient system for obtaining haploids from thin-skinned melon ovules has been established. This method significantly outperforms the efficiency of haploid generation through gynogenesis, allowing for the effective innovation of melon germplasm resources.