Production of doubled haploid (DH) plants is an efficient tool in genetic and plant breeding programs; however, sweet pepper (Capsicum annuum L.) is recalcitrant to microspore embryogenesis and DH production. Trying to break the barrier of DH production, three independent experiments were conducted on microspore embryogenesis of sweet pepper. In the first experiment, the effect of cold (4 °C) and heat (32 °C) pretreatments were investigated on microspore embryogenesis of three genotypes of sweet pepper including "Inspiration F1," "Maratus F1," and "Magno F1" cultivars in a factorial design with three replications. Heat shock (32 °C for 7 days), applied to mannitol-starved anthers of "Inspiration F1," showed higher multinuclear microspore percent, number of multicellular structures, total embryos, cotyledonary embryos, and regenerants. In the second experiment, the effect of different concentrations of putrescine (0, 0.5, 1, 2, and 5 mg l) was evaluated on microspore embryogenesis of the three aforementioned cultivars of sweet pepper. The highest mean number of multicellular structures, cotyledonary embryos, and regenerants were achieved by applying 0.5-1 mg l putrescine during the mannitol starvation and heat shock (32 °C) treatments of isolated microspore culture of "Inspiration F1" cultivar. Significant decrease in microspore embryogenesis efficiency was observed when high levels of putrescine (2 and 5 mg l) were used. Microspore embryogenesis was prevented completely at 5.0 mg l putrescine. In the third experiment, the effect of different concentrations of ascorbic acid (0, 20, 50, 100, and 200 mg l) was investigated and the results showed that the application of ascorbic acid (20 and 50 mg l) during mannitol starvation and heat shock treatment (32 °C) caused remarkable improvement in the number of produced cotyledonary embryos and their regeneration ability compared to control treatment. However, the application of higher levels of ascorbic acid (100 and 200 mg l) inhibited microspore cell divisions and embryogenesis. In conclusion, the results indicated that both putrescine and ascorbic acid have significant effect on microspore embryogenesis efficiency of sweet pepper when they are used in appropriate concentrations.
