Enhancement of microspore embryogenesis induction and plantlet regeneration of sweet pepper (Capsicum annuum L.) using putrescine and ascorbic acid

Abstract

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.

Evaluation of General and Specific Combining Ability for Doubled Haploid Lines in Sweet Pepper (Capsicum Annuum L.)

“…Then the buds rinsed 3-4 times with sterile distilled water. Based on our previously reported protocol (Heidari-Zefreh et al, 2018), the isolation of microspores and embryogenesis induction were carried out.Chromosome doubling and creation of doubled haploid lines were performed by application of colchicine in the regenerated haploid seedlings (Gil-Humanes and Barro, 2009;Esin, et al, 2016). The ploidy level of microspores-derived lines was detected using ow cytometry analysis.…”

Androgenesis-Based Doubled Haploidy: Past, Present, and Future Perspectives

“…In contrast, Brassica isolated microspore cultures utilized 0.5-1.0 mg/L putrescine (micromolar range) with beneficial results, but higher concentrations were inhibitory (Ahmadi et al, 2014;Bhatia et al, 2021). Similarly, pepper isolated microspores were exposed to 0.5-1.0 mg/L putrescine during the temperature stress and sugar starvation stages of the protocol with improved androgenic response, and higher concentrations were inhibitory (Heidari-Zefreh et al, 2019). The much lower effective concentrations of polyamines in isolated microspore culture are likely due to their direct availability to androgenic cells, whereas anther cultures have multiple layers of cells to penetrate in order to access the androgenic cell (e.g., the microspore).…”

In Vitro Regeneration Potential of Thin Cell Layer Explants of Lentisk (Pistacia lentiscus var. Chia) Plant

“…Ascorbic acid regulates the cell cycle and affects growth through elongation growth. As ascorbic acid causes cells to transition from G1 to the S phase, its participation in growth and development is clear [33].…”