Cell and Tissue Journal

Abstract Introduction: The anther culture technique is used in vitro for many plant species as an effective tool for obtaining haploid and doubled-haploid lines.
Aims: This study aimed to investigate the influence of different concentrations of iron oxide nanoparticles and benzylaminopurine (BAP) on callus formation, embryogenesis, regeneration, and rooting in bell pepper anther culture
Materials and Methods: The experiment was conducted as a factorial experiment in a completely randomized design under in vitro culture conditions. Flower buds of appropriate size (equal sepal to petal ratio or slightly longer petal) were collected from the greenhouse, and acetocarmine solution was used to determine the growth and development stage of microspores. The results showed that the most suitable stages for embryogenesis induction were the late mononuclear and early binuclear stages. In order to sterilize the flower buds, 70% ethanol for 30 seconds and 5% sodium hypochlorite for 20 minutes were used, and after each stage, they were washed three times with sterile distilled water. Then, the anthers were separated from the flower bud and placed in C medium containing 2 mg/L naphthalene acetic acid (NAA), different concentrations of BAP (0, 0.1, 0.5, and 1 mg/L), and different concentrations of iron oxide nanoparticles (0, 1, 10, and 20 mg/L). After that, the explants cultured in C medium were kept at 35°C in a dark place for 8 days in order to apply heat treatment. Then, they were transferred to 25°C in the light for 4 days. After this period, in order to induce embryogenesis, the explants were transferred from C medium to R medium and were subcultured every three weeks until embryos emerged. For further growth and root development, the embryos were transferred to V medium.
Results: The analysis of variance showed that different concentrations of iron oxide nanoparticles had a significant effect on the percentages of embryogenesis, regeneration, and rooting, but had no significant effect on the percentage of callus formation. The results of the mean comparison showed that among the different concentrations of iron oxide nanoparticles, the 1 mg/L treatment produced the highest embryogenesis percentage (11.11%). Furthermore, the mean comparison results for regeneration indicated that the 1 mg/L treatment resulted in the highest regeneration percentage (16.66%). The results of the interaction effects showed that among the different concentrations of iron oxide nanoparticles and BAP, the highest percentage of embryogenesis was observed in the treatment of 20 mg/L iron oxide nanoparticles and 0 mg/L BAP. Also, the treatment of 20 mg/L iron oxide nanoparticles and 0 mg/L BAP had the highest percentage of regeneration (33.33%). After sufficient growth and root formation, the obtained plants were removed from the glass culture containers and transferred to pots containing sterilized culture medium and watered for adaptation. The tops of the pots were covered with plastic cups, and after three days, the cups were pierced, and the plastic was gradually removed from the plant for further adaptation. Ploidy levels were determined by chromosome counting after staining the root tip cells. The results showed that out of the 23 obtained plants, 21 were diploid and had 2n=2x=24 chromosomes, and 2 were haploid and had n=x=12 chromosomes.
Discussion: Applying different concentrations of iron oxide nanoparticles in medium C had a positive effect on the regeneration of bell pepper plants. Iron is a vital micronutrient for several key cellular processes in plants. In addition, iron is essential for ensuring the structural integrity of proteins. Iron nanoparticles positively affect plant growth in tissue culture by increasing morphological parameters. The beneficial effects of nanoparticles on plants are related to their high solubility and reactivity, which affect how they interact with membranes and other cellular components, due to their large specific surface area. The treatment of 20 mg/L of nanoparticles and 0 mg/L of BAP had the highest regeneration percentage. The culture medium containing nano-iron leads to the plant benefiting from the element iron directly at all different stages of growth and its participation with other nutrients, and therefore the production of many nutrients containing it leads to the transfer of their excess to different parts of the plant. The presence of nano iron in the culture medium has led to a nutritional balance in the plant and, as a result, has improved the plant growth process by increasing the amount of nutrients and elements accumulated in the microsample. Cytokinins act as a nutrient reservoir, which leads to an increase in the percentage of elements in the microsample and an increase in the percentage of sugars in the culture medium, which is important in the structural processes in tissues and affects the accumulation of sugars in it, and is reflected in increased vegetative growth.
Conclusion: In bell pepper anther culture, different concentrations of iron oxide nanoparticles, along with plant growth regulators at different concentrations, showed a great effect on embryogenesis, regeneration, and rooting.