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.

Abstract Aim: Salinity stress is one of the most important environmental stress in the world and one of the important factors in reducing growth in many plants, especially in arid regions of the world. Salinity stress and increased sodium ion lead to the induction of oxidative stress and consequent cell death. Melatonin is a multiple function molecule spread in different plant and triggers several physiologic responses to different environmental stress. Exogenous application of melatonin to several plants can improve crop growth and development in response to many abiotic and biotic stresses with adjusting the antioxidant system of plants. Current studies reported that, the exogenous melatonin can increase plants' stress resistance by regulating both the enzymatic and non-enzymatic antioxidant defense process. In this study, the effect of melatonin on alfalfa roots under tissue culture condition was investigated. The aim of the present study was to investigate how melatonin regulated the antioxidant system and non-enzymatic antioxidants such as reduced glutathione and ascorbate under salt stress
Materials and methods: In this study, alfalfa seeds (Medicago sativa) of the Isfahani variety were used and we studied the effect of melatonin and salinity stress on the alfalfa root. In order to sterilize the seeds, they were placed in a 70% ethanol solution for one minute and then in a 20%  sodium hypochlorite solution for 20 minutes. After disinfecting the seeds, seeds were placed in each culture dish containing MS (Murashige and Skoog) culture medium. After germination, alfalfa seeds were transferred to MS culture medium containing concentrations of 0, 0.1, 10, and 15 micromolar melatonin and concentrations of 0, 150 and 200 mM salt. After 10 days of growth, total antioxidant capacity,
the activity level of catalase, ascorbate peroxidase, superoxide dismutase, guaiacol peroxidase, and glutathione reductase, ascorbate and glutathione levels in alfalfa roots were measured.
 
Results: Also, in the salinity stress, melatonin treatment significantly increased the total antioxidant power, while no significant difference was observed between different concentrations of melatonin, 0.1µM melatonin 82 درصد  and 62 درصد  raised antioxidant activity under 150 and 200 mM NaCl, so melatonin can reduce the levels of reactive oxygen species by scavenging of them through antioxidant enzyme or non- antioxidant system. Based on the results Melatonin treatment caused a significant increase in antioxidant power, the activity of CAT, APX, POD, SOD, GR enzymes, and antioxidant compounds in the glutathione-ascorbate cycle including DHA, ASC/DHA, GSH, and GSH/GSSG. along with increasing salinity concentration. On the other hand, salt stress increased oxidized compounds including DHA and GSSG in alfalfa roots. The data were carried out by two-way analysis of variance (ANOVA), followed by Duncan’s multiple range tests.




Conclusion: In addition to its direct role in clearing free radicals, melatonin activates the antioxidant defense system of the root, i.e. both antioxidant enzymes and antioxidant compounds in the ascorbate-glutathione cycle, which increases the resistance to damage oxidative effects caused by salinity stress in alfalfa root. These findings proposed that exogenous melatonin utilization dramatically activated ROS scavenging systems including enzymatic and non-enzymatic antioxidants to keep a relatively low amount of ROS and increased the tolerance of alfalfa root against salinity stress.

Abstract

Abstract Aim: In the current study, the effect of growth regulators, type and composition of the culture medium, genotypes of the used cultivars and type of explants were investigated on the ability of callusing and regeneration of wheat cultivars.
Material and methods: two culture media (N6, MS), three immature embryos, mature embryos and leaf cuttings were used. N6 medium containing growth regulator 2, 4-D was used for callusing of embryo and the leaf pieces, but for regeneration N6 medium containing growth regulators NAA, BAP and Kin was used. For calligraphy and regeneration of immature embryos, MS medium containing a variety of growth regulators were used.
Results: Callus induction and regeneration differed based on the genotype, type of explants and media composition. Chamran variety had the highest rate of callus and regeneration from immature embryo. For the adult embryo, the highest level of calligenesis and regeneration related to the C-D-9 line. In the case of leaf parts, the highest level of callus related to the C-D-9 at the level of 2, 4-D (2.4mg/L). The highest percentage of shoots belonged to the C-D-9 line that was obtained in N6 medium (6) containing IAA (1mg/L) + BA (1mg/L).
Conclusion: Response to tissue culture in wheat is affected by several factors such as genotype, type of explants, growth regulators. Results proved that the type and concentration of used growth regulators in the culture medium differ among cultivars for callus induction and wheat regeneration. The most important factor affecting wheat cultivation was immature embryo explant and the genotype type of the used cultivar.
 

Abstract Aim: This study attempts to optimize callus induction and analyze of yeast extract and nano-silver elicitors on phenol/flavonoid content in black cumin under tissue culture conditions.
Material and Methods: The experiment was conducted a factorial design based on CRD with three replications. Factors included: explants (root, hypocotyledon, leaf and Cotyledon), 2, 4-D (1, 2, 4 and 8 Mg/L) and BAP (0.25, 0.5, and 1 Mg/L) in MS base medium. Elictor's including yeast extract (100, 250 and 500 Mg/L) and nano-silver (30, 60 and 90 Mg/L) in two time (3 and 7 days) periods.
Results: The results showed that hypocotyledon explant and interaction effect of BAP (0.25 Mg/L) and 2, 4-D (4 Mg/L) were the most effective on the callus induction percent. Direct regeneration was caused by the root explant and the interaction effect of BAP (0.5 Mg/L) and 2, 4-D (1 Mg/L). The most effective treatment on the total phenol content was yeast extract (250 ppm) in a 7-day period. HPLC for quercetin (a flavonoid component) indicated that the most effective treatment was the interaction effect of nanoAg particles (30 Mg) and yeast extract (250 Mg) in a 3-day period.
Conclusion: The highest amount of callus induction is obtained from the hypocotyledon explants. The best direct regeneration is recorded in the root explants. To increase total phenol, it is necessary to use yeast extracts in a 7-day period and to increase flavonoids using the interaction effect of nano-silver (30 Mg) and yeast extract (250 Mg) over a 3-day period.