فهرست مطالب رضا فرجامی نژاد

Stevia rebaudiana is an important medicinal plant that is used by people with diabetes because of its steviol glycosides such as stevioside and rebaudioside A. Considering the limitation of seed production in this plant and the time-consuming nature of its propagation by stem cuttings, the use of a hairy root culture can be a suitable strategy for the production of these compounds. In this study, after the production of hairy roots by Agrobacterium rhizogenes strain ATCC15834 and their proliferation, different concentrations of methyl jasmonate (0, 50, 100, 150, and 200 µM) and salicylic acid (0, 30, 60, 90, and 120 mg/L) were applied and sampling was done at different times (24, 48, 72, and 96 h). The use of 100 μM methyl jasmonate for 48 h resulted in the accumulation of 47.31 mg/g DW, the production of 134.30 mg/L stevioside, the accumulation of 45.11 mg/g DW, and the production of 128.41 mg/L rebaudioside A and the use of 200 μM caused the accumulation of 45.56 mg/g DW and production of 120.47 mg/L rebaudioside A. While, among the different concentrations of salicylic acid, only the use of 90 mg/L for 72 h increased the accumulation of rebaudioside A (68.36 mg/g DW), and the other concentrations had a negative effect on the accumulation and production of stevioside and rebaudioside A. In conclusion, these findings showed that methyl jasmonate and salicylic acid can inhibit the growth of hairy roots and instead enhance the accumulation and production of stevioside and rebaudioside A.

The synthetic seeds strategy is an effective method for conservation, germplasm exchange, and distribution of Rosmarinus officinalis. R. officinalis is one of the plants of the Lamiaceae family that originates from the Mediterranean region. According to our knowledge, there is no report on the production of synthetic seeds from somatic embryos obtained from shoots of R. officinalis. In this study, a method for synthetic seed production and regeneration was investigated from shoot explants of R. officinalis. Shoots of R. officinalis were cultured on the MS medium containing various concentrations of NAA and 2,4-D. The best callus induction (41.66%) was obtained on the MS medium fortified with 1 mg/L 2,4-D. The friable embryogenic calli were transferred to a half-strength MS medium with various concentrations of BAP and 2,4-D. Somatic embryos were obtained after 45 days. The highest somatic embryogenesis was obtained on the MS medium containing 1 mg/L 2,4-D and 0.5 mg/L BAP. Somatic embryos at the torpedo stage were encapsulated in sodium alginate and synthetic seeds were obtained. The highest growth ability of synthetic seeds (80.55%) was obtained in the MS medium supplemented with 1.5 mg/L kinetin and 0.5 mg/L BAP. Moreover, the germination percentage of synthetic seeds decreased with increasing storage period and temperature. However, a higher reduction in the synthetic seed germination was recorded at 20±2 °C. The highest germination percentage of the synthetic seeds (85%) was obtained 30 days after storage at 4±1 °C.

Nanoparticles have unique physicochemical properties and provide great opportunities in plant science studies. In this study, we investigated the impact of ZnO, TiO2 and CuO nanoparticles (0, 20, 40, 60, and 80 mg/L) and sampling times (2, 4, and 6 days) on cell suspension growth and azadirachtin accumulation and production. Factorial experiments based on a completely randomized design with three replications were used. Results demonstrated that different nanoparticles had a different effect on the studied characters. When ZnO nanoparticles were used, the highest fresh (540.73 g/L), dry cell weight (15.93 g/L), azadirachtin accumulation (5.15 mg/g DW) and production (68.27 mg/L) were obtained at control condition, 80 and 40 mg/L ZnO nanoparticles, and control condition after 6 days, respectively. The highest amount of fresh (526.95 g/L) and dry (17.05 g/L) cell weight and azadirachtin production (82.21 mg/L) and accumulation (5.93 mg/g DW) were observed in 20 mg/L TiO2 nanoparticles, 40 mg/L of TiO2 nanoparticles after 2 days, 20 mg/L TiO2 nanoparticles in 4 days and 60 mg/L of TiO2 nanoparticles, respectively. With applying CuO nanoparticles, the highest fresh cell weight and azadirachtin accumulation were 422.59 g/L and 4.00 mg/L, achieved in control conditions respectively. Also, the highest amount of azadirachtin production was 68.27 mg/L, observed in control conditions on the 6th day of treatment. It seems that suitable cell growth, except in some cases, occurred in the absence of elicitors, but azadirachtin accumulation and production were stimulated by nanoparticles treatment. However, the results showed that the CuO nanoparticles caused a decrease in overall azadirachtin accumulation and production in the cells.

Azadirachtin is an important secondary metabolite of neem which is used as a natural biopesticide. In this study the effect of different concentrations of sucrose (1, 2, 3, 4 and 5%), calcium chloride, monopotassium phosphate, potassium nitrate, magnesium sulfate and ammonium nitrate (0.5x, 1x, 1.5x, 2x and 2.5x concentrations of MS base medium) on neem cell suspension growth and azadirachtin production was investigated. Although there were no significant difference between some of the treatment in terms of studied indices, the highest amount of fresh and dry cells weight were obtained by using 3% sucrose, 1x calcium chloride, 1x magnesium sulfate and 1x ammonium nitrate (493.02 and 77.27 g/L), 2.5x monopotassium phosphate (500.97 and 80.77 g/L) and 0.5x potassium nitrate (495.01 and 78.41 g/L). The highest settled cell volume was obtained by application of 4% sucrose (59.69%), 1x calcium chloride and 1x ammonium nitrate (94.93%), 2.5x monopotassium phosphate (97.22%), 0.5x potassium nitrate (94.96%) and 2x magnesium sulfate (94.96%). Maximum amount of azadirachtin accumulation observed by using 4% sucrose (3.72 mg/g DW), 0.5x calcium chloride (3.80 mg/g DW), 0.5x monopotassium phosphate (3.73 mg/g DW), 1x and 1.5x potassium nitrate (3.69 mg/g DW), 1x magnesium sulfate (3.69 mg/g DW) and 1.5x ammonium nitrate (3.71 mg/g DW). The highest amount of azadirachtin production was 286.65 mg/L obtained in MS basal medium supplemented with 3% sucrose.

S. rebaudiana produces steviol glycosides including stevioside and rebaudioside A that are valuable as low calorie sweeteners. The objective of this study was to investigate the effects of salicylic acid elicitation and sampling times on the improvment of stevioside and rebaudioside A production and KA13H, UGT74G1 and UGT76G1 genes expression. The results showed that the addition of different concentrations of salicylic acid had different effects on stevioside and rebaudioside A production in a dosage-dependent manner. Among different concentrations of salicylic acid the highest amount of stevioside was 38.33 mg/g DW and the highest amount of rebaudioside A was 2.93 mg/g DW, observed 96 h after elicitation with 90 mg/L and 48 h after elicitation with 60 mg/L salicylic acid, respectively. Elicitation with salicylic acid increased KA13H and UGT74G1 genes expressions and decreased UTG76G1 gene expression. The correlation analysis indicated that, KA13H gene expression had a positive correlation with stevioside production. The UGT74G1 gene expression had a positive correlation with stevioside production and a negative correlation with rebaudioside A production. The UGT76G1 gene expression had a positive correlation with rebaudioside A and negative correlation with stevioside production. The qRT-PCR analysis indicated that, by increasing stevioside production under salicylic acid elicitation, the KA13H and UGT74G1 genes expression increased and UGT76G1 gene expression decreased.

Using of elicitors and different compounds in cell suspension culture is an effective strategy for enhancement and improvement of important secondary metabolites production in in vitro conditions.
Evaluation of the effects of L-tyrosine and its combination with biological elicitors such as chitosan and salicylic acid on thebaine production in Iranian poppy cell suspension cultures at different days after elicitation.
Different concentrations of L-tyrosine (1 and 2 mM) individually and in combination with chitosan (100 mg/L) and salicylic acid (50 mg/L) was applied on cell cultures, and then, pH and EC of medium, and also thebaine content of cells were measured on 2, 4 and 6 days after treatment.
The highest thebaine content of cells (42.30 mg/L) was obtained in 1 mM L-tyrosine on six days after treatment, which was significantly higher than the control. In addition, there was a relation between thebaine content of cells and pH and ECof culture medium. With increasing the thebaine content of cells, pH of medium was increased and EC of medium was decreased.
Feeding of Iranian poppy cell suspension cultures with L-tyrosine (precursor of alkaloids biosynthesis) was improved thebaine production. So that, 1 mM L-tyrosine increased the thebaine content of cells up to 22.26-fold compared with control.