4 research outputs found
Micropropagation of Bioencapsulation and Ultrastructural Features of Sainfoin ( Onobrychis viciifolia
To explore the potential of in vitro rapid regeneration, three varieties (Golpaygan-181, Orumieh-1763, and Gorgan-1601) of sainfoin (Onobrychis viciifolia Scop. syn. Onobrychis sativa L.) were evaluated. For the first time, an encapsulation protocol was established from somatic embryogenic callus in torpedo and cotyledonary stages to create artificial seeds. Callus derived from different concentrations of Kinetin (0–2.0 mg L−1) and Indole-3-acetic acid (0–2.0 mg L−1) was coated with sodium alginate and subsequently cultured either in Murashige and Skoog (MS) medium or in soil substrate. Adventitious shoots from synthetic beads developed into rooting in full and half strength MS medium supplemented with various concentrations of auxin and cytokinin. Prolonged water conservation of black and red soils (1 : 1) had the highest rate of survival plantlets in the acclimatization process. Diverse resistance techniques in Onobrychis viciifolia were evaluated when the plants were subjected to water deficiency. Higher frequency of epicuticular waxes was observed in in vivo leaves compared to in vitro leaves. Jagged trichomes nonsecreting glands covered by spines were only observed in the lower leaf side. Ultimately, stomata indices were 0.127 (abaxial), 0.188 (adaxial) in in vivo and 0.121 (abaxial), 0.201 (adaxial) in in vitro leaves
Stimulatory Effects of Gamma Irradiation on Phytochemical Properties, Mitotic Behaviour, and Nutritional Composition of Sainfoin ( Onobrychis viciifolia
Sainfoin (Onobrychis viciifolia Scop. Syn. Onobrychis sativa L.) is a bloat-safe forage crop with high levels of tannins, which is renowned for its medicinal qualities in grazing animals. Mutagenesis technique was applied to investigate the influence of gamma irradiation at 30, 60, 90, and 120 Gy on mitotic behavior, in vitro growth factors, phytochemical and nutritional constituents of sainfoin. Although a percentage of plant necrosis and non-growing seed were enhanced by irradiation increment, the germination speed was significantly decreased. It was observed that gamma irradiated seeds had higher value of crude protein and dry matter digestibility compared to control seeds. Toxicity of copper was reduced in sainfoin irradiated seeds at different doses of gamma rays. Anthocyanin content also decreased in inverse proportion to irradiation intensity. Accumulation of phenolic and flavonoid compounds was enhanced by gamma irradiation exposure in leaf cells. HPLC profiles differed in peak areas of the two important alkaloids, Berberine and Sanguinarine, in 120 Gy irradiated seeds compared to control seeds. There were positive correlations between irradiation dose and some abnormality divisions such as laggard chromosome, micronucleus, binucleated cells, chromosome bridge, and cytomixis. In reality, radiocytological evaluation was proven to be essential in deducing the effectiveness of gamma irradiation to induce somaclonal variation in sainfoin
Studies on micropropagation, cellular behavior, photosynthetic and biological activities of red clover (Trifolium pratense L.) / Arash Khorasani Esmaeili
Tissue culture studies of a temperate forage crop, Trifolium pratense L. were investigated in the current project. In vitro regeneration of this species was successfully achieved in this study using nodal explants cultured on Murashige and Skoog (MS) media supplemented with different hormones at various concentrations and also on MS hormone free media as a control. Complete plant regeneration of T. pratense was best achieved when the nodal explants were cultured on MS media supplemented with 1.5 mg/l BAP and 0.5 mg/l IBA, with mean number of 6.05 ± 0.28 shoots per explant, and 100% of the explant samples produced shoots. On the other hand, the best root formation was obtained on MS media supplemented with 1.5 mg/l BAP and 0.75 mg/l IBA, with the mean number of 3.3 ± 0.21 roots per explant. However, the nodal explants cultured on MS hormone free medium failed to produce any shoots or roots. Callus formation was successfully achieved when the nodal explants were cultured on MS medium containing different types of plant hormones. MS medium supplemented with 1.5 mg/l BAP and 0.5 mg/l 2,4-D was the most responsive, whereby 100% of the explants managed to produce callus.
Adaptation process to the natural environment or acclimatization, i,e. the transfer of in vitro grown plants to the ex vitro condition was successfully undertaken, with very high survival rates of plantlets (93.71 ± 4.64 %) when they were transferred to the combination of red soil and black soil with the ratio of 1:1.
Subsequently, the extracts of in vivo and in vitro grown plants as well as callus tissues of T. pratense were tested for their antioxidant activities, using different extraction solvents and different antioxidant assays. The total flavonoid and phenolic contents as well as extraction yield of the extracts were also investigated to determine their correlation with the antioxidant activity of the extracts. Among all the tested extracts, the highest amount of total phenolic and total flavonoids content were found in methanol extract from in vivo grown plants. The antioxidant activity of tested samples followed the order; in vivo plant extract ˃ callus extract ˃ in vitro extract. The highest reducing power, 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging and chelating power were found in methanol extracts of in vivo grown T. pratense. Whilst the chloroform fraction of in vivo grown plants showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, superoxide anion radical scavenging and hydrogen peroxide scavenging compared to the other tested extracts. A significant correlation was found between the antioxidant activity of extracts and their total phenolic and total flavonoid content.
The cytotoxicity of the plant extracts were examined against two human cancer cell lines (human breast carcinoma (MCF-7) and human colon carcinoma (HCT-116)), using MTT assay. Four different extraction solvents were used to examine the effect of the solvent on cytotoxic activity of the extracts. Two cancer cell lines were treated with the extracts for 24, 48 and 72 hours. All of the examined extracts exhibited toxicity on the tested cell lines in a time dependent increase, but in a lower potency than doxorubicin (positive control). The chloroform fraction of in vivo grown plants showed the highest cytotoxic activity against the MCF-7 cell line (IC50 = 66.44 ± 2.05 μg/ml) but it was not significantly different with the cytotoxic activity of chloroform fraction of callus tissue (IC50 = 69.48 ± 2.66 μg/ml). The highest cytotoxic activity against the HCT-116 cell line was shown by the chloroform fraction of callus tissue (IC50 = 79.53 ± 2.00 μg/ml).
The antimicrobial efficiency of extract derived from T. pratense (in vivo and in vitro grown plants, including callus) were examined using ethanol and methanol as solvents for extraction and tested against four bacterial pathogens (two gram negative and two gram positive) and three fungal pathogens. The antimicrobial activity of the methanol extract was found to give higher inhibition zone when compared with ethanol extract. Among the callus, in vitro and in vivo grown plants, the callus extract showed better antimicrobial activity, thus revealing a new potential use of T. pratense callus.
To compare the photosynthetic parameters, the Stomatal conductance (gs), Transpiration rate (E) and Net photosynthetic (Pn) were determined for the plants grown under in vitro and in vivo conditions. A comparison was made for the observed data for the light-saturated photosynthetic among the treatments which revealed that the maximum photosynthetic rate (PNmax) was 18.3 and 11.3 μmol (CO2)/m2/s in in vivo and in vitro plant leaves, respectively. Respiration (Rd) and Compensation point (CP) were found 1.5-folds and two-folds higher in in vitro plants, respectively. On the other hand, the in vitro grown plants exhibited higher transpiration rate and also higher stomatal conductance compared with the in vivo plants. Consequently, high levels of differentiation in terms of photosynthesis parameters exist among the in vivo and in vitro samples. Significant direct relation was observed between net photosynthetic rate and total phenolic and flavonoid content of T. pratense leaves.
The effect of optimal and supra-optimal concentrations of Sodium chloride (NaCl) on growth and antioxidant defence was also studied in the in vitro cultures of T. pretence. Seeds of T. pratense were germinated in Murashige and Skoog medium (MS) containing different concentrations of NaCl (0, 50, 100, 150, 200 mM). The lengths of roots and shoots as well as percentage of germination, free radical scavenging activity (DPPH) and Superoxide dismutase (SOD) were measured. A significant decrease in germination and growth was observed in the seeds exposed to 100, 150 and 200 mM salt. The highest percentage of germination was found in the MS medium containing 50 mM NaCl, although the highest root and shoot length were found in MS medium without NaCl. The highest antioxidant activity of methanol extract of the plants occurred in in vitro plants cultured in MS medium supplemented with 50 mM NaCl. A significant decrease in free radicals scavenging and superoxide dismutase activities were found in plants grown in media containing 100, 150 and 200 mM salt
Micropropagation, antioxidant and antimicrobial activities of Asparagus officinalis Cv. Mary washington in vivo and in vitro / Arash Khorasani Esmaeili
In recent years there has been renewed interest in natural medicines that are obtained from plant parts or plant extracts. Asparagus officinalis is a herbaceous perennial plant that belongs to Liliaceae family, a valued vegetable for its medicinal properties.
The present study was carried out in order to establish an efficient in vitro propagation protocol for Asparagus officinalis. For this purpose, the nodal explants of Asparagus officinalis Cv. Mary Washington were cultured on MS medium containing 3% sucrose and different concentrations of NAA and BAP or IBA and Kn, mixed or separately with range of 0-1.5 mg/l for BAP/Kn and 0-0.5 mg/l for NAA/IBA in order to obtain callus, shoot and root formation. In this study, indirect organogenesis was tested under 2 different conditions (In light and in dark) for the regeneration of Asparagus officinalis in vitro. After 6 weeks of culture the results showed that 100% of callus formation in 17 of treatments under dark and 3 treatments under light condition. So between dark and light condition, dark condition was found to be more efficient than light condition in promoting callus formation. Also among the two groups of hormones (BAP + NAA, Kn + IBA concentrations), Kn + IBA was reported to be more efficient that BAP + NAA in promoting callus formation. Results also showed that the highest average number of shoots (4.25) of size 4 mm or more per explant, formed under dark condition using 1.5 mg/l BAP mixed with 0.05 mg/l NAA. The formed shoots under dark condition were less developed, with abnormal thick and yellow color compared with the shoots produced under light condition. In light condition the highest average numbers of shoots (3.63) of size 4 mm or more per explant were found on the MS medium supplemented with 0.8 mg/l BAP alone, not in combination with NAA. Rooting was best induced in shoots excised from shoot cultures which were proliferated on MS medium supplemented with an optimal concentration of 0.4 mg/l IBA (2 roots per explant). In the second part of the study the antioxidant and antibacterial activities of ethanolic extracts of in vivo grown Asparagus officinalis cv. Mary Washington were investigated using superoxide dismutase, erythrocyte haemolysis and 2,2- diphenyl-1-picrylhydrazil free radical scavenging methods. The measured antioxidant and antimicrobial potential were then compared with the activities shown by the ethanolic extracts of in vitro grown A. officinalis as well as ethanolic extract of undifferentiated callus cells of A. officinalis produced on Murashige and Skoog medium containing 1.5 mg/l 6-benzylaminopurine combined with 0.5 mg/l naphthalene acetic acid. The highest antioxidant capacity was obtained from the in vivo grown plant extract followed by in vitro grown plant extract in all three examined assays. Although, no antibacterial activity was detected from both in vivo and in vitro grown plant extracts in the disc diffusion antimicrobial assay, ethanolic extract of A. officinalis offered antibacterial activity against Bacillus cereus