In the present study the extracts of in vivo and in vitro grown plants as well as callus tissue of red clover were tested for their antioxidant activities, using different extraction solvent 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 amounts of total phenolic and total flavonoids content were found in methanol extract of 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 red clover, while 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. According to the findings, the extract of in vitro culture of red clover especially the callus tissue possesses a comparable antioxidant activity to the in vivo cultured plants' extract.

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.

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.

Abstract-Antimicrobial efficiency of extracts derived from Trifolium pratense (in vivo and in vitro grown plants, including callus) were examined using ethanol and methanol as solvents 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 callus of Trifolium pretense.

The present study deals with the cytological investigations on the meristematic root cells of carnation (Dianthus caryophyllus Linn.) grown in vivo and in vitro. Cellular parameters including the mitotic index (MI), chromosome count, ploidy level (nuclear DNA content), mean cell and nuclear areas, and cell doubling time (Cdt) were determined from the 2 mm root tip segments of this species. The MI value decreased when cells were transferred from in vivo to in vitro conditions, perhaps due to early adaptations of the cells to the in vitro environment. The mean chromosome number was generally stable (2n = 2x = 30) throughout the 6-month culture period, indicating no occurrence of early somaclonal variation. Following the transfer to the in vitro environment, a significant increase was recorded for mean cell and nuclear areas, from 26.59 ± 0.09 μm2 to 35.66 ± 0.10 μm2 and 142.90 ± 0.59 μm2 to 165.05 ± 0.58 μm2, respectively. However, the mean cell and nuclear areas of in vitro grown D. caryophyllus were unstable and fluctuated throughout the tissue culture period, possibly due to organogenesis or rhizogenesis. Ploidy level analysis revealed that D. caryophyllus root cells contained high percentage of polyploid cells when grown in vivo and maintained high throughout the 6-month culture period.