Document Type : Research Paper

Authors

• M. Sarrafi ¹
• M. Mahmoodnia Meimand ²
• M. Dahajipour ³
• M.R. Dehghani ³

¹ Genetics and Plant Production Dept, Agriculture College, Vali-eAsr University of Rafsanjan, Rafsanjan, Iran

² Genetics and Plant Production Dept. Agriculture College, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran

³ Genetics and Plant Production Dept, Agriculture College, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran

Abstract

     Background and objectives: In recent years, Urtica dioica L. has attracted the attention of researchers due to its significant effect on many diseases, especially diabetes. However, more research needs to be done in the field of tissue culture and the effect of elicitors on antioxidant properties and the production of its secondary metabolites. This research aimed to investigate the best hormonal combination and light conditions effective on nettle callogenesis and the effect of methyl jasmonate elicitor on its biochemical properties.
Methodology: For this purpose, using stem explants, the effect of different levels of auxin hormones, naphthalene acetic acid (NAA) (1.5 and 3 mg/l) and 2,4-dichlorophenoxy acetic acid (2,4 D) (0.5 and 1.5 mg/l) and cytokinin hormone, benzylaminopurine (BAP) (0.5 and 1.5 mg/l) were evaluated in two light conditions (light and dark) by measuring fresh weight, dry weight and percentage of callogenesis. The nettle plant's callus growth curve was obtained using liquid culture. After starting the suspension culture, methyl jasmonate treatment was applied with three levels of 1, 0.1 and 0.01 mM, and sampling was done at 24, 48 and 96 hours. The enzyme activity of phenylalanine ammonialyase and polyphenol oxidase and total protein content by extracting in 50 mM Tris-HCl buffer with 8.8 acidity and total phenol, total flavonoid content by extracting in 80% methanol was measured using spectrophotometric and standard methods for control samples and those under different treatments of methyl jasmonate. Also, three metabolites of quercetin, kaempferol and rutin were measured using an HPLC device with a UV detector and 18C column stationary phase. Data from the callogenesis experiment were analyzed in a two-factor experiment in a completely randomized design. The data from the biochemical and HPLC experiments were analyzed in a split-plot in time design, and the means were compared with Duncan's test.
Results: According to the results, the best hormonal combination to produce fresh and dry weight in light conditions includes 1.5 mg/l of NAA and 1.5 mg/l of BAP and in dark conditions, it includes 1.5 mg/l naphthalene acetic acid and 0.5 mg/l of benzyl aminopurine. Also, according to the results, naphthalene acetic acid (62.5%) performed better than 2,4-D (43.5%) in the nettle callogenesis percentage. Lighting was determined as the best light condition for callogenesis in nettle, so the amount of fresh and dry weight and percent of callogenesis in light compared to darkness increased by 4.79, 4.91 and 1.92 times, respectively. There was an increase in polyphenol oxidase enzyme activity, total phenol and total protein in some samples treated with methyl jasmonate compared to the control, and no significant increase was observed in the activity of phenylalanine ammonialyase enzyme and total flavonoid content. According to the HPLC results, the highest amount of rutin metabolite was obtained in 96 hours after the addition of the elicitor and at the concentration of 0.01 mM methyl jasmonate.
Conclusion: The naphthalene acetic acid hormone was more effective than the 2,4-D hormone and light conditions than darkness in nettle callogenesis. Methyl jasmonate elicitor increased the antioxidant property of nettle (48 hours after treatment) by increasing the polyphenol oxidase enzyme activity. However, compared to the control, it had no significant effect on the phenylalanine ammonialyase enzyme activity and total flavonoid. It is suggested that other concentrations of methyl jasmonate be tested to reach optimal conditions for increasing the production of secondary metabolites in nettle in vitro culture conditions.

Keywords

• Auxin
• antioxidant
• polyphenol oxidase
• secondary metabolite
• rutin
• nettle (Urtica dioica L.)

Main Subjects

• Biotechnology