بهنام کارجو

Optimization of the culture medium of hairy roots in terms of the composition and type of ingredients in the medium is essential for proper growth and optimal biomass production. The greatest advantage of culturing Hairy roots is that they are often capable of producing secondary metabolites compared to the mother plant. In this research, transgenic hairy root induction was established through the mediation of the A7 strain of Agrobacterium rhizogenes. Molecular confirmation of hairy roots was done by PCR using rolB gene-specific primers. With response surface methodology (RSM) coding, the effect of three types of basal liquid MS media (MS, ½ MS and ¼ MS) with different sucrose concentrations (15, 30, and 45 g / L) and three pH levels (5.5, 5.8 and 6.1) on the growth of hairy root was investigated. The fresh and dry weight of hairy roots was measured after 4 weeks. The obtained results of response surface methods showed that the highest dry weight of hairy roots in the culture medium with a concentration of 0.56 of MS (≅ 1/2MS), 30 g of sucrose, and pH of 5.8. In optimum conditions, the utility value was 0.990, and the fresh and dry weight of hairy roots, was 8.58g and 0.317g, respectively. According to GC/MS results, the amount of atropine alkaloid in hairy roots grown in optimum culture medium was about 6.88%.

The rapid and mass production of secondary metabolites through chemical methods is mostly expensive, difficult, or impossible. Considering the economic importance of these metabolites and their small production in medicinal plants, some methods such as the cultivation of hairy roots and the use of biotic and abiotic stimuli have been suggested to increase these metabolites. The accumulation of the tropane alkaloid atropine in Atropa belladonna L. is of great importance in the pharmaceutical industry. In this research, transgenic hairy roots of A. belladonna was produced using an A7 strain of Agrobacterium rhizogenes. The transformation of hairy roots was confirmed by PCR test. The results of the PCR test showed the correctness of the amplification of the bp780 fragment for the rolB gene in hairy roots. In addition, the effect of different concentrations (0, 125, 250, and 500 μM) of salicylic acid in two times of 24 and 48 hours on the amount of atropine content, the plant growth rate and the activity of antioxidant enzymes of A. belladonna hairy roots was investigated. The data statistically analyzed using factorial experiment based on a completely random design with three replications. The results of the analysis of variance showed significant effect of salicylic acid on all the traits (P<0.01). The effect of time was significant for the amount of atropine and ascorbate peroxidase (APX). The salicylic acid by time interaction effects were significant for the amount of atropine, catalase (CAT), and guaiacol peroxidase (GPX) (P<0.01, 0.05). The results showed that by increasing salicylic acid concentrations the growth of hairy roots significantly decrease compared to the control and the lowest values of fresh and dry weight of the hairy roots (2.79 and 0.148 g) were obtained in 500 μM of salicylic acid compared to the control (6.76 and 0.273 g). However, based on GC/MS analysis, the highest amount of atropine (11 times more than the control) was obtained in the 250 μM salicylic acid in a period of 24 hours. The highest catalase and guaiacol peroxidase activities were obtained at concentrations of 250 and 125µM salicylic acid for 24 hours, respectively. The highest level of ascorbate peroxidase enzyme activity was obtained at the concentration of 250 µM salicylic acid compared to the control. It was concluded that the salicylic acid stimulant at low concentrations is useful for cell signaling and stimulating the production of secondary metabolites, but at high concentrations, it disrupts the growth of hairy roots.

Atropa Belladonna L. is the most important medicinal plant of Solanaceae family, due to important commercial sources of atropine and scopolamine as tropane alkaloids. Applications of hairy root cultures are advised because of their special properties such as fast growth, short doubling time, ease of maintenance, and ability to synthesize and continuous production of a range of chemical compounds. Effects of ozone depletion and UV irradiation have been studied by most of the investigators. In the present study, the effect of different levels of UV-B irradiation was studied on atropine production and activity of some antioxidant enzymes. UV-B irradiation during 3 days was treated for 4, 8 and 12 min that was equal to 13, 26 and 39 Kj/m2 respectively. Results showed that increase of UV-B lending to decrease hairy roots dry weight. This irradiation increased atropine content in comparison of control treat and the highest was recorded in 26 Kj/m2. The activity of antioxidant enzymes including catalase, guaiacol peroxidase, and ascorbate peroxidase was also elevated in treated hairy roots rather than of the control at 0.01% of probability. Based on obtained results in this work UV-B irradiation can be considered as one of the useful methods for Atropine and antioxidant enzymes in A. belladonna hairy roots.