Document Type : Research Paper

Authors

• Seyedeh Mohaddeseh Hosseini ¹
• Seyed Ali Andi ²
• Amir Sahraroo ¹
• Mohammad Bagher Farhangi ¹

¹ Guilan University

² Amol University of Special Modern Technologies

Abstract

    Background and objectives: Adventitious root cultures of medicinal plants represent a bountiful reservoir of valuable secondary metabolites.  The esteemed medicinal plant Echinacea purpurea (L.) Monech is highly sought after for its phytochemical properties, with a traditional use of its roots in herbal medicines and dietary supplements as an immune system stimulant for treating inflammatory, viral, and respiratory diseases. However, the heterogeneous synthesis of bioactive compounds in natural environments and the limitations of conventional plant production methods necessitate exploring alternative approaches for obtaining active plant substances and organs. Among various in vitro cultivation techniques, the cultivation of differentiated tissues, specifically roots, consistently demonstrates the ability to yield higher quantities of metabolites than undifferentiated tissues such as callus and suspension cells. 
Methodology: The primary aim of this study was to accurately determine the ideal combination of plant explants and plant growth regulators that would facilitate the successful establishment of adventitious root suspension cultures. Three distinct plant explants, namely leaf, petiole, and root, were carefully evaluated in this study. These explants were subjected to varying concentrations (0, 0.5, 1, 1.5, and 2 mg.L-1) of two types of plant growth regulators: indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA). After six weeks, the number of adventitious roots developed by the explants under different concentrations of plant growth regulators was measured. The impact of different sucrose concentrations (10, 20, 30, 40, 50, and 60 grams per liter) in the culture medium was investigated over a four-week cultivation period. Various dynamic characteristics of root growth, including fresh weight, dry weight, volume of the remaining medium, electrical conductivity, and pH, were closely examined. Furthermore, phytochemical properties such as total phenol, total flavonoid, and antioxidant activity were assessed. To administer the sucrose treatments, 0.35 grams of adventitious roots were placed inside 250 ml Erlenmeyer flasks containing 50 ml of half MS culture medium, 1 mg.L-1IBA, and varying concentrations of sucrose, maintaining a pH of 5.8. Measurements were taken from three flasks of each treatment weekly. The experiments followed a Completely Randomized Factorial Design, with statistical analysis conducted using SPSS software.
Results: Leaf explants exhibited superior root formation to root and petiole explants. The average number of adventitious roots in leaf explants reached 3.37, while petiole and root explants produced only 0.23 and no adventitious roots, respectively. The most effective root formation occurred in MS medium with 1 mg.L-1 IBA, resulting in an average of 4 roots per explant. Hormone concentrations of 0 and 2 mg.L-1 did not induce root formation. In the fourth week of cultivation, the highest fresh weight recorded was 29.8 grams per liter, marking a remarkable 3.4-fold increase from the initial inoculation weight. The culture medium's electrical conductivity and pH decreased as the cultivation progressed. Cultivation weeks did not significantly impact total flavonoid production and related antioxidant activity in adventitious roots. However, the cultivation period significantly affected the total phenol content at 57.56 mg of gallic acid per gram of dry weight. Altogether, lower sucrose concentrations proved more efficient for biomass production, bioactive compound content, and antioxidant activity. The highest biomass accumulation and total phenol content occurred at 3% sucrose. In comparison, in the fourth week of cultivation, the highest total flavonoid content and the highest free radical inhibition percentage were observed at 2% and 1% sucrose, respectively. Notably, a negative correlation was observed between the electrical conductivity of the remaining culture medium and root biomass in lower sucrose concentrations as the culture duration increased.
Conclusion: In light of the immense potential harbored by adventitious root suspension cultures of E. purpurea as an alternative source for the production of secondary metabolites, particularly caffeic acid derivatives, the discoveries made in this research have the potential to advance the field of mass production for active root organs, thereby facilitating the synthesis of these precious compounds through the implementation of appropriate laboratory bioreactor systems.

Keywords

• Purple coneflower (Echinacea purpurea (L.) Monech)
• sucrose feeding
• adventitious root culture
• biomass
• phenolic compounds

Main Subjects

• Biotechnology