**2. Materials and methods**

#### **2.1 Experimental location**

Experiments were conducted in the Department of Technology for Organic Synthesis, Institute of Chemical Engineering Ural Federal University, Ekaterinburg, Russia.

#### **2.2 Raw material location**

The raw materials included *C. intybus, S. rebaudiana Bertoni, Monarda citriodora, Rhodiola krylovii Chicory, Stevia, Cambridge scarlet (M. didyma), Rhodiola krylovii* and were all obtained within Russian Federation.

#### **2.3 Raw material consideration**

The primary raw material consideration for the *in vitro* cultivation of these plant cells includes solid and liquid culture media with enough nutrient composition required for the plant cells' growth.

#### *2.3.1 Materials/equipment for experiment*

#### *2.3.1.1 Reagents for media*

Sucrose, solution of iron chelate, macronutrients, micronutrients, vitamins, Kinetin, 1-naphthalene acetic acid (NAA), dichlorophenoxyacetic acid, agar, and distilled water 100 ml.

#### *2.3.2 Reagents for plant cells sterilisation*

Ethanol (70%), sodium hypochlorite (3%), Tween detergent, sterile water, carboxylic acid amide (CAA) fungicide (2%).

#### *2.3.3 Equipment*

Climate chamber, Petri dishes, laminar system, test tubes, autoclave, oven, stirrer, laboratory glassware (conical flask, beakers, burette), micropipette and pipette tips, knife, forceps, filter paper, foil paper, electric stove, refrigerator, pincers, Bunsen burner, thermometer, and pH meter.

#### *2.3.4 Methods*

Different methods/procedures were used to cultivate *C. intybus, S. rebaudiana Bertoni, Monarda citriodora and Rhodiola krylovii Stevia, Chicory Cambridge scarlet, and Rhodiola krylovii* to identify which sterilisation method was best for each plant type and which procedure would not incur contamination. Studying the effectiveness of various sterilisation methods for seed cultivation on solid media in tubes for callus formation (medium M&S + kinetin) and cultivating seeds on solid media in tubes to obtain sterile micro plants (medium M&S) were carried out. The germination of non-sterile/sterile seeds and obtaining non-sterile and sterile plant seedlings in the Petri dish were carried out. Growing sterile micro explants for callus formation (medium M&S + kinetin) and preparation of cell suspension cultures for wet and dry weight determination.

#### *2.3.5 Media formulation*

The media for this research was formulated from Murashige and Skoog Culture Media (1962). The Murashige and Skoog Medium is a shared medium used to cultivate laboratory plant cells. The discovery was made by two Plant scientists, Toshio Murashige and Folke K. Skoog [97], searching for a new type of plant growth regulator. Using Murashige and Skoog medium supplemented with 3 g of sucrose, 0.5 ml solution of iron chelates, 5 ml of macronutrients, 0.1 ml of micronutrients, 0.1 ml of vitamins, 0. ml of Kinetin, 0.25 ml of 1-naphthalene acetic acid (NAA), 0.8 to 0.9 ml of agar, and 100 ml distilled water for every volume of 100 ml media.

#### *2.3.6 Preparation of solid culture media using Murashige and Skoog method*

A total of 100 ml of distilled water was poured into a 250 ml round bottom flask. A weighing balance was used to measure 0.8 g of agar added to the water. The sample was placed on a heating device (infrared cooker) at the temperature of 60°C; 0.25 ml of 1-naphthalene acetic acid (NAA) was measured with a micropipette and poured

#### *Obtaining Cell Cultures of Medicinal Plants DOI: http://dx.doi.org/10.5772/intechopen.104650*

into the sample, and stirred. Then, 3 g of sucrose was added to the sample on heating and stirred, followed by adding 0.1 ml of Kinetin and 0.5 ml of chelate were added to the heating sample. After 10 mins, the sample was removed from the heating device and allowed to cool at room temperature (25°C). Then, 5 ml of macronutrients, 0.1 ml of micronutrients, and 0.1 ml of vitamin were added to the sample as the temperature reached 40°C. The sample was stirred and covered with cotton and foil paper with a plastic band around the cover and kept in the refrigerator to solidify for over 12 hours. The media was sterilised in an autoclave for 1 hour at 121°C. The sample was placed on a heating device for 20 mins at 60°C, and after heating, the sample was left to cool for 10 mins before transferring to test tubes.

#### *2.3.7 Preparation of liquid culture media using Murashige and Skoog method*

A total of 100 ml of distilled water was added to a 250 ml round bottom flask. The sample was placed on a heating device (infrared cooker) at a temperature of 60 °C and 0.125 ml of 1-naphthalene acetic acid (NAA) and 0.1 ml of dichlorophenoxyacetic acid, whereas measured with a micropipette and poured into the sample and stirred. Next, 3 g of sucrose was added to the sample while heating and stirring.

Then, 0.1 ml of Kinetin and 0.5 ml of chelate were added to the heating sample. After 10 mins, the sample was removed from the heating device and allowed to cool at room temperature before adding 5 ml of macronutrients and 0.1 ml of micronutrients. A total of 0.1 ml of the vitamin was added to the sample when the temperature was 40° C. The sample was stirred and covered with cotton and foil paper with a plastic band around the cover before it was preserved and kept in the refrigerator to solidify for more than 12 hours. The media was sterilised in an autoclave for 1 hour at 121°C. The sample was placed on a heating device for 20 mins at 60°C, and the sample was left to cool for 10 mins before transferring into a sterile flask. Different for callus cultivation.
