*2.3.8.3 Growing sterile explants for callus formation (Medium M&S + kinetin)*

I.Growth Media with Kinetin (+Kinetin)

The media for growing callus was slightly different, with a variance in supplements (Murashige and Skoog media, 1962). A 300 ml M and S media volume was prepared, and 9 ml was poured into 24 sterile test tubes. The nonsterile seedlings from the six Petri dishes containing Chicory, Rhodiola krylovii, and Cambridge scarlet were transferred to the test tubes after cutting and disinfection: First roots of seedlings were cut, and explants were disinfected using 2% carboxylic acid amide (CAA) fungicide for 2 mins, followed by the washing of explants in 70% ethanol for 2 mins. Explants were rinsed with sterile water. Seeds were washed with 3% NaOCl for 2 mins and rinsed with sterile water. Explants were transferred into labelled sterile test tubes and were incubated for 2 weeks with conditions of no light intensity, a temperature of 250C (�1 °C), and relative humidity of 50%.

### II.Growth Media without Kinetin (-Kinetin)

The volume of 600 ml of M and S media without Kinetin was prepared using the above reagents for plant cells. Then, 9 ml of media was poured into 60 sterilised test tubes each. Sterile seedlings from the six Petri dishes containing Chicory, Bee balm (Cambridge scarlet), and Rhodiola krylovii were cut and thoroughly washed. Disinfection of explants was done using 2% carboxylic acid amide (CAA) fungicide for 2 mins followed by washing with 70% ethanol for 2 mins and then with 3% NaOCl solution for 2mins. Rinsing of explants thoroughly with sterile water thrice (three times) in three separate sterilised Petri dishes for 5 mins was carefully carried out. The three plants' explants were put into 20 labelled sterilised test tubes, respectively, and the remaining media was preserved in the refrigerator. Incubation of explants was done under conditions of no light intensity, the temperature of 25°C (1°C), and relative humidity of 50% for a period of 2 weeks.@

The formula below was used to determine the percentage contamination for the above experiments:

% of Contamination ¼ ð Þ Number of Contaminated Test tubes with cells *=* ð Þ Total Number of Test tubes with cells <sup>∗</sup> <sup>100</sup> (2)

#### *2.3.8.4 Preparation of cell suspension cultures*

The callus of Chicory was transferred into two sterile flasks containing M and S cell suspension cultures of Kinetin and without Kinetin. Samples were crushed with a knife and stirred vigorously to aid the formation of more cells. The samples were incubated in plant-controlled equipment under conditions of continued shaking with 16 hours of light intensity phase at 24(�1)C and 8 hours of dark phase at 18 (�1) 0C and after 1 week, the callus was transferred to a new cell suspension culture maintaining the conditions for a week. One week later, the callus in both flasks was transferred to six sterile flasks using the same media formulation; three flasks had no kinetin, while three had Kinetin in the media. A total of 10 ml of starter culture (cell suspension) and 50 ml of liquid media were poured into the six labelled sterile flasks, and the samples were returned to the plant-controlled equipment to grow to maintain the conditions of continuous shaking with 24 hours of light at 24 °C (�1) for 4 weeks. Each week the samples were checked to ensure that they were free from contamination.

#### **2.4 Microscopy**

The overall magnification can be calculated as the product of the lenses and the distance over which the image is projected:

$$\mathbf{M} = (\mathbf{D} \ast \mathbf{M1} \ast \mathbf{M2}) / 250 \text{ mm} \tag{3}$$

where D = projection (tube) length (usually = 250 mm); M1, M2 = magnification of objective and ocular. Thus, 250 mm = minimum distance of distinct vision for 20/20 eyes [98]. Callus of Chicory grown using cell suspension culture (liquid media) of + Kinetin and – Kinetin was viewed under the microscope using a 10 mm focal point.

#### **2.5 Wet and dry weight analysis**

Drying process was achieved by initially setting the oven temperature at 60°C. 5 ml of Kinetin containing media starter was pipetted into three separate centrifuge tubes and properly labelled. Afterwards, 5 ml of media without Kinetin was pipetted into three different and well labelled centrifuge tubes. Centrifugation was done for 10 mins on 5000 rpm at 15°C; thus, the aliquots were separated, and 5 ml of sterile water was added to the supernatants and centrifugation was done twice. Six filter papers (2.5 cm) were dried in the oven for 9 hours at 60°C. Then, the empty weight of filter papers was obtained, followed by the weight of filter papers with samples. The samples were later placed in the oven for 24 hours at 60°C. The weight of the dried samples on the filter papers were obtained. The fresh and dry weights were determined one week after transferring the seeds into a new culture media.

Wet wt gð Þ¼ *=*L ð Þ Wt of wet filter paper þ cells gð Þ wt of wet filter paper gð Þ *=* ð Þ Sample vol mL ð Þ x 100

(4)

Dry wt gð Þ¼ *<sup>=</sup>*<sup>L</sup> Wt of dry filter paper <sup>þ</sup> cells gð Þ wt of dry filter paper gð Þ *<sup>=</sup>* ð Þ Sample vol mL ð Þ x 100 (5)

#### **2.6 Charts**

Bar charts and area charts were used to present the data obtained from the experiments.
