*3.1.2 Method B*

The result of sterilisation in Method B **Figure 2** shows that Chicory had 40% contamination and Stevia had 55% contamination. In contrast, Bee balm and *Rhodiola krylovii* had a 100% level of contamination. This sterilisation method of plant seeds is suitable for Chicory (40%) since the contamination percentage was few. Stevia seeds also can be sterilised using this method because the percentage level of contamination after cultivation was 55%. Nevertheless, this method is unsuitable for Bee balm and *Rhodiola krylovii* since the level was very high (100%), as shown in **Figure 2** and **Table 3**, and the agar composition in this method was 0.8 g, and this could have affected the growth rate of the plants.

**Figure 2.** *Chart showing percentage contamination of method B.*


**Table 3.**

*Growing sterile explants for callus formation (Solid Medium M&S + kinetin and – Kinetin).*

**Figure 3.**

*The chart showing percentage contamination of method C comes after the subheading method C.*

#### *3.1.3 Method C*

The percentage (%) contamination was determined for method C, and from the result, as shown in **Figure 3**, Chicory and Stevia had 30% and 50% contamination, respectively, while Bee balm and *Rhodiola krylovii* both had 100% contamination. Method C is a suitable sterilisation method for Chicory seeds. The agar composition used for media preparation in this method was 0.9 g, which could have influenced the plant seeds.

#### *3.1.4 Method D*

**Figure 4** shows the level of percentage (%) of Stevia (14.29), Bee balm (100%), Chicory (50%), and Rhodiola krylovii (100%). The result obtained using method D indicates that Stevia seeds had a minor percentage (%) contamination, which is 14.29%, and it is the best method for sterilising stevia cells. Although Chicory seeds can also be sterilised using this method, it stands an average chance of not getting contaminated than Stevia, which has a greater chance of no contamination. While Bee balm and Rhodiola krylovii had 100% contamination, thus this method of sterilisation is not suitable for both plant seeds, and the agar composition used during the media preparation was 0.9 g.

#### **Figure 4.**

*Chart showing percentage contamination of method D.*

From the four (4) methods of sterilisation used in this experiment, the result for each Method (A, B, C, and D) Stevia and Chicory cells were less prone to 100% contamination when compared to Bee balm and Rhodiola krylovii, which turned out to have 100% contamination in the four methods of sterilisation and cultivation. The most suitable method for Stevia is method D which only had 14.29% contamination, and this result can be linked to the findings of Halim et al., 2016 for the sterilisation process of Stevia, which had 15% contaminated plant cell culture. The best method for cultivating Chicory cells was method C, which had a 30% (%) contamination level. Hence, it can be stated that different plant cells have a specific method of sterilisation that enables cells/ seeds to be free from pathogenic microorganisms that may affect the cultivation process. However, the composition of culture media might have influenced the growth rate of the plants, and this agrees with the findings of Yildiz and Usha et al. [57, 99] that the higher the composition of plant regulators used, the higher the observable differences and the amount of agar used can affect the growth of plant cells. A total of 0.9 g of agar was used in methods C and D. In comparison, 0.8 g was used for methods A and B. Although, other factors, such as percentage concentration, application period, and temperature of NaOCl could, affect *in vitro* germination of seeds, regeneration potential of explants, and growth of seedlings when these factors are not taken into consideration. Hence, these results might also lead to seed contamination due to the inefficacy of NaOCl used during seed disinfection [100] (see **Figures 5**–**8**).

#### **3.2 Germination of non-sterile/sterile seeds and obtaining non-sterile and sterile plant seedlings in Petri dishes**

Two factors were considered under this experiment; a) non-sterile plant seedlings and b) sterile plant seedlings, and the plants were observed after 2 weeks of culturing. The observable differences between the plant cells of Chicory, Bee balm, and *Rhodiola krylovii* were determined by sight. The variation of growth between the plant cells was evident to the eyes. This experiment was done to obtain callus. Non-sterile Chicory showed moderate growth in the Petri dishes, while the growth of the pure Chicory was prominent. The non-sterile Bee balm showed no growth in the petri dish, unlike the sterile Bee balm was showed noticeable growth (sprouts). Non-sterile *Rhodiola krylovii* showed noticeable sprouts, while sterile *Rhodiola krylovii* showed noticeable sprouting.

**Figure 5.** *Cultured stevia seeds in solid media.*

**Figure 6.** *(i) Stevia, (ii) chicory, and (iii) Hedysarum.*

**Figure 7.** *Contaminated samples of bee balm (*Cambridge scarlet*) and* Rhodiola krylovii *plants.*
