**3.3 Growing sterile explants for callus formation (Solid Medium M&S + kinetin and - kinetin)**

Two factors were considered during the cultivation process of Callus of Chicory, Rhodiola krylovii, and Beebalm. These factors were (I) Growth Media with Kinetin

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

#### **Figure 10.**

*(i) Sterile Chicory with sprouts after 2-weeks. (ii) Sterile Cambridge scarlet with sprouts after 2-weeks. (iii) Sterile Rhodiola krylovii with sprouts after 2-weeks.*

(+ Kinetin) and (II) Growth Media without Kinetin (-Kinetin) and the determination of percentage (%)contamination of callus in test tubes after 2 weeks was done to know the percentage contamination of each plants using different media.

Growth of callus was observed with samples in media containing Kinetin. However, there was 62.5–75% contamination in the media containing Kinetin, as shown in **Figure 11**. Chicory and Rhodiola krylovii had the same level of percentage contamination, which was 62.5% and less than the percentage contamination of Bee balm.

**Figure 11.** *Percentage contamination for callus formation (Medium M&S + kinetin).*

**Figure 12.** *Percentage contamination for callus formation (Medium M&S - kinetin).*

After 2-weeks, no sign of growth was observed from any of the samples in the media with no Kinetin and all samples were contaminated. As shown in **Figure 12** of growth media without Kinetin, the level of contamination was 100% for all plants, and no growth was observed in all plant cultures. Thus, this can be due to the absence of Kinetin, a plant hormone that helps induce callus and shoot regeneration formation. While the high level of contamination can be a result of the poor method of sterilisation and the efficacy of sodium hypochlorite, this agrees with the studies of Racoppi [102], which reported that the sterilising agent NaOCl (Sodium Hypochlorite) could decrease in its activity when all the parameters, such as temperature and pH, are not stable. However, if the temperature is slightly above 10 °C, this might increase disinfection activity since this enables easy incursion into the seed coat. Higher temperatures of NaOCl affected the morphology of seedlings, resulting in abnormal growth of hypocotyls and other parts of the plant cells. The unsuccessful result of no growth must have been due to variation in room temperature. According to Yildiz [57], change in temperature of (�1°C) can have an adverse effect on the plant, resulting in no growth (see **Figures 13**–**15**).

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

**Figure 13.** *Callus of chicory in solid culture media after 8 weeks.*

**Figure 14.** *Bee balm (*Cambridge scarlet*) and* Rhodiola krylovii *in solid media with no growth of callus.*

**Figure 15.**

*Stevia, chicory and bee balm (*Cambridge scarlet*),* Rhodiola krylovi*i in solid media showing no growth sign after 4 weeks of culture.*
