# In Vitro Cultures and Volatile Organic Compound Production in _Chiliadenus montanus_ ([PERSON].) [PERSON]
## 3 Results and Discussion
### Microshoots Culture  
In this study, direct shoot regeneration from axillary buds on nodal segments cultured on half-strength MS medium supplemented with 0.05 M sucrose, 4.4 uM BAP, and 1.4 uM GA\({}_{3}\) proved to be superior when compared to half-strength White media and hormone-free MS media (Table 1; Figure 3a). This media produced the highest mean value for shoot length (1.51 cm) and the highest mean value for the number of leaves (13.8). On the other hand, all nodal segments cultured on half-strength White media produced shorter shoots (0.56 cm) with a lower number of leaves (5.1). Nodal segments cultured on hormone-free MS media showed no bud breakage and no microshoots were produced (Table 1). In general agreement with the result of this study, MS media supplemented with 4.4 uM BAP, 4.6 uM Kin, or 4.9 uM 2-isopentyladenine (2 ip) in combination with 1.4 uM GA\({}_{3}\) produced the maximum number of shoots in _Eclipta alba_[36]. Half-strength MS medium supplemented with 4.4 uM BAP and 1.4 uM GA\({}_{3}\) was more efficient in shoot culture establishment of _Baccharis antiquensis_, a member of the Asteraceae family, than hormone-free MS media [37]. On the other hand, using White media for culturing _Centaurea zeybekii_ shoots resulted in slower shoot growth and a low transplantation rate when compared to half-strength MS media [38]. The superiority of half-strength MS medium may reflect the requirement of a relatively low concentration of salts for the growth of _C. montanus_ shoots, which is in general agreement with the results of [39] who indicated that low-ion strength MS medium resulted in the high growth of shoots and roots of _Stevia rebaudiana_ [PERSON], compared to a full-strength medium. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Type of Media** & **Number of Leaves** & **Shoot Length (cm)** \\ \hline
1/2 MS media supplemented with 1.4 uM & & \\ GA\({}_{3}\) + 4.4 uM BAP & 13.80 \(\pm\) 0.36 1  & 1.51 \(\pm\) 0.024 2 \\ Half strength White media & 5.10 \(\pm\) 0.36 2  \\ Hormone-free and full-strength MS media & 0.00 \(\pm\) 0.36 3  & 0.56 \(\pm\) 0.024 \\ Hormone-free and full-strength MS media & 0.00 \(\pm\) 0.36 4  \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Effect of different media types on direct shoot bud initiation from nodal explants (n = 10) after four weeks of in vitro culture of _C. montanus_. Figure 2: Callus induction using a leaf explant of _C. montanus_ on MS media supplemented with 2.2 uM 2,4-D and 6.9 uM Kin (**a**) Callus induction after four weeks (**b**) Four-week-old callus culture growing on callus maintenance media. For the rooting experiment, from all tested treatments, only three microshoots produced roots; one of them was obtained on half-strength MS media supplemented with 0.05 M sucrose and 4.2 uM NAA (10%), another on MS media supplemented with 10.7 uM NAA (10%), and the last one on MS media supplemented with 3.9 uM IBA (10%) (Figure 3b). Similar results were obtained by [40] where rooting of _Echinacea_ spp. was unsuccessful for most of the treatments, except for 4 uM NAA. For indirect shoot regeneration using callus as an explant, successful regeneration was achieved using MS media supplemented with 0.1 sucrose and a combination of 8.8 uM BAP, 2.2 uM Zeatin, and 1.4 uM GA\({}_{3}\) (Figure 4a,b). Microshoots were allowed to grow until they reached one cm before sub-culturing on MS medium supplemented with 0.1 M sucrose and different levels of BAP (8.8, 17.7, and 26.6 uM) or Kin (9.2, 18.5, and 27.8 uM) in combination with 0.5 uM NAA. The shoot length and number of leaves of microshoots were significantly affected by cytokinin type and concentrations (Table 2). The highest mean values for shoot length (2.25 cm) and number of leaves (19.70) were obtained from microshoots cultured on MS medium supplemented with 8.8 uM BAP and 0.5 uM NAA, which was significantly different when compared to other treatments (Figure 4c; Table 2). Figure 4: (**a**) _C.