2.1.2. Preparation of Basal Medium, Aseptic Condition and Glassware

A basal medium was prepared using a formulation described by Lloyd and McCown (WPM) (1981). The basal medium was fortified with 30 g/L sucrose, 3.0 g/L gelrite as a gelling agent, and pH was adjusted to 5.7 ± 0.5 using 1 M sodium hydroxide (NaOH) or 1 M hydrochloric acid (HCl). Then, the basal medium was autoclaved at a temperature of 121 ◦C for 20 min at a pressure of 1.05 kg/cm2. The laminar airflow chamber was exposed to ultraviolet (UV) light for 30 min to sterilize the surface of the working area.

The glassware used for the culture comprised of 35 mL culture tubes for surface sterilization with 10 mL of solid WPM medium and 150 to 250 mL conical flasks, 300 mL modified jars with transparent polypropylene caps with 30 mL of solid WPM medium for the callus induction experiment. Before using the glassware, they were thoroughly washed under running tap water with liquid detergent and then rinsed with distilled water. Next, the clean glassware was sterilized by autoclaving at 121 ◦C and 104 kPa pressure for 20 min.

#### 2.1.3. Callus Induction as Affected by Cytokinin and Auxin

The survived nodal explants were selected after four weeks for callus induction under different culture conditions. The prepared basal medium was supplemented with the combination of auxin; 2,4-dichlorophenoxyacetic acid (2,4-D) at a concentration of (2.5, 5, and 7.5 μM) and cytokinin; 6-benzylaminopurine (BAP) at a concentration of (0.5, 1, and 1.5 μM). The WPM basal medium without PGRs served as control to find out and compare the influence of PGRs on callus induction of *B. glabra* under different cultural conditions. Each treatment of this experiment was replicated three times, with 9 explants per replication and every three explants cultured in a 250 mL conical flask (Figures 1 and 2A) for callus initiation, but after the callus initiated, only two calluses derived from nodal segment cultured in a 250 mL conical flask (Figures 1 and 2B–E) in order to have better access to the media and space. The data on days to callus initiation were taken every day but the data such as callus frequency, callus morphology, fresh and dry weight of the callus were taken after every four weeks of incubation four times for, in total, 16 weeks. The following Equation (1) was used for determining the callus induction frequency as given below [32]:

Callus induction frequency (%) = *Number of explants induced callus Number of explants cultured* <sup>×</sup> 100 (1)

**Figure 1.** In vitro callus induction of *Bougainvillea glabra* Choisy under light incubation condition. (**A**) Sterilized nodal segment on WPM basal medium; (**B**) callus induction from a node section on WPM medium supplemented with 5 μM 2,4-D + 1 μM BAP after 4 weeks; (**C**) callus after 6 weeks in the same medium and PGRs condition; (**D**) callus after 8 weeks; (**E**) callus induction from a node section on WPM medium fortified with 7.5 μM 2,4-D + 1.5 μM BAP after 10 weeks with a red spot.

**Figure 2.** In vitro callus induction of *Bougainvillea glabra* Choisy under dark incubation condition. (**A**) Sterilized nodal segment on WPM basal medium; (**B**) callus induction from a node section on WPM medium supplemented with 2.5 μM 2,4-D + 1.5 μM BAP after 3 weeks; (**C**) callus after 4 weeks in the same medium and PGRs condition; (**D**) callus after 8 weeks; (**E**) callus induction from a node section on WPM medium fortified with 7.5 μM 2,4-D + 1.5 μM BAP after 10 weeks with a red spot.

## 2.1.4. Culture Maintenance

All the cultures were incubated in a culture room maintained at a temperature of 25 ± 2 ◦C under 16 h light and 8 h dark using white fluorescence light irradiation of 45 μmol/m2/s or complete dark. Subculture of experimental materials (developing culture) was done every four weeks after each culture. Similar basal medium and PGRs composition from previous culture was used.
