2.2.6. Iron (II) Chelating Activity

Iron (II) chelating activity was conducted by the procedure described by Dinis et al. [38] with minor modifications. An amount of 400 μL of the extract was mixed with 50 μL of 2 mM ferrous chloride. The reaction was started by adding 200 μL of 5 mM ferrozine to the reaction liquid and incubating it at room temperature for 10 min. The mixture's absorbance was measured at 562 nm in comparison to a blank sample. The decrease in absorbance was due to increased iron chelating activity. The following Equation (2) was used to calculate the iron (II) chelating activity:

$$\text{Chelating (\%)} = \left(1 - \text{A562 Sample/A562 Control} \right) \times 100\tag{2}$$

## *2.3. Experimental Design and Statistical Analysis*

The data were analyzed using the Statistical Analysis System (SAS) ver. 9.4. All of the experiments were performed in triplicates in a two or three-factorial Completely Randomized Design (CRD) and the results are expressed as mean ± SE. Duncan's Multiple Range Test (DMRT) was used to compare means at a *p* = 0.05 level. Pearson's correlation analysis was used to determine the correlation between variables with indicator r < 0.25 indicating a weak correlation, r < 0.75 indicating an intermediate correlation, r < 1 indicating a strong correlation,r=1 indicating perfect correlation.

#### **3. Results**

#### *3.1. In Vitro Callus Induction of Bougainvillea glabra via Nodal Segment*

3.1.1. The Main Effect of 2,4-D, BAP and Light Regimes on Callus Induction of *B. glabra*

Results from the present study showed that from the different concentrations of 2,4-D, the minimum days for callus initiation was obtained in the treatment of 5 μM 2,4-D but it was not significantly different from the treatment of 2.5 μM 2,4-D. Meanwhile, the maximum days for callus initiation were recorded from the treatment of 7.5 μM 2,4-D. However, for the treatments of BAP, the minimum days for callus initiation was exhibited from the 1.5 μM BAP but it was not significantly different from 0.5 and 1 μM BAP, respectively. The type of explants to induce callus is also an important factor that needs to be considered in callus induction experiments due to various factors affecting the development of cell culture systems, such as genotype, explant type, plant growth regulators (PGR), culture medium, and culture condition. Based on the results in (Table 1), with WPM medium supplemented with various concentrations of 2,4-D, an increment of the callus frequency was observed as 2,4-D concentration increased until 5 μM. The highest callus frequency was recorded using 5 μM 2,4-D, which is significantly different from 7.5 μM 2,4-D, but it was not significantly different from 2.5 μM 2,4-D. On the other hand, there was no callus formation on the control media without plant growth regulators, indicating that growth regulators had a significant effect on callus induction on the nodal explant. Following that, there was a decrease in percent callus induction growth, followed by an increase of the concentrations of 2, 4-D, and BAP.

**Table 1.** Main effects of 2,4-D and BAP on callus induction in the nodal segment of *Bougainvillea glabra* under different light conditions.


Values are means ± SE. the same letter within the same column for each factor indicates no significant difference (*p* < 0.05). The means separation is done by using Duncan's multiple range test (DMRT). F value represented \* = <0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001 and ns = no significant differences. 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl amino purine (BAP), FW (Fresh Weight), and DW (Dry Weight).

In terms of biomass accumulation, maximum callus biomass was recorded when the nodal explants were cultured on WPM medium supplemented with 5 μM 2,4-D. However, for the treatments of BAP, 1.5 μM BAP exhibited significantly higher callus biomass, respectively. Based on the results, it was seen that there was an increase in the fresh and dry weight of callus as the concentration of BAP was increased up to 1.5 μM, but for the

treatments of 2,4-D, the callus biomass increased as the 2,4-D increased until 5 μM and significantly decreased with 7.5 μM. Primary metabolism, particularly carbon and nitrogen metabolism, is closely linked to biomass accumulation. Carbon metabolism meets the energy requirements resulting from carbohydrate synthesis, and hence, contributes to cell growth and structural components [39]. The products of nitrogen metabolism, on the other hand, are primarily amino acids and proteins, which are then used to regulate cellular processes [40]. Over all, both cellular development and division result in increased fresh and dry weight.

The callus induction of *B. glabra* was also evaluated under two growth conditions, dark and light. The results showed that the dark condition was more favorable for earlier callus initiation and higher callus frequency than the light condition. However, the calluses induced under light incubation conditions produced significantly higher callus fresh and dry weight than the calluses induced under complete dark incubation conditions. Based on the observation in this experiment, the nodal segments incubated under complete dark conditions grew faster than in a light-containing photoperiod. In the dark condition, the minimum days for callus initiation was (8.45), which is significantly different from the light condition with (14.73) days. In addition, the higher callus frequency was also obtained from the cultured incubated under the dark condition, which is significantly different from the cultured incubated under the light condition, respectively. However, callus grown under a set photoperiod presented the higher callus fresh and dry weight than callus incubated under the complete dark condition.

Based on the observation in this experiment, the interaction effects of various concentrations of 2,4-D and BAP were significant (*p* < 0.05) for all parameters, but the interaction effect between 2,4-D \*Condition and BAP \*Condition were only significant for the parameters such as days to callus initiation and callus frequency; also, the statistical analysis indicated that the triple interaction of 2,4-D, BAP, and culture condition was significantly different for days to callus initiation and callus induction rate, but was not significantly different for the biomass production (Table 1). A similar trend also was observed in the WPM medium supplemented with different concentrations of BAP. The maximum callus frequency was obtained from 0.5 μM and 1 μM BAP, respectively. However, by increasing the BAP concentration, the callus frequency decreased.

#### 3.1.2. Synergistic Effect of Cytokinin, Auxins, and Light Regime on Callus Induction

The results in Table 2 exhibit that the minimum days of callus initiation were recorded from the treatments of 2.5 μM 2,4-D + 1.5 μM BAP and 5 μM 2,4-D + 1 μM BAP under the dark incubation condition, respectively. However, the maximum days for callus initiation were recorded from the treatment of 7.5 μM 2,4-D + 0.5 μM BAP under light incubation conditions. As shown, the period of callus induction and growth of callus varied. They depend on the type and concentration of growth regulators and lighting conditions. Meanwhile, the highest callus induction rate was produced from the combination of 2.5 μM 2,4-D + 1.5 μM BAP under a dark incubation condition and 5 μM 2,4-D + 0.5 μM BAP under both light and dark incubation conditions, respectively. However, the lowest callus induction rate was obtained from the treatments of 7.5 μM 2,4-D + 1.5 μM BAP under both light and dark conditions.

In this experiment, for the WPM medium fortified with various concentrations of 2,4-D and BAP, a different trend of callus fresh and dry weight was observed. The callus fresh and dry weight was drastically increased by three-fold in the treatment of 7.5 μM 2,4-D + 1.5 μM BAP under dark incubation condition but it was not significantly different from the same treatment under the light condition. As the concentration decreased to 2.5 μM 2,4-D and 0.5 μM BAP, the callus fresh weight was decreased under dark and light incubation conditions, respectively. Overall, no significant biomass differences were observed for light regimes.


**Table 2.** Interaction effects of 2,4-D and BAP on callus induction in the nodal segment of *Bougainvillea glabra* under different light conditions.

Values are means ± SE. the same letter within the same column for each factor indicates no significant difference (*p* < 0.05). The means separation is done by using Duncan's multiple range test (DMRT). 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). W white, Y yellow, R red, B brown, F friable, and C compact.

Based on the results, the morphogenesis responses based on the intensity of callus formation, texture, types, and concentration of PGRs, cultural condition, and in vitro morphogenesis were different according to the treatment used and are presented in (Table 2) and (Figures 1 and 2A–E). The cultural condition was capable of inducing a callus that could be classified into two types. The first type, which was induced under light incubation conditions, was compact and yellow to brown and red (Figure 1A–E). The second type, which was induced under dark incubation conditions, was friable and yellow to white and brown (Figure 2A–E).
