*4.1. In Vitro Callus Induction of B. glabra*

*Bougainvillea glabra* is an important medicinal plant with high medicinal values. Plants propagated via traditional methods are vulnerable to a variety of diseases and pests, as well as weather and land availability, all of which have an adverse effect on the medicinal qualities of the harvested plants [41]. The overall objective of this study was to induce calli under different PGRs and cultural conditions and evaluate the secondary metabolites content of the in vitro-induced calli and conventionally propagated plant. In the in vitro callus induction, plant growth regulators are one of the most important factors affecting the growth of explants. Synthetic auxins, such as 2,4-D, are important PGRs that are applied in a variety of embryogenic cell and tissue culture methods, as well as callus production and cell suspension culture [42]. Meanwhile, some research has confirmed the positive effect of 2,4-D on callus formation during the physiological and molecular process in many circumstances, and those studies have shown that 2,4-D regulates the endogenous IAA metabolism, promotes specific proteins, and controls DNA methylation [43]. On the other hand, cytokinin plays an important part in inducing callus by promoting cell division and differentiation [44].

In the present study, the minimum days of callus initiation (7) was recorded from the treatments of 5 μM 2,4-D + 1 μM BAP under a dark incubation condition; however, the maximum days for callus initiation (17.67) was recorded from the treatment of 7.5 μM 2,4-D + 0.5 μM BAP under light incubation conditions. The study results also showed that the highest callus induction rate (100%) was produced from the combination of 2.5 μM 2,4-D + 1.5 μM BAP under a dark incubation condition; however, the lowest callus induction rate (73.33%) was obtained from the treatments of 7.5 μM 2,4-D + 1.5 μM BAP under both light and dark conditions. Similarly, the synergistic effect of plant growth regulators and cultural condition has been studied by Renu et al. [45] in the nodal segment of *Catharanthus roseus*, revealing the minimum days for callus initiation and higher callus frequency obtained when WPM basal medium supplemented with BAP 0.5 mg + 2,4-D 2 mg under dark condition. Meanwhile, a study conducted by Behbahani et al. [46] showed that the earlier callus initiation and higher callus frequency obtained when the nodal segment was cultured on a WPM basal medium fortified with 1 mg 2,4-D under dark incubation condition. Moreover, previous studies by Azad et al. [47] of *Phellodendron amurense*, Hoque et al. [48] of *Trapa* sp., Thammina et al. [49] of *Euonymus alatus*, Hesami and Daneshvar. [42] of *Ficus religiosa*, Pandey et al. [50] of *Boerhaavia diffusa* reported that the explants cultured on basal medium supplemented with 2.2–8.8 μM 2,4-D + 1.1–4.4 μM BAP significantly increased the callus induction rate and minimized the callus initiation under a dark condition. Meanwhile, they have reported that a higher concentration of 2,4-D with a higher concentration of BAP significantly reduced the callus frequency and increased the days for callus initiation. It was noted that a combination of 2,4-D with lower BAP concentrations was found to increase the callus induction frequency. This improved effect could be related to BAP, which helps 2,4-D influence rapid cell division and the synthesis of zeatin-like hormones in the plant for effective callus induction [51]. Furthermore, the frequency of callus induction decreased with the increase in the concentration of 2,4-D. The increase in the concentration of 2,4-D causing a significantly delayed response. Bhojwani and Dantu [52] reported that 2,4-D may have herbicidal properties at high concentrations, which could reduce callus formation.

Other than PGRs, light is one of the most important elicitors, as it affects several physiological processes such as photosynthesis; hence, light can influence the growth, development, and morphogenesis of a variety of plant species in vitro [53,54]. Light, on the other hand, is known to affect cell division rates and ethylene evolution, which can affect callogenesis and rhizogenesis. Therefore, the duration and timing of light exposure are critical in explant morphogenesis [55,56]. In our experiments, the explants exposed to light in the early stages of culture developed more severe browning than those exposed to darkness, implying that darkness is preferable for callus induction in the early stages. According to Habibah et al. [56], in a dark environment, it is thought that the release of phenolic substances can be inhibited, and the rate of callus induction can be increased. However, dark incubation conditions may be associated with negative effects in some parameters such as callus biomass. Overall, callus initiation was faster in the dark than in the light, but the callus deteriorated when sub-cultured in the dark continuously, independent of media.

Based on the callus biomass accumulation, a different trend of callus fresh and dry weight was observed. The callus fresh weight was drastically increased by three-fold in the treatment of 7.5 μM 2,4-D + 1.5 μM BAP with 5.33 g. As the concentration decreased to 2.5 μM 2,4-D and 0.5 μM BAP, the callus fresh weight was decreased to 2.11 g and 2.53 g under dark and light incubation conditions, respectively. The yield of biomass and secondary metabolites can be used to determine the success of callus culture. These can be achieved with the proper balance of plant growth regulators, nutritional media, and growing conditions. The 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].
