**5. Conclusions**

The results of this study demonstrated that the growth of *B. glabra* calli is greatly influenced by the combination of plant growth regulators and cultural conditions. No callus was induced on a PGR-free medium. The media composed of WPM supplemented with 2.5 μM 2,4-D + 1.5 μM BAP and 5 μM 2,4-D + 1 μM BAP significantly reduced the days for callus initiation (7 days) under a dark incubation condition, while the maximum days (17.67) was recorded when WPM fortified with 7.5 μM 2,4-D + 0.5 μM BAP under light incubation condition. Meanwhile, 5 μM 2,4-D + 0.5 μM BAP significantly increased the callus frequency (100%) under both light and dark incubation condition. By increasing the PGRs concentration and combination, the callus frequency significantly decreased. Moreover, the light regime did not have any influence on biomass accumulation, while the PGRs significantly influenced and the maximum biomass was recorded when the WPM basal medium was supplemented with 7.5 μM 2,4-D + 1.5 μM BAP under both dark and light conditions with FW: 5.33 g per callus and DW: 230.67 mg per callus, respectively. Furthermore, the morphology of the callus was influenced by PGRs and cultural condition; the calli that induced under a light condition had good texture and were compact in nature with yellow to brown and red colors, while the calli induced under a dark condition were friable with yellow to brown and white colors.

The study on phenolics content found that the aqueous extract of the conventionally propagated node exhibited the highest phenolic acids (21.88 mg GAE/g DW) and flavonoids content (42.05 mg RE/g DW). Furthermore, in antioxidant activities conducted, the highest DPPH free radical scavenging activity (7.64 mg TE/g DW), and ABTS scavenging activity (1.51 mg TE/g DW), were also recorded from aqueous extract of the conventionally propagated node. On the other hand, the highest iron (II) chelating activity was exhibited from the aqueous extract of the calli induced under the dark condition with 43.3% inhibition, respectively. In conclusion, higher phenolics content and antioxidant properties recorded from the conventionally propagated nodal part showed that the plant propagated through conventional propagation technique has the potential to produce high secondary metabolites. Meanwhile, both conventionally propagated parts and callus samples suggest that in addition to the plant material, the callus may also be used as a supplement raw material to obtain secondary metabolites for the pharmaceutical industry. Thus, the in vitro-derived *B. glabra* callus could plausibly act as a novel source of metabolites for food and pharmaceutical industries in their preparation of food preservatives and medicines, respectively.

**Author Contributions:** Conceptualization, M.N.N. and M.H.; methodology, M.N.N. and M.H.; resources, S.Z.S. and M.H.; data curation, M.N.N.; writing—original draft preparation, M.N.N.; writing—review and editing, M.N.N., S.Z.S. and M.H.; supervision, S.Z.S. and M.H.; funding acquisition, S.Z.S. and M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Higher Education Malaysia (Translational Research Program (TR@M) and Universiti Putra Malaysia (Putra Grant-Young Putra Initiative), grant number 5526700 and 9629000, respectively, and the APC was funded by the Universiti Putra Malaysia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the article.

**Acknowledgments:** The authors would like to thank the Higher Education Development Project (HEDP) for Afghanistan and Herat University for the scholarship.

**Conflicts of Interest:** The authors declare no conflict of interest.
