Cytotoxic, clonal fidelity, and antioxidant evaluation of in vitro propagated Vitex negundo var. cannabifolia

Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. is an important medicinal plant rich in bioactive compounds. In vitro tissue culture can be used to produce high-quality starting materials for commercial cultivation. However, the micropropagation technique, especially the comprehensive evaluation of regenerants at the metabolite level, is not well known in this plant species. Employing dormant buds as explants, we developed direct and callus mediated indirect propagation protocols for this species and then evaluated the antioxidant and cytotoxic activities of regenerants. The highest multiple shoot induction frequency of 100%, with a proliferation coefficient of 20.3, was achieved using Murashige and Skoog basal medium with 4.4 µM 6-benzylaminopurine and 1.1 µM 1-naphthylacetic acid. The same medium supplemented with 31.5 µM copper sulfate was optimal for callus differentiation, with a shoot regeneration rate of 81.10%. Inter-simple sequence repeat analysis showed a high genomic fidelity of in vitro regenerants. Chemical, physiological, and cytotoxic analyses indicated that in vitro propagated plants at the adult stage are indistinguishable from wild-grown plants. In particular, the cytotoxicity of V . negundo var. cannabifolia to breast cancer cell line SUM159 reveals the potential of this species for use in treating human breast cancer, with cytotoxic compounds beyond vitexin also apparently observed. Our results indicate that V . negundo var. cannabifolia can be reliably micropropagated in vitro. Moreover, the regenerants have high cytotoxic activity against highly metastatic human breast cancer.