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Evolvulus alsinoides (L.) is an annually growing medicinal plant used in many popular systems as a source of traditional medicine. However, fresh plant material for medicinal uses and research purposes is critically unavailable year-round due to its short life cycle. A micropropagation method for E. alsinoides was developed, and its genetic diversity was described. Surface sterilized nodal explants were inoculated onto Murashige and Skoog (MS) medium supplemented with different plant growth regulators; the best response of multiple shoot induction was observed on MS media augmented with 5 μM kinetin (Kn) and 0.1 μM Indole-3-acetic acid (IAA), which gave a maximum number of 10.27 ± 0.0118 shoots. Further, the addition of 30 μM of adenine sulfate to the MS media enhanced shoot multiplication (18). The cloned shoots were accomplished to in vitro rooting on MS medium with 0.1 μM Indole-3-butyric …