Direct shoot bud organogenesis and plant regeneration from pre-plasmolysed leaf explants in Catharanthus roseus

Abstract

A protocol for high-frequency shoot bud regeneration from the leaves of Catharanthus roseus is reported here for the first time. A 60-min pre-plasmolytic treatment of leaf explants in a cell protoplast washing medium containing 13% (w/v) mannitol followed by their plating on a half-strength Murashige and Skoog (MS) medium supplemented with 7.0 mg/l 6-benzyladenine (BA) and 3.0 mg/l α-naphthaleneacetic acid (NAA) resulted in the de novo induction and development of adventitious shoot buds in more than 75% of explants. Histological observations revealed a direct origin of these shoot buds from hypodermal tissue around the mid-rib. The rooting in the regenerated shoots was obtained in the presence of 3.0 mg/l indole-3-butyric acid (IBA) and the rooted plants could be successfully established in soil with a 70% rate of success. The relevance of the developed protocol in terpenoid indole alkaloids pathway engineering at the whole-plant level in C. roseus is discussed.