In vitro propagation and cryopreservation of the medicinal species Hovenia dulcis Thunb. (Rhamnaceae)

This study describes in vitro propagation and cryopreservation of Hovenia dulcis, a woody species used in traditional medicine. Stem and leaf explants from axenic seedlings were cultivated on Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) and kinetin (KIN) alone or in combination (0.1, 0.2, 0.5 mg L−1). For in vitro propagation, rates of regeneration (percentage of responsive explants) and proliferation (multiplication capacity of explant-derived shoots) were evaluated after 30 days and five subcultures, respectively. For cryopreservation by V Cryo-plate technique, shoot tips were excised from microcuttings cultured from in vitro-grown stock plants, or excised directly from axillary shoots of stock plants. The shoot tips were precultured in 0.3 M sucrose (24 h), exposed to loading (20 min) and to PVS2 (0–150 min) before storage in liquid nitrogen. The regrowth was assessed by plating of shoot tips on recovery medium (MS with BA + KIN), with or without a sterile filter paper over the culture medium. Cryopreservation was evaluated by survival (4-weeks) and recovery (8-weeks). The highest regeneration by direct organogenesis (100%) were reached on medium with BA + KIN (0.5 mg L−1 each). Shoots maintained multiplication capacity, showing the highest proliferation (87%) in the presence of BA. Shoot elongation and rooting were achieved on growth regulator-free MS. The most efficient cryopreservation protocol (68% survival and 62% recovery) applied exposure to PVS2 (120 min), and recovery on medium containing BA + KIN (0.5 mg L−1 each) with filter paper. The propagation and cryopreservation of H. dulcis may contribute to its conservation and that of other woody species. This study aimed to establish an in vitro propagation methodology and the first cryopreservation protocol for Hovenia dulcis, a woody species of commercial, medicinal and nutraceutical values.