Criopreservação e avaliação da capacidade biossintética de Hovenia dulcis Thunb. (Rhamnaceae)

Abstract

Hovenia dulcis is a woody species that has been subjected to several biotechnological studies due to its medicinal properties, such as hepatoprotective and antineoplastic effects. Protocols of micropropagation, callogenesis, cell suspension and compact callus clusters cultures have already been established for the species. However, the material maintained in active growth under in vitro conditions is vulnerable to contamination and loss of its morphogenetic potential. Therefore, the cryopreservation, which consist of storing biological material at ultra-low temperatures using liquid nitrogen (LN), has been applied for the long-term conservation of germplasm. This study aims to develop a cryopreservation protocol for shoot tips of H. dulcis, using the V Cryo-plate technique, as well as to evaluate the maintenance of secondary metabolites production in the cryopreserved material. In vitro propagated plants maintained on MS medium supplemented with 0.5 mg.L−1 kinetin (KIN) + 0.5 mg.L−1 benzylaminopurine (BA) were used as source of explants. The experiments were carried out using shoot tips excised from microcuttings cultured from in vitro-grown stock plants. In addition, the most suitable cryopreservation protocol was also evaluated using shoot tips excised directly from axillary shoots of stock plants. The shoot tips were pre-treated on medium containing high concentration of sucrose (0.3 M) for 24 h, and they were adhered to the cryo-plates using the solutions of sodium alginate and calcium chloride. The shoot tips were exposed to loading solution (20 min) at room temperature and then to PVS2 at 0oC for different times (0, 15, 30, 60, 90, 120, 150 min), before storage in LN. For rewarming, the cryo-plates were immersed in unloading solution (1.2 M sucrose) for 20 min at room temperature. After cryopreservation, the shoot tips were removed from the cryo-plates and transferred to MS medium containing the association of BA and KIN at the same concentration (0.2 or 0.5 mg.L−1). The use of filter paper over the culture medium for shoot tips inoculation was evaluated during the first week of recovery. The cultures were gradually exposed to light. The parameters evaluated were survival (after 4 weeks) and recovery (after 8 weeks). The longer exposure times to PVS2 resulted in increased recovery rates, highlighting the exposure to 120 minutes. The cytokinin concentration significantly affected the regeneration of shoot tips after cryopreservation. The recovery medium supplementation with BA+KIN (0.5 mg.L−1 each) with filter paper allowed the highest survival (68%) and recovery (63%) rates of shoot tips from microcuttings and shoot tips excised directly from in vitro plants (75% survival and 45% recovery). Plants regenerated from cryopreserved shoot tips showed a normal phenotype, which confirms the efficiency of the protocol. The chromatographic analysis showed a stability in the production of secondary metabolites of plants developed from cryopreserved shot tips and in vitro propagated plants suggesting that the cryopreservation process did not change plant’s biosynthetic capacity.