Criopreservação de ápices caulinares e raízes produzidos in vitro e avaliação fitoquímica de Cleome rosea Vahl ex DC. (Cleomaceae)

Abstract

Species of the genus Cleome L. have been reported by their different medicinal properties. Recent studies revealed the medicinal potential of Cleome rosea, a native species occurring in Brazilian restingas, ecosystems changed through human actions. The first plant tissue culture studies were performed at the Laboratory of Plant Biotechnology (Labplan) of UERJ, resulting in the establishment of different in vitro systems and the production of anthocyanins and carotenoids, secondary metabolites with medicinal interest. Adventitious root cultures, with high biomass accumulation, have also been established, showing the significant potential for commercial exploration of these in vitro systems. Thus, it is important to develop conservation strategies, as well as to investigate the phytochemical diversity of this species. This study aimed to evaluate cryopreservation methods for in vitro conservation of shoot tips and roots. Additionally, phytochemical analysis was performed using field-grown plants and in vitro cultures (callus and roots cultures). Cryopreservation methods based on the use of vitrification solutions to prepare the material for the preservation in liquid nitrogen were evaluated. For cryopreservation of shoot tips, efficient vitrification protocols have been established using aluminum plates (cryo-plates) with vitrification solutions PVS2 and PVS3. It was achieved a recovery rate of 100% for the cryopreserved shoot tips treated with the phytoregulator Benzylaminopurine (BA) and maintained under diffuse light during the recovery step. For cryopreservation of roots, it were evaluated the vitrification and encapsulation-vitrification techniques using the PVS3 solution. Recovery rate above 90% was achieved when cryopreserved roots were encapsulated in calcium alginate matrix, washed gradually in the unloading solution and were inoculated on solid MS medium during the recovery step. The cryopreserved roots showed multiplication capacity after recovery, which was maintained for up to four subcultures. The effect of the cryopreservation steps on the roots structural integrity was evaluated by histological and histochemical studies. Tissues alterations were more significant in the cortex of the roots, characterized by the accumulation of polysaccharides, proteins and lipidic and pectic substances in the intercellular spaces during the cryopreservation process. The vascular cylinder remained intact, ensuring the formation of new roots from the pericycle, showing the maintenance of the proliferative capacity of the cryopreserved roots. The phytochemical studies demonstrated the presence of secondary metabolites in the different extracts from field-grown plants, as well as from roots and callus cultures. The chromatographic analysis allowed to suggest the presence of different classes of secondary metabolites as phenolic compounds, alkaloids, terpenoids, steroids, saponins and fatty acids. These results demonstrate the long-term storage possibilities for in vitro cultures of C. rosea by cryopreservation and the phytochemical diversity of this species in nature and under in vitro systems