Estudos de criopreservação, análise fitoquímica e avaliação da estabilidade genética de Cleome spinosa Jacq. (Cleomaceae)

Abstract

Germplasm conservation systems based on plant tissue culture are subject to contamination and loss of their morphogenetic potential. These problems can be circumvented by the establishment of the cryopreservation, a method that allows long-term conservation by immersion of the biological material into liquid nitrogen. The objectives of this study were the cryopreservation of shoot tips from in vitro plant of Cleome spinosa using vitrification and desiccation techniques in cryo-plates (V-Cryo-Plate and D-Cryo-Plate, respectively) and evaluation of genetic and phytochemical stability. For the V-Cryo-plate technique, the recovery was evaluated using different concentrations and exposure time to benzylaminopurine (BAP). The shoot tips were treated with the PVS2 solution (25°C or 0°C), applying protocols previously established to the species. In the present work, the highest percentages of survival (83%) and recovery (76%) were achieved for shoot tips exposed to PVS2 for 90 min (0°C) and recovered on MS medium supplemented with 0.5 mg.L-1 BAP for 15 days. The D-Cryo-Plate technique was applied for the first time with C. spinosa. The effect of pre-culture treatment, sucrose concentration on polymerization solution, exposure to loading solution and to silica gel as well as the recovery medium were evaluated. The highest percentages of survival (76%) and recovery (76%) rates after cryopreservation were achieved using the followed protocol: Pre-culture with increasing concentrations of sucrose (0.25 M - 0.5 M) for 48 h, fixation to cryo-plates with the polymerization solution containing 0.6 M sucrose, followed by exposure to loading (20 min) and desiccation on silica gel for 60 min. The recovery was performed on MS medium supplemented with 0.5 mg.L-1 BAP for 15 days. Acclimatization process, molecular and phytochemical analysis were performed with plants regenerated after cryopreservation using the V-Cryo-plate technique. The cryopreserved plants achieved 100% acclimatization rate and showed a normal phenotypic aspect. The analysis of genetic stability aimed to compare the regenerated plants after the cryopreservation process (-NL and + NL) with their mother plants, using the molecular makers SCoT and ISSR. The SCoT analysis revealed only a single polymorphic fragment, while using the ISSR no polymorphisms were detected. Methanolic extracts of cryopreserved plants (+ NL), in vitro propagated plants and field collected plants were subjected to analysis by high performance liquid chromatography to compare their phytochemical profile. The results showed that the field sample presented a greater diversity of substances, while the in vitro propagated plants showed similar phytochemical profile. The studies conducted with C. spinosa demonstrated the feasibility of cryopreservation techniques for long-term conservation of biotechnological materials established with the species.