Criopreservação de ápices caulinares de Passiflora suberosa L. com a técnica de vitrificação em crioplaca e avaliação de crioinjúrias nas membranas celulares

Abstract

Passiflora suberosa L., known as maracujá-mirim or maracujazinho-cortiça-preto, is a native species, which occurs from the Northeast to the South of Brazil. In spite of its great agronomic, ornamental and medicinal potential, there are few biotechnological studies with this species, aiming at its propagation and in vitro conservation. Therefore, the goal of this work was the establishment of a cryopreservation protocol for shoot tips of P. suberosa using V-Cryo-plate, including the evaluation of the influence of the explant sources and the type and exposure time to the vitrification solutions PVS2 (0°C) and PVS3 (25°C). In addition, the evaluation of cell membrane damages was also investigated. In order to evaluate the influence of the explant source in post-freezing recovery, shoot tips were excised from microshoots produced from nodal segments cultured on MSM ½ medium for 20, 30 or 40 days. After these periods, explants were precultured in the presence of 0.3 M sucrose for 24 hours, and then transferred to aluminum plates, where they were exposed to the loading and vitrification solutions PVS2 or PVS3, for different periods (0, 30, 45, 60, 90, 120, 150 minutes), before immersion into liquid nitrogen. Survival and recovery rates were evaluated 30 and 60 days after rewarming and transference to the recovery medium, respectively. Plant recovery from cryopreserved shoot tips occurred at distinct frequencies, depending on the culture time of the source explant and exposure time to the cryoprotectant solution. Best recovery rates were observed from shoot tips excised from microshoots maintained for 20 days in culture and exposed to PVS2 for 90 minutes (50 %), and from explants excised from microshoots with 40 days and exposed to PVS3 for 45 to 90 minutes (50 to 60 %). However, shoot production occurred faster from shoot tips exposed to PVS3. The formation of friable callus was also observed at the base of the explants exposed to both cryoprotectant solutions. MDA content was higher after osmotic dehydration of the material, when compared to the observed among the control samples and to the cryopreserved material maintained for 24 hours in the dark after rewarming. These results showed that the V-Cryo-plate technique was efficient for the cryopreservation of P. suberosa shoot tips, and was influenced by the age of the explants and by the exposure time and type cryoprotectant solution used. The investigation of injuries in the cell membranes during the cryopreservation process allowed the preliminary identification of a critical step of the protocol. These results emphasized the need for a wide investigation of morpho-physiological alterations induced by cryopreservation, aiming at optimizing the protocol described here for the increase of plant recovery rates.