Criopreservação de plantas propagadas in vitro de Cleome spinosa Jacq. (Cleomaceae)

• Autor(a) principal: Vilardo, Anna Flávia Rodrigues Mortani
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/7965
• topic: PVS2; PVS3; Shoot tips; Sucrose; V-cryo-plate technique; Vitrification technique; Ápices caulinares; PVS2; PVS3; Sacarose; Vitrificação; Plantas medicinais - Criopreservação; Plantas - Propagação in vitro; Tecidos vegetais - Cultura e meios de cultura; CNPQ::CIENCIAS BIOLOGICAS::BOTANICA::BOTANICA APLICADA

Cleome spinosa is used in folk medicine and as garden plant. This species has been investigated by biotechnological strategies, which has resulted in the establishment of different in vitro systems by tissue culture techniques. However, long-term maintenance of in vitro cultures in active growth conditions show risks, as contamination and somaclonal variations. Cryopreservation, the storage of biological material at ultra-low temperature, is an efficient long-term conservation system, allowing the maintenance of plant germplasm for indefinite periods of time. This study aimed to establish cryopreservation protocols for in vitro plants of C. spinosa using vitrification and v-cryo-plate techniques. Explants from different regions were evaluated for their ability to regenerate from small-sized fragments. For preculture, the material was maintained for 24 hours in a medium containing 0.3 M sucrose or increasing concentrations of suc. (0.25 M - 0.50 M), remaining at each concentration for 24 hours. Then, the material was exposed to loading solution and to the vitrification solutions PVS2 (25°C and 0°C) and PVS3 (25°C) for different exposure times (15, 30, 45, 60, 90, 120 min). To rewarming, the material cryopreserved by the Vitrification technique was immersed in a water bath, while in the V-Cryo-plate technique the process was direct by immersing the cryo-plates in a high osmolarity solution (unloading). Unfrozen and cryopreserved explants were transferred to unloading solution and were inoculated in regrowth medium (MS + 0.5 mg.L-1 BAP). During regrowth the material was kept in darkness for 24 hours and followed by gradual transfer to light. After 30 days, the material was transferred to MS0 to evaluate the recovery rates. Shoot tips showed the best regeneration capacity from small fragments and were selected for cryopreservation studies. The preculture protocols showed no recovery differences in the presence of the vitrification solutions at 25°C. However, in the presence of PVS2 at 0°C, higher recovery values were reached by cryopreserved shoot tips pre-treated with a progressive increase in sucrose concentration. PVS2-based protocols with lower exposure times were more effective for cryopreservation at 25°C when compared to protocols at 0°C. To PVS3based protocols intermediates exposure times were more efficient in the vitrification technique, while higher recovery rates were achieved in the V-Cryo-plate technique after longer exposure times. The V-Crio-plate was the most effective technique for shoot tips cryopreservation in C. spinosa, mainly when it was used in association with the PVS2 at 0°C. Shoot tips pre-treated with a progressive increase in sucrose concentration and exposed to PVS2 (0°C) for 90 minutes, using the V-Cryo-plate technique, reached 70% recovery rates. The regenerated plants showed normal phenotypic aspect and maintained their regenerative capacity over time in culture