Uncaria tomentosa presents tomentum that resembles cat’s claws, hence its common name, is a plant that produces various secondary metabolites that are traditionally used in alternative medicine. The natural distribut...Uncaria tomentosa presents tomentum that resembles cat’s claws, hence its common name, is a plant that produces various secondary metabolites that are traditionally used in alternative medicine. The natural distribution of this species has been affected by indiscriminate harvesting from its habitat. In the present research, cryopreservation (liquid nitrogen, LN, -196°C) was evaluated as an option for ex situ conservation of this species. The following techniques were evaluated: vitrification and encapsulation-dehydration of apices, vitrification of cell suspensions, and seed desiccation and vitrification. Preculture conditions and exposure times to LS and PVS2 were evaluated. Apex survival was the highest (82%) with preculture in 0.25 M sucrose followed by incubation for 20 and 30 min in LS and PVS2, respectively, prior to cooling in LN. The encapsulation-dehydration technique was evaluated by using sucrose preculture and different capsule moisture contents. Survival of apices cooled in LN was not significantly different between treatments and varied from 31.8% to 52.9% for capsule moisture contents between 22.7% and 20.3%. For cell suspensions precultured in 0.5 M sucrose, cell multiplication and formation of calli with very good appearance were observed in 61.1% of the cultures following vitrification. For cryopreservation of seeds, germination was 89.5% using the desiccation technique and 67.6% to78.1% using vitrification.展开更多
Secondary somatic embryos (SSEs) of cocoa, a recalcitrant tropical, seed-producing species, were cryopreserved using a vitrification approach and Differential Scanning Calorimetry (DSC) was employed to optimise sucros...Secondary somatic embryos (SSEs) of cocoa, a recalcitrant tropical, seed-producing species, were cryopreserved using a vitrification approach and Differential Scanning Calorimetry (DSC) was employed to optimise sucrose preculture and Plant Vitrification Solution 2 (PVS2) incubation. The objective of the study was to evaluate the influence of sucrose preculture and PVS2 dehydration on water content of SSE that will enable it to survive cryostorage. SSEs were precultured for 3 or 5 days on media containing 0.5 M or 0.75 M sucrose and cryoprotected in loading solution (2 M glycerol and 0.4 M sucrose in medium) for 20 min before they were dehydrated with cold PVS2 for 0 - 90 min. Thermal analysis revealed the occurrence of ice crystallization in the SSEs with the extent declining with increasing PVS2 exposure. Maximal survival of SSEs was promoted by preculture on 0.5 M sucrose medium and dehydration with PVS2 for 45 - 60 min, which was characterised by small ice crystallization. Exposure of SSEs beyond 60 min leads to excessive dehydration as characterized by no change in the thermograms. Based on these findings, preculture of SSEs on 0.5 M sucrose medium and dehydration with cold PVS2 for 60 min has been adopted for the successful cryopreservation of cocoa germplasm.展开更多
文摘Uncaria tomentosa presents tomentum that resembles cat’s claws, hence its common name, is a plant that produces various secondary metabolites that are traditionally used in alternative medicine. The natural distribution of this species has been affected by indiscriminate harvesting from its habitat. In the present research, cryopreservation (liquid nitrogen, LN, -196°C) was evaluated as an option for ex situ conservation of this species. The following techniques were evaluated: vitrification and encapsulation-dehydration of apices, vitrification of cell suspensions, and seed desiccation and vitrification. Preculture conditions and exposure times to LS and PVS2 were evaluated. Apex survival was the highest (82%) with preculture in 0.25 M sucrose followed by incubation for 20 and 30 min in LS and PVS2, respectively, prior to cooling in LN. The encapsulation-dehydration technique was evaluated by using sucrose preculture and different capsule moisture contents. Survival of apices cooled in LN was not significantly different between treatments and varied from 31.8% to 52.9% for capsule moisture contents between 22.7% and 20.3%. For cell suspensions precultured in 0.5 M sucrose, cell multiplication and formation of calli with very good appearance were observed in 61.1% of the cultures following vitrification. For cryopreservation of seeds, germination was 89.5% using the desiccation technique and 67.6% to78.1% using vitrification.
文摘Secondary somatic embryos (SSEs) of cocoa, a recalcitrant tropical, seed-producing species, were cryopreserved using a vitrification approach and Differential Scanning Calorimetry (DSC) was employed to optimise sucrose preculture and Plant Vitrification Solution 2 (PVS2) incubation. The objective of the study was to evaluate the influence of sucrose preculture and PVS2 dehydration on water content of SSE that will enable it to survive cryostorage. SSEs were precultured for 3 or 5 days on media containing 0.5 M or 0.75 M sucrose and cryoprotected in loading solution (2 M glycerol and 0.4 M sucrose in medium) for 20 min before they were dehydrated with cold PVS2 for 0 - 90 min. Thermal analysis revealed the occurrence of ice crystallization in the SSEs with the extent declining with increasing PVS2 exposure. Maximal survival of SSEs was promoted by preculture on 0.5 M sucrose medium and dehydration with PVS2 for 45 - 60 min, which was characterised by small ice crystallization. Exposure of SSEs beyond 60 min leads to excessive dehydration as characterized by no change in the thermograms. Based on these findings, preculture of SSEs on 0.5 M sucrose medium and dehydration with cold PVS2 for 60 min has been adopted for the successful cryopreservation of cocoa germplasm.
