Articles
Effect of different dehydration conditions on osmolyte accumulation, thermal events and survival of vanilla shoot-tips following droplet-vitrification approach
Article number
1297_14
Pages
99 – 106
Language
English
Abstract
The impact of different osmodehydration conditions was studied using droplet-vitrification procedure, and Vanilla insignis shoot-tips as a study case.
By gas chromatography-mass spectrometry the accumulation of some compatible osmolytes was analyzed after dehydration conditions which resulted in survival higher than 50%. Differential Scanning Calorimetry (DSC) was used to determine the removal of osmotically active water from treated shoot-tips.
Survival after each dehydration step (preconditioning, loading and exposure to PVS) was evaluated by the regeneration of new shoots after 3 months of recovery.
Vanilla shoot-tips up to 3 mm long isolated from in vitro propagated plantlets were preconditioned on standard semi-solid MS medium for 7 days, followed by additional 7 days on MS medium supplemented with 0.3 M sucrose or trehalose.
After preconditioning, shoot-tips were loaded for 30 min in solutions containing 2 M glycerol and 0.4 M sucrose or trehalose and exposed to vitrification solutions PVS2 or PVS3 for 30 min at room temperature prior to immersion in liquid nitrogen.
Out of 12 amino acids, 9 carbohydrates and 5 polyols detected in untreated shoot-tips (controls), proline, sucrose and glycerol varied in all the dehydration steps, regardless whether osmolytes were exogenously supplied or not.
Trehalose was only detected when supplied during preconditioning and/or loading.
Osmotically active water was reduced from 0.9 g g-1 FW until around 0.28 g g-1 FW (32%) after preconditioning with trehalose and exposure to PVS2 regardless the sugar used in loading solution.
The highest survival rates prior to liquid nitrogen immersion ranged between 53 and 65% mostly using pretreatments with trehalose.
Freezing events were recorded after all studied dehydration conditions, indicating that they might be provoking the loss of survival after cryopreservation.
By gas chromatography-mass spectrometry the accumulation of some compatible osmolytes was analyzed after dehydration conditions which resulted in survival higher than 50%. Differential Scanning Calorimetry (DSC) was used to determine the removal of osmotically active water from treated shoot-tips.
Survival after each dehydration step (preconditioning, loading and exposure to PVS) was evaluated by the regeneration of new shoots after 3 months of recovery.
Vanilla shoot-tips up to 3 mm long isolated from in vitro propagated plantlets were preconditioned on standard semi-solid MS medium for 7 days, followed by additional 7 days on MS medium supplemented with 0.3 M sucrose or trehalose.
After preconditioning, shoot-tips were loaded for 30 min in solutions containing 2 M glycerol and 0.4 M sucrose or trehalose and exposed to vitrification solutions PVS2 or PVS3 for 30 min at room temperature prior to immersion in liquid nitrogen.
Out of 12 amino acids, 9 carbohydrates and 5 polyols detected in untreated shoot-tips (controls), proline, sucrose and glycerol varied in all the dehydration steps, regardless whether osmolytes were exogenously supplied or not.
Trehalose was only detected when supplied during preconditioning and/or loading.
Osmotically active water was reduced from 0.9 g g-1 FW until around 0.28 g g-1 FW (32%) after preconditioning with trehalose and exposure to PVS2 regardless the sugar used in loading solution.
The highest survival rates prior to liquid nitrogen immersion ranged between 53 and 65% mostly using pretreatments with trehalose.
Freezing events were recorded after all studied dehydration conditions, indicating that they might be provoking the loss of survival after cryopreservation.
Authors
O. Flores-Castaños, M.T. González-Arnao, F. Hernández-Ramírez, J. Molina-Torres, M.P. Rascón-Díaz
Keywords
vanilla shoot-tips, osmodehydration, osmolytes, osmotically active water, DSC
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