Glassy state and cryopreservation of mint shoot tips

ALINE SCHNEIDER TEIXEIRA; MARIA ELENA GONZALEZ BENITO; ANTONIO D. MOLINA-GARCÍA

BIOTECHNOLOGY PROGRESS

AMER CHEMICAL SOC

Lugar: Washington; Año: 2013 vol. 29 p. 707 - 717

8756-7938

Vitrification refers to the physical process by which a liquid supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Thus, vitrification is an effective freeze-avoidance mechanism and living tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly viscous and stops all chemical reactions that require molecular diffusion, its formation leads to metabolic inactivity and stability over time. In order to investigate glassy state in cryopreserved plant material, mint shoot tips were submitted to the different stages of a frequently employed cryopreservation protocol (droplet-vitrification) and evaluated for water content reduction and sucrose content, as determined by ion chromatography (IC), frozen water fraction and glass transitions occurrence by differential scanning calorimetry (DSC), and investigated by low-temperature scanning electron microscopy (cryo-SEM), as a way to ascertain if their cellular content was vitrified. Results show how tissues at intermediate treatment steps develop ice crystals during liquid nitrogen cooling, while specimens whose treatment was completed become vitrified, with no evidence of ice formation. The agreement between calorimetric and microscopic observations was perfect. Besides finding a higher sucrose concentration in tissues at the more advanced protocol steps, this level was also higher in plants precultured at 25ºC/-1ºC than in plants cultivated at 25ºC.