Comparative Studies for Cryopreservation of Shoot Tips by Droplet-Vitrification.

The objective of this work was to assess the suitability of the Droplet-vitrification protocol previously developed with shoot tips for the cryopreservation of six species. Shoot tips were precultured for 1 day on a medium with 0.3 M sucrose in the dark, loaded in a solution with 1.6 M glycerol and 0.4 M sucrose for 20 min, and dehydrated by exposure to Plant Vitrification Solution 2 (PVS2) at 0 °C for 20 min. Complementary studies using histological analysis, Differential scanning calorimetry (DSC), and evaluation of morphological characteristics in cryo-derived plants were performed. Survival rates ranged from 84% to 100% and from 76% to 97% before and after cryopreservation regardless of the species belonging to two taxonomic subgenera. Thermal analysis of shoot tips subjected to the successive steps of the Droplet-vitrification protocol identified ice crystal formation after loading treatment and glass transition after osmotic dehydration with PVS2. The average glass transition temperature (Tg) was -55.44 °C based on the results of four species. The histological studies showed the anatomical differences that could be found in the meristematic structures depending on the loss of apical dominance. This is the most advanced research on cryopreservation of shoot tips.