Cryopreservation of rye protoplasts by vitrification

Area of Science:

• Plant Cell Biology
• Cryobiology
• Agricultural Science

Background:

• Cryopreservation of plant protoplasts is essential for germplasm conservation and genetic manipulation.
• Winter rye (Secale cereale L.) seedlings are valuable for studying cold acclimation and stress tolerance.
• Developing efficient vitrification protocols for protoplasts is critical for preserving plant genetic resources.

Purpose of the Study:

• To establish and optimize a vitrification procedure for winter rye mesophyll protoplasts.
• To evaluate the impact of different ethylene glycol concentrations and osmotic unloading solutions on protoplast survival.
• To compare the cryosurvival rates of nonacclimated and cold-acclimated winter rye protoplasts.

Main Methods:

• Isolation of mesophyll protoplasts from nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings.
• Vitrification procedure involving ethylene glycol (EG) loading, dehydration in a vitrification solution (EG, sorbitol, BSA), quenching in liquid nitrogen (LN(2)), and unloading.
• Testing various EG concentrations (1.5-2.0 M) for loading and hypertonic/isotonic sorbitol solutions for unloading.

Main Results:

• Optimal survival for NA protoplasts (47±1%) was achieved with 1.75 M EG loading and hypertonic unloading (2.0 osm).
• ACC protoplasts showed higher survival (51±9%) with 2.0 M EG loading and hypertonic unloading (1.5 osm) compared to isotonic unloading (34±10%).
• Osmotic excursions during the unloading step significantly influence protoplast survival rates.

Conclusions:

• The developed vitrification procedure offers a method for cryopreserving winter rye protoplasts.
• Careful control of osmotic conditions during unloading is critical for maximizing cryosurvival.
• The findings provide insights into optimizing cryopreservation protocols for plant cells under varying physiological states.