A mixture of innate cryoprotectants is key for freeze tolerance and cryopreservation of a drosophilid fly larva.

Insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on the mechanisms of individual CP interactions, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Here, we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata, which survives long-term cryopreservation in liquid nitrogen. The CPs proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine and sarcosine accumulate in hemolymph in a ratio of 313:108:55:26:6:4:2.9:0.5 mmol l-1. Using calorimetry, we show that artificial mixtures, mimicking the concentrations of major CPs in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we show that during slow extracellular freezing trehalose becomes concentrated in partially dehydrated hemolymph where it stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it probably forms a layer of dense viscoelastic liquid. We propose that amorphous glass and viscoelastic liquids may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen.

Analysis of Thermal Characteristics of Potato and Hop Pollen for Their Cryopreservation and Cross-Breeding.

This study investigated the thermal properties of potato and hop pollen for cryopreservation and subsequent cross-breeding. Phase transitions and frozen water content in selected pollen samples were measured using a differential scanning calorimeter (DSC). Unlike hop pollen, potato pollen showed high variability in thermal properties and water content. Three specific types of pollen samples based on their thermal characteristics and water content were distinguished by DSC in potato: (1) 'glassy', with a water content lower than 0.21 g water per g dry matter; (2) 'transient', with a water content between 0.27 and 0.34 g of water per g of dry matter; (3) 'frozen', with a water content higher than 0.34 g of water per g of dry matter. Only the 'glassy' pollen samples with a low water content showed suitable properties for its long-term storage using cryopreservation in potato and hops. Cryopreservation of pollen did not significantly reduce its viability, and cryopreserved pollen was successfully used to produce both potato and hop hybrids. The results indicate that cryopreservation is a feasible technique for the preservation and utilization of pollen of these crops in the breeding process.

Vitrification Ability of Combined and Single Cryoprotective Agents.

Cryoprotective agents (CPA) are an important part of many current vitrification methods. The vitrification ability of CPAs influences the probability of the glass transition and water crystallization occurrence. Thermal characteristics and the vitrification ability of two combined CPAs (PVS2 and PVS3), common plant vitrification solutions, and four single CPAs (ethylene glycol, DMSO, glycerol, and sucrose), the components of the mentioned PVSs, were evaluated utilizing a differential scanning calorimetry (DSC) during standard cooling/warming rates of 10 °C min-1. The effect of solute concentration on their vitrification ability was shown in the CPAs tested. Four typical concentration regions at which the glassy state and/or crystallization occurred were defined. We suggest the solute concentration of 0.7 g g-1 as the universal vitrification concentration, characterized by an actual Tg of CPA solution and limited water crystallization. Knowledge of the thermal properties of CPAs allows the design of new combined CPAs with the required vitrification ability respecting the cryopreservation method used and the characteristics of the cryopreserved sample.

The Survival of Pear Dormant Buds at Ultra-Low Temperatures.

Currently, there is a varietal diversity decline in pear orchards of the Czech Republic. Thus, the safe storage of their gene pool collections is becoming increasingly important. Therefore, the ultra-low temperature survival after two-step cryopreservation treatment of dormant buds was tested for a safe and rapid way to conserve pear germplasm in a broader range of varieties. The following varieties crucial for cultivation in the Czech Republic were tested; 'Amfora', 'Beurré Hardy', 'Bosc', 'Clapp's Favourite', 'Conference', 'Dicolor', 'Erika', 'Lucas', 'Williams' and 'Williams Red'. In 2011 and 2012, dormant pear buds were dehydrated to 40.1% and 36.0% water content, respectively, before cryopreservation. The average regeneration of the dormant pear buds after cryopreservation by the two-step cryoprotocol in 2011 and 2012 was 54.3% and 16.1%, respectively. The mentioned cryopreservation procedure is suitable for the safe storage of dormant buds in most tested pear varieties.

Vitrification Solutions for Plant Cryopreservation: Modification and Properties.

Many plants cannot vitrify themselves because they lack glassy state-inducing substances and/or have high water content. Therefore, cryoprotectants are used to induce vitrification. A cryoprotectant must have at least the following primary abilities: high glass-forming property, dehydration strength on a colligative basis to dehydrate plant cells to induce the vitrification state, and must not be toxic for plants. This review introduces the compounds used for vitrification solutions (VSs), their properties indicating a modification of different plant vitrification solutions, their modifications in the compounds, and/or their concentration. An experimental comparison is listed based on the survival or regeneration rate of one particular species after using more than three different VSs or their modifications. A brief overview of various cryopreservation methods using the Plant Vitrification Solution (PVS) is also included. This review can help in alert researchers to newly introduced PVSs for plant vitrification cryoprotocols, their properties, and the choice of their modifications in the compounds and/or their concentration.

Cryotolerance of apple tree bud is independent of endodormancy.

Increasing interest in cryopreservation of dormant buds reveals the need for better understanding of the role of dormancy in cryotolerance. Dormancy stage and low-temperature survival of vegetative apple buds (Malus domestica Borkh.), cultivars 'Sampion' and 'Spartan', collected from orchard were evaluated during three seasons contrasting in temperature and precipitation throughout the arrested plant growth period. During each season, the cultivars differed either in the onset of the endodormancy or in the length of the endodormant period. A simple relation between endodormancy of the buds and their water content was not detected. The cryosurvival of vegetative apple buds of both cultivars correlated with their cold hardening without direct regard to their particular phase of dormancy. The period of the highest bud cryotolerance after low-temperature exposure overlapped with the endodormant period in some evaluated seasons. Both cultivars had the highest cryosurvival in December and January. The presented data were compared with our previous results from a dormancy study of in vitro apple culture. Endodormancy coincided with the period of successful cryosurvival of apple buds after liquid nitrogen exposure, but as such, it was not decisive for their survival and did not limit their successful cryopreservation.