Amorphous state stability of plant vitrification solutions

INTRODUCTION

The deterioration of the environmental quality parameters urges preservation of plant biodiversity (Volis, 2016). Nowadays, development of new plant germplasm cryopreservation methods is very important, since it provides a possibility of long-term survival of the gene pool of valuable species (Pritchard et al., 2014). Vitrification is a convenient cryopreservation method for practical use. It comprises maximum high cooling rates usually achieved by immersion of samples directly into liquid nitrogen, as well as high concentrations of cryoprotectants to prevent intracellular crystallization (Kim  et  al., 2009; Ruzic et al., 2013). Multicomponent media are often used in order to reduce the  cell damage during saturation with vitrification solutions. Such combined cryoprotective media for plant cryopreservation by vitrification are called PVS (plant vitrification solution; Uragami  et  al., 1989). As a  rule, these media have high toxicity to plant explants, therefore the  saturation time, the  object structure and size, the equilibration temperature, and the PVS concentration should be considered in order to reduce the damaging effect.

The modifications of the  available composition of PVS for various plants, as well as the  development of new combined solutions (Kim et al., 2009; Vіcenja et al., 2010) are currently being continued. Dilution of PVS with water is one of the approaches to avoid its pronounced toxicity to plant explants (Nishizawa et al., 1993). These aqueous solutions could enable saturation of larger objects with cryoprotectant at room temperature for a longer time, thereby providing better penetration of the cryoprotectant into plant cells without viability affection.

Due to the high concentrations of various substances in their composition, PVSs do not crystallize when cooled, and when they reach a certain temperature they are transferred into the  solid amorphous state. However, the  absence of crystallization at the  cooling stage does not exclude its occurrence while heating above the  glass transition temperature. Crystal formation at the  temperatures above the glass transition as well as the temperature and the  intensity of crystallization characterize the stability of the amorphous state (Baudot et al., 1996; Baudot, Boutron, 1998; Baudot  et  al., 2000). During cryopreservation by vitrification, the achievement of a stable glassy state and the  avoidance of ice formation are two critical factors for obtaining high viable biological samples. However, the  stability of the  amorphous state and the  glass-forming ability of a number of concentrated and diluted aqueous PVSs have not yet been clarified. The  development of cryogenic technologies is facilitated by biophysical studies capable of monitoring glass stability during cryopreservation (Zámečník et al., 2012).

The purpose of the study was to investigate the  glass-forming ability and the  amorphous state stability of PVSs, which are promising for plant cryopreservation, using differential scanning calorimetry (DSC).

MATERIALS AND METHODS

For the preparation of different PVSs, the following cryoprotectants were used: dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (1,2-PD), glycerol, and sucrose (Sigma Aldrich, Germany) (Table  1). 100% and 80% solutions of PVSs were prepared by diluting cryoprotectants with Murashige and Skoog medium: PVS1 modified, PVS2, PVS3 modified, PVS4, and PVSN.

The  phase and glass transitions of PVSs in the temperature range from –196°C to complete media melting were investigated using low-temperature DSC (Zinchenko  et  al., 2015). Glass transition temperature (Т_g) was determined as a  midpoint between the  onset and endset of the inflectional tangent. Crystallization (Т_с) and melting (T_m) temperatures were determined as an  extrapolated onset-temperature (the designed point of intersection of the extrapolated baseline and the inflectional tangent at the beginning of the melting or crystallization peak). The samples were frozen by immersion into liquid nitrogen, with an average cooling rate of 200 degrees/min. The thermograms were recorded during warming with the  rate of 0.5 degrees/ min. The weight of all the investigated samples was 1 g.

RESULTS AND DISCUSSION

DSC-thermograms of PVS1 modified, PVS2, and PVS3 modified (Fig.  1, Table  2) revealed only one heat capacity jump at temperature T_g associated with a reverse glass transition process (transition from the  solid amorphous state to the state of supercooled liquid). Neither exo- nor endothermic peaks were recorded, indicating no crystallization, at the cooling and warming stage. This fact testifies that at the cooling stage, PVS1 modified, PVS2, and PVS3 modified are completely transformed into the  glassy state with a highly stable amorphous phase, which does not crystallize even under slow warming above the glass transition temperature.

Fig. 1. DSC-thermograms of PVS

*  –  statistically significant difference relative to other PVS; ^#  –  statistically significant difference relative to PVSN (p = 0.05), n = 4.

In the thermograms of PVS4 and PVSN, in addition to the glass transition, an exothermic crystallization from the  amorphous state (devitrification) peak and an endothermic peak of melting were registered (Fig. 1, Table 2). A superposition of peaks is observed in the PVS4 thermogram: the melting (Т_m = –50.5°С) begins before complete crystallization (T_c = –64.0°C). It should be noted that for PVS4 and PVSN, the  area under crystallization and melting curves does not differ significantly (Fig.  2). This indicates that crystallization occurs only at the warming stage. The fact that crystallization and melting enthalpies for PVSN are 2.8 times greater than for PVS4 is an evidence of larger crystalline phase formation during PVSN warming. Moreover, the  temperature interval between reverse glass transition and crystallization of the liquid phase in the samples is significantly broader for PVS4 (Fig. 1 and Table 2). Thus, the differences between the glass transition and crystallization temperature of PVS4 and PVSN are 47.5°C and 39.1°C, correspondingly. This result demonstrates higher stability of the amorphous phase of PVS4 compared to PVSN. Probably, replacement of other cryoprotective compounds with sucrose in PVSN (Vіcenja et al., 2010) affects negatively the stability of the amorphous phase of the resulting PVSN.

It should be noted that crystallization in PVS4 and PVSN may be related to the  low warming rates used for calorimetric assays. The crystallization will most probably be avoided at higher warming rates. Previously, it has been reported that reducing the warming rate of PVS leads to an increased crystallization level, while glass transition is not significantly affected by the cooling or warming rate (Teixeira et al., 2014).

Fig. 2. Crystallization and melting enthalpy (ΔH) of PVSs; # – statistically significant difference relative to other PVSs (p = 0.05), * – statistically significant difference relative to crystallization enthalpy of the same PVSs (p = 0.05), n = 4

Analyzing the glass transition temperature and heat capacity of the studied PVSs, we revealed no significant differences in the  glass transition temperatures for PVS2, PVS4, and PVSN, while this parameter was significantly higher for PVS3 modified (Fig. 2 and Table 2). This may be either due to the absence of DMSO and EG, which have low glass transition temperatures, or higher sucrose concentration in PVS3 modified.

Saturation of such large objects as shoot tips or dormant buds with 100% PVSs is rather a difficult task. To ensure effective saturation of the  plant genetic resources with cryoprotectant, long time incubation is required. However, due to toxicity of PVSs, the cells on the sample surface may be damaged. It has been found that in some cases 80% concentration of PVS is sufficient for successful plant vitrification (Nishizawa et al., 1993). Therefore, the stability of the amorphous phase in 80% PVSs was also investigated in this research. As Table 2 shows, crystallization occurs in all 80% solutions of PVSs under slow warming. But the  crystallization temperature differs for all the studied solutions. The amorphous phase stability was the highest for 80% PVS1 modified and for 80% PVS3 modified. In these solutions, the amorphous phase crystallization does not occur immediately after the  appearance of the  liquid phase, but at higher temperatures that are closer to the melting point. Crystallization of 80% PVS1 modified and 80% PVS3 modified can be prevented by changing the sample volume and the warming rate. Melting enthalpy for all the studied 80% PVSs was significantly higher than crystallization enthalpy (Fig.  2). We assume that the liquid in PVSs was partly crystallized at the cooling stage.

Analyzing heat capacity jumps in 80% and 100% PVSs, it was demonstrated that the glass-forming ability for PVS2, PVS3 modified, and PVS4 did not differ significantly (Fig. 3). For 80% PVS1 modified and 80% PVSN, heat capacity jumps were significantly lower than for the  corresponding 100% PVSs. This may be due to lower concentration of cryoprotectant components in these solutions.

Fig. 3. Heat capacity jumps at the glass transition (ΔCp), * – statistically significant difference relative to 100% PVS (p = 0.05), n = 4

CONCLUSIONS

The higher glass-forming ability and amorphous state stability of PVS1 modified, PVS2, and PVS3 modified compared to PVS4 and PVSN suggest these cryoprotective media to be more promising during plant cryopreservation.

Received 26 June 2019
Accepted 11 December 2019

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Krystyna Vozovyk, Olena Bobrova, Anton Prystalov, Nadiya Shevchenko, Larisa Kuleshova

AUGALŲ VITRIFIKACIJOS TIRPALŲ AMORFINĖS BŪSENOS STABILUMAS

Santrauka

Norint gauti gyvybingus biologinius mėginius po kriokonservavimo vitrifikacijos metodu, yra svarbūs du veiksniai – krioprotektanto gebėjimas virsti ledu aušinimo metu ir jo amorfinės būsenos stabilumas atšilimo metu. Šio darbo tikslas – ištirti augalų vitrifikacijos tirpalų (plant vitrification solutions, PVS) amorfinės būsenos stabilumą diferencinio nuskaitymo kalorimetrijos metodu. Tyrimo objektai buvo modifikuoti PVS1, PVS2, PVS3, PVS4 ir PVSN. Skystame azote užšaldytų tirpalų termogramos buvo užfiksuotos esant 0,5 laipsnio / min. atšilimo greičiui. PVS1, PVS2 ir PVS3 suledėjimo temperatūra buvo atitinkamai  –109,0–115,3  °C ir –93,9 °C. Egzoterminių ir endoterminių pikų, patvirtinančių nesikristalizavimą tiek aušinimo, tiek atšilimo metu, neužregistruota. PVS4 ir PVSN termogramos, be suledėjimo (atitinkamai –111,5 °C ir –110,0 °C), rodė amorfinės būsenos (devitrifikacija) kristalizaciją ir ledo tirpimo smailes. PVSN kristalizacijos entalpija buvo 2,8 karto didesnė, palyginti su PVS4. Atitinkamai PVSN kristalinė fazė taip pat buvo didesnė. Reikėtų pažymėti, kad PVS4 ir PVSN kristalizacija gali būti susijusi su nedideliu atšilimo greičiu eksperimento metu. Esant didesniam atšilimo greičiui, šios kristalizacijos greičiausiai būtų galima išvengti. Taigi PVS1, PVS2 ir PVS3 pasižymi gebėjimu formuoti ledą ir stabilia amorfine būsena. PVS4 ir PVSN amorfinės būsenos stabilumas yra kur kas mažesnis, palyginti su PVS1, PVS2 ir PVS3.

Raktažodžiai: augalų vitrifikacijos tirpalai, suledėjimas, amorfinės būsenos stabilumas, devitrifikacija, ledo tirpimas

* Corresponding author. Email: helen.bobrova.77@gmail.com