KR20100110524A - Vitrification solutions and loading solutions applicable for droplet-vitrification procedures - Google Patents

Abstract

The present invention relates to a composition of a cryoprotectant mixture vitrification solution and loading solution which is essential for cryogenic preservation of nutrient gene sources using liquid nitrogen.

According to the present invention, cryopreservation by developing a cryoprotectant vitrification solution and loading solution which is indispensable for survival after cryopreservation in cryopreservation of nutrient gene sources such as garlic and chrysanthemum at cryogenic temperature of liquid nitrogen using liquid nitrogen It is widely used as a general purpose cryoprotectant mixture solution that can improve post survival and regeneration rate and can be applied to a wide range of materials across various plant species. Long-term preservation of genetic resources such as high-quality varieties through long-term preservation of nutrients that are easily lost during packaging preservation can be widely used in the genetic resources and seed industries.

Description

Lyoprotectant Mixture Vitrification Solutions and Loading Solutions Suitable for Droplet Vitrification

The present invention relates to a composition of a cryoprotectant mixture vitrification solution and loading solution which is essential for cryogenic preservation of nutrient gene sources using liquid nitrogen.

Preservation of seed propagation resources (e.g. rice, etc.), which has been used the most as a method of preserving agricultural genetic resources, has been extended by minimizing moisture after freezing and drying them, but nutrition propagation resources (e.g. garlic, etc.) Dryness in the natural state loses its vitality, so it must be maintained and renewed through packaging growth and storage every little time, and it is expensive to maintain, and is exposed to the risk of loss and genetic erosion due to pathological and physiological degradation and meteorological disasters. Long-term, stable, and effective conservation studies are urgently needed, and cryogenic cryopreservation has been recognized as the only long-term preservation of these resources (IPGRI). In some developed countries such as the US and Germany, and international institutions such as the International Potato Research Institute (CIP), the cryopreservation technology using vitrification has been applied to some crops. Cases are increasing worldwide.

So-called nutrients such as garlic and chrysanthemum do not multiply as seeds, but because they grow as nutrients, they cannot be stored for a long time in the cellar. There is a risk that genetic resources, such as superior varieties, will be lost. It is also reported that 1.5% of the resources are lost each year in the conservation of garlic genetic resources in Germany (Keller et al., 2008).

Cryopreservation techniques of plant tissues were mainly used in micro cell lines such as suspension culture cells by slow freezing (two-step freezing) method, but the vitrification method was tried (Fahy, Cryobiology, 1984). In particular, relatively large tissues, such as shoots, can obtain regenerated plants without undergoing retardation such as callus formation. Cryogenic preservation has been recognized as the only option for long-term preservation of nutrients and recalcitrant seeds. Recently, large-scale implementations of cryopreservation have been known in inflight seedlings such as potatoes and bananas. have.

The droplet-vitrification method combines the advantages of the known droplet-freezing and solution-based vitrification methods. It has been developed in the field of garlic and garlic, and it is a new technology that is in the spotlight worldwide. The main advantage of this preservation method is to increase the regeneration rate by improving the cooling and thawing speed. In the conventional vitrification method, the solution and the sample are cooled and thawed by adding the solution and the sample to the carrier vial. It is placed on a small strip of aluminum foil, cooled directly with liquid nitrogen, immersed directly in the unloading solution and thawed. The rate of cooling and thawing is one of the major factors in determining the regeneration rate of the sample. In the case of incomplete vitrification during the cooling process, de-vitrification can be prevented only when the thawing speed is high. For this purpose, instead of bulky cry vials, thin, low volume straws, open-pulled straws, grids, etc. are used, which can be applied only to small samples, which are somewhat inconvenient to maintain and costly. There is an expensive disadvantage. In contrast, when aluminum foil is used, heat transfer can be performed quickly and uniformly easily and inexpensively.

Until now, the response to cryoprotectant such as vitrification solution varies greatly depending on the species and varieties of plants, and there is also a big difference in the regeneration rate after cryopreservation, which is one of the most influential factors for regeneration rate in order to realize the practical application of cryopreservation on a large scale. Selection of the vitrification solution is a priority. The development of the droplet-vitrification method has resulted in some improvement in regeneration rate, but it requires a low viability and high efficiency suitable vitrification solution and loading solution applicable to a wide range of materials across a wide variety of species.

In cryopreservation protocols based on vitrification, a dehydration process that removes almost all frozen water in a sample before freezing has been recognized as the key to success (Block, 2003; Dussert et al., 2001; Engelmann, 2000). The vitrification process therefore involves dehydration with a high concentration of cryoprotectant mixture (vitrification solution) before immersion in liquid nitrogen. Application of a suitable vitrification solution is a key factor in determining the success or failure of cryopreservation in a vitrification-based process. This is because the composition of the vitrification solution and the dehydration time in the solution are the most influential factors in the regeneration rate of cryopreserved shoots (Kim et al., 2006a).

Cryoprotectants are substances that act to protect cells or tissues by inhibiting freezing formation. For cryopreservation based on the vitrification method, a high concentration of cryoprotectant is usually used. Usually several types of permeable and non-invasive protective agents are used in combination. Cryoprotectants are classified according to their permeability to biological samples. According to Tao & Li (1986) classification, sucrose, a group of sucrose that crosses cell walls and plasma membranes due to small molecular weight such as glycerol, dimethyl sulfoxide and ethylene glycol. Is classified into two groups that pass through the cell wall but do not pass through the plasma membrane. In general, sucrose has a high molecular weight together with polyethylene glycol, which does not penetrate inside the cell, and stays in the cell gap or solution to induce dehydration by osmotic pressure. It has been classified as a protective agent.

The vitrification solution is a freeze protection agent mixture in an aqueous solution that can be cooled slowly to the temperature below the glass transition temperature (Tg) without freezing between cells or within the cells, and maintains an amorphous glass state by avoiding freezing during cooling and thawing. (Fahy et al., 1987). In order to dehydrate the sample, to reduce toxicity, and to protect the cells from freezing of residual water molecules, the vitrification solution usually consists of a mixture of a permeable cryoprotectant and a non-invasive cryoprotectant. Traditional cryobiological studies have recommended the use of permeable cryoprotectants such as dimethylsulfoxide or glycerol to protect cells from freezing disorders, but noninvasive cryoprotectants such as sucrose were more effective than permeability protectors for protecting mouse sperm. (Sztein et al., 2001).

The vitrification solution used for cryopreservation of plants has been reported by Steponkus Group (1993), Towill Group (1990), Sakai Group (1990; 1993), but PVS2 (30%) designed to freeze citrus callus by Sakai et al. Glycerol + 15% Dimethylsulfoxide + 15% Ethylene Glycol + 0.4M Sucrose, Sakai et al., 1990) has been the most widely used, as it is applied to freezing over 200 species / cultivars (Sakai and Engelmann, 2007) . On the other hand, PVS3 (50% glycerol + 50% sucrose, Nishizawa et al., 1993) has higher survival rates for some plants, such as bulbs, which are relatively large and resistant to dehydration. (Matsumoto et al., 1995), asparagus (Nishizawa et al., 1993), garlic (Makowska et al., 1999; Kim et al., 2004a) and the like.

PVS2, which is widely used in the past, contains a large amount of highly toxic and highly toxic protective agent, so it cannot be dehydrated for a sufficient time, so it is mainly applicable only to small samples having a size of 1 ~ 2mm or less, Samples over 3 mm have been shown to have very low regeneration (Kim et al., 2004; Makowska et al., 1999). Due to the toxicity of PVS2 solution, the dehydration time that can be exposed to this solution is short, so that constituent cryoprotectants such as glycerol, dimethyl sulfoxide, ethylene glycol, and sucrose do not have enough time to penetrate into the cytoplasm of shoot cells. (Sakai, 2000; Sakai et al., 1990; Steponkus et al., 1992). Fahy et al. (2004) found that cryoprotectant toxicity is the greatest barrier to successful vitrification in complex and spatially expanded biological tissues. Therefore, discriminating whether the nature of toxicity is biochemical or osmotic is essential for finding an appropriate method of reducing toxicity, and it is known that toxicity depends mainly on permeability protection agent rather than non-invasive cryoprotectant (Fahy et al., 1984; 1990).

The cryopreservation process based on vitrification dehydrates the sample in a vitrified solution, a high concentration of cryoprotectant mixture, before immersion in liquid nitrogen to remove most or all of the water that can freeze from the sample. The loading solution is a relatively low concentration of cryoprotectant solution used prior to dehydration to overcome the toxic effects of direct exposure. As a loading solution for cryopreservation of plants, a stepwise treatment of 20-60% dilution of the vitrification solution PVS2 solution is used (Sakai et al., 1990; Towill and Jarret, 1992; Sarkar and Naik, 1998). In wasabi, 0.5M glycerol + 0.3M sucrose was incubated until morning (Matsumoto et al., 1998), but most of them were 2M glycerol + 0.4M shoe used by Nishizawa et al. (1993) and Matsumoto et al. (1994). Cross solution has been widely used.

Lyoprotectant loading treatment primarily contributes to minimizing the disturbances that occur during dehydration in vitrified solutions, which prevent destabilization induced by dehydration of cell membranes and proteins. The loading process also increases the solution concentration of the cytoplasm and also contributes to the vitrification of the cytoplasm during immersion in liquid nitrogen (Steponkus et al., 1992). Nishizawa et al. (1993) also found that loading treatment was effective in inducing freeze-dehydration resistance or dehydration resistance. During the short loading treatment cells were proliferated in plasma by significant dehydration, but little penetration of glycerol into the cytoplasm (Matsumoto et al., 1998).

The present invention is trying to develop a cryoprotectant which is one of the most influential factors for survival after cryopreservation in order to develop a method for effectively long-term preservation of nutrients such as garlic and chrysanthemum by using liquid nitrogen (-196 ℃) In addition, the present invention seeks to provide cryoprotectant mixed liquor vitrification solutions and loading solutions that are widely applicable across a variety of plant species.

In order to solve the above problems, the present invention provides any vitrification solution selected from the following table as a cryoprotectant suitable for the small-drop vitrification method.

Composition (%, w / v) density(%) Glycerol 42.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 17.5 90 Glycerol 37.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 22.5 90 Glycerol 32.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 27.5 90 Glycerol 35.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 20.0 85 Glycerol 37.5 + Dimethylsulfoxide 10 + Ethylene Glycol 10 + Sucrose 32.5 90 Glycerol 30.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 25.0 85 Glycerol 30.0 + Dimethylsulfoxide 20 + Ethylene Glycol 20 + Sucrose 15.0 85 Glycerol 40.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 15.0 85 Glycerol 50 + Sucrose 40 90 Glycerol 45 + Sucrose 45 90 Glycerol 40 + Sucrose 50 90 Glycerol 40 + Sucrose 40 80

On the other hand, the present invention provides a loading solution of any one selected from the following table as a cryoprotectant suitable for the small droplet vitrification method.

Loading solution composition (M) Loading solution composition
(%, w / v) Creation costs density(%) 1.0M Glycerol + 0.70M Sucrose 9.2 + 23.96 0.38: 1 33.16 2.0M Glycerol + 0.65M Sucrose 18.4 + 22.25 0.83: 1 40.65 1.63M Glycerol + 0.44M Sucrose 15.0 + 15.0 1: 1 30.0 1.9M Glycerol + 0.51M Sucrose 17.5 + 17.5 1: 1 35.0 2.17M Glycerol + 0.58M Sucrose 20.0 + 20.0 1: 1 40.0 2.50M Glycerol + 0.50M Sucrose 23.0 + 17.1 1.3: 1 40.1 2.45M Glycerol + 0.66M Sucrose 22.5 + 22.5 1: 1 45.0

The present invention is to change the composition of glycerol and sucrose from the known PVS2 (A1) or PVS3 (B1) by changing the composition, such as glycerol + dimethyl sulfoxide + ethylene glycol + sucrose A solution and glycerol + The composition of vitrification solution such as B-series solution composed of sucrose was devised, and the loading solution was devised 30% -45% solution composed of glycerol and sucrose to analyze the physicochemical characteristics of each solution and applied it to model crops such as garlic and chrysanthemum. Applied. Differential Scanning Calorimetry was used to analyze the thermal properties of these solutions.The thermal properties of the solutions themselves were compared with those of garlic and chrysanthemum samples incubated in the loading and vitrification solutions. The effectiveness of the solution was examined through comparative analysis.

Disorders in the cryopreservation process are mainly caused by the toxicity of the cryoprotectant during direct cooling and thawing with liquid nitrogen and by direct freezing formation. To prevent freezing formation, selection of a suitable cryoprotectant is necessary. Speed also has a significant impact on survival. In the present invention, the sample is placed on a piece of aluminum foil and rapidly cooled in liquid nitrogen to be stored in a cryovial containing liquid nitrogen, and during thawing, the unloading solution (0.8M) is heated to 37-40 ° C. By immersing in sucrose, the survival rate can be stably increased by improving the cooling and thawing speed.

According to the present invention, in the cryopreservation of nutrient gene sources such as garlic and chrysanthemum at the cryogenic temperature of liquid nitrogen using liquid nitrogen, a cryoprotectant vitrification solution and a loading solution which are indispensable for survival after cryopreservation have been developed. It provides a method for long-term preservation by applying to cryopreservation of nutrient shoots such as garlic and chrysanthemum.

The vitrification solution and loading solution of the present invention are highly applicable as a general purpose cryoprotectant mixture solution applicable to a wide range of materials across various plant species, and the long-term preservation method of chrysanthemum by small droplet-vitrification method applied to chrysanthemum improves survival rate and regeneration rate. By long-term preservation of genetic resources such as high-quality varieties that are easily lost in the packaging, it will be widely used in the genetic resources and seed industry.

Example  1: small drops Vitrification  Suitable vitrification solution ( vitrification solution ) Selection

  PVS2 solution (30% glycerol + 15% dimethylsulfoxide + 15% ethylene glycol + 0.4M sucrose), widely used in plants, contains a large amount of highly toxic protective agents such as dimethylsulfoxide and ethylene glycol, which are highly toxic. Therefore, dehydration can not be performed for a sufficient time, so the sample size can be applied only to small samples of 1 ~ 2mm or less, and the sample having a size of 2 ~ 3mm or more, such as aperture, has shown very low regeneration rate. On the other hand, PVS3 solution can be used for weak toxicity and strong osmotic stress, but its concentration is too high, making it difficult to use for materials with low dry resistance.

To compensate for this drawback, a solution that increases the amount of glycerol and sucrose in PVS2 solution is formulated to provide sufficient dehydration in a short period of time without increasing chemical toxicity, thereby improving regeneration rate and applying a wide range of materials to a wide range of target materials. Vitrification solution was selected. PVS3 lowers the concentration of glycerol and sucrose so that it can be applied to materials with low dry resistance. Table 1 is a composition table of these vitrification solutions.

Table 1 Composition of Vitrification Solution

                                             ※ Con. : control solution (existing solution)

solution Composition (%, w / v) density(%) A1 (Con.) Glycerol 30.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 13.7 73.7 A2 Glycerol 42.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 17.5 90 A3 Glycerol 37.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 22.5 90 A4 Glycerol 32.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 27.5 90 A5 Glycerol 35.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 20.0 85 A7 Glycerol 37.5 + Dimethylsulfoxide 10 + Ethylene Glycol 10 + Sucrose 32.5 90 A8 Glycerol 30.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 25.0 85 A9 Glycerol 30.0 + Dimethylsulfoxide 20 + Ethylene Glycol 20 + Sucrose 15.0 85 A10 Glycerol 40.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 15.0 85 B1 (Con.) Glycerol 50 + Sucrose 50 100 B2 Glycerol 50 + Sucrose 40 90 B3 Glycerol 45 + Sucrose 45 90 B4 Glycerol 40 + Sucrose 50 90 B5 Glycerol 40 + Sucrose 40 80

Example  2: vitrification solution of  characteristic

  To analyze the performance of the selected vitrification solution, the endothermic heat of the 80% dilution of the vitrification solution (Ensol), the endothermic heat (Ensam) and water content of garlic shoots dehydrated for 30 minutes in the A-based vitrification solution or 150 minutes in the B-based vitrification solution. (MC) was investigated (Table 2).

[Table 2] Endothermic heat of 80% dilution of vitrification solution (Ensol), endothermic heat (Ensam) and water content of garlic shoots dehydrated for 30 minutes in A-based solution or 150 minutes in B-based solution

VS TC
(%) GSC
(%) GC
(%) SC
(%) DEGS
(%) DEC
(%) Ensol
(J / g FW) Ensam
(J / g FW) MC
(%) A1
A2
A3
A4
A5
A7
A8
A9
A10
B1
B2
B3
B4
B5 73.7
90
90
90
85
90
85
85
85
100
90
90
90
80 43.7
60
60
60
55
70
55
45
55
100
90
90
90
80 30
42.5
37.5
32.5
35
37.5
30
30
40
50
50
45
40
40 13.7
17.5
22.5
27.5
20
32.5
25
15
15
50
40
45
50
40 68.6
50
50
50
54.5
28.6
54.5
88.9
54.5
0
0
0
0
0 30
30
30
30
30
20
30
40
30
0
0
0
0
0 -35.9 ± 1.8d
-0.0 ± 0.0a
-0.3 ± 0.1a
-1.6 ± 0.3ab
-2.9 ± 2.4ab
-4.2 ± 1.4ab
-2.1 ± 0.5ab
-0.0 ± 0.0a
-2.0 ± 0.6ab
-6.9 ± 1.5b
-31.1 ± 5.4c
-42.1 ± 1.4e
-40.9 ± 0.8e
-48.3 ± 8.9f -16.7 ± 16.1
-15.4 ± 8.8
-3.1 ± 6.6
-14.7 ± 14.9
-10.3 ± 8.3
-19.7 ± 13.8
-8.8 ± 7.5
-6.8 ± 8.1
-13.1 ± 12.5
-3.3 ± 6.6
-1.5 ± 2.5
-4.4 ± 4.8
-2.9 ± 2.2
-12.2 ± 10.3 67.0 ± 3.0a
62.0 ± 2.4b
60.8 ± 1.9b
60.1 ± 1.1bc
62.1 ± 1.5b
57.0 ± 2.3c
60.9 ± 3.4b
63.5 ± 2.4b
62.8 ± 3.3b
37.6 ± 1.9e
40.4 ± 2.2e
39.7 ± 1.5e
40.9 ± 2.1e
45.2 ± 1.7d P <0.0001 ns P <0.0001

(VS, vitrification solution; TC, total concentration; GSC, glycerol + sucrose concentration; GC, glycerol concentration; SC, sucrose concentration; DEGS,% of DMSO + EG to glycerol + sucrose; DEC, DMSO + EG concentration; Ensol, endothermic enthalpies of 80% -diluted vitrification solutions tested; Ensam, endothermic enthalpies of garlic shoots dehydrated with PVS2 and variants for 30 min or with PVS3 and variants for 150 min MC, moisture content of dehydrated garlic shoots)

The moisture content of dehydrated garlic shoots and the calorie content of 80% diluents in vitrified solutions differed significantly between the solutions (P <0.0001), while the calories of dehydrated garlic shoots (Ensam) in the solutions were significant between the solutions. It wasn't. All the parameters of TC, GSC, GC, SC, DEGS, DEC, etc. of the vitrification solutions showed a highly significant difference (P <0.0001) in MC, Ensol, and Ensam, etc., so the design of the solution is considered appropriate. High significance (P <0.001) was found between MC and Ensol (correlation coefficient -0.59). The water content of garlic shoots decreased to 84.1% after pre-culture in 0.3M sucrose solid medium, to 72.1% after 40 min loading in 2M glycerol + 0.6M sucrose solution, and to 37.6-67.0% after dehydration in vitrified solutions. .

Calorimetric analysis of A3 solution (glycerol 37.5% + dimethylsulfoxide 15% + ethylene glycol 15% + sucrose 22.5%, w / v) to compare the performance of vitrification solution A3 selected in the first calorimetry As a result, the exothermic reaction of the 67-100% A3 solution did not occur in the cooling process, but calories were generated only in the 80-100% A3 solution during the thawing process (FIGS. 1A and 1B). This suggests that the unstabilized glass crystals in the 67-80% A3 solution re-freeze during the thawing process. During the cooling process, B1 solution (50% glycerol + 50% sucrose, w / v, PVS3) showed a similar tendency to A1 and showed similar freezing ability. During the thawing process, the 80% B1 solution showed a significant endothermic peak (-5.75 J / g FW), while the 80% A3 solution had a slight peak (-0.02 J / g FW), which was somewhat superior in the ability to freeze (FIG. 2A). , 2b).

As the concentration of A3 solution increased from 0% to 80%, the onset temperature of endothermic peak decreased linearly from 2.7 ℃ to -43 ℃ (P <0.01), 50% A3 solution and 70% A3 solution. The onset temperature of -27.1 ℃ and -43.0 ℃ was lower than -20.6 ℃ and -35.7 ℃ of B1 solution, respectively, A3 solution was slightly better than B1 solution (Fig. 3a, 3b).

Thermal analysis using vitrification solution 80% diluents to compare the freezing ability of the vitrification solutions, the exothermic peak was not detected during cooling in all 80% dilution (Fig. 4). During thawing, all A-based solutions (A2-A10) showed lower endothermic peaks than B-based solutions (B1-B5), while A-based solutions contained 0 (A2, A9) to 4 J / g FW (A7). An endothermic peak of was observed, but a peak of 37 J / g FW was observed in the original PVS2 solution (A1). Glycerol concentration solutions (A2 and A3) in PVS2 mutant solutions were more effective in inhibiting freezing formation than those in which sucrose concentration was increased (A4), and the solution of dimethylsulfoxide and ethylene glycol (A9) was higher. It was more effective than the solution (Gl) that raised glycerol.

Among the B-based solutions, the original PVS3 (B1) solution showed 8 J / g FW calories, while the PVS3 modified solutions showed relatively large peaks of 31-48 J / g FW.

Thermal analysis of garlic shoots dehydrated with PVS2 and its modified solutions for 30 minutes or PVS3 and its modified solutions for 150 minutes (Fig. 5) showed that PVS3 and modified solutions showed opposite PVS2 and 80% dilution results. It was more effective in inhibiting freezing formation of garlic shoots than in and modified solutions. These results were attributed to the fact that the moisture content of shoots dehydrated in PVS3 and modified solution (37.6-45.2%) was lower than the moisture content (60.1-67.0%) of shoots dehydrated in PVS2 and modified solution. Among PVS2 and its modified solutions, the smallest endothermic peak was observed in the order of A9 solution (increased dimethyl sulfoxide and ethylene glycol concentration) <A3 and A5 solution (balanced increase in glycerol and sucrose).

Example  3: selected vitrification solution of  garlic In the beginning  apply

  Survival and regeneration rate after dehydration were not significantly different between the two groups, but the solutions of the B-series showed higher survival and regeneration rate after cryopreservation than the solutions of the A-series (FIG. 6). Survival and regeneration rate after cryopreservation of shoots dehydrated in solution A (A2-A10) were higher than those of PVS2. There was no peak at 80% dilution and A9 solution showed fine peak when dehydrated garlic shoots showed 61.6% survival and 56.2% regeneration after cryopreservation. On the other hand, A7 solution with dimethylsulfoxide and ethylene glycol concentrations showed a higher regeneration rate after cryopreservation, despite a large peak after dehydration (-19.7 J / g FW). Furthermore, the B5 solution, 80% solution lower than the PVS2 modifications (85-90%), showed significantly higher survival and regeneration rate than the PVS2 modifications (P <0.0001). The recovery rate after freezing preservation seems to be lowered due to the toxicity of PVS2 and the modified solution, as well as the freezing failure due to freezing formation during cooling and thawing.

Among the parameters of the vitrification solution, TC (total concentration) and GSC (glycerol + sucrose concentration) had a significant (P <0.0001) effect on the survival and regeneration rate of cryopreserved garlic shoots, reaching 80-100%. The high concentration of glycerol + sucrose is thought to be the result of increased regeneration rate by allowing sufficient amount of cryoprotectant penetration and sufficient dehydration.

The moisture content of garlic shoots dehydrated with various VSs affected the survival and regeneration rate of cryopreserved samples (P <0.01). The calories of dehydrated shoots affected the survival and regeneration of all samples before (LN−) (P <0.05) and after (LN +) (P <0.001) cryopreservation.

The relationship between the parameters of the solution when the selected cryoprotectant vitrification solutions were applied to garlic small leaf shoots is shown in FIG. 7. The moisture content of shoots dehydrated in various vitrification solutions was negatively correlated with the survival rate (r = -0.82) and regeneration rate (r = -0.79) of cryopreserved shoots, indicating that the degree of dehydration of the sample is an appropriate indicator of successful cryopreservation. It is. GSC was in good agreement with the regeneration rate of the cryopreserved samples. PVS2 strains (A2-A10) are more resistant to freezing than PVS3 strains (B2-B5), but garlic shoots dehydrated with PVS3 strains are more dehydrated than PVS2 strains. Suppressed.

Example  4: small drops Vitrification  Suitable vitrification solution of  Application to chrysanthemum

  Survival and regeneration rates before and after cryopreservation (LN (-) and LN (+)) in chrysanthemum shoots were more varied than garlic depending on the concentration and composition of VS (Figure 8). Sucrose at high concentrations (32.5% -50.0%) had a positive effect on the recovery of cryopreserved samples. The balance of glycerol and sucrose in PVS3 and its modified solutions had a positive effect on the regeneration of cryopreserved samples, and higher concentrations of glycerol were more effective than high concentrations of sucrose.

As the DEGS and DEC of VS were increased in PVS2 and its modified solutions, the survival and regeneration rates of chrysanthemum shoots decreased before and after cryopreservation. A7 with decreased DMSO and EG showed high recovery before and after cryopreservation in PVS2 modified solution. On the other hand, A9 (increase in DMSO and EG) showed the lowest resilience before cryopreservation treatment. As a result, chrysanthemum was very sensitive to permeable cryoprotectants such as DMSO, EG and glycerol.

Example  5: small drops Vitrification  Selection of suitable loading solution

  Dehydration in high concentration cryoprotectant solution is essential for cryogenic preservation by small-drop vitrification method. In order to alleviate dehydration shock caused by high concentration solution, cryoprotectant infiltration and gentle dehydration are performed in one level of low concentration solution. The solution used at the time of induction is called a loading solution. A series of loading solutions that can be applied to a wide range of materials with improved survival compared to 1M glycerol + 0.8M sucrose, 2M glycerol + 0.4M sucrose, which has been used as a loading solution Was selected (Table 3).

Table 3 Composition of Loading Solution

※ Con. : control solution (existing solution)

solution Loading solution composition (M) Loading solution composition (%, w / v) Creation costs density(%) C1 1.0M Glycerol + 0.70M Sucrose 9.2 + 23.96 0.38: 1 33.16 C2 2.0M Glycerol + 0.65M Sucrose 18.4 + 22.25 0.83: 1 40.65 C3 1.63M Glycerol + 0.44M Sucrose 15.0 + 15.0 1: 1 30.0 C4 1.9M Glycerol + 0.51M Sucrose 17.5 + 17.5 1: 1 35.0 C6 2.17M Glycerol + 0.58M Sucrose 20.0 + 20.0 1: 1 40.0 C7 (Con.) 2.00M Glycerol + 0.40M Sucrose 18.4 + 13.69 1.3: 1 32.9 C8 2.50M Glycerol + 0.50M Sucrose 23.0 + 17.1 1.3: 1 40.1 C9 2.45M Glycerol + 0.66M Sucrose 22.5 + 22.5 1: 1 45.0

The freezing protection effect of these solutions was investigated using differential scanning calorimetry.The calorimeter scanning conditions were cooled at 10 ° C / min from 25 ° C to -85 ° C, stopped for 2 minutes, and then at 10 ° C / min. While thawing, the calorific value and the endothermic amount were measured (Table 4, FIG. 9, FIG. 10).

[Table 4] The osmotic pressure of the loading solution according to the concentration of the loading solution and the glycerol / sucrose ratio, the endothermic heat of the loading solution, the endothermic heat of garlic shoots incubated for 40 minutes in each loading solution

LS TC
(% w / v) GSR
(%) Osm
(Osmol) Ensol
(J / g FW) Onsol
(J / g FW) Ensam
(J / g FW) C1
C2
C3
C4
C6
C7
C8
C9 33.2
40.7
30.0
35.0
40.0
32.1
40.1
45.0 38.4
82.7
100.0
100.0
100.0
134.3
134.5
100.0 0.92 ± 0.06f
1.44 ± 0.01c
1.16 ± 0.02e
1.39 ± 0.01c
1.58 ± 0.01b
1.25 ± 0.00d
1.60 ± 0.00b
1.83 ± 0.04a -137.6 ± 3.4c
-117.9 ± 6.0b
-149.4 ± 7.3d
-134.2 ± 5.9c
-122.7 ± 4.4b
-139.9 ± 7.0c
-116.1 ± 3.1b
-100.2 ± 4.4a -12.8 ± 0.1a
-16.8 ± 0.2d
-12.9 ± 0.4a
-15.2 ± 0.4c
-17.5 ± 0.4de
-14.2 ± 0.5b
-17.9 ± 0.5e
-20.1 ± 0.5f -193.0 ± 3.7e
-148.2 ± 7.8b
-194.5 ± 4.1e
-165.2 ± 6.7c
-149.7 ± 2.4b
-174.2 ± 7.6d
-170.9 ± 7.0d
-129.7 ± 5.2a P <0.0001 P <0.0001 P <0.0001 P <0.0001

(LS, loading solutions TC, total concentration; GSR,% of glycerol concentration to sucrose concentration; Osm, osmolarity (osmol) of 50% -diluted LSS Ensol, endothermic enthalpies of LS tested; Onsol, onset temperature of recrystallization of the LS tested ; Ensam, endothermic enthalpies of garlic shoots apices loaded with LS for 40 min; MC, moisture content of garlic shoot apices loaded with LS for 40 min)

These loading solutions were applied to real garlic and chrysanthemum shoots to select promising solutions by examining the survival and regeneration rates before and after cryopreservation (FIGS. 11, 12, 13a, and 13b). The freezing capacity of the loading solution itself was determined by the concentration rather than the composition ratio of glycerol and sucrose, and high concentrations of C2 (2M glycerol + 0.65M sucrose), C8 (2.5M glycerol + 0.5M sucrose), C6 ( 2M glycerol + 0.5M sucrose) was excellent in the freeze protection effect.

Example  6: small drops Vitrification  Suitable loading solution of  Application to garlic

  Loading treatment did not affect survival and regeneration before cryopreservation in garlic shoots. Those that were not immersed in the loading solution after cryopreservation showed a survival rate of 84.5% and a regeneration rate of 82.7%, which was clearly lower than the samples that were loaded (survival rate 98.9-100%, regeneration rate 98.5-100%) (P <0.0001). After freezing and storing garlic shoots in various loading solutions, there was no significant difference in survival and regeneration rates. This process means that the loading treatment has a positive effect on the recovery of cryopreserved garlic shoots with various concentrations of loading solutions without any preference between the tested loading solutions.

Example  7: small drops Vitrification  Suitable loading solution of  Application to chrysanthemum

  Application of these loading solutions to cryopreservation of real chrysanthemum buds revealed that C4 (1.9M glycerol + 0.51M sucrose), 35% dilution of PVS3, before and after cryopreservation had the highest survival and regeneration rates (FIG. 18). This was followed by C6 (2.17M glycerol + 0.58M sucrose), a 40% dilution of PVS3. These solutions have the same weight ratio of glycerol and sucrose.

By utilizing these results, it is possible to obtain a higher regeneration rate than the conventional loading solution by diluting the PVS3 solution to around 35% without having to prepare a loading solution separately.

1A and 1B are graphs showing the change in calories (J / g FW) during the cooling process (FIG. 1A) and the thawing process (FIG. 1B) according to the concentration of the dilute solution of the vitrification solution A3 solution (0-100% solution). (Calorimeter scanning conditions: cooled at 10 ° C./minute from 25 ° C. to −85 ° C., thawed at 10 ° C./min after stopping for 2 minutes)).

2A and 2B are graphs showing the change in calories (J / g FW) during the cooling process (FIG. 2A) and the thawing process (FIG. 2B) according to the concentration of the vitrification solution B1 solution dilutions (0-100% solution).

Figure 3a is a graph showing the change in starting temperature (° C) during the thawing process according to the concentration of the vitrification solution A3 solution dilutions (0-100%), B1 solution dilutions (0-100%).

Figure 4 is a graph showing the calories (J / g FW) during the thawing of the 80% dilution of the vitrification solution.

FIG. 5 is a graph showing the endothermic heat (J / g FW) during thawing of garlic vinegar dehydrated for 30 minutes in PVS2 and modified solutions or 150 minutes in PVS3 and modified solutions.

Figure 6 shows the survival rates before (LN-) and after (LN +) freezing preservation of garlic small leaf shoots dehydrated for 30 minutes in PVS2 and modified solutions (series A) or 150 minutes in PVS3 and modified solutions (series B). surv) and refresh rate.

Figure 7 shows the concentration of glycerol + sucrose (gsr) in vitrification solutions, calorie (ensol) in 80% dilutions of vitrification solution, and water content (mc) of garlic after dehydration for 30 minutes in A-based solution or 150 minutes in B-based solution. And ensam, the regeneration of garlic shoots before (LN−) and after (LN +) cryopreservation after dehydration in these solutions.

Figure 8 shows the survival rate (surv) before and after cryopreservation (LN_) and after (LN +) of chrysanthemum shoots dehydrated with PVS2 and modified solutions (series A) for 30 minutes or with PVS3 and modified solutions (series B) for 60 minutes. The refresh rate is shown.

9 shows calorimetry (endothermic amount due to freezing formation) during thawing of a loading solution.

10 shows the refreezing start temperature during thawing of the loading solution.

FIG. 11 shows the calories (J / gFW) during thawing of garlic shoots loaded with a loading solution for 40 minutes.

Figure 12 shows the calories (J / gFW) during the thawing process of garlic shoots for each treatment step (pre-culture, loading, dehydration) loaded for 40 minutes in the loading solution C3, C4 and C6.

FIG. 13A shows the preservation (rev) and recovery (LN +) of the chrysanthemum buds before loading (LN−), and FIG.

Claims (2)

A vitrification solution suitable for the droplet-vitrification method, wherein the vitrification solution selected from the table below. Composition (%, w / v) density(%) Glycerol 42.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 17.5 90 Glycerol 37.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 22.5 90 Glycerol 32.5 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 27.5 90 Glycerol 35.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 20.0 85 Glycerol 37.5 + Dimethylsulfoxide 10 + Ethylene Glycol 10 + Sucrose 32.5 90 Glycerol 30.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 25.0 85 Glycerol 30.0 + Dimethylsulfoxide 20 + Ethylene Glycol 20 + Sucrose 15.0 85 Glycerol 40.0 + Dimethylsulfoxide 15 + Ethylene Glycol 15 + Sucrose 15.0 85 Glycerol 50 + Sucrose 40 90 Glycerol 45 + Sucrose 45 90 Glycerol 40 + Sucrose 50 90 Glycerol 40 + Sucrose 40 80 Any loading solution selected from the table below as a cryoprotectant suitable for the droplet-vitrification method. Loading solution composition (M) Loading solution composition
(%, w / v) Creation costs density(%) 1.0M Glycerol + 0.70M Sucrose 9.2 + 23.96 0.38: 1 33.16 2.0M Glycerol + 0.65M Sucrose 18.4 + 22.25 0.83: 1 40.65 1.63M Glycerol + 0.44M Sucrose 15.0 + 15.0 1: 1 30.0 1.9M Glycerol + 0.51M Sucrose 17.5 + 17.5 1: 1 35.0 2.17M Glycerol + 0.58M Sucrose 20.0 + 20.0 1: 1 40.0 2.50M Glycerol + 0.50M Sucrose 23.0 + 17.1 1.3: 1 40.1 2.45M Glycerol + 0.66M Sucrose 22.5 + 22.5 1: 1 45.0

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