CN104255706A - Method for optimizing vitrification ultra-low temperature preservation effect of arabidopsis seedlings - Google Patents

Abstract

The invention discloses a method for optimizing a vitrification ultra-low temperature preservation effect of arabidopsis seedlings. According to the invention, the arabidopsis seedlings are processed by adopting a vitrification solution which contains a carbon nanometer material so as to improve the preservation effect of the arabidopsis seedlings. The method specifically comprises the steps of loading liquid treatment, vitrification solution treatment and liquid nitrogen preservation, wherein the vitrification solution is the MS culture solution containing 0.1-0.5 g/L of grapheme quantum dots. The method disclosed by the invention optimizes the preservation effect of the arabidopsis seedlings remarkably; as the grapheme quantum dots are added to serve as an allogenic material, the vitrification ultra-low temperature preservation of plants is facilitated.

Description

A method to optimize the cryopreservation effect of vitrification of Arabidopsis seedlings

technical field

The invention relates to the field of preservation of plants or parts thereof, in particular to a method for optimizing the effect of vitrification cryopreservation of Arabidopsis seedlings.

Background technique

Cryopreservation is a modern in vitro preservation technology for germplasm resources developed in the 1970s. Usually stored in liquid nitrogen, the substance metabolism and growth activities in the preserved material cells are almost completely stopped, and they are in a relatively stable biological state, achieving the purpose of long-term preservation of germplasm. Ultra-low temperature preservation is currently the only method that does not require continuous subculture. Medium and long-term preservation method. Vitrification cryopreservation is to place cells or tissues in a vitrification solution composed of a certain proportion of permeable and non-permeable protective agents, so that the material and its vitrification solution solidify into an amorphous state at a sufficiently fast cooling rate. Vitrified state, and preserved at low temperature in this glassy state. The vitrification method has the advantages of simple and fast operation, low cost, wide variety of suitable preservation materials, stable heredity of preservation materials, and good preservation effect. It has been the preferred method for medium and long-term preservation of high-quality germplasm resources in the past ten years.

In the method of vitrification cryopreservation of plants and their tissues, when some preservation methods are used, the survival rate of plants and their tissues is not high or can not survive at all after recovery culture; in addition, when the same preservation method is used, different plants and their tissues Whether they can survive after recovery and the survival rate are not the same. Adding certain exogenous substances in the prior art is beneficial to improve the survival rate of cryopreservation, but it is unknown which exogenous substances to choose for specific plants and tissues to improve their preservation effect.

Arabidopsis thaliana belongs to the angiosperm phylum and dicotyledonous plant class. It is a biennial herb. The advantage of Arabidopsis thaliana as a model plant is that the plant is small and has many seeds. It is also a self-pollinating plant with a high degree of gene homozygosity. In the field of cryopreservation, Arabidopsis has an important position and research significance. Arabidopsis thaliana seedlings are very good research materials. It has been found in existing studies that Arabidopsis thaliana seedlings germinated for 60 hours can screen the effects of different exogenous additives on the effect of cryopreservation, and can also be used as preservation materials to study the effects of cryopreservation during cryopreservation. It is a very important material for physiology and molecular biology research.

Contents of the invention

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a method for optimizing the effect of vitrification cryopreservation of Arabidopsis seedlings, so as to overcome the shortcoming of the low recovery growth rate of Arabidopsis seedlings cryopreservation in the prior art, And optimized the physiological and biochemical response research of plant vitrification cryopreservation.

In order to achieve the above objects or other objects, the present invention is achieved through the following technical solutions.

A method for optimizing the effect of vitrification cryopreservation of Arabidopsis thaliana seedlings. The method of vitrification cryopreservation is used to preserve Arabidopsis thaliana seedlings. The specific steps are as follows:

1) Loading solution treatment: soak Arabidopsis seedlings in the loading solution for 20-30 minutes at room temperature, and then remove the loading solution;

2) Vitrification solution treatment: immerse Arabidopsis thaliana seedlings in vitrification solution at 0-25°C for 40-60 minutes;

3) liquid nitrogen storage: keep Arabidopsis seedlings soaked in the vitrification solution, and place them in liquid nitrogen for storage;

The vitrification solution is a vitrification cryoprotective solution containing 0.1-0.5 g/L graphene quantum dots.

The MS culture solution in the present invention contains 1900mg/L KNO ₃ , 1650mg/L NH ₄ NO ₃ , 170mg/L KH ₂ PO ₄ , 370mg/L MgSO ₄ 7H ₂ O, 440mg/L CaCl ₂ 2H ₂ O , 37.3mg/L Na ₂ -EDTA, 27.8mg/L FeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride , 2mg/L Glycine, 0.83mg/L KI, 6.2mg/L H ₃ BO ₃ , 22.3mg/L MnSO ₄ ·4H ₂ O, 8.6mg/LZnSO ₄ ·7H ₂ O, 0.25mg/L Na ₂ MoO ₄ · 2H ₂ O, 0.025mg/L CuSO ₄ ·5H ₂ O, 0.025mg/LCoCl ₂ ·6H ₂ O, the balance is water. The pH of the MS culture solution is 5.8.

Preferably, the Arabidopsis seedlings are Arabidopsis seedlings cultivated on MS solid medium, and the method of cultivating on MS medium is as follows: Arabidopsis seeds are placed on MS solid medium, and first carried out every day Light culture for 8-12 hours, light intensity 100-200 μmol/m ² , temperature 20-27°C, dark culture for the rest of light culture every day, dark culture temperature 20-25°C, light culture and dark culture every day cycle.

The MS solid medium in the present invention contains 1900mg/L KNO ₃ , 1650mg/L NH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/L MgSO ₄ 7H ₂ O, 440mg/L CaCl ₂ 2H ₂ O , 37.3mg/L Na ₂ -EDTA, 27.8mg/L FeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride , 2mg/L Glycine, 0.83mg/L KI, 6.2mg/L H ₃ BO ₃ , 22.3mg/L MnSO ₄ 4H ₂ O, 8.6mg/L ZnSO ₄ 7H ₂ O, 0.25mg/L Na ₂ MoO ₄ · 2H ₂ O, 0.025 mg/L CuSO ₄ · 5H ₂ O, 0.025 mg/L CoCl ₂ · 6H ₂ O, 30 g/L sucrose, 10 g/L agar powder, the balance is water. The pH of the MS solid medium is 5.8.

Preferably, the time for culturing the Arabidopsis seeds on the pre-medium is 24-72 hours.

More preferably, the time for culturing the Arabidopsis seeds on the pre-medium is 60 hours.

The method for culturing Arabidopsis thaliana seeds on the MS medium in the present invention is to adopt the form of light culture and dark culture alternately every day until reaching 60h. For example, 8 hours of light culture is carried out every day, and then 16 hours of dark culture is carried out, and this cycle is repeated until the third day for another 12 hours, and the 12 hours of the third day is light culture.

Preferably, Arabidopsis thaliana seedlings are soaked in the loading solution for 20 minutes at room temperature, and then the loading solution is removed.

Preferably, Arabidopsis seedlings are soaked in a vitrification solution at 0°C for 50 minutes.

More preferably, the method for culturing the Arabidopsis seedlings on MS medium is as follows: 8 hours of light cultivation at a light intensity of 100-200 μmol/m ² at a temperature of 25°C; followed by 16 hours of dark cultivation at a temperature of 20 ℃. More preferably, the light intensity is 150 μmol/m ² .

Preferably, the Arabidopsis seeds are Arabidopsis seeds that have been subjected to vernalization treatment, and the vernalization treatment is placing the Arabidopsis seeds at 0-10° C. for 24-72 hours.

Preferably, the vernalization treatment is placing the Arabidopsis seeds at 4-6° C. for 40-50 hours.

More preferably, the vernalization treatment is placing the Arabidopsis seeds at 4° C. for 48 hours.

Preferably, the Arabidopsis seeds are disinfected before the vernalization treatment, and the disinfection treatment process is as follows: soak the Arabidopsis seeds in 65-75% ethanol for 10-30s, then rinse them with sterile water, and then use Soak in an aqueous solution of 15-25 wt% NaClO and 0-0.03 wt% Tween 20 for 5-15 minutes, and rinse with sterile water. The 65-75% ethanol mentioned in the present invention refers to the aqueous solution of ethanol, wherein the percentage content is the volume percentage content.

More preferably, the disinfection treatment process is: disinfect the Arabidopsis seeds with 70% ethanol for 15 seconds, rinse them with sterile water for 4-6 times, and then use 15-25wt% NaClO and 0-0.03wt% Tween 20 water solution for 10 minutes, and rinse with sterile water 5 to 6 times.

More preferably, the disinfection treatment process is: disinfect the Arabidopsis seeds with 70% ethanol for 15 seconds, rinse them with sterile water 4 times, and then soak them in an aqueous solution containing 20 wt% NaClO and 0.01 wt% Tween 20 for 10 minutes , and rinsed 6 times with sterile water.

Preferably, the loading liquid is MS culture liquid containing 1-2 mol/L glycerol and 0.3-0.5 mol/L sucrose.

Preferably, the Arabidopsis thaliana seeds are Colombian ecotype Arabidopsis seeds such as Arabidopsis thaliana ecotype Col-0.

Preferably, the vitrification solution contains 300g/L glycerol, 150g/L ethylene glycol, 150g/L dimethyl sulfoxide, 0.4mol/L sucrose and 0.1-0.5g/L graphene quantum dots MS medium.

More preferably, the vitrification solution contains 300g/L glycerol, 150g/L ethylene glycol, 150g/L dimethyl sulfoxide, 0.4mol/L sucrose and 0.1-0.3g/L graphene quantum dots MS culture medium.

More preferably, the vitrification solution is MS containing 300g/L glycerol, 150g/L ethylene glycol, 150g/L dimethyl sulfoxide, 0.4mol/L sucrose and 0.1g/L graphene quantum dots Culture fluid, or described vitrification solution is the MS that contains 300g/L glycerol, 150g/L ethylene glycol, 150g/L dimethyl sulfoxide, 0.4mol/L sucrose and 0.3g/L graphene quantum dot culture medium.

The invention also discloses a method for thawing and recultivating Arabidopsis seedlings, wherein the Arabidopsis seedlings are preserved by the method described above, and the method for thawing and recultivating Arabidopsis seedlings is as follows: Arabidopsis thaliana seedlings were taken out of liquid nitrogen, thawed in a water bath, then removed from the vitrification solution, washed with washing solution, and finally transferred to MS solid medium for recovery.

Preferably, the condition for thawing in a water bath is to thaw in a water bath at 30-40°C for 60-120 seconds, and the process of washing with washing liquid is as follows: soak in washing liquid for 30-50 minutes at room temperature, and change it every 5-10 minutes Washing liquid, the washing liquid is MS culture liquid containing 1.0-1.5 mol/L sucrose.

More preferably, the condition for thawing in a water bath is to thaw in a water bath at 40° C. for 90 seconds, and the process of washing with a washing solution is as follows: soak in the washing solution for 40 minutes at room temperature, and change the washing solution every 10 minutes. It is MS culture fluid containing 1.2mol/L sucrose.

More preferably, a shaking-accelerated thawing solution is used for thawing in a water bath.

Preferably, the re-cultivation method is to cultivate Arabidopsis seedlings under light for 8-12 hours every day, the light intensity is 100-200 μmol/m ² , the culture temperature is 20-27°C, and the rest of the day is cultivated in the dark. It is 20-25°C.

More preferably, the recovery culture process is to cultivate Arabidopsis seedlings under light for 8 hours every day, the light intensity is 150 μmol/m ² , the culture temperature is 25°C, and the rest of the day is dark culture, and the dark culture temperature is 20°C

More preferably, in order to prove the preservation effect of the method in the present invention, the recovery growth rate is used for statistics, specifically, the number of surviving Arabidopsis seedlings is counted on the 15th day of the recovery culture, and the recovery growth rate is calculated, and the recovery Growth rate: number of surviving seedlings/total number of seedlings×100%.

According to the principles of nanoscience, adding nanomaterials to the cryoprotectant can effectively increase the viscosity and thermal conductivity of the cryoprotectant, change the formation of ice crystals, and reduce the damage to cells. The present invention treats Arabidopsis thaliana seedlings, especially seedlings of Arabidopsis thaliana seeds germinated for 60 hours in the ultra-low temperature preservation of vitrification method, firstly treats them with loading solution, then uses vitrification solution, and finally stores them in ultra-low temperature in liquid nitrogen, wherein the vitrification solution In addition, the addition of graphene quantum dots to the vitrification solution, the addition of this exogenous substance and other processes in the method, can effectively improve the preservation effect of Arabidopsis seedlings. The method disclosed in the present invention is significantly optimized for the preservation effect of Arabidopsis thaliana seedlings, and the addition of graphene quantum dots as exogenous substances can promote the ultra-low temperature preservation of plant vitrification.

Description of drawings

Fig. 1 is the photo of recovery growth of Arabidopsis seedlings in the experimental group and the control group after 60h cryopreservation.

The left column in Fig. 1 is the picture of recovery growth of 60h Arabidopsis seedling cryopreservation in the control group;

The right column in Fig. 1 is the photo of 60h Arabidopsis seedling cryopreservation recovery growth in the experimental group;

The contents of graphene quantum dots added in the vitrification solution from the first row to the third row in Fig. 1 are respectively 0.1g/L, 0.3g/L and 0.5g/L.

Detailed ways

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

The Arabidopsis thaliana seeds used in the examples of the present invention are Arabidopsis thaliana ecotype seeds, Arabidopsis thalianaecotype Col-0 type.

The formula of experimental reagent in the embodiment of the present invention is as follows:

1) MS culture medium: MS culture medium contains 1900mg/L KNO ₃ , 1650mg/L NH ₄ NO ₃ , 170mg/L KH ₂ PO ₄ , 370mg/L MgSO ₄ 7H ₂ O, 440mg/L CaCl ₂ 2H ₂ O, 37.3mg/L Na ₂ -EDTA, 27.8mg/L FeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L hydrochloric acid Thiamine, 2mg/L Glycine, 0.83mg/L KI, 6.2mg/L H ₃ BO ₃ , 22.3mg/L MnSO ₄ 4H ₂ O, 8.6mg/L ZnSO ₄ 7H ₂ O, 0.25mg/LNa ₂ MoO ₄ ·2H ₂ O, 0.025 mg/L CuSO ₄ ·5H ₂ O, 0.025 mg/L CoCl ₂ ·6H ₂ O, the balance is water, and the pH of the MS culture solution is 5.8.

2) MS solid medium: MS solid medium contains 1900mg/L KNO ₃ , 1650mg/L NH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/L MgSO ₄ 7H ₂ O, 440mg/L CaCl ₂ . 2H ₂ O, 37.3mg/L Na ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L hydrochloric acid Thiamine, 2mg/L Glycine, 0.83mg/L KI, 6.2mg/L H ₃ BO ₃ , 22.3mg/L MnSO ₄ 4H ₂ O, 8.6mg/L ZnSO ₄ 7H ₂ O, 0.25mg/L Na ₂ MoO ₄ ·2H ₂ O, 0.025mg/L CuSO ₄ ·5H ₂ O, 0.025mg/L CoCl ₂ ·6H ₂ O, 30g/L sucrose, 10g/L agar powder, the balance is water, the MS solid culture The pH of the base is 5.8.

3) The loading solution is: MS culture solution containing 2mol/L glycerol and 0.4mol/L sucrose.

4) The vitrification solution is: MS culture containing 300g/L glycerol, 150g/L ethylene glycol, 150g/L dimethyl sulfoxide, 0.4mol/L sucrose and 0.1-0.5g/L graphene quantum dots liquid.

5) The washing solution is MS culture solution containing 1.2mol/L sucrose.

Example 1

1) Acquisition of Arabidopsis seedlings: Disinfect Arabidopsis seeds with 70% ethanol for 15 seconds, rinse them with sterile water for 4 times, then disinfect them with an aqueous solution containing 20 wt% NaClO and 0.01 wt% Tween 20 for 10 min, rinse them with sterile water 6 times; then the sterilized seeds were vernalized at 4°C for 48 hours, and cultured on MS solid medium under the conditions of light culture for 8 hours per day, light intensity of 150 μmol/m ² , and culture temperature of 25°C; then dark culture for 16 hours, The culture temperature was 20° C., and the seeds were cultured for 60 hours in total to obtain Arabidopsis thaliana seedlings with a seedling age of 60 hours.

2) Divide the 60h Arabidopsis seedlings into the experimental group and the control group, and set up 3 parallel experiments in each group, put 50 seedlings into a 2mL cryovial containing 1mL loading solution, and treat them at room temperature for 20min; Remove by suction, add vitrification solution, and treat at 0°C for 50 minutes; put the cryovial into liquid nitrogen and store for 1 hour.

The vitrification solutions of the experimental groups contained 0.1g/L, 0.3g/L, and 0.5g/L graphene quantum dots respectively.

The vitrification solution of the experimental group 1 contains 0.1g/L graphene quantum dots; the control group 1 does not contain graphene quantum dots, and the others are the same as the experimental group 1;

The vitrification solution of the experimental group 2 contains 0.3g/L graphene quantum dots; the control group 2 does not contain graphene quantum dots, and the others are the same as the experimental group 2;

The vitrification solution of the experimental group 3 contained 0.5g/L graphene quantum dots; the control group 3 did not contain graphene quantum dots, and the others were the same as the experimental group 3.

3) After storing the cryovial in liquid nitrogen for 1 hour, take out the cryovial, quickly put it into a 40°C water bath, thaw it for 90 seconds, and shake it gently from time to time; absorb the vitrification solution, add washing solution, and treat it at room temperature for 40 minutes. Change the washing solution every 10 minutes; move the washed seedlings to MS solid medium, and put them into a plant incubator. The parameters of the incubator are the same as the seed germination conditions, that is, 8 hours of light cultivation per day, light intensity of 150 μmol/m ² , and cultivation temperature 25°C; then cultivated in the dark for 16 hours at a temperature of 20°C; on the 15th day of recovery of the Arabidopsis seedlings, calculate and compare the recovery growth rate of the Arabidopsis seedlings in the experimental group and the control group within 60 hours.

See Table 1 for the recovery growth rate of Arabidopsis thaliana seedlings in the experimental group and the control group.

Table 1

3. Experimental results

As can be seen from Table 1, graphene quantum dots with a concentration of 0.1g/L, 0.3g/L, and 0.5g/L were added to the vitrification solution of Arabidopsis seedlings germinated for 60h in cryogenic storage, and the 60h Arabidopsis seedlings were respectively made The recovery growth rate increased from 25.54% to 38.97%, 46.80% and 41.52%, and the improvement effect of graphene quantum dots with a concentration of 0.3g/L was the most significant, as shown in Figure 1 for details.

Example 2

This example is mainly to test and analyze the parameters of Arabidopsis thaliana seedlings at various stages of cryopreservation, so as to demonstrate the regulation of exogenous graphene quantum dots on membrane damage and oxidative stress during 60h cryopreservation of Arabidopsis seedlings.

1) Experimental material: the material in group 1 is: the seedling of Arabidopsis thaliana germinated according to the method described in Example 1 of the present invention for 60h

Materials in group 2 are: seedlings treated with vitrification solution in Example 1;

Group 3 is: the seedlings after thawing after vitrification cryopreservation according to the method in Example 1;

Group 4 is the seedling after washing with washing liquid in embodiment 1;

Group 5 is the seedlings after recovery culture treatment in Example 1.

1) Experimental materials: Arabidopsis thaliana seedlings germinated for 60 hours and seedlings after the key steps of dehydration, thawing, washing, and recovery culture in the cryopreservation process.

2) Experimental method:

1. Determination of relative conductivity during cryopreservation of Arabidopsis thaliana seedlings for 60 hours

Arabidopsis thaliana seedlings treated in the above 5 stages were taken and repeated 3 times. Rinse several times with double distilled water, then dry the surface moisture with filter paper, put it into a 50mL beaker, add 50mL double distilled water and soak at 25°C for 2h, and measure its conductance R with a Thermo Orion FE30 conductivity meter. After the measurement, seal each beaker with a plastic film, put it in a boiling water bath and boil for 15 minutes, take it out and cool it to 25°C, shake it well, measure the conductivity value R ₀ , and calculate the relative conductivity according to the following formula:

2. MDA determination during cryopreservation of Arabidopsis thaliana seedlings for 60 hours

Weigh 0.2g of the material and put it into a pre-cooled mortar, add 5mL of 10% trichloroacetic acid (TCA) to grind until homogenized, pour it into a 10mL centrifuge tube and centrifuge at 5000rpm for 10min, and the supernatant is the MDA test solution. Draw 2 mL of the supernatant and add 2 mL of 0.6% thiobarbituric acid (TBA) solution, mix well, react in a boiling water bath for 30 min, rapidly cool and centrifuge at 5000 rpm for 10 min, and measure colorimetrically at 600 nm, 532 nm, and 450 nm wavelengths respectively. Adjust to zero with 2 mL of distilled water.

The concentration of MDA in the extract C ( _μ moL/L) = 6.45 (OD ₅₃₂ -OD ₆₀₀ ) - 0.56OD ₄₅₀ , converted into the content of MDA per gram of material:

3. Determination of hydrogen peroxide content during cryopreservation of Arabidopsis thaliana seedlings for 60 hours

According to the instructions of Nanjing Jiancheng Hydrogen Peroxide Determination Kit.

3) Experimental results

The regulation of exogenous graphene quantum dots on cell membrane damage, membrane lipid peroxidation and oxidative stress during cryopreservation of Arabidopsis thaliana seedlings for 60 h was studied by relative conductivity, malondialdehyde and hydrogen peroxide content. The experimental results show that the addition of 0.3g/L graphene quantum dot material significantly reduces the degree of cell membrane damage during cryopreservation, effectively reduces the content of hydrogen peroxide, thereby weakening the degree of membrane lipid peroxidation. The protective effect of 0.3g/L graphene quantum dots on 60h Arabidopsis seedlings during cryopreservation was confirmed from the perspectives of physiology, biochemistry and cytology. The specific results are shown in Table 2, Table 3 and Table 4. Wherein the control group in Table 2, Table 3 and Table 4 is the control group in Example 1, and the experimental group is the experimental group added with 0.3g/L graphene quantum dots in Example 1.

Table 2 Changes in relative conductivity between the control group and the experimental group

Relative conductivity (%) group 1 group 2 group 3 group 4 Group 5 control group 29.78 80.09 86.78 84.23 83.72 test group 29.78 74.32 79.89 77.14 76.56

Table 3 Changes in MDA content between the control group and the experimental group

Table 4 Changes in the content of hydrogen peroxide between the control group and the experimental group

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (10)

1. optimize a method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect, it is characterized in that, the method adopting vitrification ultra-low temperature to preserve is preserved Arabidopsis thaliana Seedlings, and concrete steps are as follows:

1) load liquid process: under room temperature, Arabidopsis thaliana Seedlings immersion treatment in loading liquid was removed loading liquid after 20 ~ 30 minutes;

2) vitrification solution process: use vitrification solution immersion treatment Arabidopsis thaliana Seedlings 40 ~ 60 minutes at 0 ~ 25 DEG C;

3) Liquid nitrogen storage: keep Arabidopsis thaliana Seedlings to be immersed in state in vitrification solution, and be placed in liquid nitrogen and preserve;

Described vitrification solution is the glass freezing protection liquid containing 0.1 ~ 0.5g/L graphene quantum dot.

2. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 1, it is characterized in that, described Arabidopsis thaliana Seedlings is the Arabidopsis thaliana Seedlings after cultivating on MS solid culture medium, the method that MS solid culture medium is cultivated is: be placed in by arabidopsis seed on MS solid culture medium, every day first carries out illumination cultivation 8 ~ 12h, light intensity 100 ~ 200 μm of ol/m ², temperature 20 ~ 27 DEG C, every day, illumination cultivation carried out dark culturing remaining time, and dark culturing temperature is 20 ~ 25 DEG C, circulation illumination cultivation every day and dark culturing.

3. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 2, it is characterized in that, described arabidopsis seed is the arabidopsis seed first crossed through vernalization, and described vernalization is that described arabidopsis seed is placed 24 ~ 72h at 0 ~ 10 DEG C.

4. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 3, it is characterized in that, described vernalization is that described arabidopsis seed is placed 40 ~ 50h at 4 ~ 6 DEG C.

5. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 3, it is characterized in that, described arabidopsis seed is first sterile-processed before vernalization, disinfecting technique is: by arabidopsis seed 65 ~ 75% alcohol immersion 10 ~ 30s, re-use aseptic water washing, then use the aqueous solution soaking 5 ~ 15 minutes containing the NaClO of 15 ~ 25wt% and the polysorbas20 of 0 ~ 0.03wt%, and use aseptic water washing.

6. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 1, it is characterized in that, described loading liquid is the MS culture fluid containing 1 ~ 2mol/L glycerine and 0.3 ~ 0.5mol/L sucrose.

7. optimize the method for Arabidopsis thaliana Seedlings vitrification ultra-low temperature preservation effect as claimed in claim 1, it is characterized in that, described vitrification solution is for containing 300g/L glycerine, 150g/L ethylene glycol, the MS culture fluid of 150g/L dimethyl sulfoxide (DMSO), 0.4mol/L sucrose and 0.1 ~ 0.5g/L graphene quantum dot.

8. the thawing and cultural method again an of Arabidopsis thaliana Seedlings, described Arabidopsis thaliana Seedlings is for adopting the Arabidopsis thaliana Seedlings that as described in claim as arbitrary in claim 1 ~ 7, method is preserved, described Arabidopsis thaliana Seedlings thaw and again cultural method for Arabidopsis thaliana Seedlings is taken out from liquid nitrogen, first water-bath is thawed, then wash with cleaning solution after removing vitrification solution, finally proceed to renewal cultivation in MS solid culture medium.

9. thaw as claimed in claim 8 and cultural method again, it is characterized in that, the condition that water-bath is thawed is the 60 ~ 120s that thaws in the water-bath of 30 ~ 40 DEG C, by the technique of cleaning solution washing be: at room temperature soak 30 ~ 50 minutes with cleaning solution, and changing once washing liquid every 5 ~ 10 minutes, described cleaning solution is the MS culture fluid containing 1.0 ~ 1.5mol/L sucrose.

10. thaw as claimed in claim 8 and cultural method again, it is characterized in that, described cultural method is again by Arabidopsis thaliana Seedlings every day first illumination cultivation 8 ~ 12h, light intensity 100 ~ 200 μm of ol/m ², cultivation temperature is 20 ~ 27 DEG C, and in every day, remaining time adopts dark culturing, and dark culturing temperature is 20 ~ 25 DEG C.