CN106613838A - Method for increasing regeneration rate after Arabidopsis thaliana stem tips are preserved at ultra-low temperature - Google Patents

Abstract

The invention belongs to the technical field of refrigeration regeneration, and particularly relates to a method for increasing the regeneration rate after Arabidopsis thaliana stem tips are preserved at ultra-low temperature. The method includes the main steps that the Arabidopsis thaliana stem tips are obtained, loading treatment is carried out, plant microdrop vitrification treatment is carried out, ultra-low temperature preservation is carried out, unfreezing and unloading treatment is carried out, and renewing culturing is carried out. The method compensates for the research blank in ultra-low temperature preservation research about the aspect of cytology changes, meanwhile provides method support for increasing the regeneration rate of the plant stem tips preserved at the ultra-low temperature, and has important meaning for research on an ultra-low temperature preservation technology.

Description

A method to improve the regeneration rate of shoot tips of Arabidopsis thaliana after cryopreservation

technical field

The invention belongs to the technical field of freeze regeneration, and in particular relates to a method for improving the regeneration rate of Arabidopsis shoot tips after cryopreservation.

Background technique

Plant cryopreservation (cryopreservation) is a method of preserving plant material at liquid nitrogen temperature (-196°C). Since all cellular activity is temporarily halted at liquid nitrogen temperatures, plant material can theoretically be preserved indefinitely without modification. Plant shoot apical meristem has the ability to regenerate intact plants, and is the main object of plant cryopreservation. An important bottleneck in the development of plant ultra-low temperature technology is the limited research on the cellular level of the ultra-low temperature treatment process. The main object of the ultra-low temperature treatment is the plant tissue with regenerative ability, such as the isolated shoot tip (meristematic tissue). During the processing, the isolated shoot tips will be subjected to vigorous manual treatments such as pre-cultivation, osmoprotection, dehydration, liquid nitrogen treatment, thawing, rehydration and recovery culture. During the above process, the plant cell membrane system and cytoskeleton will undergo drastic changes and responses. These changes and their recovery are the key to the success of the ultra-low temperature treatment program. However, what is the change mode of the cytoskeleton during the cryopreservation of plant shoot tips, and the relationship between the state of microtubule structure and the regeneration rate of plant shoot tips after cryopreservation is still unclear.

Contents of the invention

In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for improving the regeneration rate of Arabidopsis shoot tips after cryopreservation, aiming at solving the problem of cryopreservation of plant materials. The present invention takes Arabidopsis thaliana shoot tips as the research object, analyzes the microtubule change mode of cells during cryopreservation, and changes the microtubule state by adding the microtubule stabilizer paclitaxel and the drug oryzalin that promotes microtubule depolymerization, and then verifies Effect of microtubule structure on regeneration rate of plants after cryopreservation. The method of the invention fills up the research gap on cytological changes in the cryopreservation technology, and at the same time provides method support for improving the regeneration rate of the plant shoot tip cryopreservation, and is of great significance to the study of the cryopreservation technology.

In order to achieve the above object, the present invention adopts the following technical solutions to achieve.

A method for improving the regeneration rate of Arabidopsis shoot tips after cryopreservation, the method comprising the following steps:

(1) sowing the sterilized Arabidopsis seeds on MS medium, and culturing them under the culture conditions of 22-25°C 12h/12h day and night cycle for one week to obtain Arabidopsis seedlings after low temperature treatment at 4-6°C, and then cut the shoot tip under a dissecting microscope;

(2) Select 90 shoot tips and divide them into three groups on average, with 30 shoot tips in each group. The first group is added with 20 μmol/L paclitaxel and treated for 20 minutes, which is labeled as T group; The oryzalin was treated for 20 minutes, which was marked as group O; the shoot tips of the third group were directly marked as group C.

(3) Loading treatment, placing the shoot tips of the T group, the O group and the C group in the loading solution at the same time, and treating them for 18-25 minutes at 25°C;

(4) plant droplet vitrification treatment, after the loading solution treatment ends, three groups of shoot tips are transferred to the precooled PVS2 solution simultaneously, and are processed on ice for 25-30 minutes;

(5) For ultra-low temperature storage, transfer the PVS2-treated shoot tip to a sterile aluminum foil strip and insert it into liquid nitrogen. When there are no more bubbles, transfer the aluminum foil strip to a cryopreservation tube and place it in liquid nitrogen for 30 minute;

(6) Thawing and unloading treatment, remove the aluminum foil strip from the liquid nitrogen and directly transfer it to the unloading solution, and treat it at 25°C for 20-30 minutes;

(7) Recovery culture, transfer the shoot tip after thawing to the recovery medium for recovery culture, the first 7 days of recovery culture is dark culture; after dark culture, continue to cultivate under light, and regularly count the shoot tip survival of A. thaliana Rate.

Further, the loading solution in the step (3) is MS culture solution containing 2M glycerol and 0.4M sucrose.

Further, in the step (4), the PVS2 solution is MS culture fluid containing 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) DMSO and 0.4M sucrose .

Further, the unloading solution in the step (6) is MS culture solution containing 1.2M sucrose.

Further, the recovery medium in the step (7) is MS solid medium containing 30g/L sucrose, 1.0mg/L BA and 3g/L phytogel.

Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

(1) Analyzing the changes in the microtubule structure of cells during cryopreservation, filling the research gap in cytology during cryopreservation;

(2) Adding a microtubule stabilizer before the loading process is beneficial to improve the regeneration rate of the plant, and improves the existing plant shoot tip cryopreservation technology;

(3) Adding microtubule depolymerization reagents before loading treatment reduced the regeneration rate of plant shoot tips cryopreserved.

Description of drawings

Figure 1 is the effect of microtubule reagents on the regeneration rate of Arabidopsis shoot tips after cryopreservation, wherein: a, regeneration of Arabidopsis shoot tips after cryopreservation; b, plants after regeneration;

Figure 2 is the statistical situation of the regeneration rate of Arabidopsis shoot tips after cryopreservation;

Figure 3 shows the changes in microtubule structure during cryopreservation;

Figure 4 is the effect of microtubule reagents on microtubule structure during cryopreservation;

specific embodiment

The present invention will be described in further detail below in conjunction with specific examples, but the present invention is not limited to these examples.

Example 1

(1) Acquisition of Arabidopsis shoot tip

Arabidopsis thaliana seeds expressing α-tublin-GFP fusion protein were sown on MS medium after being sterilized, treated at a low temperature of 4-6°C, and cultured at 22-25°C for 12h/12h day and night cycle for one week. Arabidopsis thaliana seedlings, and cut the shoot tips under a dissecting microscope;

(2) Droplet vitrification cryopreservation

Get 90 shoot tips and divide them into three groups on average, with 30 shoot tips in each group. The first group was added with a microtubule fixative containing 20 μmol/L paclitaxel (the microtubule fixative contained 50mmol/L Pipes, 2mmol/L EGTA, 2mmol /L 0.05% MgSO4) for 20 minutes, marked as T group; the second group added the microtube fixative containing 20μmol/L oryzalin (microtube fixative contains 50mmol/L Pipes, 2mmol/L EGTA, 2mmol /L 0.05% MgSO4) for 20 minutes, marked as O group; the shoot tips of the third group were directly marked as C group, after the T group and O group were processed, three groups of shoot tips were processed simultaneously.

Transfer the shoot tips of the three groups into cryopreservation tubes, add 1 mL of loading solution, and treat at 25°C for 20 minutes. The loading solution was MS broth containing 2M glycerol and 0.4M sucrose. After the loading solution treatment was completed, the loading solution was replaced with 1 mL of ice-cooled PVS2 solution and treated on ice for 25-30 minutes. PVS2 solution is MS broth containing 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) DMSO and 0.4M sucrose. 1 min before the end of PVS2 treatment, transfer shoot tips and a drop of PVS2 solution (15 μL) onto an 8 × 25 mm strip of sterile aluminum foil. After the PVS2 treatment, use a pair of fine tweezers to insert the aluminum foil strip directly into the liquid nitrogen, and when there are no more bubbles (about 5-20 seconds), transfer the aluminum foil strip to the cryopreservation tube placed in the liquid nitrogen , and kept in liquid nitrogen for 30 min. To thaw, remove the aluminum foil strips from the liquid nitrogen and quickly insert into 10 mL of unloading solution in a 6 cm diameter Petri dish and process at 25 °C for 20 min. The unloading solution was MS broth containing 1.2M sucrose. After the end of the unloading solution treatment, the shoot tips were transferred to recovery medium. The recovery medium was MS medium containing 30g/L sucrose, 1.0mg/L BA and 3g/L phytogel. The first 7 days of recovery culture were dark culture. After dark culture, continue to culture under light, and count the survival rate regularly.

(3) Changes in microtubule structure during droplet vitrification cryopreservation and the effect of microtubule reagents on microtubule structure during cryopreservation

The GFP (green fluorescent protein) signal of the Arabidopsis shoot tip was detected by laser scanning confocal microscopy, and then the microtubule structure (488nm excitation light and 520nm emission light) of the Arabidopsis shoot tip under different treatment conditions was observed.

The sampling points for microscopic inspection are arranged as follows:

Group C: before loading treatment, after loading treatment, after PVS2 treatment, and after unloading treatment, a total of four detection points;

Group T: after loading treatment, after PVS2 treatment, after unloading treatment, a total of three sampling points;

Group O: after loading treatment, after PVS2 treatment, after unloading treatment, a total of three sampling points;

10 shoot tips for each sampling point were placed on a glass slide for microscopic examination.

In the present invention, the effects of pretreatment of shoot tips with the microtubule stabilizer Paclitaxel and the drug Oryzalin, which promotes microtubule depolymerization, for 20 minutes before the loading treatment in the droplet vitrification approach were tested. . The results showed that the corresponding regeneration rates of group T, group O and group C were 77.2%, 32.3% and 39.5% respectively (see Fig. 1 and Fig. 2). It can be seen that compared with group C, the addition of paclitaxel, a microtubule stabilizer, in group T is beneficial to improve the survival rate and regeneration rate of shoot tips in cryopreservation, while the addition of oryzalin, a drug that promotes microtubule depolymerization in group O, reduces the rate of cryopreservation. Shoot tip survival and regeneration. During cryopreservation, microtubules were completely depolymerized after loading treatment and PVS2 solution treatment, and microtubules began to aggregate again after recovery (see Figure 3); during cryopreservation, microtubule stabilizers were added before loading treatment The depolymerization of microtubules after paclitaxel was significantly relieved compared with that without paclitaxel; during cryopreservation, the depolymerization of microtubules after adding oryzalin, a reagent that promotes microtubule depolymerization before loading treatment, was worse than that without oryzalin The treatment is obviously more severe (refer to Figure 4).

The above is only a preferred case of the present invention, and does not make any limitation to the present invention. Any simple modification, change, and imitation made to the above implementation cases according to the technical content of the present invention belong to the protection of the technical solution of the present invention. scope.

Claims (5)

1. it is a kind of improve arabidopsis stem apex Excised Embryos after regeneration rate method, it is characterised in that the method include following step Suddenly：

(1) the arabidopsis seed disinfected is sowed on MS culture mediums, at 22-25 DEG C after Jing 4-6 DEG C K cryogenic treatments Cultivate one week under the condition of culture of 12h/12h daily cycles, subsequently cut stem apex under anatomical lens；

(2) 90 stem apexs are chosen, is divided into three groups, per group of 30 stem apexs, the taxol of first group of addition, 20 μm of ol/L is simultaneously located Reason 20 minutes, is labeled as T groups；The oryzalin oryzalin of second group of addition, 20 μm of ol/L is processed 20 minutes, is labeled as O groups；The Three groups of stem apex is directly labeled as C groups.

(3) loading processing, T groups, O groups and C groups stem apex are placed in loading solution simultaneously, and 18-25 minutes are processed at 25 DEG C；

(4) plant droplet vitrifying is processed, and after loading solution process terminates, three groups of stem apexs is proceeded into the PVS2 of precooling simultaneously Solution, 25-30 minutes are processed on ice；

(5) Excised Embryos, the stem apex processed through PVS2 is transferred on sterile aluminum foil bar and is inserted in liquid nitrogen, waits there is no longer Aluminum foil strip is proceeded into cryopreservation tube when bubble is produced, and is placed on holding 30 minutes in liquid nitrogen；

(6) thaw and unloading is processed, aluminum foil strip is removed and directly proceeded in unloading solution from liquid nitrogen, and process at 25 DEG C 20-30 minutes；

(7) renewal cultivation, the stem apex after defrosting is transferred on recovery media carries out renewal cultivation, first 7 days of renewal cultivation For light culture；After light culture, continuation is cultivated under light illumination, and periodic statistical south mustard stem apex survival rate.

2. it is according to claim 1 improve arabidopsis stem apex Excised Embryos after regeneration rate method, it is characterised in that institute It is the MS nutrient solutions containing 2M glycerine and 0.4M sucrose to state loading solution in step (3).

3. it is according to claim 1 improve arabidopsis stem apex Excised Embryos after regeneration rate method, it is characterised in that institute It is containing 30% (w/v) glycerine, 15% (w/v) ethylene glycol, 15% (w/v) DMSO and 0.4M sugarcanes to state PVS2 solution in step (4) The MS nutrient solutions of sugar.

4. it is according to claim 1 improve arabidopsis stem apex Excised Embryos after regeneration rate method, it is characterised in that institute It is the MS nutrient solutions containing 1.2M sucrose to state unloading solution in step (6).

5. it is according to claim 1 improve arabidopsis stem apex Excised Embryos after regeneration rate method, it is characterised in that institute It is the MS solid mediums containing 30g/L sucrose, 1.0mg/L BA and 3g/L plant gels to state recovery media in step (7).