CN109752226B - Method for quickly quantifying pollen activity at normal temperature or high temperature and application - Google Patents

Abstract

The invention belongs to the field of application of plant physiological and biochemical technology. In particular to a method for quickly quantifying pollen activity at normal temperature or high temperature and application thereof. The formula, dyeing concentration, optimal dyeing time and application range of the TTC dyeing solution for the pollen at normal temperature and high temperature are determined, and the formula of the combined dyeing solution is prepared as follows: 5.23g of 1mol/L potassium dihydrogen phosphate and 14.03g of dipotassium hydrogen phosphate, and the pH value of the potassium phosphate buffer is 7; 8g of 2,3, 5-triphenyltetrazolium chloride (TTC) was dissolved in the obtained potassium phosphate buffer solution, and the volume was adjusted to 1L with distilled water to a final concentration of 8 g/L. The method can be used for quantitatively detecting the pollen activity of a large batch of materials, omits microscopic examination operation after dyeing, simplifies the flow of identifying the pollen activity of the large batch of materials, and is suitable for identifying the pollen activity of various crops such as cotton, rice and the like.

Description

Method for quickly quantifying pollen activity at normal temperature or high temperature and application

Technical Field

The invention belongs to the field of application of plant physiological and biochemical technology. In particular to a method for quickly quantifying pollen activity at normal temperature or high temperature and a scheme for applying the method to carry out quantification by dyeing and extracting through a dyeing solution and measuring absorbance. The method greatly simplifies the operation process on one hand, and can accurately quantify the vitality of the pollen on the other hand.

Background

As global climate warms, high temperature stress has become one of the major factors affecting crop production. Plant organ development is very sensitive to changes in environmental temperature, and high temperatures can cause abnormal development of reproductive organs. Male reproductive organs are more sensitive to environmental temperature changes than female reproductive organs, particularly stamens, and extreme temperatures during development can cause dysplasia of the stamens or male gametes (pollen) to cause male sterility. There are many reports on male sterility caused by high temperature stress, such as barley, Arabidopsis thaliana, rice and cotton, of which abortion due to high temperature influence on pollen activity accounts for the most. Pollen activity is one of the key factors determining whether a plant female organ functions to be normally fertilized, and is directly related to whether the plant can produce seeds or viable seeds.

The effect of high temperature on pollen activity varies with the time of stressThere are differences, in the review article by levodandan et al, several pollen viability staining methods commonly used at the present stage are summarized, including I₂KI, fluorescent dye double staining method, acid magenta staining method, and the like. The principle of these detection methods is basically that a series of active contents in the pollen are stained by using a dye, microscopic examination is carried out, and then the fertility and sterility are further determined by counting the number of fertile pollen and sterile pollen in a visual field.

Another detection method is to perform pollen tube germination test, place the pollen tube on the germination culture medium, induce pollen tube germination, and identify sterility by counting the number of germinated pollen and the number of ungerminated pollen.

The methods have some defects, such as qualitative activity, quantitative activity, large-batch sample identification and analysis, large influence of artificial subjective factors on counting, and the like. Therefore, developing a simple and accurate qualitative and quantitative scheme of pollen viability plays an important role in accurately evaluating pollen viability.

The 2,3, 5-triphenyltetrazolium chloride (TTC) dyeing method is a commonly used pollen activity identification method at present, and the TTC is an oxidation-reduction pigment and is a colorless solution when dissolved in water. TTC can enter plant cells and is reduced by dehydrogenase in the plant cells to generate water-insoluble tritylhydrazone (TTF), and the TTF is stable at room temperature, is not easy to be oxidized and has little toxicity to the cells, so the TTC is often used as a coloring agent for identifying plant tissues and cells, including pollen activity.

TTF can be generated in the pollen after the TTC enters the pollen cell and is reduced by dehydrogenase, and the TTF in the pollen can not penetrate through a cell membrane to enter a stain, so that the stained pollen can be subjected to microscopic examination under a microscope to count the activity of the pollen. Viable pollen can be stained red and easily identified under a microscope, but the viability of each pollen is different and the ability to produce TTF is also different. Among the pollens tested, there are many moderately active pollens. Under a microscope, such pollen activity is very difficult to distinguish with the human eye. Meanwhile, due to microscopic examination, the pollen activity statistical result in the selected field of view cannot be guaranteed to accurately represent the pollen activity of the material, and time and labor are wasted when performing activity statistics on a large batch of materials. More problems derive from this, leading to the widespread use of TTC staining in pollen activity identification.

The TTC staining quantitative method is based on a microscopic examination method after the TTC staining of pollen. By optimizing the protocol, the phosphate (preferably potassium phosphate) buffer used in the present invention allows staining of TTC pollen at ambient temperature and further determines the optimal staining duration. Meanwhile, by comparing the pollen quantification with the anther quantification, the invention determines that the pollen activity can be accurately evaluated through the anther activity quantification, and the two methods can meet the qualitative and quantitative detection of the pollen activity in large batch.

Disclosure of Invention

The invention aims to overcome the defect that the pollen activity identification cannot be rapidly carried out on a large scale by various dyeing methods at present, and the pollen activity is evaluated by a method for extracting The Triphenylmethylzone (TTF) for quantification after dyeing the pollen or the whole anther, and the method is used for the pollen activity identification on a large scale so as to simplify the quantitative operation process and improve the detection accuracy.

The invention relates to a formula of a combined staining solution (reagent) used for quantitative analysis, optimal staining time, a staining solution extraction method and application in a process of massively identifying the activity of anther/pollen. The quantitative treatment scheme of the invention can be used for qualitative and quantitative experiments of the activity of various plant anthers/pollen.

The technical scheme of the invention is as follows:

generally, 2,3, 5-triphenyltetrazolium chloride (TTC) staining of plant tissues needs to be carried out at 37 ℃, and the applicant firstly searches for the optimal osmotic pressure of the staining solution at room temperature and carries out pollen staining and settlement tests by respectively adopting 0mol/L, 0.05mol/L, 0.1mol/L, 0.2mol/L, 0.5mol/L and 1mol/L potassium phosphate buffer solution. The test results are shown in fig. 2 and 3. Through a plurality of attempts, the formula of the combined dyeing solution of the method for rapidly quantifying the activity of the plant pollen by TTC at normal temperature or high temperature is determined: namely: 1mol/L potassium dihydrogen phosphate (38.5mL, 5.23g), dipotassium hydrogen phosphate) 61.5mL, 14.03g), pH 7 potassium phosphate buffer (this buffer can be used to maintain the normal osmotic pressure of the cells), 8g 2,3, 5-triphenyltetrazolium chloride (TTC) was dissolved in the above obtained potassium phosphate buffer, and the volume was adjusted to 1L with distilled water to make the final concentration 8g/L (preservation protected from light).

The applicant adopts the combined staining solution to perform pollen staining on pollen of a cotton temperature-sensitive material identified in the early stage of a key laboratory in the China of Huazhong agriculture university crop genetic improvement of the applicant at the normal temperature control and high temperature treatment period (see figure 4). The pollen of dicotyledonous crops such as rape and monocotyledonous crops such as rice is dyed by adopting the combined dyeing solution, the combined dyeing solution is determined to be applicable to rape and rice, the pollen without viability can not be dyed red, and the pollen is easy to distinguish and has certain wide application potential (see figure 5).

The TTF quantitative detection in the invention is completed on the microplate reader, and the applicant finds that the maximum loading amount of the single-hole sample TTF does not exceed 30 mu g in the process of standard curve formulation, and the absorbance read by the microplate reader does not increase in a linear form after the maximum loading amount exceeds 30 mu g, and the result is shown in FIG. 6.

At the same time the applicant carried out a comparative test of two protocols: namely, scheme 1: pure pollen was stained in the dark, followed by quantitative staining for TTF, and the amount of TTF was found to increase linearly with the increase in pollen mass, with quantitative results shown in fig. 7 and 8. Since cotton anther tissue is not stained with color in the staining solution (as shown in fig. 9), further protocol 2 was performed: the anthers were taken into the staining solution, stained in the dark and then subjected to TTF quantification. The amount of TTF also increased linearly with the mass of anthers (quantitative results are shown in fig. 10 and 11).

The invention has the beneficial effects that:

by analyzing the pollen quantification and the whole anther quantification, the applicant integrally optimizes the test operation including dyeing duration, sampling mode, quantification method and the like, and simplifies a simple, convenient and accurate quantification scheme. The invention has the advantages that:

1: the formula of the combined staining solution is determined, so that the TTC staining of the pollen can be carried out at room temperature, and the pollen does not need to be placed in an incubator but needs to be protected from light.

2: the optimal dyeing time of the invention for the plant pollen or the anther is 1h, and the TTF in the plant pollen or the anther can not increase in a linear form according to the time after the optimal dyeing time exceeds 1 h.

3: the amount of TTF generated per unit time (the time of the invention is 1h) is increased linearly with the increase of the mass of pollen or anther, and after high-temperature treatment, the amount of TTF generated per unit time is still linear with the increase of the mass of anther and pollen, but high temperature affects the activity of anther and pollen, and the amount of TTF generated per unit time is reduced.

4: the amount of TTF produced per unit mass of anthers per unit time can be used as another reliable indicator for assessing pollen viability, reducing or eliminating errors due to incomplete shake-off of pollen or damage to pollen during shake-off.

5: the method can be widely applied to the identification of the activity of the pollen of different plants.

Drawings

FIG. 1: the invention relates to a dyeing quantitative technical route block diagram.

FIG. 2: and (3) comparing the dyeing degrees of the plant pollen after 1h of dyeing in a combined dyeing solution prepared from potassium phosphate buffer solutions with different concentrations. And simultaneously shows the sedimentation effect after sedimentation at different times. It was found that the staining of pollen in the 0.1mol/L combined staining solution was the deepest, indicating that the pollen was the most viable at this osmotic pressure, while the plant pollen settled faster than the pollen at several other osmotic pressures using 0.1mol/L potassium phosphate buffer.

FIG. 3: and (3) performing microscopic examination on the pollen after dyeing for 1h by using the combined dyeing solution under different osmotic pressures. It was found that the vigor of the plant pollen remained the best in the 0.1mol/L combined staining solution.

FIG. 4: and performing microscopic examination on the pollen dyeing of the temperature-sensitive cotton material under normal-temperature and high-temperature treatment. At room temperature, substantially all pollen can stain red. However, at high temperature, the cotton temperature-sensitive material generates a large amount of pollen without vitality.

FIG. 5: and (3) dyeing test results of pollen of dicotyledonous crop rape and monocotyledonous crop rice. Viable pollen was stained red in the microscopic pictures.

FIG. 6: is a standard curve drawing process. Preparing by using 8 g/L2, 3, 5-triphenyltetrazolium chloride (TTC) combined dyeing solution: 0 mu g/L (only potassium phosphate buffer), 5mg/L, 10mg/L, 25mg/L, 50mg/L, 100mg/L, 150mg/L, 250mg/L of 2,3, 5-triphenyltetrazolium chloride (TTC) containing combined staining solution (i.e., staining combined staining solution) of 1mL each, adding excess sodium hyposulfite (5-10 mg sodium hyposulfite is added to 1mL combined staining solution), after full reduction, adding equal volume of ethyl acetate, shaking for extraction for 5min, centrifuging, sucking 200 mu L of supernatant, and measuring with a microplate reader, wherein each absorbance corresponds to 0 mu g, 1 mu g, 2 mu g, 5 mu g, 10 mu g, 20 mu g, 30 mu g, 50 mu g of TTF. It can be seen that the absorbance linearly increased with the change in the amount of TTF when the amount of the sample was 30. mu.g or less, but did not increase linearly any more when the amount exceeded 30. mu.g.

FIG. 7: the amount of TTF generated by pollen with the same mass at normal temperature in different dyeing time is shown to increase in a linear form when the amount is less than or equal to 60min, but the amount does not increase in a linear form when the amount exceeds 60 min.

FIG. 8: is the same mass of plant pollen at high temperature, and the amount of TTF produced in different dyeing time. High temperature has a large effect on the activity, but also at less than or equal to 60min, the TTF increases in a linear form, but beyond 60min it no longer increases in a linear form.

FIG. 9: the result of separate staining of anther tissue and pollen of different test materials shows that anther tissue of cotton on the day of flowering is not stained red in TTC, while pollen is stained red, indicating that the entire anther can be used for staining.

FIG. 10: the material is the same-mass anther at normal temperature, the TTF amount generated in different dyeing time is similar to the pollen dyeing result, and the activity of the pollen of the material can be evaluated in a quantitative mode of anther dyeing.

FIG. 11: is the same mass of plant anthers at high temperature, the amount of TTF produced during different dyeing times. After high temperature, the amount of TTF produced per unit time is significantly lower than that produced by anthers at normal temperature, and the TTF still increases in a linear form within 60 min.

FIG. 12: the amount of TTF produced by different quality plant anthers in the same time (60min) at room temperature. The amount of TTF increases linearly with anther mass.

FIG. 13: the amount of TTF produced by different quality plant pollen in the same time (60min) at room temperature. The amount of TTF increases linearly with increasing pollen mass.

FIG. 14: the amount of TTF produced by different quality plant anthers was within the same time (60min) at high temperature. The amount of TTF increases linearly with the anther mass, and plant anthers per unit mass produce less TTF than normal temperature, indicating that the activity is affected.

FIG. 15: the amount of TTF produced by different quality plant pollen at the same time under high temperature (60 min). The amount of TTF increases linearly with the increase of the pollen mass, and the TTF produced by the plant pollen per unit mass is less than that produced at normal temperature, which indicates that the activity of the plant pollen is indeed influenced by high temperature.

FIG. 16: the amount of TTF after extraction from the experiments of fig. 7-8 and fig. 10-15.

FIG. 17: after dyeing a large batch of materials, directly crushing plant anthers to extract an extraction result after TTF.

Detailed Description

Example 1: preparing optimal concentration buffer solution and TTC dye solution

The 2,3, 5-triphenyltetrazolium chloride (TTC) combined staining method employed in the present invention is based on an improvement of the method (Min, L., Zhu, L., Tu, L., Deng, F., Yuan, D., and Zhang, X. (2013) Cotton GhCKI dispersions normal reproduction by y deletion of taperated cell programmed tissue stain of vitamin a inactivation stage synthesis.the Plant J. for cell and molecular biology 75, 823. 835) already reported by the national emphasis laboratory of the university of Huazhong agriculture crop where the applicant is located, in which the applicant employs a method of staining a 37 ℃ incubator in the absence of light, considering the possible conditions without incubator, with reference to the general buffer catalog, phosphate suspension buffers of different concentrations are respectively designed, pollen suspension buffer of 0.0 mol/L, 0.0.05/L/0.0 mol/0.05L/0.0 mol/L, 1 mol/L. Meanwhile, the concentration of TTC dye liquor commonly used for active dyeing of plant root systems is referred to (refer to: Wangchui, Huangliang, plant physiological and biochemical experiment principle and technology, third edition, advanced education Press, 1 month in 2015, Beijing), and the invention adopts TTC dye liquor concentration of 8g/L to carry out dyeing test. As a result, it was found that in potassium phosphate buffer at a concentration of 0.1M and pH 7, cotton pollen rapidly fell to the bottom and the pollen cells were maintained in a normal state without disruption and inactivation (see FIG. 2). After the optimum potassium phosphate buffer solution is determined, the activity of the pollen can be clearly distinguished by the staining test of the combined staining solution with the concentration (as shown in figure 3 and figure 4), and finally, the activity is stained by using 0.1M potassium phosphate buffer solution and preparing 8g/L TTC staining solution.

The formula of the combined dyeing solution is as follows: 5.23g of monopotassium phosphate; 14.03g of dipotassium phosphate, adding distilled water to a constant volume of 1L (adjusting the pH to 7), and then adding 8g of TTC; .

Example 2: staining test for pollen of different crops

By using the combined staining solution in example 1, the staining test was performed on the pollen of dicotyledonous crops such as rape and monocotyledonous crops such as rice, and microscopic examination of the stained pollen revealed that viable pollen in both crops could be stained red, indicating that the combined staining solution of the present invention has a certain wide applicability (as shown in fig. 5).

Example 3 optimal microplate Loading amount test

In order to determine the precise measurement range of the microplate reader, the combined staining solution of example 1 is diluted by different times to prepare 1mL (as shown in fig. 3) of combined staining solutions of 0 μ g/L (only potassium phosphate buffer), 5mg/L, 10mg/L, 25mg/L, 50mg/L, 100mg/L, 150mg/L and 250mg/L, after sufficient reduction by adding excess sodium hyposulfite (5-10 mg added to 1 mL), equal volume of ethyl acetate is added to shake and extract for 5min, and the supernatant is centrifuged by absorbing 200 μ L of the microplate reader, wherein each measured absorbance corresponds to 0 μ g, 1 μ g, 2 μ g and 5 μ g, TTF amount of 10. mu.g, 20. mu.g, 30. mu.g, 50. mu.g. It can be seen that the absorbance linearly increased with the change in the amount of TTF when the amount of the above-mentioned sample was 30. mu.g or less, but did not linearly increase any more when it exceeded 30. mu.g. The upper limit of the amount of the sample to be finally selected is 30. mu.g (see FIG. 6 for the results).

Example 4: optimum staining time test

After the optimum concentrations of potassium phosphate buffer and TTC staining solution were determined in example 1, it was necessary to further confirm the optimum staining time. The difficulty of microscopic examination is increased due to insufficient and overlong dyeing time of plant pollen, and the activity is difficult to distinguish, which is mainly caused by uncertain dyeing time. In this example, according to the results of (Min, L, etc., sub and automatic signaling pathway response to high-temperature strain degradation as modified by trading profiling in cotton etc. (2014) Plant Physiology 164, 1293. banding 1308), the high temperature sensitive material in the report was used, and the optimum dyeing time was determined at normal temperature. In the embodiment, tests of crushing and quantifying TTF after dyeing for 5min, 10min, 15min, 20min, 30min, 60min and 90min are designed for pollen with certain mass. The specific steps are that firstly pollen with the same mass is respectively added into 7 clean 2mL centrifuge tubes, 1mL of the combined staining solution prepared in the embodiment 1 is added, and the timing is started by shading and staining. After the reaction time is up, 2 mu L of diluted concentrated sulfuric acid (5mol/L) is added into each centrifuge tube to stop the reaction, the pollen is extracted by 300-500 mu L of ethyl acetate after being crushed, diluted by 2-3 times, and 200 mu L of enzyme label plate is absorbed to measure the absorbance. More than three times of repeated experiments show that the TTF generated by the pollen increases linearly with the time within 60min, and is not linear after exceeding 60min, so that the finally selected dyeing time is 60min (the result is shown in figure 7).

Example 5: quantitative test of pollen Activity in anther after high temperature treatment

In this example, a quantitative test was conducted on the pollen after the high-temperature treatment by TTC staining using a quantitative method. As a result, it was found that the activity of pollen was significantly affected after high temperature stress, but the optimal staining time was still 60min, and after 60min, TTF production did not increase linearly (see FIG. 8).

Example 6: quantitative test of anther activity at ordinary and high temperatures

The result of example 2 shows that it is feasible to express the activity by using TTF quantification after dyeing with a certain quality of pollen combined dyeing solution, and the invention considers that damage may be caused to the pollen in the process of obtaining the pollen, and the damage may have a certain influence on the activity of the pollen, so that TTF is extracted after dyeing the whole anther and quantified for evaluating the activity.

The method comprises the following specific steps: it was first confirmed whether the presence of anther tissue affected the quantification of pollen viability. 5 different cotton varieties are taken, anthers which open the day are stained for 1 hour, and pollen and anther tissues are separated. It can be seen that the anthers are not stained red substantially, and the pollen is stained red. In this example, a quantitative test of TTF was carried out by directly crushing and extracting the whole of anthers of a certain mass (for example, 15mg) without collecting pollen after dyeing. The test steps also adopt the steps of dyeing for 5min, 10min, 15min, 20min, 30min, 60min and 90min, and then crushing for TTF quantitative test. As a result, it was found that the best test for staining with the whole anther was 60min, and over 60min, the test results were no longer linear (see FIG. 1).

Example 7: quantitative test of pollen and anther with different qualities after normal temperature and high temperature stress

Examples 4, 5 and 6 were conducted with the same quality of anthers or pollen in anthers for identification tests, and in consideration of the different reduction capacities of anthers or pollen of different qualities, in this example, dyeing tests were conducted for 60min with anthers of different qualities and pollen of anthers of different qualities, and two comparative tests under normal temperature and high temperature stress were set. In this example, 7 different quality test groups of 1mg, 2mg, 5mg, 10mg, 15mg, 20mg, 40mg, etc. were designed to perform quantitative tests of staining on anthers and pollen, respectively. As a result, it was found that the production of TTF increased linearly with the increase in the mass of anthers or pollen. At the same time, high temperature has a large effect on the ability of pollen or anthers to produce TTF, but the amount of TTF produced is still linear with quality (see fig. 12-15). The results show that even anthers or pollen of different qualities can be identified and evaluated for activity using the present invention. This can provide convenience for the anther quantification under the different treatments of the large quantities of different materials in the later stage, and all anthers of a whole flower can be quantified without worrying about the influence on the quantification result caused by the different qualities of a whole anther taken by different materials.

Example 8: extraction tests were performed on different materials after high temperature treatment in bulk

According to the results of the examples, the quantitative results of anthers and pollen at the dyeing time of 60min were accurate to evaluate the activity of the material at normal and high temperatures, while the amount of TTF produced by anthers or pollen of the same material at the dyeing time of 60min was linearly increased with the mass (see fig. 16). In the embodiment, a scheme of weighing the anther and then dyeing and extracting is adopted to quantitatively test a large batch of materials, the activity of different materials at normal temperature and high temperature is evaluated according to the TTF amount generated by the anther or pollen of unit mass in unit time (see figure 17), and the TTF generation amount of different materials at high temperature is greatly different and can be identified by naked eyes, so that the method can be used for identifying the activity of the large batch of materials.

The method is improved from a TTC dyeing method of the pollen, although the TTC dyeing method of the pollen is applied in a large number and has quite good identification effect on the pollen with extreme phenotype (extremely strong vitality and extremely weak vitality), the influence of high temperature on the activity of each pollen is different, and the activity identification effect on the pollen with the activity in an intermediate state through TTC dyeing is not ideal. The invention improves the traditional TTC dyeing method, so that the pollen still has better activity in the combined dyeing solution, meanwhile, the dyeing operation can be carried out at room temperature, and the activity of the pollen of the corresponding material under normal temperature or high temperature stress can be accurately evaluated by carrying out quantitative TTF instead of microscopic examination on the dyed pollen. Meanwhile, the invention discovers that the activity of the pollen of the corresponding material can still be accurately evaluated by a method for quantifying the whole anther, so that the operation process is greatly reduced, and the damage to the pollen in the process of obtaining the pollen is also reduced. Furthermore, the invention can carry out activity identification on the dyed anther in a large batch by a method for quantifying the anther used by the corresponding material. Therefore, the operation flow is greatly omitted, the evaluation can be rapidly and accurately carried out, and the evaluation method can be used for evaluating the pollen or the anther activity of various crops except cotton.

Claims (2)

1. A dyeing method suitable for plant anther or pollen at normal temperature or high temperature is characterized in that: the combined staining solution consists of potassium phosphate buffer solution and 2,3, 5-triphenyltetrazolium chloride (TTC);

wherein the formula of the potassium phosphate buffer solution in the combined staining solution is 5.23g of 1mol/L monopotassium phosphate and 14.03g of 1mol/L dipotassium phosphate, and the volume is determined to 1L by using distilled water to obtain the potassium phosphate buffer solution; dissolving 8g of 2,3, 5-triphenyltetrazolium chloride (TTC) in 1L of potassium phosphate buffer solution to make the final concentration be 8 g/L;

the optimal dyeing time of the combined dyeing solution in the dyeing process at normal temperature or high temperature is 1 h;

the plant is rape, rice or cotton.

2. Use of the method of claim 1 for the identification of activity of canola pollen or anthers, rice pollen or anthers, cotton pollen or anthers.

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