A method for separating and collecting rice anther tissue

Abstract

This invention belongs to the field of biological tissue separation technology, and particularly relates to a method for separating and collecting rice anther tissue. The method includes: 1) Sampling: After removing the husks from a rice panicle, the anthers are peeled off, using the anthers as a sample; 2) Pollen extraction: The sample is mixed with Hank's incubation solution, and the anther tissue is mechanically broken. After the anther wall is completely ruptured, pollen grains are released to obtain a sample solution; 3) Separation of pollen grains and anther wall: The sample solution is filtered in stages to separate pollen grains and anther wall. The filtrate is centrifuged to separate pollen grains and anther chamber fluid, thus completing the separation and collection of rice anther tissue. This invention allows for highly mechanized operation, saving manpower, improving the efficiency of separating and collecting anther tissue, significantly improving the purity of pollen grains, and controlling the loss rate to a low level.

Description

Technical Field

[0001] This invention belongs to the field of biological tissue separation technology, and in particular relates to a method for separating and collecting rice anther tissue. Background Technology

[0002] In my country, rice anther culture technology is a very important agricultural research technique. It is mainly applied in three areas: first, combining anther culture technology with conventional intervarietal hybridization breeding to cultivate new varieties; second, using anther culture technology for interspecific hybridization between indica and japonica rice subspecies; and third, using anther culture technology for the purification and rejuvenation of hybrid rice, and for purifying and renewing sterile lines and maintainer lines. Currently, the theoretical basis for using plants obtained from anthers for breeding has been established, and rice has become a crop that can be rapidly bred using anther culture technology.

[0003] However, anther culture technology requires prior separation and collection of anther tissues. Existing separation and collection methods are prone to anther contamination, are complex, and result in low pollen purity and yield. Ultimately, this leads to low callus induction rates and poor actual anther culture outcomes. Summary of the Invention

[0004] To address the shortcomings of existing anther tissue separation technologies, such as high contamination rates, complex processes, and the need for extensive manual operations, this invention provides a method for separating and collecting rice anther tissue.

[0005] The purpose of this invention is:

[0006] I. Reduce manual operation steps to achieve fast and efficient mechanized operation;

[0007] II. Improve pollen grain separation efficiency and reduce pollutants;

[0008] 3. Improve pollen yield.

[0009] To achieve the above objectives, the present invention adopts the following technical solution.

[0010] A method for isolating and collecting rice anther tissue.

[0011] The method includes:

[0012] 1) Sampling: After removing the glumes from rice panicles, the anthers are peeled off and used as samples;

[0013] 2) Pollen collection: After mixing the sample with Hank's incubation solution, mechanically break up the anther tissue. Once the anther wall is completely dehisced, release the pollen grains to obtain the sample solution.

[0014] 3) Separation of pollen grains and anther walls: The sample solution is filtered in stages to separate pollen grains and anther walls. The filtrate is centrifuged to separate pollen grains and anther chamber fluid, thus completing the separation and collection of rice anther tissue.

[0015] In this invention, highly mechanized operations reduce errors caused by manual labor. For example, mechanical crushing, grading filtration, and centrifugal separation can all be automated using equipment to achieve a stable, production-line process for pollen grains. Furthermore, this invention separates and collects pollen grains layer by layer through grading, and with the addition of impurity removal, effectively improving pollen grain purity and reducing contaminants.

[0016] Preferably, the anthers described in step 1) are placed in liquid nitrogen for cold embrittlement treatment before the powder is collected in step 2), and then the samples are placed in step 2) or stored at ≤-80℃ for later use.

[0017] Because anthers possess a certain degree of flexibility, direct mechanical crushing is ineffective, necessitating manual separation, which is inefficient. Furthermore, even with flat-tipped tweezers, manual separation can easily lead to uneven stress on the anther tissue, resulting in some anther walls completely shattering while others remain intact. The powdery residue from the completely shattered anther walls is difficult to separate and remove effectively, causing contamination. However, liquid nitrogen treatment to induce brittleness makes the anther walls extremely brittle and easily broken, allowing for effective separation and removal via mechanical crushing.

[0018] As a preferred option

[0019] The cold embrittlement process is carried out for 4 to 6 seconds.

[0020] Cold embrittlement treatment requires controlling the anther wall to effectively become brittle while avoiding damage to the internal pollen grains; therefore, an appropriate cold embrittlement treatment duration must be selected.

[0021] As a preferred option

[0022] The specific process of the cold embrittlement treatment is as follows:

[0023] Take a liquid nitrogen insulated container and control the liquid nitrogen level in the container. Sprinkle the anther sample evenly on the liquid nitrogen surface and let the anther sample fall naturally. Then separate the anther sample.

[0024] Using the above methods can avoid the problem of uneven cold embrittlement caused by sampling and watering, and at the same time reduce the ineffective volatilization of liquid nitrogen and improve the utilization rate of liquid nitrogen.

[0025] As a preferred option

[0026] Step 2) The samples are mixed at a ratio of 1-3 mL of Hank's incubation solution for each sample;

[0027] The temperature of the Hank's incubation solution is ≤8℃.

[0028] Controlling the ratio of sample to incubation solution and the temperature of the incubation solution can help improve the crushing effect.

[0029] As a preferred option

[0030] The mechanical crushing method is crushing with a vibrator, and zirconium beads are added to the vibrator to control the vibratory grinding frequency to 15-25Hz and continue grinding for 15-25s.

[0031] The amount of zirconium beads used is 1 to 2 zirconium beads with a particle size of 0.3 to 0.5 mm per milliliter of Hank's incubation solution;

[0032] The sample liquid is obtained by breaking and separating the zirconium beads using the oscillator.

[0033] Using a shaker, the movement of the adapter in the shaker can be superimposed with the movement of the zirconium beads, which can thoroughly mix the sample. The impact and friction between the balls can effectively break the anther tissue evenly, promote the complete cracking of the anther wall, and completely release the pollen grains, so that the two are completely dispersed and separated. Multiple samples can be processed at the same time.

[0034] As a preferred option

[0035] Step 3) describes the graded filtration process for separating pollen grains and anther walls as follows:

[0036] First, a 100-300 μm cell sieve is used for preliminary filtration to separate the anther walls and obtain the initial filtrate. The initial filtrate is then slowly dripped onto an inclined silica glass panel, allowing it to flow downwards along the panel and be collected at the bottom to obtain the impurity-removed liquid. The impurity-removed liquid is then filtered through a 100-300 μm cell sieve to obtain the filtrate, which contains pollen grains.

[0037] As mentioned in the foregoing process, the technical solution of this invention eliminates the need for manual operation steps, and a cold embrittlement treatment was performed in the aforementioned steps. However, several inconveniences brought about by the cold embrittlement treatment were also mentioned, such as the anther wall exhibiting a high degree of powdering, which is difficult to remove in the actual filtration process. Furthermore, experiments have shown that the highly powdered anther wall can even reach the micro-nano scale, with the smallest reaching below 10 μm, and its actual size largely overlaps with the pollen size, making it impossible to remove by simple filtration methods. However, considering the characteristics of the anther wall and pollen grains, researchers found that silica with abundant hydroxyl groups (hydroxyl content ≥150ppm) can actually generate intermolecular forces with the anther wall, while pollen grains lack these forces or have weaker ones. This results in a certain amount of extremely fine solid matter appearing on the silica glass panel after the initial filtrate is slowly flowed through it via dripping. This solid matter consists of powdered anther wall cells captured by the hydroxyl groups. Although a very small amount of pollen grains may remain, the loss rate is extremely low and negligible compared to the increase in purity. Therefore, using a tilted silica glass panel for flow guidance is a key step in improving the purity of the finally collected pollen grains.

[0038] Preferably, the tilt angle of the silica glass panel is 25-35°, the distance between the drop position of the initial filtrate and the bottom of the silica glass panel is controlled to be ≥25cm, and the drop rate of the initial filtrate is controlled to be 1-2 drops / s.

[0039] By controlling the above parameters, the purity and loss rate of pollen grains can be optimized.

[0040] Preferably, the centrifugal separation process controls the centrifugal temperature to ≤4℃ and the relative centrifugal force to be 80~110rcf, with centrifugation lasting 4~6 min.

[0041] Controlling the temperature during centrifugation is to ensure the biological activity of intracellular RNA, proteins (such as enzymes), etc., while controlling the relative centrifugal force is to maintain the integrity of pollen grain cells.

[0042] The beneficial effects of this invention are:

[0043] Except for the process of peeling off the glumes, the entire process can be mechanized and automated, which greatly saves manpower and improves the efficiency of separating and collecting anther tissues. Furthermore, by using the flow plate method, the abundant hydroxyl groups on the surface of silica enable the effective separation of the powdered ultrafine anther wall from the pollen grains, which greatly improves the purity of the pollen grains and controls their loss rate to be low. In addition, the whole process does not require complex or expensive equipment, and the industrialization cost is low and the difficulty is low, making it suitable for widespread application. Attached Figure Description

[0044] Figure 1 The results of fluorescence microscopy observation of the pollen grains isolated and collected in Example 1;

[0045] Figure 2 The results of fluorescence microscopy observation of the pollen grains isolated and collected in Comparative Example 1 are shown.

[0046] Figure 3 The fluorescence microscopy observation results of the separated and collected products of Comparative Example 2 are shown.

[0047] Figure 4 The fluorescence microscopy observation results of the separated and collected products in step 2) of Example 1;

[0048] Figure 5 The results of fluorescence microscopy observation of pollen grains isolated and collected in Comparative Example 3 are shown. Detailed Implementation

[0049] The present invention will be further described clearly and in detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0050] In the description of this invention, it should be understood that the terms "thickness," "upper," "lower," "horizontal," "top," "bottom," "inner," "outer," "circumferential," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified, and "several" means one or more.

[0051] In the present invention, unless otherwise specified or limited, the terms "installed," "connected," "connect," "fixed," etc. should be understood in a broad sense. For example, they can refer to fixed connection, detachable connection, or integration; mechanical connection, electrical connection, or communication; direct connection or indirect connection through an intermediate medium; internal communication between two elements or interaction between two elements, unless otherwise specified. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0052] Unless otherwise specified, all raw materials used in the embodiments of the present invention are commercially available or obtainable by those skilled in the art; unless otherwise specified, all methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0053] Unless otherwise specified, the following examples use rice anthers at the microspore stage as materials.

[0054] Example 1

[0055] A method for isolating and collecting rice anther tissue.

[0056] The method comprises:

[0057] 1) Sampling: After removing the glumes from rice ears, separate the anthers. Take a liquid nitrogen insulated container and control the liquid nitrogen level in the container. Sprinkle the anther sample evenly on the liquid nitrogen surface. After the anther sample falls naturally for 5 seconds, separate the anther sample and use the anther as the sample.

[0058] 2) Pollen collection: Take a sample and mix each sample with 2 mL of Hank's incubation solution (5℃) and place it in a shaker. Add 2 0.5 mm zircon beads to each sample and treat at a frequency of 20 Hz for 20 s until the anther wall is completely dehisced to release pollen grains. Separate the zircon beads to obtain the sample solution.

[0059] 3) Separation of pollen grains and anther walls: A 200μm cell sieve was used for preliminary filtration to separate the anther walls and obtain the initial filtrate. The initial filtrate was slowly added dropwise at a rate of 1 drop / s onto a 30° inclined silica glass panel, allowing it to flow down the silica glass panel for 30cm before being collected at the bottom to obtain the impurity-free liquid. The impurity-free liquid was then filtered through a 200μm cell sieve to obtain the filtrate containing pollen grains. The filtrate was centrifuged at 4℃ with the centrifugation parameters controlled at 90rcf for 5 minutes to separate the pollen grains and anther chamber fluid, thus completing the separation and collection of rice anther tissue.

[0060] The isolated and collected pollen grains were observed using a fluorescence microscope, and the results are as follows: Figure 1 As shown. From Figure 1 It can be clearly seen that the pollen grains separated in this embodiment are numerous and dense, and there are basically no anther wall residues and / or powdered anther wall fragments, indicating a high degree of cleanliness and low degree of contamination.

[0061] Example 2

[0062] A method for isolating and collecting rice anther tissue.

[0063] The method comprises:

[0064] 1) Sampling: After removing the glumes from rice ears, separate the anthers. Take a liquid nitrogen insulated container and control the liquid nitrogen level in the container. Sprinkle the anther sample evenly on the liquid nitrogen surface. After the anther sample falls naturally for 6 seconds, separate the anther sample and use the anther as the sample.

[0065] 2) Pollen collection: Take a sample and mix each sample with 3 mL of Hank's incubation solution (5℃) and place it in a shaker. Add 3 0.5 mm zircon beads to each sample and treat at a frequency of 15 Hz for 25 s until the anther wall is completely dehisced to release pollen grains. Separate the zircon beads to obtain the sample solution.

[0066] 3) Separation of pollen grains and anther walls: A 200μm cell sieve was used for preliminary filtration to separate the anther walls and obtain the initial filtrate. The initial filtrate was slowly added dropwise at a rate of 2 drops / s onto a 25° inclined silica glass panel, allowing it to flow down the silica glass panel for 35cm before being collected at the bottom to obtain the impurity-free liquid. The impurity-free liquid was then filtered through a 200μm cell sieve to obtain the filtrate containing pollen grains. The filtrate was centrifuged at 4℃ with the centrifugation parameters controlled at 80rcf for 6 minutes to separate the pollen grains and anther chamber fluid, thus completing the separation and collection of rice anther tissue.

[0067] The pollen grains that were separated and collected were observed using a fluorescence microscope. The results were similar to those in Example 1. The pollen grains separated in this example were more numerous and denser, and there were virtually no anther wall residues or powdered anther wall fragments. The pollen grains were clean and had a low degree of contamination.

[0068] Example 3

[0069] A method for isolating and collecting rice anther tissue.

[0070] The method includes:

[0071] 1) Sampling: After removing the glumes from rice ears, separate the anthers. Take a liquid nitrogen insulated container and control the liquid nitrogen level in the container. Sprinkle the anther sample evenly on the liquid nitrogen surface. After the anther sample falls naturally for 4 seconds, separate the anther sample and use the anther as the sample.

[0072] 2) Pollen collection: Take a sample and mix each sample with 1 mL of Hank's incubation solution (5℃) and place it in a shaker. Add one 0.3 mm zircon bead to each sample and treat at a frequency of 25 Hz for 15 s until the anther wall is completely dehisced to release pollen grains. Separate the zircon beads to obtain the sample solution.

[0073] 3) Separation of pollen grains and anther walls: A 300μm cell sieve was used for preliminary filtration to separate the anther walls and obtain the initial filtrate. The initial filtrate was slowly added dropwise at a rate of 1 drop / s onto a 35° inclined silica glass panel, allowing it to flow down the silica glass panel for 25cm before being collected at the bottom to obtain the impurity-free liquid. The impurity-free liquid was then filtered through a 100μm cell sieve to obtain the filtrate containing pollen grains. The filtrate was centrifuged at 4℃ with the centrifugation parameters controlled at 110rcf for 4 minutes to separate the pollen grains and anther chamber fluid, thus completing the separation and collection of rice anther tissue.

[0074] The pollen grains that were separated and collected were observed using a fluorescence microscope. The results were similar to those in Example 1. The pollen grains separated in this example were more numerous and denser, and there were virtually no anther wall residues or powdered anther wall fragments. The pollen grains were clean and had a low degree of contamination.

[0075] Comparative Example 1

[0076] The specific process is the same as in Example 1, except that:

[0077] Step 3) The specific process of separating pollen grains from anther walls is as follows: a 200 μm cell sieve is used for preliminary filtration, and then the filtrate is obtained by filtering through a 200 μm cell sieve. The filtrate contains pollen grains. The filtrate is centrifuged at 4°C. The centrifugation parameters are controlled at 90 rcf and continued for 5 minutes to separate the pollen grains and anther chamber fluid, thus completing the separation and collection of rice anther tissue.

[0078] The isolated and collected pollen grains were observed using a fluorescence microscope, and the results are as follows: Figure 2 As shown. From Figure 2 As can be seen, the separated pollen grains still contain a lot of powdered anther wall fragments, with a low degree of cleanliness and some foreign matter contamination. This results in poor performance and interference when used for anther culture experiments or industrialization.

[0079] Comparative Example 2

[0080] The specific process is the same as in Example 1, except that:

[0081] Step 1) Without cold embrittlement treatment, proceed directly to the crushing process in Step 2). Observe the sample solution obtained after Step 2) using a fluorescence microscope. The observation results are as follows: Figure 3 As shown, and with as Figure 4 Compare the sample liquid obtained in step 2) of Example 1 shown in the figure.

[0082] from Figure 3 and Figure 4The comparison results show that the anther wall integrity in the sample solution obtained in step 2) of Comparative Example 2 is higher, the actual pollen grain release degree is lower, and the release amount is relatively small. In contrast, the pollen grain release after treatment in step 2) of Example 1 is significantly greater than that of Comparative Example 2, and the anther wall breakage rate is higher, indicating that the cold embrittlement treatment has a significant impact on the actual pollen grain yield.

[0083] Since the actual collection rate of pollen grains could not be determined, anther samples of equal batches and mass (accurate to 1 mg) were processed according to the methods described in Example 1 and Comparative Example 2, respectively, and the relative collection rate of Example 1 relative to Comparative Example 2 was calculated.

[0084] The formula for calculating the relative collection rate is as follows:

[0085] In the formula: Ca is the relative collection rate of Example 1 relative to Comparative Example 2, m1 is the mass of pollen grains collected in Example 1, m2 is the mass of pollen grains collected in Comparative Example 2, and m is the mass of the anther sample used.

[0086] The calculation results after five parallel experiments each showed that Ca max =2.21, Ca min =1.76, and using the pollen grain mass obtained for the first time using the method of Example 1 as the standard, the following formula was used for calculation: In the formula: n = 1 or 2 or 3 or 4 or 5, Ca n m represents the relative pollen collection rate of the nth parallel experiment conducted according to the method of Example 1 compared to the first parallel experiment. n m1 represents the mass of pollen grains collected in the nth parallel experiment, m1 represents the mass of pollen grains collected in the 1st parallel experiment, and m represents the mass of the anther sample used.

[0087] Based on the above calculations, Ca n = [0.97, 1.01], which means that the method has relatively stable returns and does not produce significant fluctuations. It also shows that the comparative example 2, which did not undergo cold embrittlement treatment, has a large fluctuation in the collection rate, mainly due to the inability to guarantee the degree of fragmentation of the anther wall.

[0088] Comparative Example 3

[0089] Pollen grains are collected and separated using existing artificial processing methods:

[0090] After removing the glumes from rice panicles, the anthers were peeled off. Using the anthers as samples, each sample was mixed with 2 mL of Hank's incubation solution (5℃), crushed with flat-mouthed forceps, and filtered through a 200 μm cell sieve to obtain the filtrate. The filtrate was then centrifuged at 4℃, with the centrifugation parameters controlled at 90 rcf for 5 minutes to separate pollen grains and anther chamber fluid, thus completing the separation and collection of rice anther tissue.

[0091] The pollen grains collected in Comparative Example 3 were observed using a fluorescence microscope. The results are as follows: Figure 5 As shown.

[0092] Because only one filtration is performed, some anther walls puncture the cell sieve and enter the final product. A close examination of the anther walls in Figure 5 reveals that they still contain a significant amount of pollen grains, and the number of released pollen grains is relatively small, indicating a very poor actual effect. Furthermore, the manual processing method is inefficient.

[0093] Through the observation and comparison of the above embodiments and comparative examples, it can be clearly seen that the technical solution of the present invention can significantly improve the collection efficiency and collection effect of pollen grains, ensure that the collected pollen grains have high purity, and at the same time, the collection rate is stable and relatively higher, which has a very significant effect.

Claims

1. A method for isolating and collecting rice anther tissue, characterized in that, The method includes: 1) Sampling: After removing the glumes from rice panicles, the anthers are peeled off and used as samples; 2) Pollen collection: After mixing the sample with Hank's incubation solution, mechanically break up the anther tissue. Once the anther wall is completely dehisced, release the pollen grains to obtain the sample solution. 3) Separation of pollen grains and anther walls: The sample solution is filtered in stages to separate pollen grains and anther walls. The filtrate is centrifuged to separate pollen grains and anther chamber fluid, thus completing the separation and collection of rice anther tissue. The anthers described in step 1) are used as samples for step 2) before powder collection. They are placed in liquid nitrogen for cold embrittlement treatment. After cold embrittlement treatment, they are placed in step 2) or stored at ≤-80℃ for later use. Step 3) describes the graded filtration process for separating pollen grains and anther walls as follows: First, a 100-300 μm cell sieve is used for preliminary filtration to separate the anther walls and obtain the initial filtrate. The initial filtrate is then slowly dripped onto an inclined silica glass panel, allowing it to flow downwards along the panel and be collected at the bottom to obtain the impurity-removed liquid. The impurity-removed liquid is then filtered through a 100-300 μm cell sieve to obtain the filtrate, which contains pollen grains.

2. The method for isolating and collecting rice anther tissue according to claim 1, characterized in that, The cold embrittlement process is carried out for 4 to 6 seconds.

3. The method for isolating and collecting rice anther tissue according to claim 2, characterized in that, The specific process of the cold embrittlement treatment is as follows: Take a liquid nitrogen insulated container and control the liquid nitrogen level inside the container. Sprinkle the anther sample evenly on the surface of the liquid nitrogen and let the anther sample fall naturally. Then separate the anther sample.

4. The method for isolating and collecting rice anther tissue according to claim 1, characterized in that, Step 2) The samples are mixed at a ratio of 1 to 3 mL of Hank's incubation solution for each sample; The temperature of the Hank's incubation solution is ≤8℃.

5. A method for isolating and collecting rice anther tissue according to claim 1 or 4, characterized in that, The mechanical crushing method is crushing with a vibrator, and zirconium beads are added to the vibrator to control the vibratory grinding frequency to 15-25Hz and continue grinding for 15-25s. The amount of zirconium beads used is 1 to 2 zirconium beads with a particle size of 0.3 to 0.5 mm per milliliter of Hank's incubation solution; The sample liquid is obtained by breaking and separating the zirconium beads using the oscillator.

6. The method for isolating and collecting rice anther tissue according to claim 1, characterized in that, The tilt angle of the silica glass panel is 25-35°, the distance between the drop position of the initial filtrate and the bottom of the silica glass panel is controlled to be ≥25cm, and the drop rate of the initial filtrate is controlled to be 1-2 drops / s.

7. A method for isolating and collecting rice anther tissue according to claim 1 or 6, characterized in that, The centrifugation process controls the centrifugation temperature to ≤4℃ and the relative centrifugal force to be 80~110rcf, with centrifugation lasting 4~6min.

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