Method for determining whether pollen viability of rice obtained by biological breeding technology and receptor material of rice is different

Abstract

The invention belongs to the field of environmental safety evaluation of rice obtained by a biological breeding technology, and discloses a method for determining whether the pollen viability of the rice obtained by the biological breeding technology is different from that of a receptor material of the rice. The method comprises the following steps: culturing transgenic or gene-edited rice pollen and receptor rice pollen in a culture medium, and detecting the difference between the germination rate of the transgenic rice pollen and the germination rate of the receptor rice pollen; the culture medium consists of the following substances: 175 g / L of sucrose, 40 mg / L of boric acid, 20 mg / L of calcium chloride, 10 mg / L of monopotassium phosphate, 10 mg / L of magnesium sulfate and the balance of distilled water. The method has the characteristics of high operability, simple data processing, objective and reliable result and the like. The invention provides a method for measuring pollen viability in environmental safety detection of rice cultivated by using a biological breeding technology, and the method plays an important role in scientific safety evaluation of transgenic and gene-edited rice.

Description

Technical Field

[0001] The invention belongs to the field of environmental safety assessment of transgenic organisms in biobreeding technology, and relates to a method for determining whether there is a difference in pollen viability between rice obtained through biobreeding technology and its recipient material. Background Art

[0002] Biobreeding is a general term for modern agricultural biotechnology breeding, primarily utilizing technologies such as transgenic technology, gene editing, and synthetic biology to conduct efficient, precise, and targeted genetic improvement and variety development in plants and animals. Biobreeding, represented by transgenic technology, is at the forefront of agricultural science and technology development and is leading a new round of agricultural science and technology revolution.

[0003] Research and development of genetically modified rice is booming. Genetically modified rice rich in vitamin A precursors, herbicide-tolerant rice, and insect-resistant rice have received safety certificates for production and application. Genetically modified rice for producing pharmaceutical proteins, improving quality, and resisting diseases has also entered the field trial stage. Meanwhile, gene editing, a recent emerging technology, has also been applied to rice, such as regulating disease resistance and tillering, contributing to increased stress tolerance and yield.

[0004] Before genetically modified or gene-edited rice is commercialized, an environmental safety assessment needs to be conducted. Analyzing the survival competitiveness of genetically modified rice by evaluating pollen viability is a major part of the environmental safety assessment.

[0005] The pollen germination method is a commonly used method for measuring pollen viability. Its principle is that mature pollen grains germinate and form pollen tubes when grown on a medium containing the required nutrients and under suitable culture conditions, thus recreating the pollen germination and growth process. Viable pollen germinates and grows normally on the pollen germination medium; inactive pollen fails to germinate. By measuring the germination rate of pollen from transgenic plants and their corresponding non-transgenic recipients under different treatment conditions, differences in pollen viability can be determined. Currently, the in vitro pollen germination method is also used to evaluate pollen viability in transgenic corn, soybeans, and cotton, and corresponding standards have been established. These standards assess whether the transgenic operation alters the competitiveness of the crop by analyzing the germination rate differences between transgenic and non-transgenic pollen at different in vitro times and temperatures.

[0006] While there are some reports on in vitro germination of rice pollen, there are no systematic studies on the minimum and maximum temperatures that rice pollen can tolerate, or on pollen viability under different in vitro conditions. Nor have there been established methods and standards for assessing pollen viability in transgenic rice. Based on this, the inventors have developed a method for assessing the viability of transgenic rice pollen by optimizing existing rice pollen germination culture media, selecting appropriate observation and pollen collection times, and systematically analyzing pollen viability and its extreme temperature tolerance. This method is of great significance for scientifically evaluating the environmental safety of transgenic rice. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a method for evaluating the viability of rice pollen obtained through biobreeding techniques. This method can determine whether genetic transformation operations result in changes in the viability of transgenic or gene-edited rice pollen, and can be used for environmental safety assessment of transgenic or gene-edited rice. The technical problem to be solved is not limited to the technical subject matter described herein. Other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The present invention provides a method for measuring the difference in germination rate between pollen of rice obtained through biological breeding technology and its recipient material, comprising the following steps: culturing pollen of transgenic rice or gene-edited rice and recipient rice pollen in a culture medium, and detecting the difference in germination rate between the pollen of the transgenic rice or gene-edited rice and the pollen of the recipient rice; the transgenic rice or gene-edited rice is rice obtained through biological breeding technology;

[0010] The culture medium is composed of the following substances: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate and 10 mg / L magnesium sulfate, with the balance being distilled water.

[0011] The transgenic or gene-edited rice pollen is derived from transgenic rice or gene-edited rice, the recipient rice pollen is derived from recipient rice, and the transgenic rice or gene-edited rice is obtained by performing transgenic or gene-editing operations on the recipient rice.

[0012] If the difference in germination rate between the transgenic rice or gene-edited rice pollen and the recipient rice pollen is significant, it indicates that the viability of the transgenic rice or gene-edited rice pollen (specifically manifested as the germination rate) has been changed by the transgenic or gene-editing operation.

[0013] If the difference in germination rate between the transgenic rice or gene-edited rice pollen and the recipient rice pollen is not significant, it indicates that the transgenic rice or gene-edited rice is no different from the recipient rice, and the viability of the transgenic rice or gene-edited rice pollen (specifically manifested in the germination rate) has not been changed by the transgenic or gene editing operation.

[0014] The rice obtained by biobreeding technology includes but is not limited to transgenic rice or gene-edited rice.

[0015] The germination rate mentioned in the above method is the percentage of germinated pollen to the number of pollen used for statistics.

[0016] In the above method, the culturing time in the culture medium is 15 minutes; the culturing temperature in the culture medium is 25-37°C (specifically 25°C, 28°C, 31°C, 34°C, 37°C).

[0017] The culture temperature in the culture medium is preferably 31°C.

[0018] The present invention also provides a method for determining whether there is a difference in germination rate between pollen of transgenic or gene-edited rice and its recipient material, comprising the following steps: isolating the pollen of the transgenic or gene-edited rice and the recipient rice pollen in vitro and then culturing them in a culture medium, and detecting the difference in germination rate between the pollen of the transgenic or gene-edited rice and the recipient rice pollen;

[0019] The culture medium is composed of the following substances: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate and 10 mg / L magnesium sulfate, and the balance is distilled water;

[0020] The recipient rice pollen comes from recipient rice, and the transgenic rice or gene-edited rice is rice obtained through biological breeding technology.

[0021] If the difference in germination rate between the transgenic rice or gene-edited rice pollen and the recipient rice pollen is significant, it indicates that the viability of the transgenic rice or gene-edited rice pollen (specifically manifested as the germination rate) has been changed by the transgenic or gene-editing operation.

[0022] If the difference in germination rate between the transgenic rice or gene-edited rice pollen and the recipient rice pollen is not significant, it indicates that the transgenic rice or gene-edited rice is no different from the recipient rice, and the viability of the transgenic rice or gene-edited rice pollen (specifically manifested in the germination rate) has not been changed by the transgenic or gene editing operation.

[0023] The germination rate mentioned in the above method is the percentage of germinated pollen to the number of pollen used for statistics.

[0024] In the above method, the culturing time in the culture medium is 15 minutes; the culturing temperature in the culture medium is 25-37°C (specifically 25°C, 28°C, 31°C, 34°C, 37°C).

[0025] The culture temperature in the culture medium is preferably 31°C.

[0026] In the above method, the environmental temperature of the pollen during in vitro separation is 28°C, and the environment can be provided by an incubator, and the temperature of the incubator can be set to 28°C; the time for in vitro separation is 1-15 minutes (specifically 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 9 minutes, 12 minutes, or 15 minutes).

[0027] The above method specifically includes the following steps:

[0028] Pollen collection: The effects of pollen collection time on the test results were analyzed and compared, and it was found that the most appropriate pollen for vitality determination was collected in the late stage of anther dehiscence preparation, that is, when the anther grew to the upper half of the spikelet and was about to shed pollen outwards. The specific collection method was to take the spikelet that was about to shed pollen, cut off the top of the glume with scissors, and use tweezers to pick out the anther and place it directly in the culture medium.

[0029] Preparation of culture medium: By analyzing and comparing the germination rates of rice pollen under different culture media and culture conditions, the appropriate culture medium formula was determined: using distilled water as the solvent, each 100 mL of culture medium contains 17.5 g of sucrose, 4 mg of boric acid, 2 mg of calcium chloride, 1 mg of potassium dihydrogen phosphate and 1 mg of magnesium sulfate.

[0030] In vitro culture: Place the culture dish in a constant temperature incubator and culture for 15 minutes.

[0031] Observation: Place the Petri dish under a microscope and observe using the microscope. Observe three fields of view per dish. Count the number of germinated pollen and the total number of pollen in each field. The total number of pollen counted per field should be no less than 20, and the total number of pollen observed per treatment should be no less than 180. Pollen is considered germinated if the pollen tube length is greater than half the diameter of the pollen grain.

[0032] Statistical analysis of pollen germination rate: Take photos using a microscope, count using ImageJ software, and calculate the pollen germination rate.

[0033] Secondly, in order to evaluate the effect of in vitro time on pollen vitality, the present invention further analyzed and compared the pollen germination rate after different in vitro times, and found that as the in vitro time increased, the pollen germination rate decreased. There was no obvious difference in the pollen germination rate after 1 minute, 2 minutes, 3 minutes and 4 minutes in vitro, and there was no difference in pollen vitality. As the in vitro time continued to increase, the germination rate continued to decrease, and it decreased significantly after more than 12 minutes, and was completely inactivated after 30 minutes.

[0034] Furthermore, in order to determine the temperature range that rice pollen can tolerate, the present invention analyzed and compared the effects of temperature conditions on the vitality of rice pollen. According to the temperature range of rice flowering period, multiple temperatures such as 16°C, 22°C, 25°C, 28°C, 31°C, 34°C, 37°C, and 40°C were set. The results showed that the most suitable temperature for pollen germination was 31°C. When the temperature was lower than 16°C or higher than 40°C, the pollen would lose its vitality and could not germinate.

[0035] The present invention also provides a method for germinating in vitro rice pollen, comprising placing the in vitro rice pollen in a culture medium and culturing the pollen until the pollen germinates;

[0036] The culture medium is composed of the following substances: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate and 10 mg / L magnesium sulfate, and the balance is distilled water;

[0037] The temperature of the culture medium until the pollen germinates is 31°C;

[0038] The time of culturing in the culture medium until pollen germinates is 15°C;

[0039] The time for isolating the rice in vitro pollen is less than or equal to 5 minutes.

[0040] The time when the rice in vitro pollen is separated from the rice body refers to the time when the pollen leaves the rice body.

[0041] The application of the above culture medium in improving the germination rate of rice pollen also falls within the scope of protection of the present invention.

[0042] Specifically include:

[0043] 1. Sowing genetically modified or gene-edited rice and its recipient rice varieties at the same time.

[0044] 2. During the rice flowering period, select transgenic or gene-edited rice and non-transgenic or gene-edited rice plants with normal growth and consistent status for collecting test pollen.

[0045] 3. Pollen Collection: Three transgenic or gene-edited plants and three corresponding non-transgenic or gene-edited control plants were selected as three biological replicates, with three technical replicates for each biological replicate. Pollen was collected from the late stage of the anther dehiscence preparation phase for viability testing. The specific collection method was to take a spikelet that was about to shed pollen, cut off the tip of the glumes with scissors, remove the anthers with tweezers, and place them directly in culture medium. Morphological observation was performed first. If the pollen was normal, in vitro culture was performed.

[0046] 4. In vitro pollen culture: First, add 20 ml of distilled water to a 90 mm dish. Place dry filter paper in the center of a 35 mm dish. Place nine pollen samples from transgenic or gene-edited plants or their corresponding non-transgenic controls on the dry filter paper in each 35 mm dish. Incubate in a 28°C incubator for 0, 1, 2, 3, 4, 5, 6, 9, 12, 15, 20, or 30 minutes. Transfer the pollen to a 35 mm dish containing liquid culture medium and gently shake to evenly distribute the pollen throughout the dish. Incubate the dishes in a 25°C, 31°C, or 37°C incubator for 15 minutes. Observe pollen germination in each dish using a microscope.

[0047] 5. Pollen germination rate statistics: Observe three fields of view per culture dish and count the number of germinated pollen and the total number of pollen in each field. The total number of pollen counted per field should be no less than 20, and the total number of pollen counted per treatment should be no less than 180. Pollen is considered germinated if the pollen tube length is greater than half the diameter of the pollen grain.

[0048] 6. Results analysis:

[0049] Analysis of control treatment results: In the initial pollen germination test, if the pollen germination rate of the non-GM control group reached 70% or higher, it indicated that the detection system was normal; otherwise, the test was repeated. A one-way analysis of variance was performed on the in vitro germination rates of the GM or gene-edited and non-GM rice pollen under different conditions. If there was no significant difference, it indicated that the GM or gene-editing operation had no effect on pollen viability. If there was a significant difference, it indicated that the GM or gene-editing operation had affected pollen viability.

[0050] Data processing: The average pollen grain size and pollen germination rate of transgenic or gene-edited plants and their corresponding non-transgenic controls were calculated based on the experimental records.

[0051] GraphPad Prism software was used for T-test analysis to compare the differences in pollen grain size and pollen germination rate between transgenic or gene-edited plants and their corresponding non-transgenic controls under the same treatment conditions.

[0052] 7. Presentation of Results: Based on the statistical analysis results, evaluate the difference in pollen viability between the transgenic or gene-edited rice plants and their corresponding non-transgenic controls. Presentation of the results: If the difference in germination rate between the transgenic or gene-edited rice pollen and the recipient rice pollen is significant, it indicates that the viability of the transgenic or gene-edited rice pollen has been altered by the transgenic or gene-editing procedure.

[0053] If the difference in germination rate between the transgenic or gene-edited rice pollen and the recipient rice pollen is not significant, it indicates that the transgenic or gene-edited rice is no different from the recipient rice, and the viability of the transgenic or gene-edited rice pollen has not been changed by the transgenic or gene-editing operation.

[0054] The present invention also provides a culture medium for culturing rice pollen, which is composed of the following substances: 175g / L sucrose, 40mg / L boric acid, 20mg / L calcium chloride, 10mg / L potassium dihydrogen phosphate and 10mg / L magnesium sulfate, with the balance being distilled water.

[0055] Compared with the prior art, the present invention has the following advantages and effects:

[0056] 1. The rice pollen germination medium of the present invention provides the necessary environmental conditions for pollen germination and growth. At the same time, the medium components are simple, and the operation is simple, easy, and fast.

[0057] 2. The present invention provides a method for measuring the viability of in vitro pollen of transgenic or gene-edited rice. It determines three temperatures for comparing pollen viability and three in vitro times. It can be used to evaluate whether there is a difference in pollen viability between transgenic or gene-edited rice and its recipient material, and further used for transgenic environmental safety monitoring, providing assistance for the safety evaluation of transgenic or gene-edited rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The anther positions in four states.

[0059] Figure 2 Pollen germination in four states.

[0060] Figure 3 This is the germination of rice pollen cultured in medium No. 22 at 31°C for 15 minutes.

[0061] Figure 4 Pollen diameters of ZH11, G2-6 and CYP81A6. DETAILED DESCRIPTION

[0062] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0063] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0064] The data in the following examples were processed using GraphPad Prism 8 statistical software. The experimental results were expressed as mean ± standard deviation, and one-way analysis of variance and T-test were used. P < 0.05 (*) indicated a significant difference.

[0065] In the following examples, the pollen germination rate was calculated according to formula (1).

[0066]

[0067] (1) Where:

[0068] Y—pollen germination rate, %;

[0069] n1—number of germinated pollen;

[0070] N—The total number of pollen counted.

[0071] The average pollen grain size was calculated according to formula (2).

[0072]

[0073] (2) Where:

[0074] X—average pollen grain size;

[0075] Xi—diameter of the i-th pollen;

[0076] n—total number of pollen grains.

[0077] Herbicide-tolerant transgenic rice G2-6 and the corresponding non-transgenic rice receptor ZH11: described in the non-patent literature "Yufeng Dong 1 ,Xi Jin 2 ,Qiaoling Tang 1 ,Xin Zhang 1 ,Jiangtao Yang 1 ,XiaojingLiu 1,Junfeng Cai 1 ,Xiaobing Zhang 3 ,Xujing Wang 1 ,Zhixing Wang 1 .Development and Event-specific Detection of Transgenic Glyphosate-resistant Rice Expressing the G2-EPSPS Gene. Front Plant Sci. 2017May 30:8:885.doi:10.3389 / fpls.2017.00885.eCollection 2017. ", which is publicly available from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. This biological material is only for use in repeating the experiments related to the present invention and cannot be used for other purposes.

[0078] Herbicide-tolerant gene-edited rice CYP81A6: recorded in the non-patent literature "Analysis of the Effects of CRISPR-Cas System in Gene-Edited Rice and Corn" DOI: 10.27630 / d.cnki.gznky.2022.000081, the public can obtain it from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0079] As a self-pollinating crop, the in vitro viability of rice pollen is significantly lower than that of cross-pollinating crops such as corn, and it is greatly affected by external factors. Therefore, the selection of pollen with consistent state is crucial to the repeatability and accuracy of the experiment. The inventors have done a lot of work on selecting the state of pollen in the early preliminary experiments and found that weather, time, pollen collection site, etc. can all affect the germination rate of rice pollen. Therefore, the experimental operating conditions were finally determined to be from 11 am to 2 pm on a sunny day. The inventors divided the pollen into four states (four different periods of pollen development) according to the position of the anthers, that is, the anthers are located below the husk ( Figure 1 Middle a), anthers are located above the glumes ( Figure 1 Middle b), the glume has just cracked, revealing the anthers ( Figure 1 Middle c), the anthers are fully exposed and have begun to shed pollen naturally ( Figure 1 d). The preliminary experimental results are as follows Figure 2 Shown, indicating Figure 1 The pollen germination rate in the middle b state is the highest ( Figure 2 b), so this study selected pollen in this state for all experiments. Figure 1 -e, cut off the top of the husk without damaging the anthers, and then use tweezers to gently remove the anthers from the husk.

[0080] Example 1. Screening of Rice Pollen Germination Medium

[0081] Using 175 g / L sucrose, 40 mg / L boric acid, and 20 mg / L calcium chloride as the basic culture medium (the remainder is distilled water), on the basis of the basic culture medium, by adjusting the concentrations of sucrose, calcium chloride, boric acid, potassium dihydrogen phosphate, or magnesium sulfate, different concentrations of sucrose, calcium chloride, boric acid, potassium dihydrogen phosphate, or magnesium sulfate were set to conduct a single-factor experiment. The details are as follows:

[0082] Table 1. Germination rate of rice pollen in culture medium with different sugar concentrations

[0083]

[0084] Table 2. Germination rate of rice pollen in culture medium with different boric acid and calcium chloride concentrations

[0085]

[0086]

[0087] Table 3. Germination rate of rice pollen in culture medium with different PEG4000 concentrations

[0088]

[0089] Table 4. Germination rate of rice pollen in culture medium with different concentrations of potassium dihydrogen phosphate and magnesium sulfate

[0090]

[0091] As shown in Tables 1 to 4, the optimal composition of the culture medium is as follows: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate and 10 mg / L magnesium sulfate, with the remainder being distilled water.

[0092] Prepare mother solutions based on the above-mentioned optimal culture medium. Mother solution I: Weigh 1 g of boric acid, dissolve it in distilled water, and dilute to 1 L. Filter through a 0.22 μm filter, then store in a conical flask. The boric acid concentration is now 1 g / L. Mother solution II: Concentrate calcium chloride 1000-fold. Accurately weigh 20 g of calcium chloride, dissolve it in distilled water, and dilute to 1 L. Filter through a 0.22 μm filter, then store in a conical flask. The calcium chloride concentration is now 20 g / L. Mother solution III: Weigh 1 g of potassium dihydrogen phosphate, dissolve it in distilled water, and dilute to 1 L. Filter through a 0.22 μm filter, then store in a conical flask. The potassium dihydrogen phosphate concentration is now 1 g / L. Mother solution IV: Weigh 1 g of magnesium sulfate, dissolve it in distilled water, and dilute to 1 L. Filter through a 0.22 μm filter, then store in a conical flask. The magnesium sulfate concentration is now 1 g / L. After the mother liquor is prepared, it is stored in a refrigerator for future use without adding sucrose and PEG4000.

[0093] Example 2: Determination of in vitro survival time and pollen viability of rice pollen

[0094] In order to evaluate the effect of in vitro time on pollen viability, this example further analyzed and compared pollen germination rates after different in vitro time periods.

[0095] 1. Between 11:00 and 14:00 on a sunny day, select healthy, disease-free plants, remove the glumes, and collect the anthers that are about to shed pollen.

[0096] 2. Prepare liquid culture medium: Take 40 ml of 1 g / L boric acid (mother solution I), 1 ml of 20 g / L calcium chloride (mother solution II), 10 ml of 1 g / L potassium dihydrogen phosphate (mother solution III), 10 ml of 1 g / L magnesium sulfate, and 175 g of sucrose, add 700 ml of distilled water, stir to dissolve, mix well, and adjust the volume to 1 L.

[0097] 3. In vitro pollen culture: First, add 20 ml of distilled water to a 90 mm dish. Place dry filter paper in a 35 mm dish and place it in the center of the 90 mm dish. Place pollen on the dry filter paper and incubate in a 28°C incubator for 0 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 9 min, 12 min, 15 min, 20 min, and 30 min. Then, transfer the pollen to a 35 mm dish containing liquid culture medium and gently shake to evenly distribute the pollen in the dish. Place the dish in a 31°C incubator for 15 min.

[0098] 4. Microscopic examination: Place the dish under a stereoscope and take photos with a microscope. The pollen germination standard is that the length of the pollen tube is greater than half of the diameter of the pollen grain. Observe three fields of view at a time, with about 20 pollen grains in each field. Statistical analysis of the pollen germination rate is based on three biological replicates for each sample. The result is the pollen germination rate determination result.

[0099] 5. Evaluation: One-way ANOVA was performed on pollen viability at different in vitro times. The results are shown in Table 5.

[0100] Table 5. Germination rate of rice pollen after different in vitro times and 15 minutes of culture on medium 22

[0101] serial number In vitro time (min) Pollen germination rate (%) 26 0 88.57±3.83ab 27 1 85.76±6.19bc 28 2 85.90±2.65b 29 3 83.60±3.68bc 30 4 79.07±3.57bc 31 5 75.84±5.15cd 32 6 67.20±3.19de 33 9 58.58±4.59e 34 12 38.18±6.27f 35 15 17.57±4.21g 36 20 6.38±2.95h 37 30 0.00±0.00h

[0102] The results, as shown in Table 5, show that the pollen germination rate decreased with increasing in vitro time. The results show that the pollen germination rate decreased with time. Compared with pollen isolated at 0 minutes, the germination rates of pollen isolated at 1, 2, 3, and 4 minutes were not significantly different, and there was no difference in pollen viability. The germination rate of pollen isolated after 5 minutes was significantly lower than the initial germination rate, and the germination rate decreased with increasing in vitro time. The germination rate dropped sharply after 12-15 minutes of in vitro isolation, and after 30 minutes of in vitro isolation, the viability was lost and the germination rate dropped to zero. Therefore, the in vitro isolation time for evaluating pollen environmental safety was set at 0 minutes, 5 minutes, and 15 minutes.

[0103] Example 3: Determination of Temperature Conditions for Rice Pollen Germination

[0104] In order to evaluate the effect of temperature on the viability of in vitro pollen, the present invention further analyzed and compared the pollen germination rates after culture at different temperatures, as follows:

[0105] 1. Between 11:00 and 14:00 on a sunny day, select plants that are normally developed, free of pests and diseases, and growing healthily. Cut off the glumes and collect the anthers that are about to shed pollen.

[0106] 2. Prepare liquid culture medium: Take 40 ml of 1 g / L boric acid (mother solution I), 1 ml of 20 g / L calcium chloride (mother solution II), 10 ml of 1 g / L potassium dihydrogen phosphate (mother solution III), 10 ml of 1 g / L magnesium sulfate, and 175 g of sucrose, add 700 ml of distilled water, stir to dissolve, mix well, and adjust the volume to 1 L.

[0107] 3. Pollen Culture: Add 10 ml of liquid culture medium to a 35 mm dish. Gently break the anther with tweezers, quickly place it in the culture medium, and shake it to evenly distribute the pollen in the dish. Perform three replicates for each pollen sample. Place the dish in a constant temperature incubator at 16°C, 22°C, 25°C, 28°C, 31°C, 34°C, 37°C, or 40°C for 15 minutes.

[0108] 4. Microscopic examination: Place the dish under a stereoscope and take photos with a microscope. The pollen germination standard is that the length of the pollen tube is greater than half of the diameter of the pollen grain. Observe three fields of view at a time, with about 20 pollen grains in each field. Statistical analysis of the pollen germination rate is based on three biological replicates for each sample. The result is the pollen germination rate determination result.

[0109] To conduct subsequent safety evaluations of transgenic or gene-edited rice pollen, it is necessary to compare the in vitro viability of transgenic or gene-edited rice pollen with that of conventional rice pollen. To determine the temperature for comparison, a temperature gradient experiment was conducted, as shown in Table 6.

[0110] Table 6. Germination rate of rice pollen after culturing for 15 min at different culture temperatures in medium 22

[0111] serial number Culture temperature (℃) Pollen germination rate (%) 38 16 0±0f 39 22 9.42±2.52e 40 25 39.53±4.57c 41 28 78.18±6.30a 42 31 88.21±5.30a 43 34 64.30±4.54b 44 37 26.90±3.27d 45 40 0.00±0.00f

[0112] The results showed that pollen could not germinate when the culture temperature was 16°C; at 16-31°C, the pollen germination rate increased with increasing temperature, and reached the highest germination rate at 31°C. At 31-40°C, the pollen germination rate decreased with increasing temperature, and pollen could not germinate when the temperature reached 40°C. Therefore, 31°C was selected for subsequent experiments. At the same time, the germination rate of pollen was extremely low at 22°C; the difference between the germination rate at 28°C and the germination rate at 31°C was small. However, the germination rates at 25°C, 34°C and 37°C were significantly different from those at 31°C, among which the difference between 25°C and 37°C was greater than that at 31°C. Therefore, these two temperatures were selected as low-temperature and high-temperature treatment conditions to test the vitality of rice in vitro pollen. The temperature settings for evaluating the safety of the pollen environment were determined to be 25°C, 31°C, and 37°C. The germination of rice pollen cultured at 31°C for 15 minutes in culture medium No. 22 is shown in the figure below. Figure 3 shown.

[0113] Example 4. Pollen Viability Determination and Safety Assessment of Transgenic Rice G2-6 and Gene-Edited Rice CYP816

[0114] In this example, herbicide-tolerant transgenic rice G2-6, gene-edited rice CYP816, and the corresponding non-transgenic rice receptor ZH11 were used as test varieties. They were sown simultaneously at the Nanbin Farm in Yazhou Bay, Sanya, Hainan, with a row spacing of 30 cm and a plant spacing of 12 cm. The purpose was to verify whether transgenic or gene-editing operations had an impact on pollen viability. The specific process was as follows:

[0115] 1. Between 11:00 and 14:00 on a sunny day, select ZH11 and G2-6 rice plants that are developing normally, free of pests and diseases, and about to shed pollen. Cut off the husks and collect the anthers.

[0116] 2. Prepare liquid culture medium: Take 40 ml of 1 g / L boric acid (mother solution I), 1 ml of 20 g / L calcium chloride (mother solution II), 10 ml of 1 g / L potassium dihydrogen phosphate (mother solution III), 10 ml of 1 g / L magnesium sulfate, and 175 g of sucrose, add 700 ml of distilled water, stir to dissolve, mix well, and adjust the volume to 1 L.

[0117] 3. In vitro pollen culture: Add 20 ml of distilled water to a 90 mm dish. Place dry filter paper in a 35 mm dish and place it in the center of the 90 mm dish. Place pollen on the dry filter paper. Place the dish in a 28°C incubator for 0, 5, and 15 minutes. Transfer the pollen to a 35 mm dish containing liquid culture medium and gently shake to evenly distribute the pollen throughout the dish. Incubate for 15 minutes in a 25°C, 31°C, or 37°C incubator.

[0118] 4. Microscopic examination: Place the dish under a stereoscope and take photos with a microscope. The pollen germination standard is that the length of the pollen tube is greater than half of the diameter of the pollen grain. Observe three fields of view at a time, with about 20 pollen grains in each field. Statistical analysis of pollen germination rate and pollen diameter is performed by three biological replicates for each sample.

[0119] 5. Evaluation: Variance analysis was performed on the in vitro pollen germination rates of transgenic rice G2-6, gene-edited rice CYP81A6, and non-transgenic rice ZH11 at different culture temperatures and different in vitro isolation times. The results are shown in Table 7.

[0120] Table 7. In vitro pollen germination rates of transgenic rice G2-6, gene-edited rice CYP81A6, and non-transgenic rice ZH11 after 15 min of incubation in medium 22 at different incubation temperatures and in vitro time.

[0121]

[0122]

[0123] The results in Table 7 show that under nine combinations of culture temperatures of 25°C, 31°C, and 37°C and in vitro time of 0 min, 5 min, and 15 min, there was no significant difference in the in vitro pollen germination rate among transgenic rice GG6-2, gene-edited rice CYP81A6, and non-transgenic rice ZH11 (P>0.05). Therefore, it can be determined that the transgenic or gene editing operation has no effect on the pollen viability of the rice.

[0124] Pollen germination when anther is located below the glumes ( Figure 2 Middle a), Pollen germination when the anther is located above the glume ( Figure 2 Middle b), Pollen germination when the glume just cracks and the anther is exposed ( Figure 3 Middle c), pollen germination when the anthers are fully exposed and have begun to shed pollen naturally ( Figure 4 (d) Pollen diameter Figure 4As shown, a is ZH11 (39.86±2.67μm), b is G2-6 (40.05±2.88μm), and c is CYP81A6 (40.45±2.44), all of which are 37-43μm, with no significant difference.

[0125] It can be seen from this that the safety of transgenic rice can be evaluated by analyzing whether there is a significant difference in the germination rate of in vitro pollen between transgenic rice and conventional rice under different in vitro time and temperature culture conditions through appropriate culture medium.

[0126] It can be seen from this that the safety of genetically modified rice can be evaluated by analyzing whether there is a significant difference in the germination rate of in vitro pollen between gene-edited rice and conventional rice under different in vitro time and temperature culture conditions through appropriate culture medium.

[0127] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for determining the difference in germination rate between pollen of rice obtained by biobreeding technology and that of its recipient material, characterized in that: The method comprises the following steps: culturing pollen of transgenic rice or gene-edited rice and pollen of recipient rice in a culture medium, and detecting the difference in germination rate between the pollen of the transgenic rice or gene-edited rice and the pollen of the recipient rice; the transgenic rice or gene-edited rice is rice obtained through biological breeding technology; The culture medium is composed of the following substances: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate and 10 mg / L magnesium sulfate, with the balance being distilled water.

2. The method according to claim 1, characterized in that The culturing time in the culture medium is 15 minutes; the culturing temperature in the culture medium is 25-37°C.

3. A method for germinating rice pollen in vitro, characterized in that: placing the rice in vitro pollen in a culture medium and culturing it until the pollen germinates; The culture medium is composed of the following substances: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate and 10 mg / L magnesium sulfate, and the balance is distilled water; The temperature of the culture medium until the pollen germinates is 31°C; The time of culturing in the culture medium until pollen germinates is 15°C; The time for isolating the rice in vitro pollen is less than or equal to 5 minutes.

4. A method for determining the difference in germination rate between pollen of rice obtained by biobreeding technology and that of its recipient material, characterized in that: The method comprises the following steps: isolating transgenic rice or gene-edited rice pollen and recipient rice pollen in vitro and then culturing them in a culture medium, and detecting the difference in germination rate between the transgenic rice or gene-edited rice pollen and the recipient rice pollen; The culture medium is composed of the following substances: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate and 10 mg / L magnesium sulfate, and the balance is distilled water; The recipient rice pollen comes from recipient rice, and the transgenic rice or gene-edited rice is rice obtained through biological breeding technology.

5. The method according to claim 4, characterized in that The temperature at which the transgenic rice or gene-edited rice pollen and the recipient rice pollen are placed during the separation is 28° C.; and the separation time is 1-15 minutes.

6. The method according to claim 4, characterized in that The culturing time in the culture medium is 15 minutes; the culturing temperature in the culture medium is 25-37°C.

7. A culture medium for germinating rice pollen, characterized in that: The culture medium is composed of the following substances: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate and 10 mg / L magnesium sulfate, with the balance being distilled water.

8. Use of the culture medium according to claim 7 in improving the germination rate of rice pollen.

9. A method for germinating rice pollen, characterized in that: The isolated rice pollen was placed in a culture medium and cultured until the pollen germinated. The culture medium consisted of the following substances: 175 g / L sucrose, 40 mg / L boric acid, 20 mg / L calcium chloride, 10 mg / L potassium dihydrogen phosphate, and 10 mg / L magnesium sulfate, with the balance being distilled water.

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