Rice stigma vitality determination device and method based on micro gas quantification

Abstract

The application discloses a device and method for determining rice stigma vitality based on micro-gas quantification. The device comprises a reaction tube with a conical bottom and a micro-injection port with an embedded self-healing silica gel pad; an elastic sealing plug sealingly embedded in the open end of the reaction tube to form a closed reaction chamber; a capillary tube vertically penetrating the sealing plug and provided with volume scale lines; and a colored indicator liquid drop preloaded in the capillary tube. The determination method is as follows: placing the stigma to be measured on the conical bottom of the reaction tube, sealing and adjusting to zero, injecting hydrogen peroxide solution through the micro-injection port, recording the volume scale difference of the indicator liquid drop rising due to gas production caused by enzymatic reaction within a predetermined time period, and then calculating the stigma vitality index. The application replaces the traditional subjective observation by microscope with objective physical volume displacement in the closed capillary tube, gets rid of the dependence on large instruments, and realizes the field in-situ, rapid and accurate quantitative determination of rice stigma vitality.

Description

Technical Field

[0001] This invention relates to the fields of agricultural testing instruments and rice physiological technology, and more specifically, to a device and method for measuring rice stigma vigor based on quantitative determination of trace gases. Background Technology

[0002] Stigma vigor refers to the ability of the stigma to receive pollen and perform normal fertilization after flowering. In rice production and breeding, stigma vigor determines the duration of pollen reception after the glume closes and the pollination efficiency, and can jointly affect the final yield of hybrid seed production along with stigma exposing rate. Studies have shown that rice stigma vigor generally reaches its peak on the day of flowering, begins to decline significantly on the 3rd day after flowering, and approaches loss by the 5th to 7th day. Especially in the context of frequent extreme heat weather in recent years, accurate assessment of stigma vigor is particularly important. Under normal circumstances, the heat tolerance threshold for pollen vigor is about 35℃, while the heat tolerance threshold for stigma vigor is relatively strong, at about 41℃. Therefore, under high-temperature stress, the ability to accurately and quickly assess the stigma vigor of different varieties is of great practical significance for screening heat-resistant varieties and guiding field hybrid seed production.

[0003] However, current methods for measuring stigma vigor in rice still have many limitations in practical applications, mainly manifested in the following two types of conventional technical approaches and their inherent defects: The first category is the traditional seed setting rate method: This method mainly involves artificially pollinating the rice plants under test, counting the number of grains after about 15 days, and using the seed setting rate as a measure of stigma vigor. Although this method is the most widely used classic measurement method to date, it has a very serious time lag. Breeders cannot obtain vigor data and make breeding decisions on the day the rice flowers. In addition, the final seed setting rate is not only affected by stigma vigor, but is also easily interfered with by a variety of variables such as pollen vigor, pollen quantity, and external environment. Solely using this to measure stigma vigor has unavoidable systematic errors.

[0004] The second method is microscopic hydrogen peroxide observation: Industry research has found that rice stigmas contain catalase (EC 1.11.1.6), which can catalyze the decomposition of hydrogen peroxide and release oxygen. Based on this biochemical principle, current technology attempts to place the removed stigmas in a hydrogen peroxide solution on a single concave slide, and then time the observation under a 4x field of view of an optical microscope, observing the time required for the bubbles to fill the entire field of view, and using the reciprocal of the time as the stigma viability index. Although this method has been proven to have a positive correlation with seed setting rate based on biochemical principles, its practical operation and measurement methods have the following obvious defects: It heavily relies on precision optical instruments, making in-situ field detection impossible; the microscope is bulky and delicate, making it unsuitable for portable use in complex field environments, requiring samples to be brought back to the laboratory after collection. This not only increases operational complexity but may also lead to stigma dehydration and decreased enzyme activity over time; the judgment criteria are subjective, with significant human error. Using the filling of the entire field of view as the judgment point relies heavily on the operator's subjective visual judgment, making consistency difficult between different operators or batches, resulting in poor data reproducibility; it lacks objective quantitative dimensions, limiting accuracy. Using the reciprocal of time as a viability indicator is a semi-quantitative evaluation system, lacking direct physical measurement of the actual gas volume produced, thus failing to achieve true quantitative determination; and the measurement throughput is extremely low, with long single-measurement times, making it completely unsuitable for the rapid parallel screening needs of modern large-scale breeding materials.

[0005] Given the numerous shortcomings of existing assessment methods, such as evaluation lag, susceptibility to interference, reliance on expensive and inconvenient instruments, subjective judgment, and lack of direct gas volume dimensions, there is an urgent need in this field for a rice stigma vigor measurement device and method that can eliminate reliance on microscopes, eliminate subjective interpretation errors, and provide rapid, objective, quantitative, and portable field use. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide a rice stigma vigor measuring device based on trace gas quantification, so as to solve the technical problems in the prior art that the measurement of rice stigma vigor relies heavily on microscopes, which makes it impossible to use in the field, and that bubble observation has subjective errors and lacks direct objective quantitative means.

[0007] To overcome the shortcomings of the prior art, the present invention provides a device for determining rice stigma vigor based on quantitative determination of trace gases, comprising: The reaction tube has an internal cavity with one end open, the bottom of the reaction tube is conical, the lower side wall of the reaction tube is provided with a micro-injection port, the micro-injection port is embedded with a self-healing silicone pad, and the effective gas space of the inner cavity of the reaction tube is 0.5 mL-10 mL. An elastic sealing plug is fitted into the open end of the reaction tube to seal the internal cavity and form a closed reaction chamber. A capillary tube is vertically inserted into the elastic sealing plug and forms an airtight fit with it. The lower end of the capillary tube is suspended in the reaction chamber and does not contact the bottom of the reaction tube. The upper end of the capillary tube is connected to the outside atmosphere. The capillary tube wall is provided with volume scale lines. A colored indicator droplet is pre-placed in the inner cavity of the capillary and located within a tube section with the volume scale lines.

[0008] Compared with existing technologies, the present invention provides a method for determining rice stigma vigor based on quantitative determination of trace gas volume. This invention replaces the subjective interpretation method of relying on microscopic observation of microscopic bubbles in existing technologies with objective quantitative determination of gas volume based on a sealed capillary. The reaction tube and the elastic sealing plug are fitted together to construct a sealed reaction chamber. When the injected reagent reacts with the stigma to produce oxygen through an enzymatic reaction, the gas pressure in the sealed chamber increases accordingly, directly pushing the colored indicator droplet in the capillary vertically inserted in the sealing plug to move upward. The macroscopic physical volume displacement of the droplet accurately reflects the biochemical reaction rate, eliminating the dependence on large optical instruments and eliminating human visual interpretation errors. The conical structure at the bottom of the reaction tube causes the tiny stigmas to naturally settle and converge at the center under gravity, avoiding reactant dispersion and dead zones where air bubbles accumulate at the bottom. This structure works in conjunction with the micro-injection port, which has a self-healing silicone pad embedded in the lower sidewall. This allows the operator to inject liquid to initiate the reaction after assembling the capillary tube and sealing plug and adjusting the droplet to achieve zero pressure. This effectively eliminates initial pressure fluctuations caused by assembly pressure or top-opening injection, ensuring the absolute accuracy of the zero point measurement. The lower end of the capillary tube, suspended inside the reaction chamber, avoids direct contact with the bottom reaction liquid, preventing blockage or siphoning. The close collaboration of the various structural features of this invention gives the overall structure extremely high portability and data stability. It can rapidly obtain objective and quantitative results of rice stigma vigor measurement in an in-situ field environment, effectively overcoming the application difficulties of traditional seed setting rate methods and conventional biochemical methods that cannot be applied in the field.

[0009] In one possible implementation, the structural parameters of the reaction tube include: an inner diameter of 5 mm-15 mm and a tube length of 30 mm-80 mm.

[0010] Compared with existing technologies, this embodiment can control the sensitivity of the measurement system to trace gases and the stability of the measurement range in an optimal dynamic balance: when the effective gas space is less than 0.5 mL, the system is extremely sensitive to interference from the external environment. The micro-pressure fluctuations generated at the moment of injection by the micro-syringe can easily cause the indicator droplet to rush out of the capillary range, resulting in measurement failure. When the effective gas space is greater than 10 mL, the gas chamber buffer volume of the entire closed system is too large, and the contribution of the weak enzymatic gas production of the column head to the overall gas pressure increment of the system will be severely diluted, resulting in the actual displacement of the droplet being lower than the minimum scale accuracy that can be distinguished by the naked eye. With the inner diameter limited to 5 mm-15 mm and the tube length to 30 mm-80 mm, it is ensured that the limited gas production of the rice column head can be accurately and stably converted into a macroscopic liquid column displacement visible to the naked eye in the capillary, thereby improving the true accuracy of physical quantification of trace biochemical reactions.

[0011] In one possible implementation, the cone angle of the cone-shaped bottom is 45°-90°; the elastic sealing plug is an elastic plug with a Shore hardness of 30A-50A.

[0012] Compared with existing technologies, in this embodiment, the cone angle design of 45° to 90° allows the extremely small amount of added hydrogen peroxide solution to be completely submerged at the maximum liquid depth, sinking to the tiny column at the bottom by gravity. This avoids insufficient enzymatic reaction due to a shallow liquid surface. At the same time, this slope threshold effectively prevents capillary adhesion to the device wall and dead zones of bubble stagnation caused by an excessively small cone angle, ensuring that the generated oxygen floats smoothly into the upper gas chamber. The elastic plug with a Shore hardness limited to 30A to 50A provides moderate deformation rebound force, ensuring that it forms a long-term reliable pressure-resistant sealing barrier with the inner wall of the rigid reaction tube, preventing the escape of micro-pressure gas from interfering with the measurement value. It also ensures that the operator can smoothly insert and remove the plug by hand without any auxiliary tools in complex field environments. The structural parameters of both components work together to ensure the measurement accuracy and smoothness of the instrument under harsh field conditions.

[0013] In one possible implementation, the size range of the capillary includes an inner diameter of 0.3 mm to 1.0 mm and the volume scale range is 10 μL to 100 μL.

[0014] Compared with existing technologies, the above-mentioned technical solution can linearly amplify extremely small changes in the volume of a confined gas space into a highly significant physical displacement. The capillary inner diameter of 0.3 mm to 1.0 mm maintains extremely low fluid resistance while overcoming the surface tension of the droplets. An excessively large inner diameter would result in insufficient gas pressure thrust, causing reading lag; an excessively small inner diameter would easily cause droplets to split or adhere to the wall under pressure impact. This specific inner diameter range ensures excellent displacement sensitivity, allowing even a small increase in gas volume to be converted into a significant liquid column rise of several millimeters. Furthermore, the 10 μL-100 μL scale range precisely covers the peak gas production range of stigmas at different vigor states during the rice flowering period within the conventional measurement time. This avoids frequent boundary crossings caused by an excessively small range or dense, indistinguishable scale lines caused by an excessively large range, providing breeders with the most suitable visual and fluid dynamics basis for quickly reading objective data. 。

[0015] In one possible implementation, the colored indicator droplet is a low surface tension silicone oil droplet with added food-grade pigment, the surface tension of the low surface tension silicone oil droplet being less than 20 mN / m and the length being 2 mm-5 mm.

[0016] Compared with existing technologies, the silicone oil material with a surface tension of less than 20 mN / m greatly reduces the wetting friction between the droplet and the inner wall of the capillary, ensuring that the droplet can exhibit extremely high physical follow-up when facing slight changes in air pressure in a closed chamber. It will not produce sliding resistance or hysteresis due to adhesion to the tube wall. The droplet length of 2 mm-5 mm provides sufficient visual observation area and color saturation while strictly limiting the mass of the droplet itself to the microgram level. Combined with the characteristics of low surface tension, it ensures that in the normal handheld operation state, the component of the droplet's own weight along the tube axis is much smaller than the wetting friction between it and the tube wall, preventing the droplet from spontaneously slipping and losing its position due to the tilt of the device.

[0017] Another technical problem to be solved by the present invention is to provide a method for determining rice stigma vigor based on trace gas quantification, so as to solve the problems of serious time lag and susceptibility to environmental and pollen interference in the existing seed setting rate method, and the cross-temporal and spatial measurement difficulties of biochemical detection methods that rely on large instruments and lack objective quantitative calculation standards.

[0018] To overcome the shortcomings of the prior art, this invention provides a method for determining rice stigma vigor based on trace gas quantification. Using the aforementioned trace gas quantification method for determining rice stigma vigor, the method includes the following steps: S1. Sampling and loading: Place N stigmas of the rice to be tested at the conical bottom of the reaction tube, where N is a positive integer not less than 3; S2, Sealing and Zeroing: The elastic sealing plug is sealed and fitted into the open end of the reaction tube to form a sealed reaction chamber. The colored indicator droplet in the capillary is adjusted so that its lower edge is aligned with the starting zero mark of the volume scale line. S3. Injection activation: Use a microsyringe to pass through the self-healing silicone pad of the microsyringe inlet. After inserting the needle, visually confirm that the colored indicator droplet is still at the zero mark before quantitatively injecting hydrogen peroxide aqueous solution into the sealed reaction chamber. After the hydrogen peroxide aqueous solution immerses the column head, remove the microsyringe. S4. Quantitative reading: Start timing immediately after the liquid injection is completed, and record the volume difference of the colored indicator droplet rising in the capillary tube during a predetermined time T seconds to obtain the gas production volume V. S5. Calculate vitality: Calculate the rice stigma vitality index I based on the gas production volume V, the predetermined duration T, and the number of stigmas N.

[0019] Compared with existing technologies, the present invention provides a quantitative method for determining rice stigma vigor based on trace gas, which has the following advantages: The present invention replaces the existing methods of delayed evaluation (requiring a 15-day wait for counting the number of grains) and semi-quantitative methods (requiring subjective judgment of bubble area using a microscope) with an instantaneous quantitative calculation method that directly measures the objective physical displacement within a closed capillary tube in the field. In stage S2, the gas chamber of the system is pre-sealed and aligned with the zero point of the droplet. Then, in stage S3, the reaction is activated by puncture and injection. This sequential logic of first sealing and zeroing, then dynamically activating, cleverly eliminates the cavity-related issues caused by the instantaneous pressing of the sealing plug. The interference of sudden changes in internal gas pressure on the initial zero point ensures the absolute isolation and stability of the initial state of the reaction system. In the S4-S5 stage, by strictly controlling the reaction time and the number of stigma samples, the rate of enzymatic gas production at the biochemical microscale is transformed into a macroscopic physical volume difference that is visible to the naked eye, and finally the vigor index is calculated with a unified dimension. The entire measurement process reduces the time taken from several days to less than 5 minutes, completely eliminating external interference variables such as pollen quantity and temperature fluctuations. It provides breeders with an objective and highly reproducible standardized testing procedure for assessing the degree of damage from high temperature stress and screening heat-resistant varieties on the day of rice flowering.

[0020] In one possible implementation, in step S1, the N stigmas of the rice to be tested are florets that have been pollinated and taken from any day from the day the rice plant blooms to the 5th day after blooming, and are stigmas with the style portion removed.

[0021] Compared with existing technologies, the above-mentioned technical solution can accurately pinpoint the entire effective biological cycle of rice stigma vigor changes and minimize biochemical interference from non-target tissues. In this embodiment, the sampling time window is set from the day of flowering to the 5th day after flowering, which fully covers the critical decline process of rice stigma from the peak of vigor to the tendency to lose it. This provides the most distinguishable time dimension data for the screening of heat-resistant varieties. The requirement to strictly remove the style and take the spikelets in a specific state directly eliminates the influence of pollen itself and other endogenous enzymes in the style, ensuring that the oxygen ultimately catalyzed in the closed system comes entirely and exclusively from the target stigma tissue, thus improving the biological specificity and longitudinal comparability of the test results of different batches.

[0022] In one possible implementation, in step S3, the hydrogen peroxide aqueous solution is a hydrogen peroxide aqueous solution with a volume fraction of 1%-5%, and the injection volume of the quantitative injection is 100 μL-500 μL.

[0023] Compared with existing technologies, in this embodiment, the concentration of hydrogen peroxide aqueous solution with a volume fraction of 1%-5% ensures that the substrate in the reaction system is fully saturated, so that the gas production rate of the enzymatic reaction can truly reflect the absolute content of the enzyme in the column head. It also avoids protein denaturation and inactivation or violent bubble boiling interference caused by excessive concentration. With the quantitative injection of 100 μL-500 μL, the liquid volume is precisely matched with the volume space of the conical bottom of the reaction tube, ensuring that all test columns are completely submerged to achieve a reaction without dead zones, while not excessively occupying the effective gas space reserved above, thus maintaining the extremely high hydrodynamic sensitivity of the entire sealed gas chamber to physical volume displacement.

[0024] In one possible implementation, step S4, after obtaining the gas production volume V, further includes a temperature correction step, wherein the temperature correction step is: according to formula V 校正 =V 读数 ×(273+T0) / (273+T 测定 The readings are corrected for temperature, where T0 is the reference temperature, and T... 测定 This is the actual measured ambient temperature.

[0025] Compared with existing technologies, the above-mentioned technical solution can effectively eliminate the physical errors caused by the thermal expansion and contraction of gases due to drastic fluctuations in field environmental temperature. In this embodiment, based on the principle of the ideal gas law, the measured field environmental temperature varies at different times or locations. The reading volume defined by the indicator droplet in the closed capillary tube not only includes the actual amount of oxygen generated by the enzymatic reaction, but also contains the false displacement caused by the thermal expansion or contraction of the gas in the original closed chamber. By introducing this temperature correction formula, the measured readings are uniformly converted to the set reference temperature, eliminating the pressure variables caused by non-biochemical factors. This allows for parallel and objective comparison of large-scale field measurement data across days and regions on the same baseline, ensuring the rigor of vitality assessment data under specific meteorological conditions such as high-temperature stress.

[0026] In one possible implementation, in step S5, the rice stigma vigor index I is calculated using the formula I = V / (T × N), where the unit of I is μL·s. -1 ·stigma -1 V is the gas production volume in μL, T is in seconds, and N is the total number of column heads used in a single measurement.

[0027] Compared with existing technologies, this embodiment uses the above-mentioned technical solution to establish a completely standardized objective quantitative measurement system for stigma vigor. The total volume of macroscopic gas production is normalized to the reaction time and the total number of stigma samples, directly converting the absolute volume reading, which is limited by specific operating conditions, into an index characterizing the inherent gas production rate of a single stigma per unit time. The establishment of this biochemical dimension in this embodiment not only allows breeders to flexibly adjust the measurement time span and sampling quantity to obtain the most readable volume range according to the enzyme activity level of different varieties or the weakening of vigor in the later stages of flowering, but also ensures that the final calculated vigor index has a high degree of consistency and universality, breaking the calculation bottleneck of the traditional time reciprocal method, which cannot achieve accurate conversion across experimental groups.

[0028] In one possible implementation, after the measurement is completed, the device cleaning step is also included: rinsing the inner cavity of the reaction tube with distilled water 3 times and then air-drying it, rinsing the capillary tube with anhydrous ethanol and then blowing it dry, and replacing the self-healing silicone pad of the micro-injection port after a total of 10 punctures.

[0029] The present invention also includes the application of the rice stigma vigor assay device based on trace gas quantification in the field for rapid on-site quantitative detection of rice stigma catalase activity under field conditions without microscope equipment.

[0030] The present invention also includes the method for determining rice stigma vigor based on trace gas quantification, which allows for real-time evaluation of stigma vigor on the day of high temperature stress during rice flowering, to assist in the selection of heat-resistant varieties or in the decision-making of flowering management in hybrid seed production. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the main structure of a portable device provided in an embodiment of the present invention; Figure 2 A longitudinal cross-sectional view of the portable device provided in an embodiment of the present invention along its central axis; Figure 3 This is a three-dimensional exploded view of the portable device provided in an embodiment of the present invention; Figure 4 This is a top view of the portable device provided in an embodiment of the present invention.

[0032] Figure label: 1. Reaction tube; 11. Conical bottom; 12. Micro-injection port; 2. Elastic sealing plug; 3. Capillary tube; 31. Volume scale line; 32. Colored indicator droplet; 4. Reaction chamber. Detailed Implementation

[0033] First, those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make adjustments as needed to adapt to specific application scenarios.

[0034] In the description of the embodiments of the present invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "connected" and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of the present invention based on the specific circumstances.

[0035] In embodiments of the present invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0036] This invention provides a device for determining rice stigma vigor based on trace gas quantification, comprising: The reaction tube 1 has an internal cavity with one end open. The bottom of the reaction tube 1 is a conical bottom 11. The lower side wall of the reaction tube 1 is provided with a micro-injection port 12. The micro-injection port 12 is embedded with a self-healing silicone pad. The effective gas space inside the cavity of the reaction tube 1 is 0.5 mL-10 mL. An elastic sealing plug 2 is fitted into the open end of the reaction tube 1 to seal the internal cavity and form a closed reaction chamber 4; The capillary tube 3 is vertically inserted into the elastic sealing plug 2 and forms an airtight fit with it. The lower end of the capillary tube 3 is suspended in the reaction chamber 4 and does not contact the bottom of the reaction tube 1. The upper end of the capillary tube 3 is connected to the external atmosphere. The tube wall of the capillary tube 3 is provided with volume scale lines 31. A colored indicator droplet 32 ​​is pre-placed in the inner cavity of the capillary tube 3 and located in the tube section with the volume scale line 31.

[0037] As a preferred embodiment, the reaction tube 1 includes specific structural parameters, namely: an inner diameter of 5 mm-15 mm and a tube length of 30 mm-80 mm.

[0038] As a preferred embodiment, the cone angle of the conical bottom 11 is 45°-90°; the elastic sealing plug 2 is an elastic plug with a Shore hardness of 30A-50A.

[0039] As a preferred embodiment, the capillary 3 includes a specific size range, wherein the inner diameter is 0.3 mm to 1.0 mm and the volume scale line 31 has a range of 10 μL to 100 μL.

[0040] As a preferred embodiment, the colored indicator droplet 32 ​​is a low surface tension silicone oil droplet with added food-grade pigment. The surface tension of the low surface tension silicone oil droplet is less than 20 mN / m, and its length is 2 mm-5 mm.

[0041] This invention provides a method for determining rice stigma vigor based on trace gas quantification. Using the aforementioned trace gas quantification method for determining rice stigma vigor, the method includes the following steps: S1. Sampling and loading: Place N stigmas of the rice to be tested at the conical bottom 11 of the reaction tube 1, where N is a positive integer not less than 3; S2, sealing and zeroing: The elastic sealing plug 2 is sealed and fitted into the open end of the reaction tube 1 to form the sealed reaction chamber 4, and the colored indicator droplet 32 ​​in the capillary tube 3 is adjusted so that its lower edge is aligned with the starting zero mark of the volume scale line 31. S3. Injection activation: Use a microsyringe to pass through the self-healing silicone pad of the microsyringe inlet 12. After inserting the needle, visually confirm that the colored indicator droplet 32 ​​is still at the zero mark before quantitatively injecting hydrogen peroxide aqueous solution into the sealed reaction chamber 4. After the hydrogen peroxide aqueous solution immerses the column head, remove the microsyringe. S4. Quantitative reading: Start timing immediately after the liquid injection is completed, record the volume difference of the colored indicator droplet 32 ​​rising in the capillary 3 at a predetermined time T seconds, and obtain the gas production volume V. S5. Calculate vitality: Calculate the rice stigma vitality index I based on the gas production volume V, the predetermined duration T, and the number of stigmas N.

[0042] As a preferred embodiment, in step S1, the N stigmas of the rice to be tested are florets that have been pollinated and taken from any day from the day the rice plant blooms to the 5th day after blooming, and are stigmas with the style removed.

[0043] As a preferred embodiment, in step S3, the hydrogen peroxide aqueous solution is a hydrogen peroxide aqueous solution with a volume fraction of 1%-5%, and the quantitative injection volume is 100 μL-500 μL. As a preferred embodiment, in step S4, after obtaining the gas production volume V, a temperature correction step is further included, wherein the temperature correction step is: according to formula V... 校正 =V 读数 ×(273+T0) / (273+T 测定 The readings are corrected for temperature, where T0 is the reference temperature, and T... 测定 This is the actual measured ambient temperature.

[0044] As a preferred embodiment, in step S5, the rice stigma vigor index I is calculated using the formula I = V / (T × N), where the unit of I is μL·s. -1 ·stigma -1 V is the gas production volume in μL, T is in seconds, and N is the total number of column heads used in a single measurement.

[0045] As a preferred embodiment, after the measurement is completed, the device cleaning step is also included: rinsing the inner cavity of the reaction tube 1 with distilled water 3 times and then air-drying it, rinsing the capillary tube 3 with anhydrous ethanol and then blowing it dry, and replacing the self-healing silicone pad of the micro-injection port 12 after a total of 10 punctures.

[0046] The present invention also includes the application of the rice stigma vigor assay device based on trace gas quantification in the field for rapid on-site quantitative detection of rice stigma catalase activity under field conditions without microscope equipment.

[0047] The present invention also includes the method for determining rice stigma vigor based on trace gas quantification, which allows for real-time evaluation of stigma vigor on the day of high temperature stress during rice flowering, to assist in the selection of heat-resistant varieties or in the decision-making of flowering management in hybrid seed production.

[0048] The following are embodiments incorporating specific data to further elaborate on the above-described technical solutions of the present invention: Example 1 This embodiment provides a specific overall structure of a rice stigma vigor determination device based on trace gas quantification.

[0049] Please see Figures 1 to 4 ,like Figure 1 Assembly overall view and Figure 2 As shown in the sectional view, the device mainly consists of three parts: reaction tube 1, elastic sealing plug 2, and capillary tube 3. Its three-dimensional explosive assembly relationship is shown in the figure. Figure 3 See the top view section relationship. Figure 4 .

[0050] Specifically, the reaction tube 1 is made of high-transparency polycarbonate (PC) material, with the following structural parameters: outer diameter 10 mm, inner diameter 8 mm, and total tube length 50 mm; the total internal cavity volume is approximately 2.51 mL. The bottom of the reaction tube 1 is a conical bottom 11 with a cone angle of 60°. After injecting 200 μL of hydrogen peroxide solution, the effective gas space sealed above the liquid surface is approximately 2.0 mL-2.2 mL. A micro-injection port 12 is provided on the lower side wall of the reaction tube 1 near the conical bottom. The micro-injection port 12 is embedded with a 3 mm thick self-healing medical silicone pad, which can withstand repeated punctures by a 25-gauge needle and automatically seals, without leakage at working pressures below 0.2 MPa.

[0051] The elastic sealing plug 2 is made of natural rubber with a Shore hardness of 40A. It is shaped like a frustum, narrow at the top and wide at the bottom, and is fitted into the opening end of the reaction tube 1 to seal the internal cavity and form the reaction chamber 4.

[0052] The capillary tube 3 is made of borosilicate glass and is sealed through the center of the elastic sealing plug 2. Its structural dimensions are: inner diameter 0.5 mm, outer diameter 1.5 mm, and effective scale section length 80 mm. The lower end of the capillary tube 3 is suspended at a height of ≥5 mm from the lowest point of the conical bottom 11 (without touching the bottom of the tube), and the upper end is connected to the external atmosphere. The volume scale line 31 on the tube wall has a range of 0-50 μL and a minimum division value of 1.0 μL, and is calibrated by a micro-syringe quantitative gas injection method (1 μL corresponds to a liquid column displacement of approximately 5.1 mm).

[0053] The inner cavity of the capillary 3 is pre-filled with a 3 mm long colored indicator droplet 32. This droplet is a low surface tension dimethyl silicone oil droplet (viscosity 5 cSt, surface tension approximately 19.7 mN / m, density 0.91 g / cm³) containing 0.1% food-grade red pigment. 3 It weighs approximately 0.54 mg and is located within the 31-range of the volume scale.

[0054] Example 2 This embodiment provides a method for determining rice stigma vigor based on trace gas quantification. The method utilizes the measuring device described in Embodiment 1 and includes the following steps: S1. Sampling and loading: During the peak flowering period of rice, collect the spikelets of rice plants that have been naturally pollinated on the day of flowering. Use tweezers to remove the stigmas of 5 spikelets and remove the style. Place the N stigmas (N=5) of the rice to be tested into the conical bottom 11 of the reaction tube 1. The stigmas will naturally sink to the center of the bottom under the action of gravity.

[0055] S2. Sealing and Zeroing: The elastic sealing plug 2 is sealed and fitted into the open end of the reaction tube 1 to form a sealed reaction chamber 4. The colored indicator droplet 32 ​​in the capillary tube 3 is adjusted by gently blowing air into the upper end of the capillary tube 3 so that its lower edge is aligned with the starting zero mark of the volume scale line 31, thus completing the zeroing.

[0056] S3. Injection Activation: Using a microsyringe equipped with a 25-gauge needle, slowly and vertically insert it through the self-healing silicone pad of the microsyringe inlet 12. After inserting the needle, visually confirm that the colored indicator droplet 32 ​​is still at the zero mark. Precisely and quantitatively inject 200 μL of 3% hydrogen peroxide aqueous solution into the sealed reaction chamber 4, ensuring that the hydrogen peroxide aqueous solution completely submerges the column head. Then, remove the microsyringe needle, and the silicone pad will automatically seal.

[0057] S4. Quantitative reading: Start timing immediately after the liquid injection is completed, and record the volume difference of the colored indicator droplet 32 ​​rising in the capillary 3 during a predetermined time T seconds (T=180 s) to obtain the gas production volume V (in μL).

[0058] S5. Calculate Vigor: Based on the gas production volume V, the predetermined duration T, and the number of column heads N, calculate according to the formula... The rice stigma vigor index I (unit: μL·s) is calculated using the formula I = V / (180 × 5). -1 ·stigma -1 ).

[0059] In practical applications, based on the experimental verification data of the microscopic hydrogen peroxide method (see Tables 2 and 3), the stigma vigor index of each material in the Japonica rice male-sterile line on the day of flowering was approximately 28.89-34.23 s. -1 The theoretical gas production rate for a single column head is approximately 0.12–0.17 μL·s. -1 ·stigma -1 When N=5 and T=180 s, the expected total gas production volume is approximately 108 μL-153 μL. The operator can shorten T to 60 s (expected to be approximately 36 μL-51 μL) to achieve a reading within the 0 μL-50 μL range, or adjust N appropriately to obtain the optimal reading range. After the measurement, rinse the inside of reaction tube 1 three times with distilled water and air dry. Rinse capillary tube 3 with anhydrous ethanol and then blow dry for later use.

[0060] To further verify the scientific validity and accuracy of the underlying core biochemical principle of "characterizing rice stigma vigor by using the rate of gas production induced by catalase" upon which this invention is based, the applicant conducted a systematic background benchmark verification and correlation analysis.

[0061] First, the applicant used the traditional "seed setting rate method," a widely accepted method in the agricultural field, as the verification benchmark, and selected stable two-line male-sterile japonica rice TS948, TS992, TS9211, TS256, TS248, and DH9070 (with DR901 as the male parent) as experimental materials. From the day of flowering to the 5th day after flowering, 5 main panicles were collected daily for artificial pollination. The seed setting rate was calculated 15 days after pollination, and the results are shown in the table below: Table 1. Results of stigma vigor determination after flowering in rice using the seed setting rate method (%) Note: The above data are the average seed setting rate of 5 spikelets. The seed setting rate of each material reaches its maximum on the day of flowering and then decreases over time.

[0062] Concurrently, the applicant used the microscopic hydrogen peroxide method to observe the gas production rate of the same material under the same time gradient. The stigmas of pollinated spikelets after flowering on the same day were placed on a single concave slide, and a 3% hydrogen peroxide solution was added. The time it took for the bubbles to fill the field of view was recorded under a 4x optical microscope, and the viability index was set at 10000 / time (s). The results are shown in the table below: Table 2. Determination of stigma vigor index of rice after flowering (10000 / hour, s) by microscopic hydrogen peroxide method -1 ) Note: The stigma vigor index decreases monotonically with the number of days after flowering, which is highly consistent with the trend of the fruit set rate in Table 1.

[0063] Furthermore, a CORREL correlation analysis was performed on the data obtained by the two methods described above, and the results are shown in the table below: Table 3. Correlation analysis results between seed setting rate and stigma vigor index Note: ** indicates a highly significant positive correlation at the 0.01 significance level (CORREL function, two-tailed test). The above results indicate that the stigma vigor determination by the microscopic hydrogen peroxide method is closely correlated with the traditional seed setting rate method (total r = 0.9186, p < 0.01), demonstrating high reliability and consistency in describing rice stigma vigor. This data fully confirms the absolute reliability of the biochemical determination principle upon which this invention is based. Examples 1 and 2 of this invention are based on this scientific foundation, cleverly transforming microscopic subjective visual interpretation into macroscopic objective physical volume displacement, thereby achieving high-precision quantification in the field.

[0064] Furthermore, to verify the stability of the measuring device of this invention under complex field conditions, a corresponding theoretical force analysis was conducted. The mass of the pre-placed colored indicator droplet 32 ​​in this invention is approximately m ≈ 0.54 mg = 5.4 × 10⁻⁶. -7 kg, when tilted at 15°, the component of gravity along the tube axis F = m × g × sin15° = 5.4 × 10 -7 ×9.8×0.259≈1.4×10 -6 Therefore, within the normal handheld operation range of ±15°, the droplet will not spontaneously shift due to the tilt of the device, and the reading stability is theoretically guaranteed.

[0065] Finally, regarding the specificity of the detection reagent, the applicant also conducted a comparative verification of the benzidine enzyme method. This comparative example used benzidine solution (0.5%-5% by volume, with anhydrous ethanol as the solvent) as the detection reagent to detect benzidine enzyme activity in rice stigmas. In the concentration gradient experiments from 0.5% to 5%, no blue substance was observed, indicating that rice stigmas do not contain benzidine enzyme or its activity is too low, making this detection method infeasible. The experimental results of this comparative example further confirm the biological specificity and uniqueness of using catalase as an indicator of stigma activity detection.

[0066] The above embodiments and verifications further demonstrate that this invention successfully bridges the technological gap between laboratory biochemical theory and in-situ field detection. The preliminary background verification and comparative experiments not only solidified the scientific foundation of the high correlation between enzymatic gas production rate and the true viability of rice stigmas at the underlying logic level, but also confirmed the specificity of this particular detection system and its mechanical stability in field operation. With this solid data and theoretical support, the core innovation of this invention is fully revealed: it cleverly abandons the subjective and spatially limited visual interpretation of traditional microscopes, creatively introducing the physical means of sealed micro-gas volume replacement to objectively quantify microscopic biochemical reactions; through the cone-shaped bottom's seamless convergence of tiny stigmas, the anti-interference mechanism of first sealing and zeroing, followed by stimulation through a self-healing silicone pad, and the highly sensitive physical homing of low surface tension droplets, the components work closely together in structure and fluid dynamics, completely eliminating systematic errors caused by sudden changes in gas pressure and human vision. This design, which directly transforms microscopic enzymatic reactions into macroscopic and intuitive intra-tube liquid column displacement, frees breeders from dependence on expensive large instruments. With just a lightweight and portable device, they can quickly, accurately, and objectively obtain quantitative data in the field, effectively filling the technological gap of previous detection methods that were severely lagging behind, subjective in judgment, and unable to be measured in the field.

[0067] In the description of the embodiments of the present invention, it should be noted that the terms "inner" and "outer" and other terms indicating the direction or positional relationship are based on the direction or positional relationship shown in the drawings. This is only for the convenience of description and does not indicate or imply that the device or component must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of the present invention.

[0068] In the description of this invention, the references to "one embodiment," "some embodiments," "in this embodiment," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0069] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A device for determining rice stigma vigor based on trace gas quantification, characterized in that, include: The reaction tube (1) has an internal cavity with one end open. The bottom of the reaction tube (1) is a conical bottom (11). The lower side wall of the reaction tube (1) is provided with a micro-injection port (12). The micro-injection port (12) is embedded with a self-healing silicone pad. The effective gas space inside the cavity of the reaction tube (1) is 0.5 mL-10 mL. An elastic sealing plug (2) is fitted into the opening end of the reaction tube (1) to seal the internal cavity and form a closed reaction chamber (4). The capillary tube (3) is vertically inserted into the elastic sealing plug (2) and forms an airtight fit with it. The lower end of the capillary tube (3) is suspended in the reaction chamber (4) and does not contact the bottom of the reaction tube (1). The upper end of the capillary tube (3) is connected to the external atmosphere. The capillary tube (3) has volume scale lines (31) on its tube wall. Colored indicator droplets (32) are pre-placed in the inner cavity of the capillary (3) and located in the tube section with the volume scale line (31).

2. The rice stigma vigor determination device based on trace gas quantification according to claim 1, characterized in that, The structural parameters of the reaction tube (1) include: an inner diameter of 5 mm-15 mm and a tube length of 30 mm-80 mm.

3. The rice stigma vigor determination device based on trace gas quantification according to claim 1, characterized in that, The cone angle of the cone-shaped bottom (11) is 45°-90°; the elastic sealing plug (2) is an elastic plug with a Shore hardness of 30A-50A.

4. The rice stigma vigor determination device based on trace gas quantification according to claim 1, characterized in that, The size range of the capillary (3) includes an inner diameter of 0.3 mm to 1.0 mm and a volume scale (31) range of 10 μL to 100 μL.

5. The rice stigma vigor determination device based on trace gas quantitative analysis according to claim 1, characterized in that, The colored indicator droplet (32) is a low surface tension silicone oil droplet with added food-grade pigment. The surface tension of the low surface tension silicone oil droplet is less than 20 mN / m, and the length is 2 mm-5 mm.

6. A method for determining rice stigma vigor based on trace gas quantification, characterized in that, Using the rice stigma vigor determination device based on trace gas quantification as described in any one of claims 1-5, the method for determining rice stigma vigor includes the following steps: S1. Sampling and loading: Place the N stubs of the rice to be tested at the conical bottom (11) of the reaction tube (1), where N is a positive integer not less than 3; S2, sealing and zeroing: the elastic sealing plug (2) is sealed and fitted into the open end of the reaction tube (1) to form the sealed reaction chamber (4), and the colored indicator droplet (32) in the capillary tube (3) is adjusted so that its lower edge is aligned with the starting zero mark of the volume scale line (31). S3, Injection activation: Use a microsyringe to pass through the self-healing silicone pad of the microsyringe inlet (12). After inserting the needle, visually confirm that the colored indicator droplet (32) is still at the zero mark before quantitatively injecting hydrogen peroxide aqueous solution into the sealed reaction chamber (4) so ​​that the hydrogen peroxide aqueous solution immerses the column head and then pull out the microsyringe. S4. Quantitative reading: Start timing immediately after the liquid injection is completed, record the volume difference of the colored indicator droplet (32) rising in the capillary (3) during the predetermined time T seconds, and obtain the gas production volume V; S5. Calculate vitality: Calculate the rice stigma vitality index I based on the gas production volume V, the predetermined duration T, and the number of stigmas N.

7. The method for determining rice stigma vigor based on trace gas quantification according to claim 6, characterized in that, In step S1, the N stigmas of the rice to be tested are stigmas taken from pollinated spikelets on any day from the day the rice plant blooms to the 5th day after blooming, and the style portion of the stigma has been removed.

8. The method for determining rice stigma vigor based on trace gas quantification according to claim 6, characterized in that, In step S3, the hydrogen peroxide aqueous solution is a hydrogen peroxide aqueous solution with a volume fraction of 1%-5%, and the injection volume of the quantitative injection is 100 μL-500 μL.

9. The method for determining rice stigma vigor based on trace gas quantification according to claim 6, characterized in that, In step S4, after obtaining the gas production volume V, a temperature correction step is further included. The temperature correction step is as follows: according to formula V 校正 =V 读数 ×(273+T0) / (273+T 测定 The readings are corrected for temperature, where T0 is the reference temperature and T... 测定 This is the actual measured ambient temperature.

10. The method for determining rice stigma vigor based on trace gas quantification according to claim 6, characterized in that, In step S5, the rice stigma vigor index I is calculated using the formula I = V / (T × N), where the unit of I is μL·s. -1 ·stigma -1 V is the gas production volume in μL, T is in seconds, and N is the total number of column heads used in a single measurement.

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