A method for evaluating rice root bleeding potential based on regenerated stem length

Abstract

This invention discloses a method for evaluating root wound flow potential in rice based on regenerated stem length, relating to the field of rice cultivation technology. It effectively improves detection throughput and reduces labor and material costs. By combining regenerated stem length with the number of effective stems per plant and employing a three-level grading system, root wound flow potential can be evaluated. Experimental data shows that regenerated stem length and the number of effective stems per plant can explain 77.8% of the inter-varietal variation in root wound flow potential. For special genotypes with short regenerated stems but a large number of stems, an identification and verification mechanism is established to avoid missing excellent breeding materials. The method also clarifies the timing of removing the sealed packaging after wound sap collection, resolving the physical conflict between wound sap collection and regenerated stem germination. A complete set of quality control measures, including standardized sowing date, heading date, water, fertilizer, and accumulated temperature conditions, removal of severely root-damaged plants, and differentiation between regenerated stem sections and original tillers, reduces systematic errors.

Description

Technical Field

[0001] This invention relates to the field of rice cultivation technology, specifically a method for measuring root wound flow potential in rice based on regenerated stem length. Background Technology

[0002] Rice roots perform key physiological functions such as water and mineral nutrient absorption and nutrient synthesis. The strength of the root system directly determines the seedling recovery rate, stress resistance, and dry matter accumulation level, ultimately affecting rice yield. Root wound flow potential is a core physiological indicator recognized in the industry for evaluating rice root vitality. Root wound flow originates from the active water absorption of the roots driven by root pressure and can objectively reflect the metabolic activity of living root cells.

[0003] Traditional root wound flow potential detection method: Cut the base of the rice stem 10cm above the ground, stuff the stem section with absorbent cotton, wrap and seal it with plastic film, fix it with rubber band, collect the wound fluid for 1 hour, and weigh the increase in weight of the absorbent cotton to obtain the root wound flow potential of a single plant.

[0004] This method has several practical shortcomings: the operation is complex, requiring cutting, wrapping and sealing, sample recovery, and weighing, resulting in a large amount of manual labor; the number of samples tested per batch is limited, making it unable to meet the high-throughput phenotypic identification needs of hundreds of breeding populations; there are many human interference factors: the tightness of the cotton wrapping, the integrity of the seal, and changes in ambient temperature all contribute to detection errors; moreover, the detection is destructive, and the plants die after the test, making it impossible to continue reproducing valuable breeding materials; and the detection stability is poor in machine transplanting scenarios. Summary of the Invention

[0005] Therefore, in order to overcome the above-mentioned shortcomings, the present invention provides a method for measuring the root wound flow potential of rice based on the regenerated stem length.

[0006] This invention is achieved by constructing a method for measuring root wound flow potential in rice based on regenerated stem length. The device includes the following steps:

[0007] Step 1: Pretreatment of test materials: Select rice materials from the same batch with the same sowing date, and the heading date of the rice materials from the same batch should not differ by more than 5 days; if the rice to be tested is machine-transplanted seedlings in the field, the test should be carried out after the seedlings have fully recovered from transplanting; remove plants with severe mechanical tearing damage to the root system, which refers to a root breakage rate of more than 50% or a main root tear length of more than 1 / 3 of the main root length.

[0008] Step 2, stem cutting and root sap flow collection: 45-50 days after rice transplanting, horizontally cut the base of the rice plant stem at a height of 9-11 cm from the ground surface and collect the sap flow.

[0009] Step 3: Count the number of effective stems per plant: Count the number of effective stems per rice plant to be tested;

[0010] Step 4, Measurement of regenerated stem length: Keep the cut surface of the stem from Step 2 and allow new regenerated stems to sprout from the cut surface; select 5 regenerated stems from the cut surface on the same plant, which are of uniform growth, without deformities, and not from the original tillers, and measure the length of the regenerated stems using a ruler with a minimum scale of 1 mm, and take the average value.

[0011] Step 5: Evaluation of root wound flow potential: Calculate the root wound flow potential per unit stem, where: root wound flow potential per unit stem = root wound flow potential per plant ÷ number of effective stems per plant; utilize the highly significant positive correlation between the length of regenerated stems and the root wound flow potential per unit stem to assist in evaluating the root wound flow potential level of the rice test material.

[0012] Preferably, in step one, the rice to be tested is machine-transplanted rice, with three sowing periods: early rice season, mid-season rice season, and late rice season, and three root damage treatments: no root damage, 2cm root retention, and 1cm root retention. After the seedlings have fully recovered from transplanting, the root damage flow potential and regenerated stem length are measured. The regenerated stem length of the 2cm root retention treatment is not significantly different from that of the no-root-damage treatment, which is used to evaluate the recovery ability of the machine-transplanted root system after root damage.

[0013] Preferably, the criteria for identifying regenerated stems in step four are: only the newly sprouted young regenerated stems from the cut surface of the stem are counted, excluding low-lying tillers that originally existed on the plant, so as to avoid interference from the original tillers with the measurement results of the regenerated stem length.

[0014] Preferably, step five uses a three-level grading scoring method to achieve rapid evaluation, specifically: 1 point is awarded for regenerated stem length < 5mm, indicating weak root wound flow; 2 points are awarded for regenerated stem length ≤ 9.9mm, indicating moderate root wound flow; 3 points are awarded for regenerated stem length > 9.9mm, indicating strong root wound flow; the higher the score, the stronger the root wound flow of the rice material.

[0015] Preferably, in step five, the length of the regenerated stem and the number of effective stems per plant are used as explanatory variables, and the root wound flow potential is used as the target variable for multiple correlation analysis. The length of the regenerated stem and the root wound flow potential per unit stem are highly significantly positively correlated.

[0016] Preferably, in step four, when the length of the regenerated stem is less than 5 mm and the number of effective stems per plant is more than 20% higher than the population average, it is determined to be a special genotype material with short regenerated stems but a large number of stems. The traditional root wound flow potential measurement method is used for verification and testing to avoid missing excellent breeding materials.

[0017] Preferably, the environmental control conditions for the entire measurement process of steps one to five are as follows: the cultivation environment is kept irrigated with shallow water; the measurement stage avoids continuous drought and low temperature stress; the water and fertilizer management conditions of the same batch of test materials are kept completely consistent to ensure that the accumulated temperature conditions of the test materials are uniform during the greening stage.

[0018] Preferably, in step two, the base of the rice plant stem is cut off, a piece of absorbent cotton is attached to the cut section, the outer layer is covered with a plastic film and sealed with a rubber band, the sap flow is collected, and the weight of the absorbent cotton is measured to obtain the root sap flow potential of a single plant; after the sap flow collection is completed, the absorbent cotton and plastic film are removed to expose the cut section for the germination of regenerated stems.

[0019] Preferably, the initial screening of the rice breeding segregation population is carried out in a two-step screening process: the first step is to conduct high-throughput initial screening of a large number of materials by combining the length of regenerated stems with the number of stems, retain high-scoring candidate materials and mark special genotype single plants; the second step is to conduct traditional root wound flow potential verification and determination only on the candidate materials and marked special genotype single plants obtained from the initial screening.

[0020] The present invention has the following advantages: The present invention provides an improved method for evaluating root wound flow potential in rice based on regenerated stem length, which has the following improvements compared to similar equipment:

[0021] This invention discloses a method for evaluating root wound flow potential in rice based on regenerated stem length. This method effectively increases detection throughput and reduces labor and material costs. By combining regenerated stem length with the number of effective stems per plant and using a three-level grading system, the root wound flow potential can be evaluated. Experimental data shows that regenerated stem length and the number of effective stems per plant can explain 77.8% of the inter-varietal variation in root wound flow potential. A recognition and verification mechanism is designed for special genotypes with short regenerated stems but a large number of stems, which can prevent the omission of superior breeding materials. The method also clarifies the timing of removing the sealed packaging after sap collection, resolving the physical conflict between sap collection and regenerated stem germination. A complete set of quality control measures, including standardized sowing date, heading date, water, fertilizer, and accumulated temperature conditions, removal of severely root-damaged plants, and differentiation between regenerated stem sections and original tillers, reduces systematic errors. The experiment has high repeatability and is suitable for both pot germplasm identification and a two-step screening process from high-throughput initial screening to candidate material verification in rice breeding populations. This provides a practical technical means for screening rice germplasm with high root vigor. Attached Figure Description

[0022] Figure 1 This is a flowchart of the invention;

[0023] Figure 2 This is a diagram showing the relationship between root characteristics and regenerated stem length in this invention;

[0024] Figure 3 This is a diagram showing the relationship between root wound flow potential and regenerated stem length according to the present invention;

[0025] Figure 4 This is a diagram showing the relationship between root wound flow potential and the length and number of regenerated stems according to the present invention;

[0026] Figure 5 This invention provides a root vitality estimation diagram based on the regenerated stem length. Detailed Implementation

[0027] The following is in conjunction with the appendix Figures 1-5 The principles and features of the present invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. The invention is described more specifically in the following paragraphs by way of example with reference to the accompanying drawings. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the invention.

[0028] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0029] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" 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; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. The embodiments of this invention will now be described according to its overall structure.

[0030] Example 1:

[0031] Please see Figures 1-5 The present invention discloses a method for evaluating root wound flow potential in rice based on regenerated stem length. Eight rice varieties were used: koshihikari, fukuhibiki, haenuki, akihikari, akitakomachi, ginmasari, nihonmasari, and yumetsukushi. The heading period of each variety was concentrated between August 5 and 10, and the heading period of the tested varieties differed by no more than 5 days.

[0032] Cultivation conditions: Use cylindrical pots with a diameter of 15cm and a height of 80cm, fill each pot with 14.5kg of seedling substrate, and place the pots in a 1m×1m water trough; keep the seedlings moist for 23 days until they have 4.5 leaves; transplant on June 12, planting one variety per pot with 2 seedlings per pot; maintain shallow water irrigation after transplanting.

[0033] Measurement time: 49 days after transplanting, falling within the measurement window period of 45-50 days after transplanting.

[0034] The measurement procedure shall be strictly performed in accordance with the steps described in claim 1:

[0035] Step 1: Pretreatment of test materials: Select rice materials from the same batch with the same sowing date, and the heading dates of the tested varieties should not differ by more than 5 days; In this example, pot cultivation is used, and there is no mechanical transplanting damage. Plants with severe mechanical tearing damage to the root system are removed, that is, plants with a root breakage rate of more than 50% or large adventitious roots with a tear length of more than 1 / 3 of the root length are removed.

[0036] Step 2, Stem Cutting and Root Sap Flow Collection: Horizontally cut the base of the rice plant stem at a distance of 10cm from the ground surface; operate according to claim 8: attach absorbent cotton to the cut section, cover the outer layer with plastic film and seal it with rubber bands, collect sap flow for 1 hour, weigh the absorbent cotton to obtain the root sap flow potential of a single plant; after completing the sap flow collection, remove the absorbent cotton and plastic film to expose the cut section for the germination of regenerated stems.

[0037] Step 3: Count the number of effective stems per plant: Count the number of effective stems per rice plant to be tested and record the data.

[0038] Step 4: Measurement of regenerated stem length: Retain the cut surface and wait for new regenerated stems to sprout from the cut surface; according to the regenerated stem identification criteria in claim 3, only select the newly sprouted tender regenerated stems from the cut surface, excluding interference from the plant's original low-position tillers; select 5 regenerated stems from the cut surface, with consistent growth, no deformities, and not from the original tillers, from the same single plant, and measure the length of the regenerated stems using a ruler with a minimum scale of 1mm, and calculate the arithmetic mean of the 5 measured values ​​as the length of the regenerated stem of the single plant.

[0039] Step 5: Root wound flow potential evaluation: Calculate the root wound flow potential per unit stem: Root wound flow potential per unit stem = Root wound flow potential per plant ÷ Number of effective stems per plant; Use the length of regenerated stems and the number of effective stems per plant as explanatory variables, and root wound flow potential as the target variable to conduct multiple correlation analysis. The length of regenerated stems and the root wound flow potential per unit stem show a highly significant positive correlation; Use this correlation to evaluate the root wound flow potential level of the rice test material with the length of regenerated stems. At the same time, use the three-level grading scoring method of claim 4 for rapid evaluation: 1 point for regenerated stem length < 5 mm, indicating weak root wound flow potential; 2 points for 5 mm ≤ regenerated stem length ≤ 9.9 mm, indicating moderate root wound flow potential; 3 points for regenerated stem length > 9.9 mm, indicating stronger root wound flow potential.

[0040] This embodiment fully implements the environmental control conditions of claim 7: the cultivation environment is kept irrigated with shallow water; the testing stage avoids continuous drought and low temperature stress; the water and fertilizer management conditions of the same batch of test materials are kept completely consistent to ensure that the accumulated temperature conditions of the test materials are uniform during the greening stage.

[0041]

[0042] Significant differences were found in root wound flow potential and regenerated stem length among the eight tested varieties. Regenerated stem length was significantly positively correlated with root wound flow potential per unit stem. Multiple correlation analysis yielded a multiple correlation coefficient of 0.882 and a coefficient of determination of 0.778. The contribution rates of regenerated stem length and effective stem number per plant to root wound flow potential were 57% and 43%, respectively.

[0043] Among them, the regenerated stem length of the cultivar Koshihikari is only 4.4 mm, scoring 1 point; the number of effective stems per plant of this cultivar is 16, which is more than 20% higher than the average of this batch. According to claim 6, it is determined to be a special genotype material with short regenerated stems but a large number of stems. The root wound flow potential of this cultivar was verified by traditional root wound flow potential measurement method. The root wound flow potential of the single plant of this cultivar is at a high level, which confirms that relying solely on the length of the regenerated stem will underestimate the root wound flow potential of this type of genotype. Verification test can avoid missing excellent breeding materials.

[0044] After removing the special genotype koshihikari, the correlation coefficient r between the regenerated stem grading score and the root wound flow potential of a single plant was 0.817, which is significantly correlated, proving that the evaluation results of this method are reliable.

[0045] Example 2:

[0046] Please see Figures 1-5 The present invention provides a method for evaluating the root wound flow potential of rice based on the length of regenerated stems. Compared with Example 1, this example further includes: the genetic law of regenerated stem-root wound flow potential, and the application of simulated breeding population screening, carried out according to the two-step screening process of claim 9.

[0047] Experimental conditions: pot or paddy field cultivation; the test population was a segregating population of hybrid rice; the sowing date of the test plants was controlled to be no more than 5 days different from the heading date of the same batch of test materials; the test was carried out 45-50 days after transplanting.

[0048] Step 1: High-throughput initial screening of large batches of materials; Pretreatment of materials to be tested: Select materials sown at the same sowing date for the population, and remove plants with severe mechanical tearing damage, such as those with a root breakage rate of more than 50% or large adventitious roots with a tear length of more than 1 / 3 of the total root length; Maintain shallow water irrigation throughout the cultivation process, avoid drought and low temperature stress, and ensure that water and fertilizer management is completely consistent.

[0049] 49 days after transplanting, the base of the rice plant stem was horizontally cut 10 cm above the ground. A representative lineage in the population was selected to carry out a complete root wound flow potential collection process: the cut section was covered with absorbent cotton and sealed with plastic film, and the wound sap was collected for 1 hour. The root wound flow potential of a single plant was measured. After collection, the absorbent cotton and film were removed to expose the cut section and wait for the regenerated stem to sprout.

[0050] Count the effective stems of all individual plants to be tested; identify and exclude the original low-positioned tillers of the plant from the regenerated stems that sprout from the cut surface; select 5 regenerated stems from each plant that have sprouted from the cut surface, have uniform growth and no deformities, measure their length using a ruler with a precision of 1 mm, and take the arithmetic mean.

[0051] The root wound flow potential level was evaluated using a three-level grading scoring method; special genotype plants with regenerated stem length <5mm and effective stem number per plant more than 20% higher than the average of this batch were marked; high-scoring candidate lines with scores of 2 and 3 were retained, while most lines with a score of 1 were eliminated.

[0052] Step 2: Verification and testing; Traditional root wound flow potential verification and testing are conducted on the high-scoring candidate lines obtained in Step 1 and all marked special genotype individuals. Combining the initial screening scores with the verified root wound flow potential data, breeding materials with high root wound flow potential and high root vigor are selected. Selected lines can be retained for subsequent breeding work.

[0053] By utilizing the genetic law that the length of regenerated stems is significantly positively correlated with the root wound flow potential per unit stem, it is not necessary to carry out tedious root wound flow potential collection and weighing for the entire population. Only a small number of candidate lines need to be verified. This can greatly reduce the workload and achieve high-throughput initial screening of root vigor in breeding populations.

[0054] This invention provides an improved method for evaluating root wound flow potential in rice based on regenerated stem length. This method effectively increases throughput and reduces labor and material costs. By combining regenerated stem length with the number of effective stems per plant and employing a three-level grading system, root wound flow potential can be assessed. Experimental data shows that regenerated stem length and the number of effective stems per plant can explain 77.8% of the inter-varietal variation in root wound flow potential. A recognition and verification mechanism is designed for special genotypes with short but numerous regenerated stems, preventing the under-screening of superior breeding materials. The timing of removing the sealed packaging after sap collection is clearly defined, resolving the physical conflict between sap collection and regenerated stem germination. A comprehensive set of quality control measures, including standardized sowing date, heading date, water, fertilizer, and accumulated temperature conditions, removal of severely root-damaged plants, and differentiation between regenerated stem sections and original tillers, reduces systematic errors. The experiment has high repeatability and is suitable for both pot germplasm identification and a two-step screening process from high-throughput initial screening to candidate material verification in rice breeding populations. This provides a practical technical means for screening rice germplasm with high root vigor.

[0055] The above description shows and illustrates the basic principles, main features, and advantages of the present invention. Standard parts used in the present invention can be purchased from the market, and irregular parts can be customized according to the description and drawings. The specific connection methods of each part adopt conventional methods such as bolts, rivets, and welding that are mature in the prior art. The machinery, parts, and equipment adopt conventional models in the prior art, and the circuit connection adopts conventional connection methods in the prior art, which will not be described in detail here.

[0056] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for evaluating root wound flow potential in rice based on regenerated stem length, characterized in that: Includes the following steps: Step 1: Pretreatment of test materials: Select rice materials from the same batch with the same sowing date, and the heading date of the rice materials from the same batch should not differ by more than 5 days; if the rice to be tested is machine-transplanted seedlings in the field, the test should be carried out after the seedlings have fully recovered from transplanting; remove plants with severe mechanical tearing damage to the root system, which refers to a root breakage rate of more than 50% or a main root tear length of more than 1 / 3 of the main root length. Step 2, stem cutting and root sap flow collection: 45-50 days after rice transplanting, horizontally cut the base of the rice plant stem at a height of 9-11 cm from the ground surface and collect the sap flow. Step 3: Count the number of effective stems per plant: Count the number of effective stems per rice plant to be tested; Step 4, Measurement of regenerated stem length: Keep the cut surface of the stem from Step 2 and allow new regenerated stems to sprout from the cut surface; select 5 regenerated stems from the cut surface on the same plant, which are of uniform growth, without deformities, and not from the original tillers, and measure the length of the regenerated stems using a ruler with a minimum scale of 1 mm, and take the average value. Step 5: Evaluation of root wound flow potential: Calculate the root wound flow potential per unit stem, where: root wound flow potential per unit stem = root wound flow potential per plant ÷ number of effective stems per plant; utilize the highly significant positive correlation between the length of regenerated stems and the root wound flow potential per unit stem to assist in evaluating the root wound flow potential level of the rice test material.

2. The method for determining root wound flow potential in rice based on regenerated stem length according to claim 1, characterized in that: In step one, the rice to be tested is machine-transplanted rice. Three sowing periods were set: early rice season, mid-season rice season, and late rice season. Three root injury treatments were also set: no root injury, 2cm root retention, and 1cm root retention. After the seedlings were fully transplanted and recovered, the root injury flow potential and regenerated stem length were measured. The regenerated stem length of the 2cm root retention treatment was not significantly different from that of the no-root-injury treatment, which was used to evaluate the recovery ability of the machine-transplanted root system after root damage.

3. The method for determining root wound flow potential in rice based on regenerated stem length according to claim 2, characterized in that: The criteria for identifying regenerated stems in step four are as follows: only the newly sprouted young regenerated stems from the cut surface of the stem are counted, excluding low-lying tillers that originally existed on the plant, so as to avoid interference from the original tillers with the results of the regenerated stem length measurement.

4. The method for determining root wound flow potential in rice based on regenerated stem length according to claim 3, characterized in that: Step five employs a three-level grading scoring method for rapid evaluation. Specifically, a score of 1 is given for regenerated stem length < 5 mm, indicating weak root wound flow; a score of 2 is given for regenerated stem length ≤ 9.9 mm, indicating moderate root wound flow; and a score of 3 is given for regenerated stem length > 9.9 mm, indicating strong root wound flow. The higher the score, the stronger the root wound flow of the rice material.

5. The method for determining root wound flow potential in rice based on regenerated stem length according to claim 4, characterized in that: In step five, the length of the regenerated stem and the number of effective stems per plant are used as explanatory variables, and the root wound flow potential is used as the target variable for multiple correlation analysis. The length of the regenerated stem is highly significantly positively correlated with the root wound flow potential per unit stem.

6. The method for determining root wound flow potential in rice based on regenerated stem length according to claim 5, characterized in that: In step four, when the length of the regenerated stem is less than 5 mm and the number of effective stems per plant is more than 20% higher than the population average, it is identified as a special genotype material with short regenerated stems but a large number of stems. The traditional root wound flow potential measurement method is used for verification and testing to avoid missing excellent breeding materials.

7. The method for determining root wound flow potential in rice based on regenerated stem length according to claim 6, characterized in that: The environmental control conditions for the entire measurement process from step one to step five are as follows: the cultivation environment is kept irrigated with shallow water; the measurement stage avoids continuous drought and low temperature stress; the water and fertilizer management conditions of the same batch of test materials are kept completely consistent to ensure that the accumulated temperature conditions of the test materials are uniform during the greening stage.

8. The method for determining root wound flow potential in rice based on regenerated stem length according to claim 7, characterized in that: In step two, the base of the rice plant stem is cut off, and a piece of absorbent cotton is attached to the cut section. The outer layer is covered with a plastic film and sealed with a rubber band. The sap flow is collected, and the weight of the absorbent cotton is measured to obtain the root sap flow potential of a single plant. After the sap flow collection is completed, the absorbent cotton and plastic film are removed to expose the cut section for the regeneration of stems.

9. The method for determining root wound flow potential in rice based on regenerated stem length according to claim 8, characterized in that: The initial screening of the rice breeding segregation population involves a two-step screening process: the first step uses the regenerated stem length combined with the stem number to conduct a high-throughput initial screening of a large number of materials, retaining high-scoring candidate materials and marking individual plants with special genotypes; the second step only performs traditional root wound flow potential verification and determination on the candidate materials and marked individual plants with special genotypes obtained from the initial screening.

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