A method for determining the vitality of rice tiller buds and a method for detecting the level of active oxygen in tiller buds

Abstract

The invention discloses a method for determining the vitality of rice tiller buds and a method for detecting the level of active oxygen in tiller buds. The determination method determines the vitality of rice tiller buds by detecting the level of active oxygen ROS in the tiller buds of rice seedlings; the level of active oxygen ROS in the tiller buds of the rice seedlings is negatively correlated with the vitality of the tiller buds. When the active oxygen ROS accumulates more, the vitality of the rice tiller buds is poor; when the active oxygen ROS accumulates less, the vitality of the rice tiller buds is strong. The method for detecting the level of active oxygen ROS in tiller buds is to cultivate rice seedlings, obtain tiller bud tissues, perform active oxygen ROS staining observation, and determine and analyze the content of active oxygen ROS components superoxide anion and hydrogen peroxide, so as to evaluate the level of active oxygen ROS in rice tiller buds from qualitative and quantitative aspects. The present invention adds a new method to the study of the growth and development (tiller bud vitality) of tiller buds of rice and other crops, and the present invention also provides new ideas and insights for explaining the morphological construction of rice plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for determining the vitality of rice tiller buds and a method for detecting the level of reactive oxygen species (ROS) in the tiller buds. Background Art

[0002] Tillering is one of the important agronomic traits of rice. The formation of tillering mainly includes the formation of tiller buds and the elongation of tiller buds. Usually, an axillary bud, i.e., tiller bud, can be formed in the axil of each leaf position of rice. Whether the tiller bud is dormant or activated is affected by a series of environmental factors and hormone signals.

[0003] Reactive oxygen species (ROS) are a class of signaling molecules in plants, including: 2- , H 2 O 2 and ·OH, etc., which control the growth and development of plants. There are many methods to detect ROS, including spectrophotometry, which works based on the reaction between free radicals and redox substances. The difference in absorbance between substrates and products at different wavelengths makes the free radicals semi-quantitative; fluorescence staining, which uses fluorescent probes to enter cells to produce fluorescent products and judge information about the generation of free radicals under stimulation; chromatography, which is the reaction of ·OH with specific reagents to generate stable compounds that can be detected by chromatographic analysis; and other detection methods. The dormancy of tiller buds will lead to a reduction in effective tillering of rice plants, ultimately affecting yield.

[0004] However, whether the accumulation of ROS affects the normal growth of rice tiller buds and how to detect the ROS content in rice tiller buds have not been studied yet. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for determining the vitality of rice tiller buds and a method for detecting the level of active oxygen in tiller buds, which are used to determine the influence of the level of active oxygen (ROS) on the growth of rice tiller buds.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides a method for determining the vitality of rice tiller buds, wherein the vitality of rice tiller buds is determined by detecting the level of reactive oxygen species (ROS) in the tiller buds of rice seedlings; the level of reactive oxygen species (ROS) in the tiller buds of the rice seedlings is negatively correlated with the vitality of the tiller buds, and when more reactive oxygen species (ROS) accumulate, the vitality of the rice tiller buds is poor; when less reactive oxygen species (ROS) accumulate, the vitality of the rice tiller buds is strong.

[0008] As a further improvement of the present invention, the activity of tillering buds of different rice seedlings is compared and determined by detecting and comparing the active oxygen ROS levels in tillering buds of different rice seedlings, and then used to judge the number of rice tillers.

[0009] On the other hand, the present invention also provides a method for detecting the level of reactive oxygen species (ROS) in tiller buds in a method for determining the vitality of rice tiller buds. The method comprises culturing rice seedlings, obtaining tiller bud tissues, performing reactive oxygen species (ROS) staining observations, and determining and analyzing the contents of reactive oxygen species (ROS components) such as superoxide anions and hydrogen peroxide, thereby evaluating the level of reactive oxygen species (ROS) in rice tiller buds from both qualitative and quantitative aspects.

[0010] As a further improvement of the present invention, the reactive oxygen species ROS staining observation is observed by NBT staining and / or H2DCFDA fluorescent colorimetric solution.

[0011] The reactive oxygen species (ROS) staining observation comprises the following steps:

[0012] (1) Use seedlings grown in the field for 21 days and in good condition;

[0013] (2) sampling rice seedlings, wherein the sampling parts include the above-ground stem base tissue and the underground root crown tissue;

[0014] (3) Before placing the sample tissue into the staining solution, use tweezers to gently loosen the outermost leaf sheath at the base so that the tiller buds wrapped in the leaf sheath can fully contact the staining solution, but do not damage the leaf sheath;

[0015] (4) Place the sample tissue into NBT staining or H2DCFDA fluorescent staining solution for staining;

[0016] (5)Observation results.

[0017] In the step (2), the sampling site includes 2 cm of stem base tissue above ground and 1 cm of root cap tissue below ground;

[0018] And / or, in the step (3), the inner layer of the leaf sheath in the sample tissue is too tightly wrapped, resulting in the dye solution being unable to contact the tiller buds and unable to be stained, which serves as a negative control.

[0019] In the step (4), placing the sample tissue obtained by sampling into NBT staining or H2DCFDA fluorescent colorimetric solution comprises:

[0020] 1) Place the sample tissues obtained from the sampling into NBT staining solution and H2DCFDA fluorescent colorimetric solution respectively, place them in a vacuum pump under light-shielding conditions, and evacuate at a pressure of 0.08 MPa for 10 minutes. After one time, shake them evenly to ensure that they are fully immersed and to exclude the gas components in and around the tiller buds, and then repeat the evacuation once;

[0021] 2) Place the vacuumed sample tissue in a 28°C incubator for 6 hours in the dark; keep the experiment away from light throughout the process.

[0022] In the step (5), the NBT-stained tissue is transferred to anhydrous ethanol with a concentration of 99.5%; the H2DCFDA-stained tissue is washed with PBS to remove excess H2DCFDA residue;

[0023] The results of NBT staining were observed under a stereomicroscope and photographed and recorded;

[0024] The H2DCFDA-stained tissues were observed and photographed under a fluorescence microscope at excitation wavelengths of 488 nm and emission wavelengths of 545 nm, respectively.

[0025] As a further improvement of the present invention, in the determination and analysis of the content of the reactive oxygen species ROS components superoxide anion and hydrogen peroxide, the sample material is prepared as follows:

[0026] Use 1.5-month-old seedlings, peel off the first leaf, take the first tiller bud, then peel off the second leaf, take the second tiller bud, mix the taken tiller buds together and then divide them into two equal parts, which are used for the determination of superoxide anion and hydrogen peroxide content respectively.

[0027] Superoxide anion and hydrogen peroxide content determination includes:

[0028] One portion was homogenized in an ice bath using superoxide anion extracting solution PBS, 0.01M, pH=7.4, and centrifuged at 10000g and 4°C for 20 minutes to extract the supernatant for the determination of superoxide anion content;

[0029] The other portion was homogenized in an ice bath using acetone, a hydrogen peroxide extract, and centrifuged at 8000 g and 4° C. for 10 minutes to extract the supernatant for determination of hydrogen peroxide content.

[0030] The present invention not only verifies that the level of reactive oxygen (ROS) will affect the normal growth of rice tiller buds, but also summarizes a method for detecting the level of reactive oxygen (ROS) in rice tiller buds after multiple experimental attempts, which adds a new method to the research on the growth and development of tiller buds of rice and other crops. The present invention also provides new ideas and insights for explaining the morphological construction of rice plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0032] Figure 1This is the NBT staining result of the tiller buds of Zhonghua 11 plants. The grey arrows point to tiller buds immersed in the NBT dye solution, while the black arrows point to tiller buds wrapped in leaf sheaths and not immersed in the dye solution. The scale is 2 mm.

[0033] Figure 2 This is the result of H2DCFDA staining of tiller buds of Zhonghua 11 plants, where BF is bright field, Merged is the integrated result of bright field and luminous field, and the scale bar is 2 mm.

[0034] Figure 3 Figure 2 shows the NBT staining results of tiller buds of 9311 plants and mutant ngr5. A and B are the NBT staining results of tiller buds of 9311 plants (A) and mutant ngr5 (B), respectively. The scale is 2 mm. The tiller buds indicated by the grey arrows are immersed in the NBT dye solution, while the tiller buds indicated by the black arrows are wrapped by the leaf sheath and are not immersed in the dye solution.

[0035] Figure 4 The results of H2DCFDA staining of tiller buds of 9311 plants and mutant ngr5 are shown in Figure 1, where A and B are the results of H2DCFDA staining of tiller buds of 9311 plants (A) and mutant ngr5 (B), respectively. The scale bar is 10 cm.

[0036] Figure 5 The figures are the results of determination of superoxide anion and hydrogen peroxide content in tiller buds of 9311 and mutant ngr5, wherein A is the result of determination of superoxide anion content; and B is the result of determination of hydrogen peroxide content.

[0037] Figure 6 Figure 1 is a diagram of the tillering of 9311 and mutant ngr5, where A and B are the tillering phenotypes of 9311 plants (A) and mutant ngr5 (B), respectively; C is a comparison of the tillering numbers of 9311 and mutant ngr5; D is a comparison of the dormant buds and elongated buds of the tillering buds of 9311 and mutant ngr5, and 2nd-11th represent the second to eleventh rice leaves (counted from bottom to top), respectively.

[0038] Figure 7 The graph shows the expression of ROS-related genes in 9311 and mutant ngr5. DETAILED DESCRIPTION

[0039] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the biochemical reagents, carriers, consumables, etc. used in the examples are all commercially available products.

[0040] The present invention mainly uses a strategy combining tissue staining and reactive oxygen component determination to study the level of reactive oxygen ROS in rice tillering buds and its application. The specific experimental technical steps are as follows:

[0041] 1. Preparation of Rice Sample Materials

[0042] 1) Select 21-day-old rice seedlings grown in the field, pull them out of the soil, clean the roots of the plants, and remove excess water with absorbent paper.

[0043] 2) The sample was collected from the base tissue of the rice seedling stem, which included 2 cm of the stem base tissue above the ground and 1 cm of the root cap tissue below the ground.

[0044] 3) Use a scalpel to cut the material from the target area and set it aside.

[0045] 2. Preparation of ROS staining solution

[0046] 1) Preparation of Nitrotetrazolium Blue chloride (NBT) staining solution: The NBT staining solution was prepared by using 0.01 M PBS, pH=7.6 to prepare 0.05% NBT staining solution for subsequent staining experiments.

[0047] 2) Preparation of 2,7-dichlorofluorescein diacetate (2',7'-Dichlorodihydro fluorescein diacetate, referred to as H2DCFDA) solution: 10 mM H2DCFDA stock solution was prepared using DMSO, and then diluted to 10 μM H2DCFDA solution using 10 mM Tris-HCl, pH = 7.2 for subsequent fluorescence colorimetric experiments.

[0048] 3) The NBT staining solution and H2DCFDA fluorescent colorimetric solution were wrapped in tin foil and stored at 4°C away from light. The shelf life is three months.

[0049] 3. Observation and analysis of reactive oxygen species (ROS) staining

[0050] 1) Place the prepared sample tissue materials in NBT staining solution and H2DCFDA solution respectively, place them in a vacuum pump (VOS-30A, STIK) under light-shielding conditions, and evacuate at a pressure of 0.08 MPa for 10 minutes. After one vacuum pump is completed, shake it evenly to ensure sufficient immersion and to exclude the gas components in and around the tiller buds, and then repeat the vacuum pumping once.

[0051] 2) Place the vacuumed tissue sample in a 28°C incubator for 6 hours in the dark.

[0052] 3) The tissue samples after NBT staining were transferred to 99.5% anhydrous ethanol and then placed under a stereomicroscope (M205FA, LEICA) for photographing and recording.

[0053] 4) The H2DCFDA-stained tissue samples were washed twice with PBS solution to remove excess H2DCFDA solution, and then observed and photographed under a fluorescence microscope (Zeiss LSM710, Carl Zeiss AG) at 488 / 545 nm (excitation / emission wavelength).

[0054] 4. Determination of active oxygen components

[0055] 1) Prepare the second sample material, peel off the first leaf, take the first tiller bud, then peel off the second leaf, take the second tiller bud, mix the taken tiller buds together and divide them into two equal parts, respectively for superoxide anion O 2- and hydrogen peroxide H 2 O 2 Content determination.

[0056] 2) One portion was homogenized in an ice bath using superoxide anion extract PBS (0.01 M, pH = 7.4), and centrifuged at 10,000 g and 4°C for 20 min. The supernatant was extracted for the determination of superoxide anion content according to the instructions of the superoxide anion assay kit (Cat # SA-2-G, Ke Ming Biotechnology, China).

[0057] 3) Another portion was homogenized in an ice bath using the prepared hydrogen peroxide extraction solution acetone, centrifuged at 8000g and 4°C for 10 minutes, and the supernatant was extracted for the determination of hydrogen peroxide content. The determination method was based on the instructions of the hydrogen peroxide determination kit (Cat#H2O2-2-Y, Keming Biotechnology, China).

[0058] The following is a description of the specific embodiments:

[0059] Example 1 Reactive oxygen species (ROS) staining of tiller buds of japonica rice variety Zhonghua 11

[0060] 1. Rice material culture and dyeing material preparation

[0061] The japonica rice variety Zhonghua 11 was selected as the experimental object. First, the seeds of Zhonghua 11 were treated to break dormancy, generally in a 45℃ oven for 28 hours, and the seeds were used for standby. The treated seeds were used for seed soaking and germination, and the germinated seeds were sown in the prepared nursery fields in advance to obtain seedlings of 18 days, 21 days, 25 days and 30 days. The morphological structure and size of the tiller buds at the base of the rice stem were observed through dissection experiments. It was found that the tiller buds at the age of 21 days were just in the critical period of elongation and were of appropriate size (the tiller buds at 18 days were small and not easy to observe and analyze; while the tiller buds at 25 days and 30 days had already shown elongation characteristics). The subsequent reactive oxygen ROS staining experiments all used rice seedlings at the age of 21 days.

[0062] 2. Observation and analysis of ROS staining

[0063] 1) The influence of sample size on dyeing

[0064] During the sampling process of rice stem base tissue, the aboveground length of the stem base was set to 0.5, 1, 1.5 and 2 cm, and the underground length of the stem base was set to 0, 0.5 and 1 cm. The plant tissues were immersed in NBT staining solution and H2DCFDA fluorescent solution respectively. The preliminary experimental results showed that the aboveground length of the stem base was 1 cm, which was the most suitable. If the length was too short, the plant wound would be too close to the tiller bud, affecting staining and observation. If the length was too long, it would occupy the solution volume and cause reagent waste. The underground part of the stem base was kept at a length of 1 cm, which was the most suitable. If the length was too short, the plant damage would seriously affect staining and observation.

[0065] 2) Improvement of dyeing effect

[0066] Before placing the sample tissue into the staining solution, use tweezers to gently loosen the outermost leaf sheath at the base of the stem so that the tiller buds wrapped in the leaf sheath can fully contact the staining solution, but do not damage the leaf sheath (during the experimental operation, damaging the leaf sheath cannot ensure the integrity of the tiller buds, and will also cause uneven staining and affect photography). The inner layer of the leaf sheath is wrapped too tightly, resulting in the inability of the staining solution to contact the tiller buds and staining. Therefore, as a preferred solution, it can be used as a negative control.

[0067] The treated stem base sample tissue was immersed in NBT staining solution and H2DCFDA solution, and the staining time was set to 2, 4 and 6 hours respectively. The results showed that the tissue was not stained when the NBT staining time was 2 hours, and the tissue staining was not obvious when the staining time was 4 hours, while the staining effect was best after 6 hours. H2DCFDA staining showed fluorescent signals after 4 and 6 hours of treatment, and 6 hours of treatment was used as the treatment time for subsequent experiments.

[0068] 3) Take photos of the staining results

[0069] The tissue stained with NBT was transferred to 99.5% anhydrous ethanol, while the tissue stained with H2DCFDA was washed with PBS to remove excess H2DCFDA residue.

[0070] The results of NBT staining were observed and photographed under a stereomicroscope, while the H2DCFDA-stained tissues were observed and photographed under a fluorescence microscope with an excitation wavelength of 488 nm and an emission wavelength of 545 nm, respectively.

[0071] The NBT staining results of tiller buds of Zhonghua 11 plants are shown in Figure 1 The H2DCFDA staining results of tiller buds of Zhonghua 11 plants are shown in Figure 2 The results showed that when the NBT staining time was 2 hours, the tissue was not stained, when the staining time was 4 hours, the tissue staining was not obvious, and the staining effect was best when it was 6 hours. H2DCFDA staining showed fluorescent signals for 4 hours and 6 hours, and the 6-hour treatment was used as the treatment time for subsequent experiments.

[0072] Example 2 Reactive oxygen species (ROS) staining of tiller buds of indica rice 9311 and the low-tillering mutant ngr5

[0073] 1. Rice material culture and dyeing material preparation

[0074] The wild-type indica rice variety 9311 and the tiller bud development defect mutant ngr5 were selected as test objects, among which the mutant ngr5 plants showed slightly shorter plants and the number of tillers was significantly reduced compared with the wild-type 9311. It was preliminarily inferred that the reactive oxygen species ROS in the tiller buds of the mutant ngr5 might change. The seeds of 9311 and the mutant ngr5 were used to culture rice seedlings with a seedling age of 21 days, and the specific preparation method was the same as the rice material culture and dyeing material preparation method in Example 1.

[0075] 2. Reactive oxygen species (ROS) staining analysis

[0076] 1) Select 21-day-old, robust seedlings of indica rice 9311 and tiller bud development defect mutant ngr5, clean the plants, wipe dry with absorbent paper, and set aside for material collection. The specific experimental method is the same as the optimized method in the ROS staining analysis in Example 1. Take the base of the plant stem, including 1 cm of tissue material at the base of the aboveground stem and 1 cm of the root crown of the underground part. At the same time, use tweezers to gently loosen the outermost leaf sheath at the base of the stem, but do not damage the leaf sheath. The tissue is immersed in NBT staining solution and H2DCFDA solution; vacuum twice / 10 minutes each time, then place the sample tissue in a 28°C incubator for dark culture for 6 hours and use it for subsequent observation and analysis.

[0077] 3. Take photos of the staining results and analyze the results

[0078] The staining results were photographed in the same manner as in Example 1. The NBT-stained tissue was placed under a stereomicroscope for photographing and recording. The ROS staining results in the tiller buds of indica rice 9311 and mutant ngr5 were compared based on the color depth of the tiller buds. The results are shown in Figure 3 A and 3B. The results showed that the NBT staining in the tiller buds of the tiller bud development defect mutant ngr5 was significantly darker than that of the wild-type indica rice variety 9311, suggesting that more superoxide anions may accumulate in the tiller buds of ngr5 plants. The results of H2DCFDA fluorescence staining are shown in Figure 4 The results showed that the mutant ngr5 plants ( Figure 4 B) The fluorescence in tiller buds was significantly stronger than that in 9311 ( Figure 4 A), it is speculated that there is more ROS accumulation in the tiller buds of ngr5 plants.

[0079] In summary, there was no obvious accumulation of reactive oxygen species (ROS) in the tiller buds of the wild-type 9311, while the tiller bud defective mutant ngr5 accumulated a large amount of reactive oxygen species (ROS) in the tiller buds, indicating that the tiller bud development defect of the mutant ngr5 was directly related to the accumulation of reactive oxygen species in the tiller buds, which can be used as an important indicator for detecting the vitality of rice tiller buds.

[0080] Example 3 Determination and analysis of superoxide anion and hydrogen peroxide content in tillering buds of japonica rice variety Zhonghua 11

[0081] 1. Rice material culture and dyeing material preparation

[0082] The japonica rice variety ZH11 was selected as the test object. ZH11 seeds were used to cultivate rice seedlings with a seedling age of 1.5 months, and the unextended tiller buds in the 1.5-month-old plants were obtained as the research objects. The specific preparation method is the same as the rice material cultivation and dyeing material preparation method in Example 1. After a lot of practice, it was found that more unextended tiller buds could be obtained at 1.5 months, which was conducive to the experiment.

[0083] 2. Determination of the content of active oxygen components superoxide anion and hydrogen peroxide in tiller buds

[0084] Using ZH11 seedlings, peel off the outermost leaves, take the tiller bud tissue (peel off the first leaf, take the first tiller bud, then peel off the second leaf, take the second tiller bud), mix them and divide them into two parts, which are used for the determination of superoxide anion and hydrogen peroxide content respectively. The specific extraction and determination methods refer to the above "4. Steps 2) and 3) in the determination of active oxygen components", and three biological replicates are performed.

[0085] The results showed that the values ​​of superoxide anion in ZH11 tiller buds were 38.33, 36.62 and 37.19 nmol g-1 FW in three replicates as shown in Table 1. The values ​​of hydrogen peroxide content in three replicates as shown in Table 2 were 0.19, 0.22 and 0.22 μmol g-1 FW in three replicates.

[0086] In summary, the present invention utilizes the ROS active oxygen component superoxide anion and hydrogen peroxide determination and analysis to study the active oxygen ROS level of rice tiller buds, which can be used as an important indicator to determine the developmental activity of rice tiller buds at a quantitative level.

[0087] Table 1. Results of determination of superoxide anion content in tiller buds of Zhonghua 11 plants

[0088]

[0089] Table 2. Results of determination of hydrogen peroxide content in tillering buds of Zhonghua 11 plants

[0090]

[0091] Example 4 Determination and analysis of superoxide anion and hydrogen peroxide content in tiller buds of indica rice 9311 and mutant ngr5

[0092] The indica rice 9311 and the mutant ngr5 were analyzed according to the method for determining the content of superoxide anion and hydrogen peroxide in the tiller buds of ZH11 in Example 3.

[0093] 1. Rice material culture and dyeing material preparation

[0094] The wild-type indica rice variety 9311 and the mutant ngr5 with few tillering buds were selected as the test objects. It was preliminarily inferred that the reactive oxygen species (ROS) in the tillering buds of the mutant ngr5, including superoxide anions and hydrogen peroxide, might have changed.

[0095] 1.5-month-old rice seedlings, and the unextended tiller buds in the 1.5-month-old plants were taken as the research objects. The specific preparation method was the same as the rice material culture and dyeing material preparation method in Example 1.

[0096] 2. Determination of the content of active oxygen components superoxide anion and hydrogen peroxide in tiller buds

[0097] Prepare the second batch of indica rice 9311 and tiller bud development defect mutant ngr5 seedlings, the seedling age is 1.5 months, peel off the outermost leaves, take the tiller bud tissue (peel off the first leaf, take the first tiller bud, then peel off the second leaf, take the second tiller bud), divide it into two parts, and use it for the determination of superoxide anion and hydrogen peroxide content respectively. The specific extraction and determination methods refer to the above "4. Determination of active oxygen components 2), 3) steps", three biological replicates.

[0098] The results showed that the superoxide anion content and hydrogen peroxide content in the tiller buds of the tiller bud development defect mutant ngr5 were higher than those of the wild type 9311. Figure 5 As shown, it is consistent with the staining results of NBT and H2DCFDA, further verifying the reliability of the ROS staining results. In summary, the present invention uses two experimental techniques and methods, namely, reactive oxygen species ROS staining and ROS reactive oxygen component superoxide anion and hydrogen peroxide determination and analysis, to study the level of reactive oxygen species ROS in rice tiller buds. It is found that compared with the wild type, the tiller bud development defective mutant accumulates a large amount of reactive oxygen species ROS level, which can be used as an important indicator to judge the developmental activity of rice tiller buds.

[0099] Example 5 Analysis of tillering and ROS-related gene expression in 9311 and mutant ngr5

[0100] like Figure 6 As shown, A and B are the tillering phenotypes of 9311 plants (A) and mutant ngr5 (B), respectively; C is a comparison of the tillering numbers of 9311 and mutant ngr5; D is a comparison of dormant buds and elongated buds of tillering buds of 9311 and mutant ngr5. In summary, the number of tillers of mutant ngr5 is significantly reduced compared with that of wild-type 9311, and it is concluded that the reduction in the number of tillers is caused by problems in the elongation of tiller buds.

[0101] like Figure 7 As shown in the figure, by quantitative detection of the expression levels of ROS accumulation-related genes, several key genes involved in ROS accumulation were found, including OsLOL1, OsPLS2, OsCATA, OsCATB and OsCATC. Compared with the wild type 9311, the expression levels of the above genes in the mutant ngr5 were significantly increased, confirming that the accumulation of ROS in rice is closely related to the expression levels of ROS accumulation-related genes.

[0102] In summary, the present invention uses NBT and H2DCFDA staining solutions to perform staining analysis on rice tiller buds, and combines the content determination and analysis of active oxygen ROS components such as superoxide anions and hydrogen peroxide in the tiller buds to establish a connection between the level of active oxygen ROS in the rice tiller buds and the vitality of the tiller buds, that is, the higher the level of active oxygen ROS in the rice tiller buds, the poorer the vitality of the tiller buds, which ultimately leads to a decrease in the number of rice tillers. Therefore, the present invention provides a new technology and method for studying the vitality of rice tiller buds, and also provides new ideas and insights for explaining the morphological construction of rice plants.

[0103] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.

Claims

1. A method for determining the vigor of rice tillering buds, characterized in that: The activity of rice tiller buds was determined by detecting the level of reactive oxygen species (ROS) in tiller buds of rice seedlings. The level of active oxygen ROS in the tiller buds of the rice seedlings is negatively correlated with the tiller bud vitality. When the detected active oxygen ROS accumulation is high, it is judged that the rice tiller bud vitality is poor and the number of rice tillers is small; when the detected active oxygen ROS accumulation is low, it is judged that the rice tiller bud vitality is strong and the number of rice tillers is large; The method for detecting the level of reactive oxygen species (ROS) in tiller buds of rice seedlings comprises: culturing rice seedlings, obtaining tiller bud tissues, performing reactive oxygen species (ROS) staining observation, and measuring and analyzing the content of reactive oxygen species (ROS) components (superoxide anion and hydrogen peroxide), thereby evaluating the level of reactive oxygen species (ROS) in rice tiller buds from both qualitative and quantitative aspects; The reactive oxygen species ROS staining observation is performed by NBT staining or H2DCFDA fluorescent colorimetric solution observation; and comprises the following steps: (1) Use seedlings that have been grown in the field for 21 days and are in good health; (2) Samples were collected from rice seedlings, including the above-ground stem base tissue and the underground root crown tissue; (3) Before placing the sample tissue into the staining solution, use tweezers to gently loosen the outermost leaf sheath at the base so that the tiller buds wrapped in the leaf sheath can fully contact the staining solution, but do not damage the leaf sheath; (4) Place the sample tissue in NBT staining or H2DCFDA fluorescent staining solution for staining; (5) Observation results: Transfer the NBT-stained tissue to 99.5% anhydrous ethanol; wash the H2DCFDA-stained tissue with PBS to remove excess H2DCFDA residue; The results of NBT staining were observed under a stereomicroscope and photographed and recorded; The H2DCFDA-stained tissues were observed and photographed under a fluorescence microscope at excitation wavelengths of 488 nm and emission wavelengths of 545 nm, respectively.

2. The method for measuring rice tillering bud activity according to claim 1, characterized in that: By detecting and comparing the levels of reactive oxygen species (ROS) in the tiller buds of different rice seedlings, the vitality of the tiller buds of different rice varieties can be compared and determined, and then used to determine the number of rice tillers.

3. The method for measuring the vigor of rice tillering buds according to claim 1, characterized in that: In the step (2), the sampling parts include 2 cm of stem base tissue above ground and 1 cm of root cap tissue below ground; And / or, in the step (3), the inner layer of the leaf sheath in the sample tissue is too tightly wrapped, resulting in the dye solution being unable to contact the tiller buds and unable to be stained, which serves as a negative control.

4. The method for measuring the vigor of rice tillering buds according to claim 1, characterized in that: In the step (4), placing the sample tissue obtained by sampling into NBT staining or H2DCFDA fluorescent colorimetric solution comprises: 1) Place the sample tissues in NBT staining solution and H2DCFDA fluorescent colorimetric solution respectively, place them in a vacuum pump under light-shielding conditions, and evacuate at a pressure of 0.08Mpa for 10 minutes. After one time, shake them evenly to ensure that they are fully immersed and the gas components in and around the tiller buds are removed, and then repeat the evacuation once; 2) Place the vacuumed sample tissue in a 28°C incubator for 6 hours in the dark; keep the experiment away from light.

5. The method for measuring the vigor of rice tillering buds according to claim 1, characterized in that: In the determination and analysis of the content of superoxide anion and hydrogen peroxide of the reactive oxygen species ROS, the sample material is prepared as follows: Use 1.5-month-old seedlings, peel off the first leaf, take the first tiller bud, then peel off the second leaf, take the second tiller bud, mix the taken tiller buds together and then divide them into two equal parts, which are used for the determination of superoxide anion and hydrogen peroxide content respectively.

6. The method for determining the vigor of rice tillering buds according to claim 5, characterized in that: Superoxide anion and hydrogen peroxide content determination includes: One portion was homogenized in an ice bath using superoxide anion extract PBS, 0.01M, pH=7.4, and centrifuged at 10000g and 4°C for 20 minutes to extract the supernatant for the determination of superoxide anion content; The other portion was homogenized in an ice bath using acetone, a hydrogen peroxide extract, and centrifuged at 8000 g and 4° C. for 10 minutes to extract the supernatant for determination of hydrogen peroxide content.

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