Method for accurately and efficiently evaluating nitrogen absorption and soil nitrogen supply conditions by measuring nitrate content of bleeding sap of corn stalks

Abstract

The invention discloses a method for accurately and efficiently evaluating nitrogen absorption and soil nitrogen supply conditions by measuring the nitrate content of bleeding sap of corn stalks. The method comprises the following steps: 1) culturing corn seedlings to grow to V8-V10 periods; 2) collecting stem bleeding sap in the morning of the second day after the plant is fully irrigated: transversely cutting the vertical stem in the second internode by using a blade until the stem is completely cut off; after standing for 5-60 minutes, bleeding sap seeps from the xylem from the cross section of the stalk, and after 5 minutes, the bleeding sap is condensed into liquid drops; using a sterile syringe to suck bleeding sap and transfer the bleeding sap into a centrifugal tube; after the bleeding sap is collected once and 3-10 minutes later, the bleeding sap is condensed into liquid drops again; collecting for multiple times to obtain bleeding sap meeting NO3 <-> determination requirements; 3, the NO3 <-> content of the stem bleeding sap is determined.According to the method, used consumables are simple and easy to obtain, the operation process is simplified, the method is easy to master, time is saved, and the method is especially suitable for obtaining nitrogen absorption phenotypes of large groups of corn in the field.

Description

Technical Field

[0001] This invention relates to the field of maize germplasm breeding technology, and in particular to a method for accurately and efficiently assessing nitrogen uptake and soil nitrogen supply by measuring the nitrate content in maize stalk sap. Background Technology

[0002] Nitrogen is an essential macronutrient for maize growth and one of the most critical factors in promoting maize yield. Maize nitrogen efficiency is determined by both nitrogen absorption and utilization, with nitrogen absorption efficiency playing a major role, especially when growing in low-nitrogen environments. Therefore, nitrogen absorption phenotype has become an important trait for screening and breeding nitrogen-efficient maize germplasm.

[0003] During the V8-V10 stage, maize is in a rapid vegetative growth phase, which is a highly efficient period for nitrogen absorption. The nitrogen it absorbs from the soil is mainly nitrate nitrogen (NO3). - Therefore, the nitrate content in plant tissues can reflect their nitrogen uptake capacity. In addition, NO3 in plants... - Content, especially NO3 in stem sap. - The content can reflect the nitrogen supply in the soil and guide the application of nitrogen fertilizer in the field.

[0004] Currently, existing research methods for nitrogen absorption phenotypes mainly involve extracting and measuring NO3 from maize roots, aboveground plants, and leaves. - Content or determination of NO3 in stem sap - The former type of method often involves complex sampling processes (such as root digging and cleaning) and time-consuming sample pretreatment processes (such as drying, grinding, and weighing). The stem sap collection methods mostly use absorbent cotton or specialized equipment, which have drawbacks such as cotton saturation, material contamination, cumbersome operation, and high cost. These shortcomings make these methods unsuitable for the accurate and efficient acquisition of large amounts of phenotypic data in the field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for accurately and efficiently assessing nitrogen uptake and soil nitrogen supply by measuring the nitrate content in corn stalk sap.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for accurately and efficiently assessing nitrogen uptake and soil nitrogen supply by measuring the nitrate content in maize stalk sap includes the following steps: (1) Cultivate maize seedlings in the field or in pots until they reach stage V8-V10; (2) On the morning of the second day after the plant has been fully watered, collect the stem sap: Use a sharp blade (such as a single-edged razor blade) to make a vertical transverse cut across the stem at the second internode until the stem is completely severed, ensuring a smooth cut surface; wait for about 5-60 minutes, after which sap will seep from the xylem of the stem cut surface, and after about 5 minutes, the sap will coagulate into droplets; use a sterile syringe to draw the sap and transfer it to a centrifuge tube; after one collection, after about 3-10 minutes, the sap will coagulate into droplets again; after multiple collections, obtain the NO3- sap. - Determine the required wound fluid; (3) Refer to the salicylic acid method (Zhao L, Wang Y. Nitrate assay for plant tissues[J].Bio-protocol, 2017, 7(2): e2029-e2029.) NO3 - Standard solution determination procedure for NO3 in stem sap - content.

[0007] In step (2), the second interstic is 5-10 cm above the soil layer.

[0008] In step (2), the centrifuge tubes are sterilized under high temperature and high pressure.

[0009] In step (2), NO3 is satisfied. - The volume of the wound fluid required for a single test is 0.1 mL, and the total volume is >0.3 mL.

[0010] Specifically, step (3) involves taking 0.1 mL of the sap sap and adding it to a 15 mL centrifuge tube, adding 0.4 mL of 5% (W / V) salicylic acid-sulfuric acid solution, mixing well, and reacting at room temperature for 20 minutes; adding 9.5 mL of 8% (W / V) NaOH solution, mixing well, cooling to room temperature, and measuring the absorbance at 410 nm; and calculating the NO3- in the sap sap by referring to the standard curve. - content.

[0011] Compared with the prior art, the outstanding effect of the present invention is as follows: (1) In this invention, the NO3 content of the sap is determined by transversely cutting the corn stalks at stages V8-V10 and extracting the sap. - The method is simple and easy to use, with readily available consumables, and the operation process is simplified and easy to learn, saving time. It is especially suitable for obtaining nitrogen absorption phenotypes of large populations of corn in the field.

[0012] (2) NO3 in stem sap - Content and soil NO3 - The content shows a significant and strong correlation, and the method of this invention can quickly and accurately assess the soil nitrogen supply.

[0013] (3) The corn in the method of the present invention can be a maize inbred line or a hybrid material, and the method is also applicable to maize materials grown in the field or in pots.

[0014] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the method of the present invention for accurately and efficiently assessing nitrogen absorption and soil nitrogen supply by measuring the nitrate content in corn stalk sap. Attached Figure Description

[0015] Figure 1 Photos of plants from five inbred lines cultured to stage V8-V10 under low-nitrogen and high-nitrogen conditions.

[0016] Figure 2 The process of collecting sap from plant stem wounds.

[0017] Figure 3 NO3 in the stem sap of five inbred lines under low- and high-nitrogen conditions - content.

[0018] Figure 4 NO3 in the leaves and stem tissues of five inbred lines under low- and high-nitrogen conditions - content.

[0019] Figure 5 NO3 in soil under low-high nitrogen conditions - content.

[0020] Figure 6 NO3 in soil - Content of NO3 in leaves, stems, and stem sap - Spearman correlation analysis of content.

[0021] Figure 7 NO3 from stem sap of hybrid materials grown in the field - content. Detailed Implementation

[0022] (1) Collect the sap from maize stalks during stages V8-V10 and determine its NO3 content. - content A. Maize plant cultivation and high / low nitrogen treatment After germination, seeds of maize inbred lines Zheng 58, Chang 7-2, CML52, Clyn489, and Chuan 321 were planted in 20 cm diameter, 30 cm deep pots (with trays at the bottom), with 2 seedlings per pot, and 4 pots of each material. These were then placed outdoors for cultivation. When the seedlings reached stage V3, high and low nitrogen conditions were applied. Specifically, each inbred line was divided into two groups (2 pots each). One group was treated with 500 mL of Hoagland solution containing 0.4 mM KNO3 every other day (low nitrogen condition), while the other group was treated with 500 mL of Hoagland solution containing 10 mM KNO3 every other day (high nitrogen condition). This treatment was continued until the seedlings reached stage V8-V10. Figure 1 ).

[0023] B. Collection of sap from stem wounds The morning after watering the plants that have grown to stage V8-V10 with nutrient solution, according to Figure 2 The procedure for collecting stem sap is as follows: Using a sharp, single-edged blade, completely cut the stem perpendicularly at the midpoint of the second internode. After waiting 20-60 minutes, use a 1 mL sterile syringe to aspirate the sap droplet into a 2 mL centrifuge tube. After one collection, the sap will again coagulate into a droplet in approximately 3-10 minutes. Collect multiple times until a total of 0.3-0.5 mL is reached, then store at 4°C for later use. Three biological replicates are collected for each inbred line.

[0024] C. Sap NO3 from stem wounds - Content determination and data analysis Pipette 0.1 mL of the sap sap into a 15 mL centrifuge tube, add 0.4 mL of 5% (w / v) salicylic acid-sulfuric acid solution, mix well, and react at room temperature for 20 minutes; add 9.5 mL of 8% (w / v) NaOH solution, mix well, cool to room temperature, and measure the absorbance at 410 nm; refer to the standard curve (y=0.0042x-0.0262) to calculate the NO3 in the sap sap. - Content. For example... Figure 3 As shown, the NO3 content in the stem sap of five self-pollinated lines under low-nitrogen treatment conditions... - The concentration ranged from 20.9 to 41.0 μg / mL, while under high nitrogen conditions, it ranged from 75.2 to 120.0 μg / mL. Under both high and low nitrogen treatments, NO3... - The content showed extremely significant differences, with the content under low nitrogen conditions being approximately 26.8-39.0% under high nitrogen conditions.

[0025] (2) Collect maize leaf and stem tissues from stages V8-V10 and determine their NO3 content. - content A. Collection of leaf and stem tissue samples The first fully expanded leaf from top to bottom of the plant cut in step (1) was collected as a leaf tissue sample; the stem 10 cm above the cut in step (1) was collected as a stem tissue sample. Two to three biological replicates were collected for each inbred line.

[0026] B. NO3 in leaf and stem tissue samples - Pretreatment for content determination The leaf and stem samples obtained in step A were placed in an oven at 105℃ for 30 minutes to blanch, and then dried in an oven at 85℃ for about 2-3 days until the tissue samples were completely dry. The dried samples were then ground into powder using a sample grinder and passed through a 100-mesh sieve for later use.

[0027] C. NO3 in leaf and stem tissue samples - Content determination and data analysis Weigh 0.1 g of powder from the powder sample obtained in step B, add it to a 10 mL centrifuge tube, add 5 mL of deionized water, and mix by inversion until the powder is completely immersed in the water; boil the mixture at 100 ℃ for 30 minutes, cool it to room temperature, mix by inversion, transfer 1 mL of the mixture to a 1.5 mL centrifuge tube, and centrifuge at 12000 rpm for 10 minutes; transfer 0.1 mL of the supernatant to a 15 mL centrifuge tube, and determine and calculate the NO3 extracted from the leaf and stem tissues according to the operation procedure in step (1) C. - content.

[0028] like Figure 4 As shown, under high and low nitrogen conditions, the NO3 content in leaf tissues of four inbred lines (Zheng 58, Chang 7-2, CML52, and Chuan 321) was... - The content of NO3 in leaves differed significantly under low nitrogen conditions. - The NO3 content was approximately 73.7%-92.9% under high nitrogen conditions; NO3 content in the stem tissues of three inbred lines (Zheng 58, Chang 7-2, and CML52) - The content of NO3 in stems varied significantly under low nitrogen conditions. - The content is approximately 62.2%-86.8% under high nitrogen conditions.

[0029] (3) Collect soil samples and determine their NO3 content. - content A. Soil sample collection Use a soil drill to extract soil from 20-25 cm below the soil layer in the corn pot. Remove plant roots, vermiculite, and other impurities, then transfer the soil sample into a 10 mL centrifuge tube and freeze it at 4°C for later use.

[0030] B. Soil sample NO3 - Content determination and data analysis Weigh approximately 0.5 g of soil sample from A and add it to a 15 mL centrifuge tube. Add 5 mL of deionized water and place the tube flat on a shaker. Shake for 1 hour. Transfer 1 mL of the mixture to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 10 minutes. Transfer 0.1 mL of the supernatant to a 15 mL centrifuge tube and, following the procedure in step (1) C, determine and calculate the NO3 content of the soil sample. - content.

[0031] like Figure 5 As shown, NO3 in soil treated with low nitrogen - The content of NO3 in high-nitrogen treated soil ranged from 81.9 to 94.5 μg / g. - The content ranges from 236.5 to 284.2 μg / g.

[0032] (4) Correlation analysis NO3 from leaves, stems, and stem sap. - Content and soil NO3 - After performing correlation analysis on the content, it was found that ( Figure 6 NO3 in soil - The content of NO3- in the stem sap showed a significant and strong correlation with the content in the stem sap, with a ρ value reaching 0.872, indicating that the content of NO3- in the soil can be assessed by measuring the content of stem sap. - Level. NO3 in stem tissue extract - Content and soil NO3 - The content also showed a significant and strong correlation, but its ρ value was 0.591, indicating a lower correlation strength than that of the stem sap. NO3 extracted from leaf samples... - The content has only a weak correlation with the soil content.

[0033] (5) Collection of stem sap from hybrid seed materials planted in the field and NO3 - Content determination Two commercial hybrid materials, Zhengdan 958 and Xianyu 335, were planted in field soil. When the plants reached stage V8-V10, stem sap was collected according to step (1)B. The hybrid materials were more robust than the inbred lines, exhibiting stronger transpiration pull. Sap began to seep out and rapidly condense into droplets 5-10 minutes after the stems were cut. NO3 in the sap was measured according to step (1)C. - Content, results as follows Figure 7 As shown.

[0034] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for accurately and efficiently assessing nitrogen uptake and soil nitrogen supply by measuring the nitrate content in corn stalk sap, characterized in that, Includes the following steps: (1) Cultivate corn seedlings to grow to stage V8-V10; (2) On the morning of the second day after the plant has been thoroughly watered, collect the stem sap: Use a blade to make a transverse cut perpendicular to the second internode until the stem is completely severed; wait 5-60 minutes, and sap will seep from the xylem from the cut surface of the stem. After 5 minutes, the sap will coagulate into droplets; use a sterile syringe to draw the sap and transfer it to a centrifuge tube; after one collection, the sap will coagulate into droplets again after 3-10 minutes; after multiple collections, obtain the sap that meets the NO3 requirement. - Determine the required wound fluid; (3) Determination of NO3 in stem sap - content.

2. The method for accurately and efficiently assessing nitrogen uptake and soil nitrogen supply by measuring the nitrate content in corn stalk sap according to claim 1, characterized in that: In step (2), the second internode is 5-10 cm above the soil layer.

3. The method for accurately and efficiently assessing nitrogen uptake and soil nitrogen supply by measuring the nitrate content in corn stalk sap according to claim 2, characterized in that: In step (2), the centrifuge tubes are sterilized by high temperature and high pressure.

4. The method for accurately and efficiently assessing nitrogen uptake and soil nitrogen supply by measuring the nitrate content in corn stalk sap according to claim 3, characterized in that: In step (2), NO3 is satisfied. - The volume of the wound fluid required for a single test is 0.1 mL, and the total volume is >0.3 mL.

5. The method for accurately and efficiently assessing nitrogen uptake and soil nitrogen supply by measuring the nitrate content in corn stalk sap according to claim 4, characterized in that: Step (3) specifically involves: adding 0.1 mL of the sap extract to a 15 mL centrifuge tube, adding 0.4 mL of 5% W / V salicylic acid-sulfuric acid solution, mixing well, and reacting at room temperature for 20 minutes; adding 9.5 mL of 8% W / V NaOH solution, mixing well, cooling to room temperature, and measuring the absorbance at 410 nm; and calculating the NO3- content of the sap extract according to the standard curve. - content.

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