A method for rapidly determining the point of loss of root turgor pressure

Abstract

The present invention provides a method for rapidly determining the root turgor pressure loss point, which specifically comprises the following steps: step 1, mixing and soaking the roots of a target plant to be measured with water; step 2, removing impurities from the epidermis, wiping off moisture from the epidermis, sealing the roots, and freezing them with liquid nitrogen for more than 2 minutes; step 3, grinding, filtering, collecting the filtrate, placing the filtrate in a vapor osmometer to detect the osmotic concentration of the filtrate, and converting the osmotic concentration into a saturated osmotic potential to obtain the root turgor pressure loss point. The present invention uses fine roots with an absorptive terminal as the main measurement unit. Compared with the traditional P-V curve method, the present invention can more accurately measure roots with an absorptive function, thereby minimizing the potential impact of non-absorptive roots on data accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of root system detection, and in particular to a method for rapidly determining a root system turgor pressure loss point. Background Art

[0002] As a key organ for plant water and nutrient absorption, the root system's turgor pressure directly influences a plant's ability to acquire soil water and maintain growth. The root turgor pressure loss point represents the critical water potential at which turgor pressure is lost, a crucial physiological indicator for measuring a plant's "hydraulic fuse." Once the root water potential falls below this physiological threshold, water cannot effectively enter the root system, limiting leaf transpiration and photosynthesis, ultimately leading to overall plant function impairment. Therefore, the root turgor pressure loss point is not only an important indicator for predicting plant drought adaptability and biogeographic distribution, but also crucial for the selection of drought-tolerant crop varieties and trees. However, due to the lack of mature research methods and reliable quantitative data, it remains unclear whether the root turgor pressure loss point universally serves as a "hydraulic fuse" limiting plant function. Traditional pressure-volume curve analysis repeatedly measures the relationship between root water potential and water content during root dehydration. This method is cumbersome in terms of root sample preparation and measurement. Coarse roots are prone to breakage during repeated measurements, while the root water outlet point (water potential) is extremely difficult to accurately measure for fine roots, leading to systematic bias in measurement results. The root pressure probe method can directly measure the turgor pressure and osmotic potential of individual cells with high measurement accuracy. However, the purchase and maintenance costs of the instrument and equipment are extremely high, the operation process is complicated, and its applicability is limited to larger cells and cells without obvious cell wall thickening. It is difficult to be widely used for the determination of the turgor pressure loss point of different types of roots.

[0003] Therefore, developing a rapid and accurate method to quantify the root turgor loss point is a key technical bottleneck facing current root physiology research. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention provides a method for rapidly determining the root turgor loss point, using the root microvapor osmotic pressure method. Osmotic potential is a determining factor in the turgor loss point, and theoretically, the turgor loss point can be predicted by measuring the osmotic potential of root cortical cells. The principle of the vapor osmotic pressure method is based on Raoult's law, which describes the relationship between the drop in vapor pressure of a solution and the solute concentration. This method determines the osmotic pressure by measuring the drop in vapor pressure of a very small sample (usually a few microliters). The higher the osmotic potential of a solution, the lower its vapor pressure. The microvapor osmotic pressure method can accurately measure this tiny vapor pressure difference and convert it into an osmotic potential value. The present invention uses fine roots with an absorptive terminal as the primary measurement unit. Compared with the traditional PV curve method, the present invention can more accurately measure roots with an absorptive function, thereby minimizing the potential impact of non-absorptive roots on data accuracy.

[0005] The specific operations are:

[0006] Step 1: Soak the target plant roots in water;

[0007] Step 2: Take out the roots, remove impurities from the surface, wipe dry the surface moisture, seal the roots and freeze them with liquid nitrogen for more than 2 minutes;

[0008] Step 3: Grind the frozen roots, filter, collect the filtrate, place the filtrate in a steam osmometer to detect the osmotic concentration of the filtrate, and convert the osmotic concentration into saturated osmotic potential to obtain the root turgor loss point.

[0009] Preferably, the mass ratio of the root system to deionized water in step 1 is 1:25, and the soaking time is 1-2 hours.

[0010] Preferably, the temperature of the liquid nitrogen in step 2 is (-210° C.) to (-196° C.).

[0011] Preferably, in step 2, the operations of removing surface impurities, drying surface moisture and sealing the root system are completed within 30 seconds.

[0012] Preferably, in step three, the powder is ground to a particle size of less than 0.1 mm.

[0013] Preferably, in step three, the operations of collecting the filtrate and placing the filtrate in the vapor osmometer are completed within 30 seconds.

[0014] The present invention has the following advantages:

[0015] Fast and efficient, the measurement time for a single sample is shortened to 5-10 minutes, significantly improving measurement efficiency; accurate and reliable, based on the principle of vapor osmotic pressure, it can detect changes in trace osmotic concentrations to ensure the accuracy of the measurement results; directly measuring the osmotic potential / turgor pressure loss point of the root cortical cells, avoiding the errors caused by the traditional method that relies on extrapolation of the relationship between water potential and water content; wide applicability, the present invention is particularly suitable for fragile and juicy terminal absorbing roots, overcoming the traditional method's stringent requirements on root integrity and expanding the application range of the PV curve method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0017] Figure 1 This is a schematic diagram of the principle of the microvapor osmotic pressure measurement method of the present invention.

[0018] Figure 2 This is the main root system measurement unit diagram of the present invention.

[0019] Figure 3 This is a measurement flow chart of the present invention.

[0020] Figure 4 This is a diagram of the measurement process and calculation method of turgor pressure loss point using the traditional PV curve method.

[0021] Figure 5 This is a comparison chart of the measurement results of vapor osmotic pressure method and traditional PV curve method. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] Example 1

[0024] Step 1: Select the target plant to be monitored, either indoors or outdoors. Between 4:00 and 6:00 AM, carefully excavate the terminal absorbing roots at the base of the target plant, regardless of whether the sampling process will damage the root branching structure. Wrap the roots with absorbent paper thoroughly moistened with distilled water, place in an insulated box at 0-4°C and a relative humidity of ≥80%, and transport to the laboratory for processing within 2 hours.

[0025] Step 2: Take 1-2 grams of fresh roots and place them in a culture dish filled with distilled water, making sure the distilled water completely submerges the roots. Soak for 1-2 hours to allow them to fully absorb water and become saturated.

[0026] Step 3: Use tweezers to pick out the roots with absorption function at the end, rinse them carefully with distilled water, and remove impurities such as dirt on the root surface.

[0027] Step 4: Spread the roots flat on absorbent paper, dry them quickly (within 30 seconds), quickly wrap and seal them with tin foil, and then freeze them in liquid nitrogen at -196°C for at least 2 minutes to freeze and crack the cell walls of the root cortical cells, releasing solute molecules.

[0028] Step 5: Remove the root sample from the liquid nitrogen and place it in an agate mortar, covered with a 6 mm diameter piece of filter paper. Grind vigorously for 10-15 seconds until the sample particle size is less than 0.1 mm, fully disrupting the cells and facilitating the rapid exudation of the slurry and soaking of the filter paper.

[0029] Step 6: Place the soaked filter paper into the sensitivity-calibrated sample chamber of the vapor osmometer and start the continuous measurement mode. The sample is removed from the liquid nitrogen and placed into the vapor osmometer, a process that takes less than 30 seconds.

[0030] Step 7: Observe the continuous measurement readings. When the sample solute concentration is maintained within ±5 mmol, record the readings. A single sample takes 3-5 minutes.

[0031] In step eight, the van't Hoff equation is used to convert the osmotic concentration into the saturated osmotic potential.

[0032] π0=-iCRT

[0033] π0 is the saturation osmotic potential, i is the van't Hoff factor (dimensionless, indicating the degree of dissociation of the electrolyte in the solution, for non-electrolytes i = 1), C, osmotic concentration (mol / L), R, ideal gas constant (0.008314L MPa mol-1K-1), T, absolute temperature (K).

[0034] Step 9. Clean the osmometer sample chamber and agate mortar with an alcohol pad and prepare for the next sample.

[0035] Comparative Example 1

[0036] The difference from Example 1 is that this comparative example uses the root pressure-volume curve method (PV curve method) for measurement, which specifically includes the following steps:

[0037] Step 1: Select the target plant to be monitored, either indoors or outdoors. Between 4:00 and 6:00 AM, carefully dig out the intact root system at the base of the target plant and sever it at the base using pruning shears. Ensure that the root systems of the different branches are not broken. Wrap the intact root system in a wet paper towel and place it in a zero-degree incubator for transport back to the laboratory for processing.

[0038] Step 2: Place the roots in a culture dish filled with distilled water and soak for 2 hours. The time it takes for different plant roots to become saturated with water varies, and a pressure chamber can be used for calibration. The water potential of saturated roots is close to 0.

[0039] Step 3: Use a razor to trim the base of the roots, remove the root bark that affects water potential observation, and place the roots in a tin foil sealed bag.

[0040] Step 4: Use a pressure chamber to measure the first water potential of the roots, and use an analytical balance to weigh the roots in the tinfoil bag (tinfoil bag weight + root weight).

[0041] Step 5: Remove the roots from the tinfoil bag and let them dry naturally on the laboratory bench;

[0042] Step 6: Place the roots back into the tinfoil bag and allow them to equilibrate in the bag for 2-3 minutes.

[0043] Step 7. Repeat steps 4 and 5, trying to obtain data points of root water potential at intervals of 0.2-0.3 MPa until the leaf water potential reaches -3.0 MPa.

[0044] Step eight, weigh the tinfoil bag without roots, then dry the leaves in an oven at >70°C for at least 48 h, and then weigh the dry weight.

[0045] Step nine: Use the curve of root hydraulics and relative water content to correct the "platform effect" caused by root water saturation, and finally deduce the point of root turgor loss based on the inflection point of the curve.

[0046] Test Example 1

[0047] In order to compare different root turgor pressure loss point determination techniques, this experiment selected three tropical tree species with different root diameters and measured their roots using the methods of Example 1 and Comparative Example 1, respectively. 5-6 replicates were set for each tree species and each technical method. The results are shown in Table 1. Figure 5 .

[0048] Depend on Figure 5 As can be seen, there was no significant difference in the turgor loss point measured by vapor osmosis and the PV curve method for both coarse-rooted and fine-rooted plants. More importantly, the vapor osmosis method exhibited a smaller range of variation. This indicates that the vapor osmosis method offers higher precision, smaller measurement error, and greater stability when determining the turgor loss point in root systems.

[0049] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for rapidly determining the point at which root turgor pressure is lost, characterized in that: The following steps are involved: Step 1: soak the target plant roots in deionized water; Step 2: Take out the roots, remove impurities from the surface, wipe dry the surface moisture, seal the roots and freeze them with liquid nitrogen for more than 2 minutes; Step 3: Grind the frozen roots, filter, collect the filtrate, place the filtrate in a steam osmometer to detect the osmotic concentration of the filtrate, and convert the osmotic concentration into saturated osmotic potential to obtain the root turgor loss point.

2. The method for rapidly determining the root turgor pressure loss point according to claim 1, wherein: The mass ratio of the root system to deionized water in step 1 is 1:25, and the roots are soaked for 1-2 hours.

3. The method for rapidly determining the root turgor pressure loss point according to claim 1, wherein: The temperature of the liquid nitrogen in step 2 is (-210° C.) to (-196° C.).

4. The method for rapidly determining the root turgor pressure loss point according to claim 1, wherein: In step 2, the operations of removing surface impurities, drying surface moisture and sealing the root system are completed within 30 seconds.

5. The method for rapidly determining the root turgor pressure loss point according to claim 1, wherein: In step 3, the powder is ground to a particle size of less than 0.1 mm.

6. The method for rapidly determining the root turgor pressure loss point according to claim 1, wherein: In step 3, the operations of collecting the filtrate and placing the filtrate in the vapor osmometer are completed within 30 seconds.

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