A method for evaluating the cold tolerance of Bamboo germplasm resources

Abstract

The present invention relates to the technical field of cold resistance assessment of bamboo, and specifically to a cold resistance evaluation method for bamboo germplasm resources. The method obtains physiological indicators of various types of bamboo, and uses a membership function method to evaluate the membership of the bamboo using the obtained indicators. The method then calculates the comprehensive membership using a weighted average method based on preset weights of the physiological indicators and the membership, thereby obtaining the cold resistance coefficient of the bamboo. This method clarifies the actual cold resistance of the bamboo, allowing selection of bamboo with better cold resistance for planting. The method also introduces a risk factor, an operational abnormality factor, and a growth risk factor for constant temperature equipment, thereby obtaining more accurate physiological indicators when cultivating bamboo in constant temperature equipment. This method facilitates the calculation of the cold resistance coefficient of the bamboo, while also improving the efficiency of cold resistance coefficient assessment.

Description

Technical Field

[0001] The invention relates to the technical field of cold resistance evaluation of bamboo latifolia, and in particular to a method for evaluating the cold resistance of bamboo latifolia germplasm resources. Background Art

[0002] At present, when evaluating the cold resistance of bamboo, it is usually necessary to consider the physiological indicators of bamboo and evaluate the cold resistance of bamboo based on the physiological indicators. However, when using physiological indicators to evaluate the cold resistance of bamboo, if the physiological indicators taken are not accurate enough or are affected by external factors, errors will occur, which will affect the accuracy of the cold resistance evaluation of bamboo. Summary of the Invention

[0003] In response to the above-mentioned shortcomings of the existing technology, the present invention provides a method for evaluating the cold resistance of bamboo germplasm resources, which can effectively solve the problem that when physiological indicators are used to evaluate the cold resistance of bamboo in the existing technology, if the physiological indicators adopted are not accurate enough or are affected by external factors, errors will occur, which will affect the accuracy of the cold resistance evaluation of bamboo.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0005] The present invention provides a method for evaluating the cold resistance of bamboo germplasm resources, comprising the following steps:

[0006] Obtain the environmental parameters for the growth of bamboo species and various types of bamboo species;

[0007] An experimental area was constructed and divided into independent experimental zones. The same type of bamboo species were grouped into the same experimental zone. Constant temperature equipment was configured in each experimental zone for low-temperature cultivation of the bamboo species. Monitoring time periods were set to collect physiological indicators of the bamboo species in the constant temperature equipment according to the monitoring time periods.

[0008] Perform status monitoring on the constant temperature equipment used to cultivate bamboo species, obtain the risk frequency coefficient, operation abnormality coefficient, and growth risk coefficient of the constant temperature equipment, and obtain the status analysis value of the constant temperature equipment. Based on whether the status analysis value meets the preset value, determine whether it is a risky constant temperature equipment, where:

[0009] If so, obtain other constant temperature devices and perform status monitoring to build a constant temperature distribution set, determine other constant temperature devices in the constant temperature distribution set and replace the risk constant temperature device to re-monitor the low-temperature cultivation of the bamboo species;

[0010] The physiological indicators of the corresponding laminaria species cultivated in the constant temperature equipment whose state analysis values ​​meet the preset values ​​are obtained, and the membership of the laminaria species is evaluated according to the membership function method. The comprehensive membership is calculated by the weighted average method based on the preset weights and membership of the physiological indicators to obtain the cold resistance coefficient of the laminaria species.

[0011] Physiological indicators include relative electrical conductivity REC, malondialdehyde MDA, soluble sugar SS, soluble protein SP, catalase CAT, peroxidase POD, and leaf photosynthetic index;

[0012] Among them, the test method of relative conductivity REC is:

[0013] Collect fresh leaves of bamboo species and wash them with pure water, then accurately weigh 0.1g of leaves of bamboo species and put them into a test tube;

[0014] Add 20 mL of ultrapure water to each test tube, let it stand for 12 h, and then shake the test tube thoroughly;

[0015] The initial conductivity of the extract was measured by a conductivity meter.

[0016] Place the test tube in a boiling water bath and heat for 10 minutes. After cooling to room temperature, shake it again and measure the final conductivity. , calculated according to the following relationship:

[0017] .

[0018] The test method for malondialdehyde (MDA) is:

[0019] Weigh 0.1g of leaves from the species Dendrocalamus latifolia and grind them into powder, then add 2ml of 10% trichloroacetic acid solution;

[0020] After 30 min of extraction at room temperature, centrifuge at 10,000 rpm for 15 min at 4°C, remove 1 mL of supernatant, add 2 mL of 0.6% TBA solution, place in a boiling water bath for 15 min, and cool with ice water.

[0021] Centrifuge again at 4°C, 10,000 rpm for 10 min, and measure the absorbance at 450 nm, 532 nm, and 600 nm.

[0022] Calculated according to the following formula:

[0023]

[0024] Where: 、 、 are the absorbance values ​​at the corresponding wavelengths; is the total volume of the extract, W is the fresh weight of the sample, is the volume of the extract used for actual measurement.

[0025] The determination method of soluble sugar SS is:

[0026] Weigh 0.1g of leaves from the species Dendrocalamus latifolia and add 5ml of distilled water to grind;

[0027] After boiling in a water bath for 30 min, centrifuge at 4°C and 10,000 rpm for 10 min, collect the supernatant, and extract the precipitate with distilled water three times. Combine the supernatants and use them for soluble sugar determination.

[0028] Pipette 0.5 ml of sample solution into a test tube, add 1.5 ml of distilled water, then add 0.5 ml of anthrone ethyl acetate mixture and 5 ml of concentrated sulfuric acid solution in sequence. Mix well and boil in a water bath for 10 min. Cool to room temperature with ice water and perform colorimetric determination at a wavelength of 630 nm. According to the standard curve, calculate the mass of soluble sugar in the sample.

[0029] The calculation formula of SS content is as follows:

[0030]

[0031] Where: is the mass of soluble sugar in the extract, obtained from the standard curve; is the total volume of the extract, is the dilution factor, is the fresh weight of the sample, is the volume of the extract used for actual measurement.

[0032] The method for determining soluble protein SP is:

[0033] Weigh 0.1g of leaves from the species Dendrocalamus latifolia and add 2mL of distilled water to grind;

[0034] Centrifuge at 4°C, 10,000 rpm for 10 min. The resulting supernatant is the test solution. Pipette 1 mL of the test solution into a test tube, add 5 mL of Coomassie Brilliant Blue solution, mix thoroughly, and let stand for 2 min. Measure the absorbance at 595 nm.

[0035] The calculation formula of SP is as follows:

[0036]

[0037] Where: is the mass of soluble protein in the extract, obtained from the standard curve. is the total volume of the extract, is the dilution factor, is the fresh weight of the sample, is the volume of the extract used for actual measurement.

[0038] The determination method of catalase CAT is:

[0039] Weigh 0.1g of bamboo leaves and add 1ml of pre-cooled PBS to grind into a homogenate;

[0040] Centrifuge at 4°C, 10,000 rpm for 10 min. The supernatant is the crude CAT enzyme solution. Take 0.2 ml of the supernatant and mix it with 3 ml of PBS. Add 0.3 ml of 0.1 mol / L H2O2 and immediately perform colorimetry. Measure at 240 nm every 30 s for a total of 3 min.

[0041] Three replicates were set for each concentration, and each replicate was repeated 3 times. 、 、 ,in To boil the enzyme solution;

[0042] The calculation formula for CAT is as follows:

[0043]

[0044] Where: = , is the absorbance value of the control tube; 、 is the sample absorbance value, is the total volume of the extracted enzyme solution, is the volume of the extract used for actual measurement, is the fresh weight of the sample, For the reaction time.

[0045] The determination method of peroxidase POD is:

[0046] Weigh 0.1 g of bamboo leaves and add 1.5 mL of pre-cooled PBS to grind into a homogenate;

[0047] Centrifuge at 4℃10000r / min for 10min. The supernatant is the crude POD enzyme solution. Take 0.1ml of the supernatant, add 1.0ml0.05mol / L guaiacol and 2.9mlPBS, and add 1.0ml2% Immediately after that, perform colorimetry and measure the absorbance at 470 nm, once every 30 seconds for a total of 3 minutes;

[0048] The formula for calculating POD is as follows:

[0049]

[0050] Where: To measure the change of absorbance during the reaction time; is the total volume of the extracted enzyme solution, is the volume of the extract used for actual measurement, is the fresh weight of the sample, For the reaction time.

[0051] The calculation method of the state analysis value is:

[0052] Calculate the risk factor:

[0053]

[0054] Where, is the state analysis value, is the total number of exceptions, The number of times an environmental anomaly occurs during use. The number of times maintenance was not performed according to the preset maintenance time. is the number of characteristic parameters, 、 and are weight coefficients, is the constant correction coefficient;

[0055] Calculate the operation abnormality coefficient:

[0056]

[0057] Where: is the operating abnormality coefficient, For usage time, The time when environmental abnormalities occur during use. The maintenance time difference between the actual maintenance time and the calibrated maintenance time when maintenance is performed according to the preset maintenance time. is the number of characteristic parameters, 、 as well as is the weight coefficient;

[0058] Calculate the growth risk coefficient:

[0059]

[0060] Where: is the growth risk coefficient, The number of plants that died when the constant temperature equipment was in an abnormal state. is the initial health value of the dead plant, 、 is the weight coefficient;

[0061] Therefore, the state analysis value is obtained according to the following relationship:

[0062]

[0063] Where, is the state analysis value, 、 as well as are the corresponding weight coefficients, and as well as The sum is 1;

[0064] Thus, the state analysis value is obtained , synchronously obtain the status analysis threshold , get The thermostat device is marked as a risk thermostat device, and other thermostat devices are obtained and the other thermostat devices are calculated synchronously. , in order to integrate other thermostat equipment Other constant temperature equipment should be selected and Build a constant temperature distribution set from small to large, and gradually select other constant temperature devices in the constant temperature distribution set to replace the risky constant temperature devices.

[0065] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0066] By obtaining the physiological indicators of various types of bamboo species, and using the membership function method to evaluate the membership of the bamboo species, and calculating the comprehensive membership by weighted average method based on the preset weights of the physiological indicators and the membership, the cold resistance coefficient of the bamboo species is obtained, thereby clarifying the actual cold resistance of the bamboo species, so as to select the bamboo species with better cold resistance for planting;

[0067] By introducing the risk frequency coefficient, operation abnormality coefficient and growth risk coefficient of constant temperature equipment, more accurate physiological indicators can be obtained when cultivating bamboo species in constant temperature equipment, so as to make it more convenient to calculate the cold resistance coefficient of bamboo species and improve the evaluation efficiency of the cold resistance coefficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] 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 only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0069] Figure 1 Schematic diagram of the overall method of the present invention. DETAILED DESCRIPTION

[0070] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0071] The present invention will be further described below with reference to the embodiments.

[0072] Example 1 (see Figure 1 ): A method for evaluating the cold resistance of bamboo germplasm resources, comprising the following steps:

[0073] Obtain the environmental parameters for the growth of bamboo species and various types of bamboo species;

[0074] Environmental parameters include light characteristic parameters, humidity characteristic parameters and temperature characteristic parameters;

[0075] In this scheme, multiple types of bamboo species are obtained and cold resistance experiments are conducted to accurately determine the cold resistance of the bamboo species.

[0076] Furthermore, a test area is built, and the built test area needs to be divided into multiple independent test zones, and the same category of bamboo species are divided into the same test zone. Each experimental zone is equipped with corresponding constant temperature equipment (constant temperature box or constant temperature incubator) for cultivating and monitoring bamboo species. The constant temperature equipment is equipped with an environmental adjustment system. The environmental adjustment system is used to adjust the temperature characteristic parameters, light characteristic parameters, and humidity characteristic parameters in the constant temperature equipment. The low temperature required for growth testing is set through the constant temperature equipment, such as 0°C. The monitoring time period is used to collect the physiological indicators of the bamboo species at 0°C in the constant temperature equipment according to the monitoring time period, thereby evaluating the cold resistance of the bamboo species.

[0077] Among them, the physiological indicators of bamboo species include relative conductivity REC, malondialdehyde MDA, soluble sugar SS, soluble protein SP, catalase CAT, and leaf photosynthetic index, among which:

[0078] Relative conductivity REC, the test method is:

[0079] After collecting fresh leaves of bamboo species and washing them with pure water, 0.1 g of leaves of bamboo species were accurately weighed and placed in a test tube. 20 mL of ultrapure water was added to each test tube and the tube was allowed to stand for 12 h. The test tube was then shaken thoroughly and the initial conductivity of the extract was measured using a conductivity meter (OHAUSST300C). ;

[0080] Place the test tube in a boiling water bath and heat for 10 minutes. After cooling to room temperature, shake it again and measure the final conductivity. , the relative conductivity REC is calculated according to the following relationship:

[0081]

[0082] Malondialdehyde MDA, the test method is:

[0083] Weigh 0.1 g of leaves from the species Bambusa latifolia and grind them into powder. Add 2 ml of 10% trichloroacetic acid (TCA) solution and extract at room temperature for 30 min. Centrifuge at 4°C, 10,000 rpm, and 15 min. Take out 1 mL of the supernatant and add 2 ml of 0.6% TBA solution. Place in a boiling water bath for 15 min, cool with ice water, and centrifuge again at 4°C, 10,000 rpm, and measure the absorbance at 450 nm, 532 nm, and 600 nm.

[0084] Malondialdehyde (MDA) content calculation formula:

[0085]

[0086] Where: 、 、 are the absorbance values ​​at the corresponding wavelengths; is the total volume of the extract (mL), is the fresh weight of the sample (g), is the volume of the extract used for actual determination (mL);

[0087] Soluble sugar SS, its content determination method is:

[0088] Weigh 0.1g of bamboo leaves, add 5ml of distilled water and grind, boil in water bath for 30min, centrifuge at 4℃ 10000r / min for 10min, collect the supernatant, and extract the precipitate repeatedly with distilled water, repeat 3 times, combine the supernatants and use them for soluble sugar determination, draw 0.5ml of sample solution into a test tube (blank is distilled water), add 1.5mL of distilled water, add 0.5ml of anthrone ethyl acetate mixture and 5ml of concentrated sulfuric acid solution in sequence, mix well and boil in water bath for 10min, take out and cool to room temperature with ice water, perform colorimetric determination at a wavelength of 630nm, and calculate the mass of soluble sugar in the sample according to the standard curve;

[0089] The calculation formula of SS content is as follows:

[0090]

[0091] Where: is the mass of soluble sugar in the extract (μg), obtained from the standard curve; is the total volume of the extract (mL), is the dilution factor, is the fresh weight of the sample (g), is the volume of the extract used for actual determination (mL);

[0092] The method for determining the content of soluble protein SP is as follows:

[0093] Weigh 0.1 g of leaves from the species Dendrocalamus latifolia, add 2 mL of distilled water, grind, and centrifuge at 4°C, 10,000 rpm, and 10 min. The resulting supernatant is the test solution. Pipette 1 mL of the test solution into a test tube, add 5 mL of Coomassie Brilliant Blue solution, mix thoroughly, and let stand for 2 min. Measure the absorbance at 595 nm.

[0094] The calculation formula of SP is as follows:

[0095]

[0096] Where: is the mass of soluble protein in the extract (μg), obtained from the standard curve, is the total volume of the extract (mL), is the dilution factor, is the fresh weight of the sample (g), is the volume of the extract used for actual determination (mL).

[0097] Catalase CAT, the content determination method is as follows:

[0098] Weigh 0.1 g of leaves from Dendrocalamus latifolia and add 1 ml of pre-cooled PBS (pH 7.8) to form a homogenate. Centrifuge at 10,000 rpm for 10 min at 4°C. The supernatant is the crude CAT enzyme solution. Take 0.2 ml of the supernatant and mix it with 3 ml of PBS. Add 0.3 ml of 0.1 mol / L H₂O₂ and immediately perform colorimetry. Measure at 240 nm every 30 s for a total of 3 min.

[0099] Three replicates were set for each concentration, and each replicate was repeated 3 times. 、 、 ,in To boil the enzyme solution;

[0100] The calculation formula for CAT is as follows:

[0101]

[0102] Where: = , is the absorbance value of the control tube; 、 is the sample absorbance value, is the total volume of the extracted enzyme solution (mL), is the volume of the extract used for actual determination (mL), is the fresh weight of the sample (g), is the reaction time (min);

[0103] Peroxidase POD, its content determination method is:

[0104] Weigh 0.1g of leaves from the species of bamboo, add 1.5mL of pre-cooled PBS (pH 7.8) and grind into a homogenate. Centrifuge at 4℃ and 10000 r / min for 10min. The supernatant is the crude POD enzyme solution. Take 0.1ml of the supernatant and add 1.0ml of 0.05mol / L guaiacol and 2.9ml of PBS. Add 1.0ml of 2% Immediately after that, perform colorimetry and measure the absorbance at 470 nm, once every 30 seconds for a total of 3 minutes;

[0105] The formula for calculating POD is as follows:

[0106]

[0107] Where: To measure the change of absorbance during the reaction time; is the total volume of the extracted enzyme solution (mL), is the volume of the extract used for actual determination (mL), is the fresh weight of the sample (g), is the reaction time (min);

[0108] Leaf photosynthetic index, the determination method is:

[0109] The upper, mature and healthy functional leaves of Bamboo latifolia were selected from different directions as the measurement objects. The Li-6400XT portable photosynthetic meter was used to measure the leaves of Bamboo latifolia from different provenances. Each leaf was repeated 3 times, and 3 plants were randomly selected from each provenance.

[0110] Calculate the average value as the measured value of the source, measure the net photosynthetic rate Pn, transpiration rate Tr, intercellular CO2 concentration Ci or stomatal conductance Gs of each bamboo leaf, and select one as the measured value of the source, such as the net photosynthetic rate Pn;

[0111] Therefore, the adaptability of plants to low temperature stress can be evaluated according to the membership function method (the membership function includes linear, trapezoidal and S-type, and any one can be used. Then, for each of the above indicators, the threshold of the membership function is set, including the lower limit a, the moderate lower limit b, the moderate upper limit c and the upper limit d, which are usually preset values), and weights are assigned to different cold resistance indicators in order to quantitatively compare the cold resistance of different materials. It should be noted that the corresponding membership degree can be calculated for the trapezoidal membership function for the membership function method adopted. The calculation formula of the trapezoidal membership function is:

[0112]

[0113] Where: is the value of relative conductivity REC, malondialdehyde MDA, soluble sugar SS, soluble protein SP, catalase CAT, peroxidase POD or leaf photosynthetic index, is the degree of membership;

[0114] The comprehensive evaluation D value is calculated by calculating the standard deviation coefficient of each indicator and assigning corresponding weights to each indicator accordingly;

[0115] By analyzing the membership, weight and comprehensive evaluation D value, and finally performing cluster analysis on the comprehensive evaluation D value, we can obtain:

[0116]

[0117] Where: represents the membership degree of the jth indicator of the i-th provenance, is the cold resistance coefficient, is the minimum value of the cold resistance coefficient of the jth indicator among the test provenances, is the maximum value of the cold resistance coefficient of the jth indicator in the tested provenance. Therefore, according to the weighted calculation method, the comprehensive evaluation D value of each Dendrocalamus latifolius provenance material can be weightedly calculated through the membership value of the physiological indicators and the cold resistance coefficient, and the cold resistance of different provenances can be ranked according to the comprehensive evaluation D value to obtain the cold resistance order of Dendrocalamus latifolius species. After low temperature stress treatment (such as 0℃), the larger the comprehensive evaluation D value is, the stronger the cold resistance of the Dendrocalamus latifolius species is; conversely, the smaller the comprehensive evaluation D value is, the worse the cold resistance of the Dendrocalamus latifolius species is.

[0118] In the above, when the relative conductivity REC, malondialdehyde MDA, soluble sugar SS, soluble protein SP, catalase CAT, peroxidase POD, superoxide dismutase SOD, and leaf photosynthesis are tested, the leaves of the laminaria species are collected and tested in the corresponding monitoring time period. Herein, the monitoring time period can be set to the time after the laminaria species are planted in the test partition, and can be set at intervals of 5 days, seven days, etc. Thus, the number of collection times can be set, at least 3 times, thereby, by obtaining multiple relative conductivity REC, malondialdehyde MDA, soluble sugar SS, soluble protein SP, catalase CAT, peroxidase POD, superoxide dismutase SOD, and leaf photosynthesis indicators, more accurate cold resistance data of the laminaria species can be obtained, thereby screening and selecting laminaria species with higher cold resistance for planting.

[0119] In the above, during the low-temperature cultivation of the bamboo species by the constant temperature equipment, it is necessary to collect the physiological indicators of the bamboo species multiple times within the limitation of the monitoring time period. Therefore, in order to ensure that the constant temperature equipment provides accurate environmental parameter control for the bamboo species, so that the physiological indicators of the collected bamboo species are more accurate, so as to evaluate the cold resistance, the following steps are also included:

[0120] Obtain the constant temperature equipment configured in the test partition;

[0121] The constant temperature equipment is analyzed for its status to obtain its status analysis value. The status analysis value is obtained based on the risk frequency coefficient, operation abnormality coefficient, and growth risk coefficient.

[0122] The risk factor is calculated according to the following relationship:

[0123]

[0124] Where, is the state analysis value, is the total number of exceptions ( 、 , abnormal alarm and short circuit fault), The number of times an environmental anomaly occurs during use (after the constant temperature equipment presets the environmental parameters for cultivating the laminaria species, which include temperature parameters, humidity parameters, light parameters, etc., the total number of times the environmental parameters in the constant temperature equipment do not meet the preset environmental parameters after the preset time. If any one of the temperature parameters, humidity parameters or light parameters exists, it is counted as one time. If multiple parameters exist at the same time, they are marked as multiple times). The number of times maintenance was not performed according to the preset maintenance time. is the number of characteristic parameters (including 、 as well as ,therefore is 3), 、 and are weight coefficients, is the constant correction coefficient;

[0125] The operation abnormality coefficient is calculated according to the following relationship:

[0126]

[0127] Where: is the operation abnormality coefficient, For usage time, The time when the environment abnormality occurs during use ( is the sum of the duration of each environmental anomaly, including but not limited to the duration of abnormal temperature parameters, humidity parameters or light parameters). The maintenance time difference between the actual maintenance time and the calibrated maintenance time when maintenance is performed according to the preset maintenance time (in minutes, is the product of multiple maintenance time differences), is the number of characteristic parameters (including 、 as well as ,therefore is 3), 、 as well as is the weight coefficient;

[0128] The growth risk coefficient is calculated according to the following relationship:

[0129]

[0130] Where: is the growth risk coefficient, The number of plants that died when the constant temperature equipment was in an abnormal state ( abnormal conditions included), is the initial health value of the dead plant (the sum of the initial health values ​​of multiple dead plants. The health value of the plant is obtained by measuring the chlorophyll content, soluble sugar content, proline content, and cell membrane permeability, and assigning weights. The larger the health value, the healthier the plant). 、 is the weight coefficient;

[0131] Therefore, the state analysis value is obtained according to the following relationship:

[0132]

[0133] Where, is the state analysis value, 、 as well as are the corresponding weight coefficients, and as well as The sum is 1;

[0134] The state analysis value is obtained through the above calculation formula , obtain a large number of constant temperature equipment , and simultaneously obtain the status analysis threshold , state analysis threshold Preset value for personnel, used in When the temperature is too high, it means that the constant temperature equipment is at an abnormal critical point. Continuous use will cause abnormal risks, affecting the cultivation of bamboo species and the test accuracy and efficiency. Therefore, the test partition is obtained. The thermostat device is marked as a risk thermostat device, and other thermostat devices that are not in use are obtained, and the other thermostat devices are calculated synchronously. , so that other constant temperature equipment Other constant temperature equipment should be selected and Build a constant temperature distribution set from small to large, select other constant temperature equipment in the constant temperature distribution set to replace the risk constant temperature equipment one by one, repair and update the risk constant temperature equipment, and thus cultivate the laminaria species through other constant temperature equipment and conduct subsequent cold resistance tests, so as to improve the accuracy and efficiency of the cold resistance evaluation of the laminaria species.

[0135] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for evaluating the cold resistance of Bamboo germplasm resources, characterized in that: The steps include: Obtain the environmental parameters for the growth of bamboo species and various types of bamboo species; An experimental area was constructed and divided into independent experimental zones. The same type of bamboo species were grouped into the same experimental zone. Constant temperature equipment was configured in each experimental zone for low-temperature cultivation of the bamboo species. Monitoring time periods were set to collect physiological indicators of the bamboo species in the constant temperature equipment according to the monitoring time periods. Perform status monitoring on the constant temperature equipment used to cultivate bamboo species, obtain the risk frequency coefficient, operation abnormality coefficient, and growth risk coefficient of the constant temperature equipment, and obtain the status analysis value of the constant temperature equipment. Based on whether the status analysis value meets the preset value, determine whether it is a risky constant temperature equipment, where: If so, obtain other constant temperature devices and perform status monitoring to build a constant temperature distribution set, determine other constant temperature devices in the constant temperature distribution set and replace the risk constant temperature device to re-monitor the low-temperature cultivation of the bamboo species; Acquire physiological indicators of the corresponding laminaria species cultivated in the constant temperature equipment whose state analysis values ​​meet the preset values, evaluate the membership of the laminaria species according to the membership function method, and calculate the comprehensive membership by the weighted average method based on the preset weights of the physiological indicators and the membership, to obtain the cold resistance coefficient of the laminaria species; The calculation method of the state analysis value is: Calculate the risk factor: ; Where, is the state analysis value, is the total number of exceptions, The number of times an environmental anomaly occurs during use. The number of times maintenance was not performed according to the preset maintenance time. is the number of characteristic parameters, 、 and are weight coefficients, is the constant correction coefficient; Calculate the operation abnormality coefficient: ; Where: is the operation abnormality coefficient, For usage time, The time when environmental abnormalities occur during use. The maintenance time difference between the actual maintenance time and the calibrated maintenance time when maintenance is performed according to the preset maintenance time. is the number of characteristic parameters, 、 as well as is the weight coefficient; Calculate the growth risk coefficient: ; Where: is the growth risk coefficient, The number of plants that died when the constant temperature equipment was in an abnormal state. is the initial health value of the dead plant, 、 is the weight coefficient; Therefore, the state analysis value is obtained according to the following relationship: ; Where, is the state analysis value, 、 as well as are the corresponding weight coefficients, and as well as The sum is 1; Thus, the state analysis value is obtained , synchronously obtain the status analysis threshold , get The thermostat device is marked as a risk thermostat device, and other thermostat devices are obtained and the other thermostat devices are calculated synchronously. , in order to integrate other thermostat equipment Other constant temperature equipment should be selected and Build a constant temperature distribution set from small to large, and gradually select other constant temperature devices in the constant temperature distribution set to replace the risky constant temperature devices.

2. The cold resistance evaluation method of a bamboo germplasm resource according to claim 1, characterized in that: The physiological indicators include relative conductivity REC, malondialdehyde MDA, soluble sugar SS, soluble protein SP, catalase CAT, peroxidase POD, and leaf photosynthetic index; Among them, the test method of relative conductivity REC is: Collect fresh leaves of bamboo species and wash them with pure water, then accurately weigh 0.1g of leaves of bamboo species and put them into a test tube; Add 20 mL of ultrapure water to each test tube, let it stand for 12 h, and then shake the test tube thoroughly; The initial conductivity of the extract was measured by a conductivity meter. ; Place the test tube in a boiling water bath and heat for 10 minutes. After cooling to room temperature, shake it again and measure the final conductivity. , calculated according to the following relationship: 。 3. A method for evaluating cold resistance of bamboo germplasm resources according to claim 2, characterized in that: The testing method of the malondialdehyde MDA is: Weigh 0.1g of leaves from the species Dendrocalamus latifolia and grind them into powder, then add 2ml of 10% trichloroacetic acid solution; After 30 min of extraction at room temperature, centrifuge at 10,000 rpm for 15 min at 4°C, remove 1 mL of supernatant, add 2 mL of 0.6% TBA solution, place in a boiling water bath for 15 min, and cool with ice water. Centrifuge again at 4°C, 10,000 rpm for 10 min, and measure the absorbance at 450 nm, 532 nm, and 600 nm. Calculated according to the following formula: ; Where: 、 、 are the absorbance values ​​at the corresponding wavelengths; is the total volume of the extract, is the fresh weight of the sample, is the volume of the extract used for actual measurement.

4. A method for evaluating cold resistance of bamboo germplasm resources according to claim 2, characterized in that: The determination method of the soluble sugar SS is: Weigh 0.1g of leaves from the species Dendrocalamus latifolia and add 5ml of distilled water to grind; After boiling in a water bath for 30 min, centrifuge at 4°C and 10,000 rpm for 10 min, collect the supernatant, and extract the precipitate with distilled water three times. Combine the supernatants and use them for soluble sugar determination. Pipette 0.5 ml of sample solution into a test tube, add 1.5 ml of distilled water, then add 0.5 ml of anthrone ethyl acetate mixture and 5 ml of concentrated sulfuric acid solution in sequence. Mix well and boil in a water bath for 10 min. Cool to room temperature with ice water and perform colorimetric determination at a wavelength of 630 nm. According to the standard curve, calculate the mass of soluble sugar in the sample. The calculation formula of SS content is as follows: ; Where: is the mass of soluble sugar in the extract, obtained from the standard curve; is the total volume of the extract, is the dilution factor, is the fresh weight of the sample, is the volume of the extract used for actual measurement.

5. A method for evaluating cold resistance of Bamboo germplasm resources according to claim 2, characterized in that: The determination method of the soluble protein SP is: Weigh 0.1g of leaves from the species Dendrocalamus latifolia and add 2mL of distilled water to grind; Centrifuge at 4°C, 10,000 rpm for 10 min. The resulting supernatant is the test solution. Pipette 1 mL of the test solution into a test tube, add 5 mL of Coomassie Brilliant Blue solution, mix thoroughly, and let stand for 2 min. Measure the absorbance at 595 nm. The calculation formula of SP is as follows: ; Where: is the mass of soluble protein in the extract, obtained from the standard curve. is the total volume of the extract, is the dilution factor, is the fresh weight of the sample, is the volume of the extract used for actual measurement.

6. A method for evaluating cold resistance of Bamboo germplasm resources according to claim 2, characterized in that: The assay method of the catalase CAT is: Weigh 0.1g of bamboo leaves and add 1ml of pre-cooled PBS to grind into a homogenate; Centrifuge at 4°C, 10,000 rpm for 10 min. The supernatant is the crude CAT enzyme solution. Take 0.2 ml of the supernatant and mix it with 3 ml of PBS. Add 0.3 ml of 0.1 mol / L H2O2 and immediately perform colorimetry. Measure at 240 nm every 30 s for a total of 3 min. Three replicates were set for each concentration, and each replicate was repeated 3 times. 、 、 ,in To boil the enzyme solution; The calculation formula for CAT is as follows: ; Where: = , is the absorbance value of the control tube; 、 is the sample absorbance value, is the total volume of the extracted enzyme solution, is the volume of the extract used for actual measurement, is the fresh weight of the sample, For the reaction time.

7. A method for evaluating cold resistance of Bamboo germplasm resources according to claim 2, characterized in that: The determination method of the peroxidase POD is: Weigh 0.1 g of leaves from the species Bambusa latifolia and add 1.5 mL of pre-cooled PBS to grind into a homogenate; Centrifuge at 4℃10000r / min for 10min. The supernatant is the crude POD enzyme solution. Take 0.1ml of the supernatant, add 1.0ml0.05mol / L guaiacol and 2.9mlPBS, and add 1.0ml2% Immediately after that, perform colorimetry and measure the absorbance at 470 nm, once every 30 seconds for a total of 3 minutes; The formula for calculating POD is as follows: ; Where: To measure the change of absorbance during the reaction time; is the total volume of the extracted enzyme solution, is the volume of the extract used for actual measurement, is the fresh weight of the sample, For the reaction time.

8. A method for evaluating cold resistance of Bamboo germplasm resources according to claim 2, characterized in that: The determination method of the described leaf photosynthetic index is: The upper, mature and healthy functional leaves of Bamboo latifolia were selected from different directions as the measurement objects. The Li-6400XT portable photosynthetic meter was used to measure the leaves of Bamboo latifolia from different provenances. Each leaf was repeated 3 times, and 3 plants were randomly selected from each provenance. The net photosynthetic rate Pn, transpiration rate Tr, intercellular CO2 concentration Ci or stomatal conductance Gs of each bamboo leaf were measured, one of them was selected as the measured value of the species source, and the average value was calculated as the measured value of the species source.

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