Method for evaluating cold resistance of dendrocalamus latiflorus germplasm resources

Abstract

The invention relates to the technical field of dendrocalamus latiflorus cold resistance evaluation, in particular to a dendrocalamus latiflorus germplasm resource cold resistance evaluation method. Physiological indexes of various types of dendrocalamus latiflorus species are obtained, the membership degree of the dendrocalamus latiflorus species is evaluated through a membership function method, the comprehensive membership degree is calculated through a weighted average method according to preset weights of the physiological indexes and the membership degree, the cold resistance coefficient of the dendrocalamus latiflorus species is obtained, and therefore the actual cold resistance of the dendrocalamus latiflorus species is determined. The dendrocalamus latiflorus seeds with good cold resistance can be selected for planting; by introducing the risk frequency coefficient, the operation abnormity coefficient and the growth risk coefficient of the constant-temperature equipment, more accurate physiological indexes are obtained when the dendrocalamus latiflorus species are cultured in the constant-temperature equipment, so that the cold-resistant coefficient of the dendrocalamus latiflorus species is calculated more conveniently, and meanwhile, the evaluation efficiency of the cold-resistant coefficient is improved.

Description

Technical Field

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

[0003] 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

[0004] In view of the above-mentioned shortcomings of the prior art, 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 prior art, if the physiological indicators adopted are not accurate enough or are affected by external factors, resulting in errors that affect the accuracy of the cold resistance evaluation of bamboo.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] The present invention provides a method for evaluating the cold resistance of Bamboo germplasm resources, comprising the following steps:

[0007] Obtain the environmental parameters for the growth of bamboo species and various types of bamboo species;

[0008] The experimental area is constructed and divided into independent experimental zones, and the same type of bamboo species are divided into the same experimental zone. A constant temperature device is configured under the experimental zone for low temperature cultivation of the bamboo species. Thus, a monitoring time period is set to collect the physiological indicators of the bamboo species in the constant temperature device according to the monitoring time period;

[0009] Perform status monitoring on the constant temperature equipment for cultivating bamboo species, obtain the risk frequency coefficient, operation abnormality coefficient and growth risk coefficient of the constant temperature equipment, so as to obtain the status analysis value of the constant temperature equipment, and judge whether it is a risk constant temperature equipment based on whether the status analysis value meets the preset value, where:

[0010] If yes, obtain other constant temperature equipment and perform status monitoring to construct a constant temperature allocation set, determine other constant temperature equipment in the constant temperature allocation set and replace the risk constant temperature equipment to re-monitor the low temperature cultivation of the bamboo species;

[0011] The physiological indicators of the corresponding bamboo species cultivated in the constant temperature equipment whose state analysis values ​​meet the preset values ​​are obtained, and the membership of the bamboo species is evaluated according to the membership function method, and 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 bamboo species.

[0012] Physiological indicators include relative conductivity REC, malondialdehyde MDA, soluble sugar SS, soluble protein SP, catalase CAT, peroxidase POD, and leaf photosynthetic index;

[0013] Among them, the test method of relative conductivity REC is:

[0014] After collecting fresh leaves of bamboo species and washing them with pure water, accurately weigh 0.1g of leaves of bamboo species and put them into a test tube;

[0015] Add 20 mL of ultrapure water to each test tube, let stand for 12 h, and then shake the test tube thoroughly;

[0016] The initial conductivity of the extract was measured by a conductivity meter as S1;

[0017] Place the test tube in a boiling water bath and heat for 10 minutes. Allow it to cool to room temperature, shake it again, and measure the final conductivity as S2, which is calculated according to the following relationship:

[0018]

[0019] The test method for malondialdehyde (MDA) is:

[0020] Weigh 0.1g of leaves of bamboo species, grind into powder, add 2ml of 10% trichloroacetic acid solution;

[0021] After 30 minutes of vibration at room temperature, centrifuge at 4°C and 10,000 r / min for 15 minutes, take out 1 mL of supernatant, add 2 ml of 0.6% TBA solution, place in a boiling water bath for 15 minutes, and cool with ice water;

[0022] Centrifuge again at 4°C, 10,000 rpm for 10 min, and measure absorbance at 450 nm, 532 nm, and 600 nm.

[0023] Calculated according to the following formula:

[0024]

[0025] Where: A 450 , A 532 , A 600 are the absorbance values ​​at the corresponding wavelengths; V T is the total volume of the extract, W is the fresh weight of the sample, V S is the volume of the extract used for actual measurement.

[0026] The determination method of soluble sugar SS is:

[0027] Weigh 0.1g of leaves from the species of bamboo and add 5ml of distilled water to grind;

[0028] After boiling in a water bath for 30 min, centrifuge at 4°C and 10,000 r / min for 10 min, collect the supernatant, and extract the precipitate with distilled water for 3 times. Combine the supernatants and use them for soluble sugar determination.

[0029] Pipette 0.5 ml of sample solution into a test tube, add 1.5 ml of distilled water, 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, take out and cool to room temperature with ice water, perform colorimetric determination at a wavelength of 630 nm, and calculate the mass of soluble sugar in the sample according to the standard curve;

[0030] The calculation formula of SS content is as follows:

[0031]

[0032] Where: C is the mass of soluble sugar in the extract, obtained from the standard curve; V T is the total volume of the extract, N is the dilution factor, W is the fresh weight of the sample, V S is the volume of the extract used for actual measurement.

[0033] The determination method of soluble protein SP is:

[0034] Weigh 0.1g of leaves from the species of Bamboo Shoots and add 2mL of distilled water to grind;

[0035] Centrifuge at 4℃ and 10000r / min for 10min. The obtained supernatant is the test solution. Pipette 1mL of the test solution into a test tube, add 5mL of Coomassie Brilliant Blue solution, mix thoroughly and let stand for 2min. Measure the absorbance at 595nm.

[0036] The calculation formula of SP is as follows:

[0037]

[0038] Where: C is the mass of soluble protein in the extract, obtained from the standard curve, V T is the total volume of the extract, N is the dilution factor, W is the fresh weight of the sample, V S is the volume of the extract used for actual measurement.

[0039] The determination method of catalase CAT is:

[0040] Weigh 0.1g of leaves of Bamboo species, add 1ml of pre-cooled PBS and grind into a homogenate;

[0041] Centrifuge at 4℃10000r / min for 10min. The supernatant is the crude CAT enzyme solution. Take 0.2ml of the supernatant and mix it with 3ml PBS. Add 0.3ml 0.1mol / L H2O2 and immediately perform colorimetry. Measure at 240nm every 30s for a total of 3min.

[0042] Each concentration was repeated three times, and each of the repeated three times. S0 , A S1 , A S2 , where A S0 To boil the enzyme solution;

[0043] The calculation formula of CAT is as follows:

[0044]

[0045] Where: A S0 is the absorbance value of the control tube; A S1 , A S2 is the sample absorbance, V T is the total volume of the extracted enzyme solution, V S is the volume of the extract used for actual determination, W is the fresh weight of the sample, and t is the reaction time.

[0046] The determination method of peroxidase POD is:

[0047] Weigh 0.1 g of leaves of Bamboo species, add 1.5 mL of pre-cooled PBS and grind into a homogenate;

[0048] 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, add 1.0ml2%H2O2, and immediately perform colorimetry. Measure the absorbance at 470nm, measure once every 30s, and measure for 3min in total.

[0049] The calculation formula of POD is as follows:

[0050]

[0051] Where: ΔA 470 V is the change of absorbance during the reaction time; T is the total volume of the extracted enzyme solution, V S is the volume of the extract used for actual determination, W is the fresh weight of the sample, and t is the reaction time.

[0052] The calculation method of the state analysis value is:

[0053] Calculate the risk factor:

[0054]

[0055] In the formula, RSd f is the state analysis value, Tp sdt is the total number of abnormalities, Gd sdt is the number of environmental anomalies that occur during use, Wt sdt Npdh is the number of times maintenance is not performed according to the preset maintenance time. sdt is the number of characteristic parameters, μ1, μ2 and μ3 are weight coefficients respectively, and g is the constant correction coefficient;

[0056] Calculate the operating abnormality coefficient:

[0057]

[0058] Where: YSd x is the operation abnormality coefficient, Tk rdt is the usage time, Gd rdt Wt is the time when the environment abnormality occurs during use. rdt Npdh is the maintenance time difference between the actual maintenance time and the calibrated maintenance time when maintenance is performed according to the preset maintenance time. rdt is the number of characteristic parameters, β1, β2 and β3 are weight coefficients;

[0059] Calculate the growth risk factor:

[0060]

[0061] Where: YSd g is the growth risk coefficient, Tg bdt Gd is the number of plants that died when the constant temperature equipment was in an abnormal state. bdt is the initial health value of the dead plant, ω1 and ω2 are weight coefficients;

[0062] Therefore, the state analysis value is obtained according to the following relationship:

[0063]

[0064] Where ZRY fx is the state analysis value, δ1, δ2 and δ3 are the corresponding weight coefficients, and the sum of δ1, δ2 and δ3 is 1;

[0065] Thus, the state analysis value ZRY is obtained fx , synchronously obtain the status analysis threshold ZRY fxt , get ZRY fx ≥ZRY fxyThe thermostat device is marked as a risky thermostat device, and other thermostat devices are obtained, and the ZRY of the other thermostat devices is calculated synchronously fx , to ZRY other thermostatic equipment fx <ZRY fxy Other constant temperature equipment should be selected according to ZRY fx 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.

[0066] Compared with the known prior art, the technical solution provided by the present invention has the following beneficial effects:

[0067] By obtaining the physiological indicators of various types of bamboo species, the membership of the bamboo species is evaluated by the membership function method, and the comprehensive membership is calculated by the weighted average method according to the preset weights and membership of the physiological indicators, and 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;

[0068] 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

[0069] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0070] Figure 1 It is a schematic diagram of the overall method of the present invention. DETAILED DESCRIPTION

[0071] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0072] The present invention will be further described below in conjunction with the embodiments.

[0073] Example 1 (see Figure 1): A method for evaluating the cold resistance of Bamboo germplasm resources, comprising the following steps:

[0074] Obtain the environmental parameters for the growth of bamboo species and various types of bamboo species;

[0075] Environmental parameters include light characteristic parameters, humidity characteristic parameters and temperature characteristic parameters;

[0076] In this scheme, various types of bamboo species are obtained and cold resistance experiments are conducted to accurately determine the cold resistance of the bamboo species.

[0077] Furthermore, a test area is built, and the built test area needs to be divided into multiple independent test areas, and the same category of bamboo species are divided into the same test area, and each experimental area is configured with a corresponding constant temperature device (constant temperature box or constant temperature incubator) for cultivating and monitoring the bamboo species. The constant temperature device is configured with an environmental adjustment system, and the environmental adjustment system is used to adjust the temperature characteristic parameters, light characteristic parameters, and humidity characteristic parameters in the constant temperature device. The low temperature that needs to be tested for growth, such as 0°C, is set through the constant temperature device. The monitoring time period is used to collect the physiological indicators of the bamboo species at 0°C in the constant temperature device according to the monitoring time period, thereby evaluating the cold resistance of the bamboo species.

[0078] 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:

[0079] Relative conductivity REC, the test method is:

[0080] After collecting fresh leaves of bamboo species and washing them with pure water, accurately weigh 0.1g of leaves of bamboo species and put them into test tubes. Add 20mL of ultrapure water to each test tube and let it stand for 12h. Then shake the test tubes thoroughly and use a conductivity meter (OHAUSST300C) to measure the initial conductivity of the extract as S1.

[0081] Place the test tube in a boiling water bath and heat for 10 minutes. Allow it to cool to room temperature and shake it again. The final conductivity is S2. The relative conductivity REC is calculated according to the following relationship:

[0082]

[0083] Malondialdehyde MDA, the test method is:

[0084] Weigh 0.1g of leaves of Bamboo species and grind them into powder, add 2ml10% trichloroacetic acid (TCA) solution, extract at room temperature for 30min, centrifuge at 4℃10000r / min for 15min, take out 1mL of supernatant, add 2ml0.6% TBA solution, place in boiling water bath for 15min, cool with ice water, centrifuge again at 4℃10000r / min for 10min, and measure absorbance at 450nm, 532nm and 600nm wavelengths;

[0085] The calculation formula of malondialdehyde (MDA) content is as follows:

[0086]

[0087] Where: A 450 , A 532 , A 600 are the absorbance values ​​at the corresponding wavelengths; V T is the total volume of the extract (mL), W is the fresh weight of the sample (g), V S is the volume of the extract used for actual determination (mL);

[0088] Soluble sugar SS, its content determination method is:

[0089] Weigh 0.1g of bamboo leaves, add 5ml of distilled water and grind, boil in water for 30min, centrifuge at 4℃10000r / min for 10min, collect the supernatant, and extract the precipitate with distilled water repeatedly, repeat 3 times, combine the supernatant and use it 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 for 10min, take out and cool to room temperature with ice water, perform colorimetric determination at a wavelength of 630nm, and obtain the mass of soluble sugar in the sample according to the standard curve;

[0090] The calculation formula of SS content is as follows:

[0091]

[0092] Where: C is the mass of soluble sugar in the extract (μg), obtained from the standard curve; V T is the total volume of the extract (mL), N is the dilution factor, W is the fresh weight of the sample (g), V S is the volume of the extract used for actual determination (mL);

[0093] The method for determining the content of soluble protein SP is as follows:

[0094] Weigh 0.1g of leaves of bamboo species, add 2mL of distilled water and grind, centrifuge at 4℃10000r / min for 10min, the obtained supernatant is the test solution, draw 1mL of the test solution into a test tube, add 5mL of Coomassie brilliant blue solution, mix thoroughly and let stand for 2min, and measure the absorbance at 595nm;

[0095] The calculation formula of SP is as follows:

[0096]

[0097] Where: C is the mass of soluble protein in the extract (μg), obtained from the standard curve, V T is the total volume of the extract (mL), N is the dilution factor, W is the fresh weight of the sample (g), V S is the volume of the extract used for actual determination (mL).

[0098] Catalase CAT, the content determination method is as follows:

[0099] Weigh 0.1g of leaves of Bamboo species, add 1ml pre-cooled PBS (pH7.8) and grind into a homogenate, centrifuge at 4℃10000r / min for 10min, the supernatant is the crude CAT enzyme solution, take 0.2ml of the supernatant and mix with 3ml PBS, add 0.3ml 0.1mol / LH2O2 and immediately perform colorimetry, measure once every 30s at 240nm, for a total of 3min;

[0100] Each concentration was repeated three times, and each of the repeated three times. S0 , A S1 , A S2 , where A S0 To boil the enzyme solution;

[0101] The calculation formula of CAT is as follows:

[0102]

[0103] Where: A S0 is the absorbance value of the control tube; A S1 , A S2 is the sample absorbance, V T is the total volume of the extracted enzyme solution (mL), V S is the volume of the extract used for actual determination (mL), W is the fresh weight of the sample (g), and t is the reaction time (min);

[0104] Peroxidase POD, the content determination method is as follows:

[0105] Weigh 0.1g of leaves of bamboo species, add 1.5mL pre-cooled PBS (pH7.8) and grind into a homogenate, centrifuge at 4℃10000r / min for 10min, the supernatant is the POD crude enzyme solution, take 0.1ml of the supernatant, add 1.0ml0.05mol / L guaiacol and 2.9mlPBS, add 1.0ml2%H2O2 and immediately perform colorimetry, measure the absorbance at 470nm, measure once every 30s, and measure for 3min in total;

[0106] The calculation formula of POD is as follows:

[0107]

[0108] Where: ΔA 470 V is the change of absorbance during the reaction time; T is the total volume of the extracted enzyme solution (mL), V S is the volume of the extract used for actual determination (mL), W is the fresh weight of the sample (g), and t is the reaction time (min);

[0109] Leaf photosynthetic index, the determination method is:

[0110] The 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.

[0111] Calculate the average value as the measured value of the seed 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 seed source, such as the net photosynthetic rate Pn;

[0112] Thus, 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 one of them 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, so as 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 adopted membership function method, wherein the calculation formula of the trapezoidal membership function is:

[0113]

[0114] Where: X is the value of relative conductivity REC, malondialdehyde MDA, soluble sugar SS, soluble protein SP, catalase CAT, peroxidase POD or leaf photosynthetic index, U(X) is the degree of membership;

[0115] The comprehensive evaluation D value is calculated by calculating the standard deviation coefficient of each indicator and assigning corresponding weights to each indicator accordingly;

[0116] By analyzing the membership, weight and comprehensive evaluation D value, and finally performing cluster analysis on the comprehensive evaluation D value, we get:

[0117]

[0118] Where: U(X ij ) represents the membership degree of the jth indicator of the ith provenance, X ij is the cold resistance coefficient, X min is the minimum value of the cold resistance coefficient of the jth index among the participating provenances, X max 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 bamboo 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 is ranked according to the comprehensive evaluation D value to obtain the cold resistance order of bamboo species. After low temperature stress treatment (such as 0℃), the larger the comprehensive evaluation D value is, the stronger the cold resistance of the bamboo species is; conversely, the smaller the comprehensive evaluation D value is, the worse the cold resistance of the bamboo species is.

[0119] 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 bamboo species are collected and tested in the corresponding monitoring time period, wherein the monitoring time period can be set to the time after the bamboo species are planted in the test partition, and is set at intervals of 5 days, seven days, etc., thereby, the number of collections 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, to obtain more accurate cold resistance data of the bamboo species, thereby, the bamboo species with higher cold resistance can be screened and selected for planting.

[0120] In the above, in the process of low-temperature cultivation of bamboo species by constant temperature equipment, under the limitation of monitoring time period, it is necessary to collect physiological indicators of bamboo species for many times. Therefore, in order to ensure that the constant temperature equipment provides accurate environmental parameter control for bamboo species, so that the physiological indicators of bamboo species collected are more accurate, so as to evaluate the cold resistance, the following steps are also included:

[0121] Obtain the constant temperature equipment configured in the test partition;

[0122] And the equipment status analysis of the constant temperature equipment is carried out to obtain the status analysis value of the constant temperature equipment, wherein the status analysis value is obtained comprehensively based on the risk frequency coefficient, operation abnormality coefficient and growth risk coefficient, among which:

[0123] The risk factor is calculated according to the following relationship:

[0124]

[0125] In the formula, RSd f is the state analysis value, Tp sdt is the total number of abnormalities (Gd sdt , Wt sdt , abnormal alarm and short circuit fault), Gd sdt is the number of times that environmental abnormalities occur during use (after the constant temperature equipment presets the environmental parameters for cultivating the laminaria species, the environmental parameters include temperature parameters, humidity parameters, light parameters, etc., and after the preset time, the total number of times the environmental parameters in the constant temperature equipment do not meet the preset environmental parameters. 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 as multiple times). sdt Npdh is the number of times maintenance is not performed according to the preset maintenance time. sdt is the number of characteristic parameters (including Tp sdt , Gd sdt and Wt sdt , so Npdh sdt 3), μ1, μ2 and μ3 are weight coefficients respectively, and g is a constant correction coefficient;

[0126] The operation abnormality coefficient is calculated according to the following relationship:

[0127]

[0128] Where: YSd x is the operation abnormality coefficient, Tk rdt is the usage time, Gd rdt The time when the environment abnormality occurs during use (Gd rdt 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), Wt rdt The maintenance time difference between the actual maintenance time and the calibrated maintenance time when the maintenance is performed according to the preset maintenance time (in minutes, Wt rdt is the product of multiple maintenance time differences), Npdh rdt is the number of characteristic parameters (including Tk rdt , Gd rdt and Wt rdt , so Npdhrdt 3), β1, β2 and β3 are weight coefficients;

[0129] The growth risk coefficient is calculated according to the following relationship:

[0130]

[0131] Where: YSd g is the growth risk coefficient, Tg bdt The number of plants that died when the constant temperature equipment was in an abnormal state (Tp sdt Abnormal state included), Gd bdt 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). ω1 and ω2 are weight coefficients.

[0132] Therefore, the state analysis value is obtained according to the following relationship:

[0133]

[0134] Where ZRY fx is the state analysis value, δ1, δ2 and δ3 are the corresponding weight coefficients, and the sum of δ1, δ2 and δ3 is 1;

[0135] The state analysis value ZRY is obtained through the above calculation formula fx , get a lot of constant temperature equipment ZRY fx , and synchronously obtain the status analysis threshold ZRY fxy , state analysis threshold ZRY fxy Preset value for personnel, used in ZRY fx ≥ZRY fxy When the temperature is higher than 0.05, 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 ZRY in the test partition is obtained. fx ≥ZRY fxy The thermostat device is marked as a risky thermostat device, and other thermostat devices that are not in use are obtained, and the ZRYfx of the other thermostat devices is calculated synchronously, so as to convert the ZRY in other thermostat devices into risky thermostat devices. fx <ZRY fxy Other constant temperature equipment should be selected according to ZRY fxBuild a constant temperature distribution set from small to large, select other constant temperature devices in the constant temperature distribution set to replace the risk constant temperature devices one by one, repair and update the risk constant temperature devices, so as to cultivate the bamboo species through other constant temperature devices and conduct subsequent cold resistance tests, so as to improve the accuracy and efficiency of the cold resistance evaluation of the bamboo species.

[0136] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. 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 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; The experimental area is constructed and divided into independent experimental zones, and the same type of bamboo species are divided into the same experimental zone. A constant temperature device is configured under the experimental zone for low temperature cultivation of the bamboo species. Thus, a monitoring time period is set to collect the physiological indicators of the bamboo species in the constant temperature device according to the monitoring time period; Perform status monitoring on the constant temperature equipment for cultivating bamboo species, obtain the risk frequency coefficient, operation abnormality coefficient and growth risk coefficient of the constant temperature equipment, so as to obtain the status analysis value of the constant temperature equipment, and judge whether it is a risk constant temperature equipment based on whether the status analysis value meets the preset value, where: If yes, obtain other constant temperature equipment and perform status monitoring to construct a constant temperature allocation set, determine other constant temperature equipment in the constant temperature allocation set and replace the risk constant temperature equipment to re-monitor the low temperature cultivation of the bamboo species; The physiological indicators of the corresponding bamboo species cultivated in the constant temperature equipment whose state analysis values ​​meet the preset values ​​are obtained, and the membership of the bamboo species is evaluated according to the membership function method, and 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 bamboo species.

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 photosynthesis index; Among them, the test method of relative conductivity REC is: After collecting fresh leaves of bamboo species and washing them with pure water, 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 stand for 12 h, and then shake the test tube thoroughly; The initial conductivity of the extract was measured by a conductivity meter as S1; Place the test tube in a boiling water bath and heat for 10 minutes. Allow it to cool to room temperature, shake it again, and measure the final conductivity as S2, which is calculated according to the following relationship:

3. The cold resistance evaluation method of a Bamboo germplasm resource according to claim 2 is characterized in that: The testing method of the malondialdehyde MDA is: Weigh 0.1g of leaves of bamboo species, grind into powder, add 2ml of 10% trichloroacetic acid solution; After 30 minutes of vibration at room temperature, centrifuge at 4°C and 10,000 r / min for 15 minutes, take out 1 mL of supernatant, add 2 ml of 0.6% TBA solution, place in a boiling water bath for 15 minutes, and cool with ice water; Centrifuge again at 4°C, 10,000 rpm for 10 min, and measure absorbance at 450 nm, 532 nm, and 600 nm. Calculated according to the following formula: Where: A 450 , A 532 , A 600 are the absorbance values ​​at the corresponding wavelengths; V T is the total volume of the extract, W is the fresh weight of the sample, V S is the volume of the extract used for actual measurement.

4. The cold resistance evaluation method of a Bamboo germplasm resource according to claim 2 is characterized in that: The determination method of the soluble sugar SS is: Weigh 0.1g of leaves from the species of bamboo and add 5ml of distilled water to grind; After boiling in a water bath for 30 min, centrifuge at 4°C and 10,000 r / min for 10 min, collect the supernatant, and extract the precipitate with distilled water for 3 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, 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, take out and cool to room temperature with ice water, perform colorimetric determination at a wavelength of 630 nm, and calculate the mass of soluble sugar in the sample according to the standard curve; The calculation formula of SS content is as follows: Where: C is the mass of soluble sugar in the extract, obtained from the standard curve; V T is the total volume of the extract, N is the dilution factor, W is the fresh weight of the sample, V S is the volume of the extract used for actual measurement.

5. The cold resistance evaluation method of a Bamboo germplasm resource according to claim 2 is characterized in that: The determination method of the soluble protein SP is: Weigh 0.1g of leaves from the species of Bamboo Shoots and add 2mL of distilled water to grind; Centrifuge at 4℃ and 10000r / min for 10min. The obtained supernatant is the test solution. Pipette 1mL of the test solution into a test tube, add 5mL of Coomassie Brilliant Blue solution, mix thoroughly and let stand for 2min. Measure the absorbance at 595nm. The calculation formula of SP is as follows: Where: C is the mass of soluble protein in the extract, obtained from the standard curve, V T is the total volume of the extract, N is the dilution factor, W is the fresh weight of the sample, V S is the volume of the extract used for actual measurement.

6. The cold resistance evaluation method of a Bamboo germplasm resource according to claim 2 is characterized in that: The determination method of the catalase CAT is: Weigh 0.1g of leaves of Bamboo species, add 1ml of pre-cooled PBS and grind into a homogenate; Centrifuge at 4℃10000r / min for 10min. The supernatant is the crude CAT enzyme solution. Take 0.2ml of the supernatant and mix it with 3ml PBS. Add 0.3ml 0.1mol / L H2O2 and immediately perform colorimetry. Measure at 240nm every 30s for a total of 3min. Each concentration was repeated three times, and each of the repeated three times. S0 , A S1 , A S2 , where A S0 To boil the enzyme solution; The calculation formula of CAT is as follows: Where: A S0 is the absorbance value of the control tube; A S1 , A S2 is the sample absorbance, V T is the total volume of the extracted enzyme solution, V S is the volume of the extract used for actual determination, W is the fresh weight of the sample, and t is the reaction time.

7. The cold resistance evaluation method of a Bamboo germplasm resource according to claim 2 is characterized in that: The determination method of the peroxidase POD is: Weigh 0.1 g of leaves of Bamboo species, add 1.5 mL of pre-cooled PBS and 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, add 1.0ml2%H2O2, and immediately perform colorimetry. Measure the absorbance at 470nm, measure once every 30s, and measure for 3min in total. The calculation formula of POD is as follows: Where: ΔA 470 V is the change of absorbance during the reaction time; T is the total volume of the extracted enzyme solution, V S is the volume of the extract used for actual determination, W is the fresh weight of the sample, and t is the reaction time.

8. The cold resistance evaluation method of a Bamboo germplasm resource according to claim 2 is characterized in that: The method for measuring the photosynthetic index of the leaves is: The 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.

9. The method for evaluating cold resistance of Bamboo germplasm resources according to claim 1, characterized in that: The calculation method of the state analysis value is: Calculate the risk factor: In the formula, RSd f is the state analysis value, Tp sdt is the total number of abnormalities, Gd sdt is the number of environmental anomalies that occur during use, Wt sdt Npdh is the number of times maintenance is not performed according to the preset maintenance time. sdt is the number of characteristic parameters, μ1, μ2 and μ3 are weight coefficients respectively, and g is the constant correction coefficient; Calculate the operating abnormality coefficient: Where: YSd x is the operation abnormality coefficient, Tk rdt is the usage time, Gd rdt Wt is the time when the environment abnormality occurs during use. rdt Npdh is the maintenance time difference between the actual maintenance time and the calibrated maintenance time when maintenance is performed according to the preset maintenance time. rdt is the number of characteristic parameters, β1, β2 and β3 are weight coefficients; Calculate the growth risk factor: Where: YSd g is the growth risk coefficient, Tg bdt Gd is the number of plants that died when the constant temperature equipment was in an abnormal state. bdt is the initial health value of the dead plant, ω1 and ω2 are weight coefficients; Therefore, the state analysis value is obtained according to the following relationship: Where ZRY fx is the state analysis value, δ1, δ2 and δ3 are the corresponding weight coefficients, and the sum of δ1, δ2 and δ3 is 1; Thus, the state analysis value ZRY is obtained fx , synchronously obtain the status analysis threshold ZRY fxy , get ZRY fx ≥ZRY fxy The thermostat device is marked as a risky thermostat device, and other thermostat devices are obtained, and the ZRY of the other thermostat devices is calculated synchronously fx , to ZRY other thermostatic equipment fx <ZRY fxy Other constant temperature equipment should be selected according to ZRY fx 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.

Images