CN110612837A - Method for rapidly identifying leaf vegetable cadmium low-absorption variety by utilizing cadmium-rich matrix cultivation - Google Patents
Method for rapidly identifying leaf vegetable cadmium low-absorption variety by utilizing cadmium-rich matrix cultivation Download PDFInfo
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Abstract
The invention relates to a method for quickly identifying a leaf vegetable cadmium low-absorption variety by utilizing cadmium-rich matrix cultivation. Compared with the prior art, the method for cultivating the cadmium-rich substrate has the advantages of economy, time saving, easy operation, capability of better reflecting the real field conditions and the like, and can provide a new idea for the rapid comparison of the cadmium accumulation capacity of the vegetables.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to a screening method of heavy metal low-accumulation crop varieties. More specifically, the invention relates to a method for quickly identifying a leaf vegetable cadmium low-absorption variety by utilizing cadmium-rich matrix cultivation.
[ background of the invention ]
Cadmium (Cd) is a common toxic heavy metal element in the soil environment. Its relatively high bioavailability in soil means that it can be easily absorbed and accumulated by crops, seriously threatening the quality and safety of agricultural products. Chronic intake of Cd through various routes, including dietary Cd intake, can lead to serious health risks. Vegetables are one of agricultural products which easily cause the problem of cadmium exceeding, vegetable fields in many areas of China are polluted by soil cadmium, and the absorption capacities of different vegetables to cadmium are greatly different.
For vegetable field soil with large area and medium and light cadmium pollution, based on the difference of different types or varieties of vegetables in the accumulation capacity of cadmium in the soil, the vegetables with low accumulation capacity to cadmium are screened for alternative planting, so that the excessive accumulation of cadmium in the vegetable field can be effectively avoided while the continuous production of vegetable products is ensured. Alternative planting is also considered to be a more efficient, environmentally friendly, and promising method for reducing heavy metal accumulation in agricultural products. At present, the difference of the accumulation capacities of heavy metals of different vegetable types or varieties is mainly compared in the early stage through a pot experiment, a target variety is screened out, and then the verification is carried out through a field experiment. The screening result is accurate, and can represent the accumulation capacity of the crop variety on the target heavy metal more truly, but the screening method has the problems of long screening period, large workload, relatively low efficiency and the like. Particularly for leaf vegetables with faster variety change, the traditional field screening method undoubtedly further increases the workload, and is not beneficial to the rapid application of low-accumulation cadmium type varieties. Therefore, a set of laboratory rapid screening method for the cadmium low-absorption leaf vegetable varieties is established, the screening and application speed of the low-absorption leaf vegetable varieties is increased, and the technology of applying variety substitution on cadmium-polluted soil to guarantee the safe production of the leaf vegetables is of more practical significance.
At present, establishment of a method for rapidly screening low-absorption vegetable varieties and field application cases are rarely reported, and related researches are few. Xu et al (Xu Z M, Mei X Q, TanL, Li Q S, Wang LL, He B Y, Guo S H, Zhou C, Ye HJ. Low root/shoot (R/S) bioorganic ratio can indicator of low cadmium accumulation in the shoot of Chinese flowering cabbage (Brassica flowering L. ssp. Chinensis var. utisis Tsen Lee) cadmium enrichment of 37 varieties of water spinach [ J ]. Environmental Science and Pollution area, 2018,25(36):36328 36340) compared the cadmium enrichment capacity of 37 varieties of water spinach by pot and water cultivation experiments, and the results of the experiments show that the edible root biomass/biomass (R/S) ratio is lower for low varieties and the edible root biomass (R/S) is higher for high. Therefore, the secretion released by the root system is insufficient, the activation amount of cadmium in the rhizosphere soil is less, the cadmium absorbed by the water spinach and transferred to the edible part is less, and the edible part has a dilution effect on the cadmium due to the large biomass of the edible part, so that the edible part has a lower cadmium content. In hydroponic tests of cadmium contaminated soil, uncontaminated soil, cadmium-bearing and cadmium-free, the R/S ratio of low cadmium accumulating varieties was significantly lower than that of high cadmium varieties (p < 0.01). The R/S ratio becomes an effective index for indicating the difference of cadmium accumulation among varieties. The research result provides a new technical means for the rapid screening of the low-cadmium crop varieties. Sugiyama (Sugiyama M, Ae N, Hajika M. developing of a simple method for sensing a root system introduction mechanism to a selected gene delivery mechanism [ J ] Plant and Soil,2011,341(1-2):413-422) determines the cadmium concentration in the seedling and mature organ based on the cadmium accumulation capacity of the root system during the vegetative growth phase of the crop. The method is characterized in that the concentration of cadmium in seeds is predicted by using the ratio of the concentration of cadmium to the concentration of zinc in soybean seedlings, soybean high-cadmium accumulated varieties and soybean low-cadmium accumulated varieties are obviously distinguished, no overlapping exists between the two varieties, and finally the method for rapidly screening the soybean high-cadmium accumulated varieties and the soybean low-cadmium accumulated varieties is obtained.
Aiming at the current situation that the efficiency of screening low-cadmium-absorption varieties of leaf vegetables is low in the field test, the method for rapidly comparing the cadmium absorption capacity of different leaf vegetables is researched by utilizing a cadmium-rich substrate, the optimal cadmium concentration level and the optimal cultivation time in the substrate are obtained by comparing and verifying field results, and finally, the method for rapidly screening the low-cadmium-absorption leaf vegetables is developed. The related results can provide reference and reference for the rapid screening of the low-accumulation crops of the heavy metals in China in the future.
[ summary of the invention ]
[ problem to be solved ]
The invention aims to provide a method for quickly identifying a leaf vegetable cadmium low-absorption variety by utilizing cadmium-rich matrix cultivation.
[ solution ]
The invention is realized by the following technical scheme.
The invention relates to a method for quickly identifying a leaf vegetable cadmium low-absorption variety by utilizing cadmium-rich matrix cultivation.
The method comprises the following steps:
A. seeding
Adding 30 g of matrix into each hole of the seedling raising tray, flattening to enable the grid chambers of the hole tray to be clearly visible, then broadcasting 8-12 disinfected leaf vegetable seeds in each hole, adding deionized water for wetting, covering 1.5-2.5 mm of matrix, covering with a plastic film, germinating 2-3 days after sowing, removing the plastic film, and keeping the conditions of wetting, sufficient illumination and good ventilation;
B. management of seedling stage
After the leaf vegetable seeds grow seedlings, watering once every 1-3 days according to the illumination condition, adding 15-30 ml of deionized water into each hole every time, and simultaneously keeping the conditions of humidity, sufficient illumination and good ventilation;
C. harvesting
Harvesting the edible parts of the leaf vegetable seedlings on the 10 th, 15 th and 20 th days of seedling culture, washing the edible parts with distilled water, drying the edible parts with filter paper, weighing the edible parts, drying the edible parts at the temperature of 60 ℃ to constant weight, and weighing to obtain the dry mass of the edible parts of the leaf vegetable seedlings;
D. determination of cadmium content
Weighing 0.1000g of the leaf vegetable seedling sample dried to constant weight in the step C, placing glass beads in a triangular flask, adding 10mL of mixed acid of nitric acid and perchloric acid, digesting overnight, heating and stewing the mixture until white smoke is emitted in the next day to obtain colorless transparent or yellowish digestive juice, transferring the digestive juice into a 10mL volumetric flask, metering the volume to a scale by using ultrapure water, uniformly mixing, standing, diluting the supernatant by using deionized water by 5 times or 10 times, and detecting the cadmium content of the solution by using an inductively coupled plasma spectrometer or an inductively coupled plasma mass spectrometer;
E. statistical evaluation of data
And D, performing statistical charting on the detection result obtained in the step D by adopting the existing software, and performing data comparison evaluation by using a clustering analysis method, a correlation analysis method, a three-factor variance analysis method and a one-factor variance analysis method so as to screen and obtain the leaf vegetables of the cadmium low-absorption variety.
According to a preferred embodiment of the invention, in the step A, the diameter of each hole is 6-7 cm, the depth of each hole is 8-12 cm, and the small holes at the bottom of the seedling raising tray are sealed by black adhesive tapes.
According to another preferred embodiment of the present invention, in step a, the leafy vegetable seed variety is selected from the group consisting of jiayuan cabbage mustard, vegetable spinach No. 1, lubao 701 flowering cabbage, specially selected leaf crowndaisy chrysanthemum, preferably polished rod crowndaisy chrysanthemum, white round leaf amaranth, green superior green peduncle vegetable, sesbania herb, red wrinkled gianthyssop herb, jiayuan flower amaranth, fast growing No. 1, vegetable spinach No. 8, pure sesame oil lettuce or green cream lettuce.
According to another preferred embodiment of the invention, in the step a, the matrix is prepared from coconut coir, vermiculite and perlite according to a weight ratio of 6.0-7.5: 1.2-1.8: 1.2-1.8, has a cadmium content of 0-2.0 mg/kg and a pH of 6.5-8.0, contains a fertilizer plant growth promoting microbial inoculum, and has no peculiar smell, pathogenic bacteria and ova.
According to another preferred embodiment of the invention, in the step B, during the nursery period, the temperature of the greenhouse is controlled to be 25-30 ℃ in the daytime, and the temperature of the greenhouse is controlled to be 15-25 ℃ in the nighttime; the relative humidity of the greenhouse is controlled to be 65-75% in the daytime and at night.
According to another preferred embodiment of the present invention, in the step C, the edible parts of the seedlings of the leafy vegetables dried to a constant weight are ground into powder with a particle size of 100 to 200 meshes.
According to another preferred embodiment of the invention, in the step C, 8 disinfected leaf vegetable seeds are sown in each hole of the seedling raising tray, and the seedlings are planted when the 3 rd true leaves grow out of the leaf vegetable seedlings, and 4-5 seedlings with uniform growth are left to be cultured until the seedlings are harvested at 10 th, 15 th and 20 th days respectively.
According to another preferred embodiment of the present invention, in step D, the oven-dried constant-weight leaf vegetable seedling sample is heated in mixed acid at a temperature of 150 ℃ for 1.0 hour, then at a temperature of 180 ℃ for 0.5 hour, and then at a temperature of 220 ℃ for digestion by heating.
According to another preferred embodiment of the present invention, in step D, the mixed acid is a mixed acid of nitric acid and perchloric acid in a volume ratio of 3: 1.
According to another preferred embodiment of the present invention, in step E, the software is Excel 2013, IBM SPSS Statistics 21 or SigmaPlot 10.0 software.
The present invention will be described in more detail below.
The invention relates to a method for quickly identifying a leaf vegetable cadmium low-absorption variety by utilizing cadmium-rich matrix cultivation.
The method comprises the following steps:
A. seeding
Adding 30 g of matrix into each hole of the seedling raising tray, flattening to enable the grid chambers of the hole tray to be clearly visible, then broadcasting 8-12 disinfected leaf vegetable seeds in each hole, adding deionized water for wetting, covering 1.5-2.5 mm of matrix, covering with a plastic film, germinating 2-3 days after sowing, removing the plastic film, and keeping the conditions of wetting, sufficient illumination and good ventilation;
in the step A, the seedling raising tray is a hole tray with the length of 54cm and the width of 28cm, and the seedling raising tray used by the invention is a product produced by a Shengji plastic product factory in Shanxi province, Wen' an county. Each seedling raising plate is provided with 32 holes, the diameter of each hole is 6-7 cm, the depth of each hole is 8-12 cm, and small holes in the bottom of each seedling raising plate are sealed by black adhesive tapes so as to prevent moisture from seeping out to influence the concentration of cadmium in the matrix.
According to the invention, the variety of the leaf vegetable seeds is selected from Jiayuan cabbage mustard, vegetable spinach No. 1, Lvbao 701 flowering cabbage, specially selected leaf chrysanthemum coronarium, preferably polished rod chrysanthemum coronarium, white round leaf amaranth, green excellent green stem vegetable, sesames, red wrinkles, jadeite baby lettuce, Jiayuan flower amaranth, fast growing No. 1, vegetable spinach No. 8, pure sesame oil lettuce or tender green cream lettuce. The vegetable seeds of the variety used in the invention are seeds widely sold in the current market in Jingjin Ji area.
After the seeds are obtained, the seeds are first sterilized by a treatment method common in the art. For example, the seeds obtained are left to stand at a concentration of 25 to 35% by weight H2O2Soaking and disinfecting the seeds in the solution for 10-15 min, cleaning the seeds with deionized water, and then broadcasting the seeds.
According to the invention, the matrix is prepared from coconut coir, vermiculite and perlite according to the weight ratio of 6.0-7.5: 1.2-1.8: 1.2-1.8, has a cadmium content of 0-2.0 mg/kg and a pH of 6.5-8.0, contains a fertilizer plant growth promoting microbial inoculum, and has no peculiar smell, pathogenic bacteria and ova. Before sowing, the pH value of the matrix is adjusted to 6.5-8.0 by using a sodium hydroxide solution, and the pH condition of cadmium-polluted soil in northern China is simulated. Cadmium nitrate is used for preparing a cadmium aqueous solution, the cadmium concentration of the substrate is adjusted to 0-2.0 mg/kg by using the cadmium nitrate aqueous solution, and vegetable seeds can be sown after the cadmium aqueous solution is placed and stabilized for one week.
The coconut chaff, the vermiculite and the perlite used in the invention are all products sold in the current market.
B. Management of seedling stage
After the leaf vegetable seeds grow seedlings, watering once every 1-3 days according to the illumination condition, adding 15-30 ml of deionized water into each hole every time, and simultaneously keeping the conditions of humidity, sufficient illumination and good ventilation;
according to the invention, during the seedling raising period, the temperature of the greenhouse is controlled to be 25-30 ℃ in the daytime, and the temperature of the greenhouse is controlled to be 15-25 ℃ at night; the relative humidity of the greenhouse is controlled to be 65-75% in the daytime and at night.
C. Harvesting
Harvesting the edible parts of the leaf vegetable seedlings on the 10 th, 15 th and 20 th days of seedling culture, washing the edible parts with distilled water, drying the edible parts with filter paper, weighing the edible parts, drying the edible parts at the temperature of 60 ℃ to constant weight, and weighing to obtain the dry mass of the edible parts of the leaf vegetable seedlings;
in the step, the edible parts of the dried leaf vegetable seedlings with constant weight are ground into powder with the granularity of 100-200 meshes.
According to the invention, in order to determine the optimal seedling raising time of the method, 8 disinfected leaf vegetable seeds are sown in each hole of the seedling raising tray, and when the 3 rd true leaf of the leaf vegetable seedling grows out, 4-5 seedlings with uniform growth are left and cultured until the seedlings are harvested at 10 th, 15 th and 20 th days respectively.
D. Determination of cadmium content
Weighing 0.1000g of the leaf vegetable seedling sample dried to constant weight in the step C, placing glass beads in a triangular flask, adding 10mL of mixed acid of nitric acid and perchloric acid, carrying out cold digestion overnight, heating and stewing the mixture until white smoke is emitted in the next day to obtain colorless transparent or yellowish digestive juice, transferring the digestive juice into a 10mL volumetric flask, carrying out constant volume to scale by using ultrapure water, uniformly mixing, standing, diluting the supernatant by using deionized water by 5 times or 10 times, and detecting the cadmium content of the solution by using an inductively coupled plasma spectrometer or an inductively coupled plasma mass spectrometer;
in the invention, the total cadmium content of the vegetables is measured according to the method for measuring cadmium in GB/T5009.15-2003 food.
According to the invention, the mixed acid is a mixed acid of nitric acid and perchloric acid in a volume ratio of 3: 1.
In this step, a sample of dried constant-weight leaf vegetable seedlings was heated in a mixed acid at a temperature of 150 ℃ for 1.0 hour, further heated at a temperature of 180 ℃ for 0.5 hour, and then heated at a temperature of 220 ℃ for digestion.
If the cadmium content of the digestion solution is ppm level, detecting the cadmium content by using an inductively coupled plasma spectrometer (ICP) according to a conventional method; and if the content of the digested cadmium is ppb level, detecting the content of the cadmium by using an inductively coupled plasma mass spectrometer (ICP-MS) according to a conventional method.
E. Statistical evaluation of data
And D, performing statistical charting on the detection result obtained in the step D by adopting the existing software, and performing data comparison evaluation by using a clustering analysis method, a correlation analysis method, a three-factor variance analysis method and a one-factor variance analysis method so as to screen and obtain the leaf vegetables of the cadmium low-absorption variety.
According to the invention, the software is Excel 2013, IBM SPSS staticiscs 21 or SigmaPlot 10.0 software.
The method comprises the steps of covering all soil and plant factors related to the effectiveness of trace element plants from soil to the plants, evaluating the migration characteristics of cadmium from the soil to vegetable products by adopting a TC value, and predicting the overproof risk of cadmium-polluted soil vegetable products and the cadmium content safety threshold of the polluted soil according to the leaf vegetable cadmium content limit value (0.2mg/kg fresh mass) specified by the national food safety standard GB 2762-2017. The specific calculation method of the TC value is the ratio of the cadmium content (fresh quality) of the edible part of the vegetable to the total cadmium content of the soil;
the test data is subjected to statistical mapping by adopting Excel 2013, IBM SPSS statics 21 and SigmaPlut 10.0 software, data comparison evaluation is carried out by using cluster analysis, correlation analysis, three-factor variance analysis and single-factor variance analysis methods, and the conditions of the substrate cadmium concentration and the seedling growing days with the highest goodness of fit with field test data under the cultivation of the cadmium-rich substrate are screened out, so that the rapid screening method of the leaf vegetable cadmium low-absorption variety with the optimal conditions is established.
The detailed description section will specifically describe these analyses and the results of the analyses.
[ advantageous effects ]
The invention has the beneficial effects that: the difference between the types and varieties of crops is an important reason for determining the difference between the absorption and accumulation capacities of cadmium in the same environment. According to the invention, the accumulation capacities of different types of leaf vegetables on cadmium are rapidly compared by a cadmium-rich matrix cultivation method, so that vegetable varieties which can be safely produced in cadmium-polluted farmlands are obtained. Test results show that the method can be used for rapidly comparing the cadmium enrichment capacity of different types of leaf vegetables and has better consistency with field test results to a certain extent. In general, compared with other methods reported at home and abroad, the method for cultivating the cadmium-rich matrix has the advantages of economy, time saving, easy operation, capability of better reflecting the real conditions in the field and the like, and can provide a new idea for the rapid comparison of the cadmium accumulation capacity of vegetables.
The cultivation method of the cadmium-rich matrix can compare or predict cadmium accumulation risks under field conditions of different varieties of leaf vegetables to a certain extent, provides a feasible technical means for rapidly screening low-accumulation type leaf vegetables in the alkaline cadmium-polluted soil in northern China, and simultaneously provides reference and reference for the development of related screening methods in the future.
[ description of the drawings ]
FIG. 1 is a field test TC value for 15 leaf vegetable varieties of vegetables;
FIG. 2 shows the correlation between the TC values of the seedling test and the field test of 8 leaf vegetable varieties under the conditions of different seedling days and different substrate cadmium contents;
FIG. 3 is a cluster analysis of the TC values of cadmium migration coefficients of 8 leaf vegetable varieties under 4 cultivation conditions;
FIG. 4 is a cluster analysis of the TC values of cadmium migration coefficients based on 9 leaf vegetable varieties;
FIG. 5 is a correlation analysis of TC values of 17 leaf vegetable varieties in a field test and a seedling culture test under the optimal cultivation condition.
[ detailed description ] embodiments
The invention will be better understood from the following examples.
Example 1: method for rapidly identifying low-cadmium-absorption variety of leaf vegetable
The method comprises the following steps:
A. seeding
Adding 30 g of matrix into each hole of the seedling raising plate, flattening to enable the grid chambers of the hole plate to be clearly visible, wherein the diameter of each hole is 6.5cm, the depth of each hole is 8cm, small holes in the bottom of the seedling raising plate are sealed by black adhesive tapes, and the matrix is prepared from coconut chaff, vermiculite and perlite according to the weight ratio of 6.0: 1.8: 1.2, the cadmium content of the fertilizer is 1.5mg/kg, the pH value is 7.0, and the fertilizer contains a fertilizer plant growth promoting microbial inoculum without peculiar smell, pathogenic bacteria and ova; respectively sowing 8 disinfected Jiayuan cabbage, vegetable spinach No. 1, Lvbao 701 flowering cabbage seeds and specially selected leaf crowndaisy chrysanthemum leaf seeds in each hole, adding deionized water to moisten the seeds, covering the seeds with a matrix of 1.5mm, covering the matrix with a plastic film, germinating the seeds 2-3 days after sowing, removing the plastic film, and keeping moist, sufficient illumination and good ventilation conditions;
B. management of seedling stage
After the leaf vegetable seeds grow seedlings, watering once every 1 day according to the illumination condition, adding 15ml of deionized water into each hole every time, and simultaneously keeping the conditions of humidity, sufficient illumination and good ventilation, wherein the temperature of the greenhouse is controlled to be 28 ℃ in the daytime and 25 ℃ at night during seedling growing; controlling the relative humidity of the greenhouse at 65% in the daytime and at night;
C. harvesting
Harvesting edible parts of the leaf vegetable seedlings on the 10 th day of seedling culture, cleaning the edible parts with distilled water, sucking the edible parts with filter paper, weighing the edible parts, drying the edible parts to constant weight at the temperature of 60 ℃, grinding the dried edible parts of the leaf vegetable seedlings to be powder with the granularity of 100 meshes, and weighing to obtain the dry mass of the edible parts of the leaf vegetable seedlings;
D. determination of cadmium content
Weighing 0.1000g of the leaf vegetable seedling sample dried to constant weight in the step C, placing the sample into a triangular flask, adding 10mL of mixed acid of nitric acid and perchloric acid according to a volume ratio of 3:1, soaking overnight, heating at 150 ℃ for 1.0 hour, heating at 180 ℃ for 0.5 hour, heating at 220 ℃ for digestion until white smoke is emitted to obtain colorless and transparent digestive juice, transferring the digestive juice into a 10mL volumetric flask, fixing the volume to scale with ultrapure water, mixing uniformly, standing, diluting the supernatant by 5 times with deionized water, and detecting the cadmium content of the solution by using an inductively coupled plasma spectrometer or an inductively coupled plasma mass spectrometer;
E. statistical evaluation of data
And D, performing statistical charting on the detection result obtained in the step D by adopting the existing software Excel 2013, and performing data comparison evaluation by using a clustering analysis method, a correlation analysis method, a three-factor variance analysis method and a single-factor variance analysis method, so as to obtain the leaf vegetables of the cadmium low-absorption variety by screening, wherein the specific implementation result refers to experimental examples 1-5.
Example 2: method for rapidly identifying low-cadmium-absorption variety of leaf vegetable
The method comprises the following steps:
A. seeding
Adding 30 g of matrix into each hole of the seedling raising plate, flattening to enable the grid chambers of the hole plate to be clearly visible, wherein the diameter of each hole is 6.0cm, the depth of each hole is 12cm, small holes in the bottom of the seedling raising plate are sealed by black adhesive tapes, and the matrix is prepared from coconut chaff, vermiculite and perlite according to the weight ratio of 6.5: 1.4: 1.6, the cadmium content of the fertilizer is 2.0mg/kg, the pH value is 6.5, and the fertilizer contains a fertilizer plant growth promoting microbial inoculum without peculiar smell, pathogenic bacteria and ova; then respectively sowing 10 disinfected preferable seeds of the polish rod crowndaisy chrysanthemum, the white round leaf amaranth, the green excellent green stem vegetable and the sesamum indicum leaf vegetable in each hole, adding deionized water for wetting, covering the substrate with the thickness of 1.8mm, covering the substrate with a plastic film, germinating the seeds 2-3 days after sowing, removing the plastic film, and keeping the conditions of wetting, sufficient illumination and good ventilation;
B. management of seedling stage
After the leaf vegetable seeds grow seedlings, watering once every 2 days according to the illumination condition, adding 20ml of deionized water into each hole every time, simultaneously keeping the conditions of humidity, sufficient illumination and good ventilation, and controlling the temperature of the greenhouse to be 25 ℃ in the daytime and 18 ℃ at night during seedling culture; controlling the relative humidity of the greenhouse at 70% in the daytime and at night;
C. harvesting
Harvesting edible parts of the leaf vegetable seedlings on the 10 th day of seedling culture, cleaning the edible parts with distilled water, sucking the edible parts with filter paper, weighing the edible parts, drying the edible parts to constant weight at the temperature of 60 ℃, grinding the dried edible parts of the leaf vegetable seedlings to powder with the particle size of 200 meshes, and weighing to obtain the dry mass of the edible parts of the leaf vegetable seedlings;
D. determination of cadmium content
Weighing 0.1000g of the leaf vegetable seedling sample dried in the step C to constant weight, placing the sample in a triangular flask, adding 10mL of mixed acid of nitric acid and perchloric acid according to a volume ratio of 3:1, soaking overnight, heating at 150 ℃ for 1.0 hour, heating at 180 ℃ for 0.5 hour, heating at 220 ℃ for digestion until white smoke is emitted to obtain yellowish digestive juice, transferring the yellowish digestive juice into a 10mL volumetric flask, fixing the volume to a scale with ultrapure water, mixing uniformly, standing, diluting the supernatant by 10 times with deionized water, and detecting the cadmium content of the solution by using an inductively coupled plasma spectrometer or an inductively coupled plasma mass spectrometer;
E. statistical evaluation of data
And D, performing statistical mapping on the detection result obtained in the step D by adopting the existing software IBM SPSS Statistics 21, and performing data comparison evaluation by using a clustering analysis, correlation analysis, three-factor variance analysis and single-factor variance analysis method to screen and obtain the cadmium low-absorption variety leaf vegetables, wherein the specific implementation result refers to test examples 1-5.
Example 3: method for rapidly identifying low-cadmium-absorption variety of leaf vegetable
The method comprises the following steps:
A. seeding
Adding 30 g of matrix into each hole of the seedling raising disc, flattening to enable the grid chambers of the hole disc to be clearly visible, wherein the diameter of each hole is 7.0cm, the depth of each hole is 10cm, small holes in the bottom of the seedling raising disc are sealed by black adhesive tapes, and the matrix is prepared from coconut chaff, vermiculite and perlite according to the weight ratio of 7.0: 1.6: 1.8, has a cadmium content of 0.5mg/kg and a pH of 7.5, contains a fertilizer plant growth promoting microbial inoculum, and has no peculiar smell, pathogenic bacteria and ova; respectively sowing 11 disinfected red wrinkles, jadeite baby lettuce, Jiayuan leaf amaranth and fast-growing leaf vegetable seeds No. 1 in each hole, adding deionized water for wetting, covering with a substrate of 2.2mm, covering with a plastic film, germinating 2-3 days after sowing, removing the plastic film, and keeping the conditions of wetting, sufficient illumination and good ventilation;
B. management of seedling stage
After the leaf vegetable seeds grow seedlings, watering every 3 days according to the illumination condition, adding 25ml of deionized water into each hole every time, and simultaneously keeping the conditions of humidity, sufficient illumination and good ventilation, wherein the temperature of the greenhouse is controlled to be 28 ℃ in the daytime and 15 ℃ at night during seedling culture; controlling the relative humidity of the greenhouse at 75% in the daytime and at night;
C. harvesting
Harvesting edible parts of leaf vegetable seedlings on the 10 th day of seedling culture, cleaning the edible parts with distilled water, sucking the edible parts with filter paper, weighing the edible parts, drying the edible parts to constant weight at the temperature of 60 ℃, grinding the dried edible parts of the leaf vegetable seedlings to be powder with the granularity of 120 meshes, and weighing to obtain the dry mass of the edible parts of the leaf vegetable seedlings;
D. determination of cadmium content
Weighing 0.1000g of the leaf vegetable seedling sample dried to constant weight in the step C, placing the sample into a triangular flask, adding 10mL of mixed acid of nitric acid and perchloric acid according to a volume ratio of 3:1, soaking overnight, heating at 150 ℃ for 1.0 hour, heating at 180 ℃ for 0.5 hour, heating at 220 ℃ for digestion until white smoke is emitted to obtain colorless and transparent digestive juice, transferring the digestive juice into a 10mL volumetric flask, fixing the volume to scale with ultrapure water, mixing uniformly, standing, diluting the supernatant by 5 times with deionized water, and detecting the cadmium content of the solution by using an inductively coupled plasma spectrometer or an inductively coupled plasma mass spectrometer;
E. statistical evaluation of data
And D, performing statistical charting on the detection result obtained in the step D by adopting the existing software SigmaPlut 10.0, and performing data comparison evaluation by using a clustering analysis, correlation analysis, three-factor variance analysis and single-factor variance analysis method to obtain the cadmium low-absorption variety of leaf vegetables by screening, wherein the specific implementation result refers to test examples 1-5.
Example 4: method for rapidly identifying low-cadmium-absorption variety of leaf vegetable
The method comprises the following steps:
A. seeding
Adding 30 g of matrix into each hole of the seedling raising plate, flattening to enable the grid chambers of the hole plate to be clearly visible, wherein the diameter of each hole is 6.5cm, the depth of each hole is 11cm, small holes in the bottom of the seedling raising plate are sealed by black adhesive tapes, and the matrix is prepared from coconut chaff, vermiculite and perlite according to the weight ratio of 7.5: 1.2: 1.4, the cadmium content of the fertilizer is 1.0mg/kg, the pH value is 8.0, and the fertilizer contains a fertilizer plant growth promoting microbial inoculum without peculiar smell, pathogenic bacteria and ova; then respectively broadcasting 12 disinfected Lvbao 701 heart, vegetable and spinach No. 8, pure sesame oil and lettuce seeds and tender green cream lettuce leaf vegetable seeds in each hole, adding deionized water for wetting, covering with a matrix of 2.5mm, covering with a plastic film, germinating 2-3 days after sowing, removing the plastic film, and keeping the conditions of wetting, sufficient illumination and good ventilation;
B. management of seedling stage
After the leaf vegetable seeds grow seedlings, watering once every 2 days according to the illumination condition, adding 30ml of deionized water into each hole every time, simultaneously keeping the conditions of humidity, sufficient illumination and good ventilation, and controlling the temperature of a greenhouse at 30 ℃ in the daytime and 20 ℃ at night during seedling culture; controlling the relative humidity of the greenhouse at 72% in the daytime and at night;
C. harvesting
Harvesting edible parts of leaf vegetable seedlings on the 10 th day of seedling culture, cleaning the edible parts with distilled water, sucking the edible parts with filter paper, weighing the edible parts, drying the edible parts to constant weight at the temperature of 60 ℃, grinding the dried edible parts of the leaf vegetable seedlings to be powder with the particle size of 160 meshes, and weighing to obtain the dry mass of the edible parts of the leaf vegetable seedlings;
D. determination of cadmium content
Weighing 0.1000g of the leaf vegetable seedling sample dried in the step C to constant weight, placing the sample in a triangular flask, adding 10mL of mixed acid of nitric acid and perchloric acid according to a volume ratio of 3:1, soaking overnight, heating at 150 ℃ for 1.0 hour, heating at 180 ℃ for 0.5 hour, heating at 220 ℃ for digestion until white smoke is emitted to obtain yellowish digestive juice, transferring the yellowish digestive juice into a 10mL volumetric flask, fixing the volume to a scale with ultrapure water, mixing uniformly, standing, diluting the supernatant by 5 times with deionized water, and detecting the cadmium content of the solution by using an inductively coupled plasma spectrometer or an inductively coupled plasma mass spectrometer;
E. statistical evaluation of data
And D, performing statistical mapping on the detection result obtained in the step D by adopting the existing software IBM SPSS Statistics 21, and performing data comparison evaluation by using a clustering analysis, correlation analysis, three-factor variance analysis and single-factor variance analysis method to screen and obtain the cadmium low-absorption variety leaf vegetables, wherein the specific implementation result refers to test examples 1-5.
Test example 1:
the implementation of this example is as follows:
testing leaf vegetable varieties:
the tested leaf vegetable varieties are the following leaf vegetable varieties commonly seen in facility agriculture in Jingjin Ji areas: jiayuan cabbage mustard, vegetable spinach No. 1, Lvbao 701 flowering cabbage, specially selected leaf chrysanthemum coronarium, preferably polished rod chrysanthemum coronarium, white round leaf amaranth, green excellent green stem vegetable, sesame vegetable, red wrinkle, jadeite baby lettuce, Jiayuan flower amaranth, fast growing No. 1, vegetable spinach No. 8, pure sesame oil wheat vegetable and tender green cream lettuce. The information of specific numbers and corresponding varieties is shown in table 1.
Table 1: name and supplier of 15 kinds of leaf vegetables
The test method comprises the following steps:
the field plot experiment is carried out in a facility greenhouse of Li family, Yingcun, Qing county, Hebei in 2017, 9 months to 2017, 11 months. The average content of organic matters in soil in the test greenhouse is 23.7 g.kg-11.4 g.kg of total nitrogen-111.3 mg/kg of available phosphorus-1Quick-acting potassium 103.8 mg/kg-1And the soil pH is 7.6. The total cadmium content of the soil in the greenhouse to be tested is 0.38mg kg-1According to the evaluation standard of greenhouse vegetable production place environment quality (HJ333-2006, when the pH value of the soil is more than 7.5, the limit value of the total cadmium content of the soil is 0.4mg kg-1) The cadmium pollution level of the soil to be tested belongs to the grade of cleanness. The area of each cell in the greenhouse is 15m2(3m wide by 5m long), each leaf dish variety was treated for 3 replicates. The planting density in the residential area is set according to the planting characteristics of each leaf vegetable. Collecting edible part samples of 4-5 leaf vegetables in each cell and soil samples of corresponding root areas in the harvest period, uniformly mixing, and then bringing the mixture back to a laboratory. Weighing fresh weight of plant sample, deactivating enzyme, and dryingDry weight, grinding, and analyzing the total cadmium content of the plant; and (4) after the soil sample is air-dried and sieved, analyzing the total cadmium content of the soil. The TC value difference of the vegetables of 15 leaf vegetable varieties is calculated, and the result is shown in figure 1. The results shown in figure 1 show that the cadmium accumulation capacity of different varieties of leaf vegetables is remarkably different. The TC value of 15 leaf vegetable varieties is obvious in gradient, the TC value of the sesames is the largest, and the preferred amount of the plate leaf crowndaisy chrysanthemum is the smallest.
Test example 2: indoor plug culture test
The implementation of this example is as follows:
hole trays with the length of 54cm and the width of 28cm, which are produced by plastic product factories of Shengji in Shangan county, Hebei province, are selected as seedling raising trays, wherein each seedling raising tray is provided with 32 holes, the diameter of each hole is 6.5cm, and the depth is 10 cm. The main components of the seedling culture substrate are coconut husk, vermiculite and perlite according to the weight ratio of 7.0: 1.5: 1.5. 30 g of the matrix is added into each hole, and then the pH value of the matrix is adjusted to 7.5 by using a 1N sodium hydroxide aqueous solution so as to simulate the pH condition of the cadmium-polluted soil in the north of China. Use 1 mg.L-1Adjusting the concentration gradient of the substrate cadmium to 0, 0.5, 1.0, 1.5 and 2.0mg/kg with the cadmium nitrate aqueous solution-1. Standing for one week, and selecting 8 leaf vegetables for cultivation, wherein the vegetables are JIAOYUANJING, vegetable Spinaciae No. 1, LVBAO 701 flowering cabbage, specially selected caulis et folium Chrysanthemi Segeti, preferably caulis et folium Chrysanthemi Segeti, herba Amaranthi Tricoloris, green caulis et folium Brassicae Capitatae, and herba Sesami. Each variety was set to 3 replicates, and each 6 wells was set to 1 replicate. And (3) directly sowing 8 seeds in each hole, thinning the seedlings when the 3 rd true leaf grows out, and respectively culturing 4-5 seedlings with uniform growth vigor until 10 th, 15 th and 20 th days for harvesting. The cadmium content of the edible parts of the leaf vegetables is measured by adopting the measuring method described in the specification, the TC values of the leaf vegetables are calculated, and then the comparison and fitting are carried out on the field test data of the leaf vegetables by the statistical method. The results of the indoor plug culture test are shown in Table 2.
The results of the three-factor analysis of variance listed in Table 2 show that the variety, the culture days, the cadmium content of the substrate and the interaction among factors of the leafy vegetables have obvious influence on the TC value of the leafy vegetables. It is known from comparative analysis that the substrate cadmium content, the two-factor interaction and the three-factor interaction contribute relatively limitedly to the total variation (sum of squares and SS), and the SS value is 0.001. The leaf vegetable varieties contribute most to the total variation, are the leading factors of the TC value difference of the leaf vegetables and control the absolute level of the TC value of the leaf vegetables.
Table 2: three-factor analysis of variance of TC values of 8 leaf vegetable varieties
Significant levels were reached when P <0.01
The comparison and analysis of the TC values of the indoor test leaf vegetables and the field test TC values shows that the TC values of the 8 leaf vegetables have obvious correlation with the TC values under the field test conditions under different substrate cadmium concentrations and different culture times. The correlation coefficient R is increased along with the cadmium content of the substrate under the same seedling raising days2The values generally show a tendency to increase first and then decrease. When the culture time is 10, 15 and 20 days, the maximum correlation coefficient R2The values are 0.905, 0.751 and 0.639 respectively, the corresponding screening conditions are 10 days of culture and the substrate cadmium content is 1.0mg/kg-1Culturing for 15 days and the cadmium content of the substrate is 1.5mg/kg-1Culturing for 20 days and the cadmium content in the substrate is 1.0mg/kg-1. In comprehensive comparison, when the culture time is 10 days, the matrix cadmium is 1.0mg/kg-1In the present study, the TC values of the leaf vegetables tested had the best correlation with the field test TC values, and the culture time required under these conditions was the shortest (table 3, fig. 2).
Table 3: correlation analysis R of TC values and field data under different seedling culture time and different substrate cadmium concentrations2Value of
Note: the cadmium concentration of the soil in the field test is 0.4 mg-kg-1
In order to further analyze the reliability of the cadmium-rich matrix method and the field test in the aspect of comparing the cadmium accumulation capacities of different leaf vegetable varieties, the TC values of 8 leaf vegetables are subjected to cluster analysis (figure 3). As shown in FIG. 3, the culture was carried out for 10 days and the substrate cadmium content was 1.0mg/kg-1Clustering of TC values of leaf vegetables under conditionsThe analysis result is basically consistent with the corresponding field test result: at a distance of 3 Europe, the cadmium accumulation capacity of 8 kinds of leaf vegetables is divided into two categories, namely a high cadmium accumulation group of sesames, a low cadmium accumulation group of Jiayuan cabbage, No. 1 vegetable spinach, cabbage heart of Lvbao 701, specially selected Chrysanthemum morifolium, preferably Chrysanthemum morifolium, Amaranthus album, and Lvyouqingkucai. Culturing for 15 days under the same classification distance and the cadmium content of the substrate is 1.5mg/kg-1Culturing for 20 days and the cadmium content in the substrate is 1.0mg/kg-1Under the condition, the cadmium accumulation capacity of 8 leaf vegetables can be divided into three categories. In comparison, the culture was carried out for 10 days and the substrate cadmium content was 1.0mg/kg-1Under the condition, the result of the cadmium accumulation capacity of different leaf vegetable varieties obtained by the cadmium-rich matrix method is the most consistent with the field result.
Table 4 shows that the TC values of 8 leaf vegetables are analyzed by significant difference, the cultivation lasts for 10 days, and the cadmium content of the matrix is 1.0mg/kg-1Under the condition, the TC value sequence of 8 leaf vegetable varieties is relatively consistent with the field test result, in particular to the sesames and the amaranthus albus. Culturing for 15 days and the cadmium content of the substrate is 1.5mg/kg-1Culturing for 20 days and the cadmium content in the substrate is 1.0mg/kg-1Under the condition, compared with the TC value sorting of the 8 leaf vegetable varieties in the field test, only the sesames with the maximum TC values are sorted consistently. The results further showed that the culture was carried out for 10 days and the substrate cadmium content was 1.0mg/kg-1The cadmium-rich matrix can relatively well reflect the accumulation capacity of the leafy vegetables to the soil cadmium under the field conditions.
Table 4: analysis of single-factor variance of TC (cadmium migration coefficient) values of 8 leaf vegetable varieties on cadmium migration coefficients
Test example 3:
to further clarify the accuracy of the method, the optimum screening conditions (10 days in the nursery stage and 1.0mg/kg of exogenous cadmium in the substrate) were clarified in the above-mentioned experiment-1) And carrying out a verification test. The conditions of the used substrate, the plug and the like are the same, and 9 leaf vegetables are selected in the test: herba Selaginellae, herba Crotalariae sessiliflorae, jadeite baby lettuce, Jiayuan flower and leaf amaranth, radix rehmanniae 1, vegetable spinach 8, pure sesame oil herba MAICAI, and light green butterVegetables, preferably crowndaisy chrysanthemum. After the culture is finished, the fresh sample of the edible part of each treatment is harvested. The test is compared with the result of the enrichment capacity of the selected vegetables to the soil cadmium under the field condition at the earlier stage, so that the accuracy and the repeatability of the method are determined.
The determined cadmium-rich matrix culture method is utilized to research the cadmium-rich capacity of the alternative 9 leaf vegetables, and the accuracy of the method is verified by comparing the cadmium-rich capacity of the 9 leaf vegetables in a field test. Based on the single-factor analysis of variance of the TC values of the leafy vegetables, the TC values of the 9 leafy vegetables are basically consistent with the magnitude ordering of the field test results, and particularly, the sesames, the vegetables and the spinach 8, the red wrinkles, the fast-growing No. 1 and the preferred polished rod crowndaisy chrysanthemum show higher consistency (Table 5). The cluster analysis result shows that the culture lasts for 10 days and the cadmium content of the substrate is 1.0 mg.kg-1The TC value data of the leaf vegetables obtained under the conditions is high in matching degree with field test results (figure 4). The cadmium-rich substrate culture and 9 leaf vegetable varieties can be divided into two groups under field conditions, namely a high accumulation group (sesames) and a medium and low accumulation group (red wrinkles, jadeite baby lettuce, Jiayuan flower leaf amaranth, fast growing No. 1, vegetable spinach No. 8, pure sesame oil wheat vegetable, light green cream lettuce, and preferably polished rod crowndaisy chrysanthemum).
Table 5: analysis of single-factor variance of TC (cadmium migration coefficient) values of 9 leaf vegetable varieties on cadmium migration coefficients
Test example 4:
the quantitative prediction of the risk of cadmium accumulation of different crops can more intuitively and accurately evaluate the risk of cadmium pollution of the crops in the farmland. The invention puts 17 kinds of leaf vegetables in cadmium-rich substrate (culturing for 10 days, the cadmium content of the substrate is 1.0 mg.kg)-1) The cadmium TC value in the cadmium-containing solution is subjected to correlation analysis with the cadmium TC value obtained under field conditions (the soil pH is 7.5), and the cadmium TC value and the cadmium-containing solution are also very significantly correlated (the Pearson correlation coefficient r is 0.9277, P<0.01, the quantitative relationship between the two can be expressed by the following equation: and y is 2.0775x-0.0911, wherein y is the TC value of the leaf vegetables cultivated in the cadmium-rich medium under the field condition, and x is the TC value of the leaf vegetables cultivated in the cadmium-rich medium, and the result is shown in figure 5.
The equation can be used for further quantitatively calculating the possible cadmium accumulation risk of certain leafy vegetables when the certain leafy vegetables are planted on certain cadmium-polluted soil, and the calculation method comprises the following steps:
under the field condition, the predicted value of the cadmium content of a certain leaf vegetable is equal to the predicted value TC of the leaf vegetable under the field condition multiplied by the cadmium content of the polluted soil;
in addition, the soil environmental quality standard GB15618-2018 (when the soil pH is 7.5, the exceeding limit value of soil cadmium is 0.3 mg/kg)-1) And cadmium accumulation limit value (0.2mg kg) of leaf vegetable agricultural products-1FW) to obtain the maximum allowable value of the cadmium migration coefficient TC of the leaf vegetable agricultural products under the condition that the soil exceeds the standard, and then applying the maximum allowable value to the equation to obtain the maximum allowable cumulant of the leaf vegetables in the cadmium-rich substrate adopted by the invention, namely 0.40 mg.kg-1FW, can be used as an important reference value to predict the possible cumulative risk of the leaf vegetable variety under the soil condition.
Claims (10)
1. A method for quickly identifying a leaf vegetable cadmium low absorption variety by utilizing cadmium-rich matrix cultivation is characterized by comprising the following steps:
A. seeding
Adding 30 g of matrix into each hole of the seedling raising tray, flattening to enable the grid chambers of the hole tray to be clearly visible, then broadcasting 8-12 disinfected leaf vegetable seeds in each hole, adding deionized water for wetting, covering 1.5-2.5 mm of matrix, covering with a plastic film, germinating 2-3 days after sowing, removing the plastic film, and keeping the conditions of wetting, sufficient illumination and good ventilation;
B. management of seedling stage
After the leaf vegetable seeds grow seedlings, watering once every 1-3 days according to the illumination condition, adding 15-30 ml of deionized water into each hole every time, and simultaneously keeping the conditions of humidity, sufficient illumination and good ventilation;
C. harvesting
Harvesting the edible parts of the leaf vegetable seedlings on the 10 th, 15 th and 20 th days of seedling culture, washing the edible parts with distilled water, drying the edible parts with filter paper, weighing the edible parts, drying the edible parts at the temperature of 60 ℃ to constant weight, and weighing to obtain the dry mass of the edible parts of the leaf vegetable seedlings;
D. determination of cadmium content
Weighing 0.1000g of the leaf vegetable seedling sample dried to constant weight in the step C, placing glass beads in a triangular flask, adding 10mL of mixed acid of nitric acid and perchloric acid, carrying out cold digestion overnight, heating and stewing the mixture until white smoke is emitted in the next day to obtain colorless transparent or yellowish digestive juice, transferring the digestive juice into a 10mL volumetric flask, carrying out constant volume to scale by using ultrapure water, uniformly mixing, standing, diluting the supernatant by using deionized water by 5 times or 10 times, and detecting the cadmium content of the solution by using an inductively coupled plasma spectrometer or an inductively coupled plasma mass spectrometer;
E. statistical evaluation of data
And D, performing statistical charting on the detection result obtained in the step D by adopting the existing software, and performing data comparison evaluation by using a clustering analysis method, a correlation analysis method, a three-factor variance analysis method and a one-factor variance analysis method so as to screen and obtain the leaf vegetables of the cadmium low-absorption variety.
2. The method for rapidly identifying the leaf vegetable cadmium low-absorption variety according to claim 1, wherein in the step A, the diameter of each hole is 6-7 cm, the depth of each hole is 8-12 cm, and small holes in the bottom of a seedling raising tray are sealed by black adhesive tapes.
3. The method for rapidly identifying the leafy vegetable cadmium low absorption variety according to claim 1, wherein in the step A, the leafy vegetable seed variety is selected from kale, vegetable spinach No. 1, Lvbao 701 flowering cabbage, specially selected leaf crowndaisy chrysanthemum, preferably polished round-grained chrysanthemum, white round-grained amaranth, Lvbou green-peduncle vegetable, sesameseed, red wrinkle, jadeite baby lettuce, Jiaoyouye amaranth, red wrinkle, fast-growing No. 1, vegetable spinach No. 8, pure sesame oil wheat vegetable or green cream lettuce.
4. The method for rapidly identifying the cadmium low-absorption variety of the leaf vegetables according to claim 1, wherein in the step A, the matrix is prepared from coconut coir, vermiculite and perlite according to a weight ratio of 6.0-7.5: 1.2-1.8: 1.2-1.8, has a cadmium content of 0-2.0 mg/kg and a pH of 6.5-8.0, contains a fertilizer plant growth promoting microbial inoculum, and has no peculiar smell, pathogenic bacteria and ova.
5. The method for rapidly identifying the cadmium-low-absorption variety of the leaf vegetables according to claim 1, wherein in the step B, during the seedling raising period, the temperature of the greenhouse is controlled to be 25-30 ℃ in the daytime, and the temperature of the greenhouse is controlled to be 15-25 ℃ at night; the relative humidity of the greenhouse is controlled to be 65-75% in the daytime and at night.
6. The method for rapidly identifying the variety with low cadmium absorption of the leaf vegetables according to claim 1, wherein in the step C, the edible parts of the dried leaf vegetable seedlings with constant weight are ground into powder with the granularity of 100-200 meshes.
7. The method for rapidly identifying the leafy vegetable cadmium low absorption variety according to claim 1, characterized in that in the step C, 8 disinfected leafy vegetable seeds are sown in each hole of a seedling tray, and when the leafy vegetable seedlings grow to have 3 rd true leaves, the seedlings are left and cultured until the 4-5 seedlings with uniform growth vigor are harvested respectively at 10 th, 15 th and 20 th days.
8. The method for rapidly identifying the leafy vegetable cadmium low absorption variety according to claim 1, characterized in that in step C, the dried constant weight leafy vegetable seedling sample is heated in mixed acid at 150 ℃ for 1.0 hour, then at 180 ℃ for 0.5 hour, and then at 220 ℃ for digestion.
9. The method for rapidly identifying the leafy vegetable cadmium low absorption variety according to claim 1, wherein in the step D, the mixed acid is a mixed acid of nitric acid and perchloric acid in a volume ratio of 3: 1.
10. The method for rapidly identifying the cadmium low-absorption variety of the leaf vegetables according to claim 1, wherein in the step E, the software is Excel 2013, IBM SPSS Statistics 21 or SigmaPlut 10.0 software.
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