Application of Zn-EDTA in reducing cadmium accumulation in wheat grain

Abstract

The application belongs to the technical field of safe production in farmland, and particularly relates to application of Zn-EDTA in reducing cadmium accumulation in wheat kernels. In cadmium-polluted soil, application of Zn-EDTA can not only supplement zinc elements required by wheat growth, but also is more superior to traditional Zn fertilizer in the effect of reducing Cd concentration in wheat kernels.

Description

Technical Field

[0001] This invention belongs to the field of agricultural safety production technology, and in particular relates to the application of Zn-EDTA in reducing cadmium accumulation in wheat grains. Background Technology

[0002] Among common heavy metals, cadmium (Cd) poses one of the highest risks of translocation to edible parts of crops. Once in the edible parts of crops, Cd enters the human body through the food chain. Long-term consumption of food with excessive Cd levels can lead to many incurable diseases, such as osteoporosis, non-hypertrophic emphysema, irreversible renal tubular damage, and even induce cancer. Compared to crops like corn and barley, wheat has a stronger capacity for Cd accumulation; under neutral and slightly alkaline soil conditions, wheat grains can accumulate even more Cd than rice. Therefore, ensuring safe wheat production in cadmium-contaminated areas and identifying wheat varieties with low Cd accumulation or implementing cost-effective control measures to reduce Cd accumulation in wheat grains are of great significance.

[0003] The accumulation and transport of heavy metal Cd in the soil-wheat system have been extensively studied. The absorption and accumulation of heavy metal Cd by wheat is affected by many factors, including wheat variety, soil properties, soil pH, and water and fertilizer management.

[0004] Zinc (Zn) is a crucial micronutrient for plant growth, participating in the regulation of numerous enzyme activities and significantly influencing physiological processes such as protein synthesis, hormone synthesis, carbohydrate metabolism, and photosynthesis. In crops like wheat, insufficient Zn supply can lead to stunted growth, inhibited chlorophyll synthesis, and reduced yield and quality. Conversely, for Zn-deficient farmland, breeding methods can increase the Zn content of wheat varieties, and applying Zn fertilizer can significantly improve crop yield and quality. Cadmium (Cd) is a non-essential element for plants, not participating in plant structure or any metabolic activities. Plants have not evolved specific absorption channels for Cd; Cd enters plants primarily through absorption and transport channels associated with Zn, Fe, and Mn. Therefore, Zn and Cd compete for absorption in plant roots. Increasing the effective utilization concentration of Zn in the soil can inhibit Cd absorption by crops and reduce its accumulation within the plant. The zinc fertilizer used in existing technologies is generally zinc sulfate. When zinc sulfate is applied to the soil, it dissolves rapidly. Most of the zinc ions that are not absorbed by plants in the short term are adsorbed and fixed by clay minerals, carbonates, iron oxides and other substances in the soil, which reduces the availability of zinc in the soil and the antagonistic effect of zinc against cadmium.

[0005] Zinc disodium ethylenediaminetetraacetate (Zn-EDTA) is commonly used in agricultural production as a foliar fertilizer or fertigation for fruit trees. Compared to zinc sulfate, EDTA-Zn can be better absorbed and utilized by crops, and it is more stable, with weaker adsorption and fixation by soil particles. However, the application of EDTA-Zn in reducing cadmium in wheat grains is not mentioned in the existing technology. On the one hand, EDTA-Zn can maintain higher activity of Zn in the soil for plant absorption, thus playing an antagonistic role in cadmium absorption. On the other hand, in dryland soils, EDTA is often used as an exogenous activator to promote the absorption of cadmium in the soil by hyperaccumulating plants (non-food crops). See the published literature "Song Bo, Zhang Yunxia, ​​Tian Meiling, et al. Potential of applying grain amaranth to remediate cadmium-contaminated farmland soil [J]. Journal of Environmental Engineering, 2019, 13(7): 1711-1719". If EDTA is applied to dryland soils of wheat, there is a risk of activation and promotion of cadmium absorption. Summary of the Invention

[0006] The inventors of this application have discovered that in cadmium-contaminated soil, the application of Zn-EDTA can not only supplement the zinc required for wheat growth, but also achieve the same effect as traditional Zn fertilizers in reducing Cd concentration in wheat grains. Furthermore, this study found that while EDTA-Zn can activate cadmium in the soil to some extent, its effect on increasing available zinc in the soil far exceeds the increase in cadmium, resulting in an increase in the ratio of available zinc to available cadmium (Zn / Cd) in the soil by 1.09 to 2.41 times. This demonstrates superior effectiveness in reducing Cd concentration in wheat grains compared to traditional Zn fertilizers. Therefore, the purpose of this invention is to provide the application of Zn-EDTA in reducing cadmium accumulation in wheat grains.

[0007] The technical solution adopted in this invention is:

[0008] This invention discloses the application of Zn-EDTA in reducing cadmium accumulation in wheat grains; the application includes the following steps: Zn-EDTA is applied in three stages. The first stage is before wheat sowing, when a portion of Zn-EDTA is applied as a base fertilizer simultaneously with nitrogen, phosphorus, and potassium compound fertilizer. After the base fertilizer is applied, Zn-EDTA is applied again in the second stage, namely the wheat jointing stage, and the third stage, the booting stage, and the mass ratio of Zn-EDTA fertilizer applied in the first, second, and third stages is 3:1:1.

[0009] Preferably, the wheat variety is a low Cd accumulation variety, and more preferably, the low Cd accumulation variety is Ningmai 11.

[0010] Preferably, the zinc addition amount is calculated per kg of dry soil, and the zinc addition amount of the Zn-EDTA fertilizer is 20-40 mg / kg.

[0011] Preferably, the amount of nitrogen, phosphorus, and potassium compound fertilizer added is calculated per kg of dry soil, and the amount of nitrogen, phosphorus, and potassium compound fertilizer added is 1.2 g / kg; more preferably, the mass ratio of N:P2O5:K2O in the nitrogen, phosphorus, and potassium compound fertilizer is 15:15:15.

[0012] Preferably, urea is applied to each pot to supplement nitrogen during the jointing and booting stage. More preferably, the amount of urea added is 0.2 g / kg, calculated per kg of dry soil.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] This invention demonstrates through experiments that, compared to traditional ZnSO4 fertilizer, Zn-EDTA not only achieves the effects of traditional Zn fertilizer, but also has a more significant effect on increasing the Zn / Cd ratio in rhizosphere soil and reducing the expression of Cd transporter protein-related genes. It also has a better efficiency in inhibiting Cd accumulation in wheat grains. At the same time, the application of Zn-EDTA also has a positive impact on sustainable planting in the soil. Attached Figure Description

[0015] Figures 1 to 27 In the figure, all values ​​are mean ± standard deviation (SD); lowercase and uppercase letters indicate statistical significance (P < 0.05) between different treatments of NM11 and YM27, respectively, and the x-axis represents different treatment groups.

[0016] Figure 1 This refers to the number of grains per plant in mature wheat under zinc treatment in the experimental example.

[0017] Figure 2 The dry weight of a single wheat plant at maturity under zinc treatment in the experimental example;

[0018] Figure 3 The dry weight of a single wheat straw at maturity under zinc treatment in the experimental example;

[0019] Figure 4 The thousand-grain weight of mature wheat under zinc treatment in the experimental example;

[0020] Figure 5 The concentration of Cd in mature wheat grains under zinc treatment in the experimental example;

[0021] Figure 6 The concentration of Cd in mature wheat straw under zinc treatment in the experimental example;

[0022] Figure 7 The concentration of Cd in the roots of mature wheat under zinc treatment in the experimental example;

[0023] Figure 8 The concentration of Zn in mature wheat grains under zinc treatment in the experimental example;

[0024] Figure 9 The concentration of Zn in the roots of mature wheat under zinc treatment in the experimental example;

[0025] Figure 10 The pH of the rhizosphere soil of mature wheat under zinc treatment in the experimental example;

[0026] Figure 11 The pH of the non-rhizosphere soil of mature wheat under zinc treatment in the experimental example;

[0027] Figure 12 The effective Cd concentrations in the rhizosphere and non-rhizosphere soils of mature wheat under zinc treatment in the experimental example;

[0028] Figure 13 The effective Zn concentrations in the rhizosphere and non-rhizosphere soils of mature wheat under zinc treatment in the experimental example;

[0029] Figure 14 The ratio of available Zn / Cd in the rhizosphere soil to the non-rhizosphere soil during the mature stage of potted plants in the experimental example;

[0030] Figure 15 The Cd translocation coefficient between wheat straw and grain at maturity under zinc treatment in the experimental example;

[0031] Figure 16 The Cd translocation coefficient between wheat roots and straw at maturity under zinc treatment in the experimental example;

[0032] Figure 17 This represents the relative expression level of the Cd transporter gene TaHMA2 in wheat roots during the flowering stage under zinc treatment in the experimental example.

[0033] Figure 18 This represents the relative expression level of the Cd transporter gene TaZIP5 in wheat roots during the flowering stage under zinc treatment in the experimental example.

[0034] Figure 19 This represents the relative expression level of the Cd transporter gene TaIRT1 in wheat roots during the flowering stage under zinc treatment in the experimental example. Figure 20 The relative expression level of the Cd transporter gene TaNRAMP3 in wheat roots during the flowering stage under zinc treatment in the experimental example.

[0035] Figure 21 The relative expression level of the Cd transporter gene TaZIP3 in wheat roots during the flowering stage under zinc treatment in the experimental example. Figure 22 The relative expression level of the Cd transporter gene TaNRAMP1 in wheat roots during the flowering period under zinc treatment in the experimental example;

[0036] Figure 23 The figure represents the relative expression level of the Cd transporter gene TaZIP7 in the first node of wheat at the flowering stage under zinc treatment in the experimental example.

[0037] Figure 24 The relative expression level of the Cd transporter gene TaZIP7 in the flag leaf of wheat during the flowering stage under zinc treatment in the experimental example; Figure 25 The relative expression level of the Cd transporter gene TaHMA2 in the flag leaf of wheat during the flowering stage under zinc treatment in the experimental example;

[0038] Figure 26 The figure shows the relative expression level of the Cd transporter gene TaHMA2 in the first node of wheat at the flowering stage under zinc treatment in the experimental example.

[0039] Figure 27 The relative expression level of the Cd transporter gene TaZIP3 in the flag leaf of wheat during the flowering stage under zinc treatment is shown in the experimental example. Detailed Implementation

[0040] The invention will be further described below with reference to the accompanying drawings.

[0041] Experimental example: Pot experiment

[0042] 1. Cultivation of potted materials

[0043] The experiment was conducted at the Comprehensive Experimental Base of the Institute of Resources and Environment, Jiangsu Academy of Agricultural Sciences, during the 2022-2023 wheat growing season. Based on the team's previous pot experiment results and the field experiment results in this paper, the stable low-Cd-accumulating wheat variety NM11 and the high-Cd-accumulating wheat variety YM27 were selected as the experimental materials for this study. The tested soil was collected from the topsoil (0-20cm) of an experimental field in Taicang, Jiangsu Province (121°5′37″N, 31°23′38″E). The soil was air-dried, crushed, and sieved. A 100g sample was taken for determination of the basic physicochemical properties. The pH was 7.55. The total Cd content was 1.28 mg / kg, total Fe content was 15.3 g / kg, total Zn content was 0.13 g / kg, total nitrogen content was 0.21 g / kg, organic matter content was 37.1 g / kg, available nitrogen content was 164.7 mg / kg, available phosphorus content was 23.1 mg / kg, and available potassium content was 98.0 mg / kg. The Taicang soil was classified as weakly alkaline, slightly Cd-contaminated soil. Five treatment groups were set up: blank control (CK), ZnSO4 with 20 mg Zn / kg soil, ZnSO4 with 40 mg Zn / kg soil, Zn-EDTA with 20 mg Zn / kg soil, and Zn-EDTA with 40 mg Zn / kg soil. The applied zinc-based fertilizer ZnSO4 (AR) was purchased from Sinopharm Chemical Reagent Co., Ltd., and the zinc-based fertilizer Zn-EDTA (≥98%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Each treatment had six replicates, with three replicates used for destructive sampling during flowering. The pots used in the pot experiments were made of polypropylene (PP), with an inner diameter, outer diameter, and height of 20.5 cm, 24 cm, and 22 cm, respectively. Each pot was filled with 5 kg of test soil, with 0.5 kg of soil placed in a root bag (300 mesh, 10 cm opening × 20 cm height). 6 g of NPK compound fertilizer (N:P2O5:K2O = 15:15:15) was applied per pot as base fertilizer. This NPK compound fertilizer was purchased from Jiangsu Aolaite Ecological Fertilizer Co., Ltd. The compound fertilizer and zinc base fertilizer (60% of the total application) were mixed thoroughly with the soil before potting. NM11 and YM27 seeds of similar plumpness and size were selected and sterilized by soaking in a 10% hydrogen peroxide solution for 10 minutes. The seeds were then rinsed thoroughly with deionized water and soaked in a moistened petri dish in the dark for 1 day. Seeds showing signs of sprouting were transferred to pots, with 4 seeds sown evenly per pot. After 3 weeks, thinning was performed, leaving 2 wheat seedlings of similar size and growth per pot. Zinc fertilizer was applied as topdressing at the jointing stage (20% of the total application rate) and the booting stage (20% of the total application rate). Additionally, 1g of urea was applied per pot during the jointing and booting stages to supplement nitrogen. The urea was purchased from Anhui Hongsifang Fertilizer Co., Ltd. (total N ≥ 46%). Wheat growth management followed local practices.

[0044] 2 Sample collection

[0045] Fresh wheat sample collection: Mature wheat plants are nearing wilting and have weak metabolism, while flowering plants have vigorous metabolism. Furthermore, the transcriptional levels of relevant genes in wheat plants during flowering directly affect Cd uptake during the grain-filling stage. Therefore, samples were collected during flowering to detect the relative expression levels of genes in wheat plant samples. Six wheat plants from three replicates of each treatment were collected at flowering. Root, first node, and flag leaf samples were collected separately, flash-frozen in liquid nitrogen, and stored at -80°C for RNA extraction.

[0046] Soil samples were collected at different times: soil inside the root bags was collected as rhizosphere soil, and soil outside the root bags was collected as non-rhizosphere soil. After air drying, crushing and sieving, the soil pH and available metal content were tested.

[0047] Wheat samples were collected at maturity: all six wheat plants from three replicates of each treatment in the potted material were harvested. The samples were divided into three parts: roots, straw, and grains. The roots, straw, and grains were washed with tap water and deionized water, respectively. After blanching at 105℃ for 30 min, the samples were dried at 70℃ to constant weight. The samples were then pulverized and digested for determination of metal element content.

[0048] 3. Determination of soil physicochemical properties

[0049] Soil pH was determined under a water-to-soil ratio of 2.5:1 (v / w); organic matter content was determined using the potassium dichromate-external heating method.

[80] Available metals in the experimental soil from Taicang were extracted using DTPA: 10.0 g of soil was weighed and 20 mL of extraction solution (0.005 M DTPA, 0.01 M CaCl2, 0.1 M TEA; pH = 7.3) was added. The mixture was shaken at 25 °C for 2 h, centrifuged at 4000 r / min for 10 min, and the supernatant was filtered through a 0.45 μm pore size filter. The concentration of available metal elements in the samples was detected using inductively coupled plasma mass spectrometry (ICP-MS; NexION-2000, Perkin Elmer, USA). The sample was analyzed after the linear fitting parameter of the elemental standard solution reached 0.999.

[0050] 4. Determination of Metal Element Content in Plants

[0051] After drying, pulverizing, and mixing, 0.20 g of the sample was weighed and placed in a digestion tube. Two reagent blanks and two plant standard reference samples (Hunan rice biocomposition analysis standard reference, GBW 10045a) were prepared for each batch of digested samples. Electrothermal digestion was performed using HNO3-H2O2 (5:1, v / v). First, 5 mL of HNO3 was added, covered with plastic wrap, and allowed to stand for 12 h. Then, using an automated digestion device, the sample was heated to 80℃ for 30 min, 100℃ for 30 min, 120℃ for 60 min (after cooling, 0.5 mL of H2O2 was added), and 140℃ for 60 min (after cooling, 0.5 mL of H2O2 was added). Digestion was then performed at 160℃ until the solution was clear and transparent. The digestion solution was brought to a final volume of 50 mL, filtered through a 0.45 μm filter, and the concentration of metal elements in the sample was detected using ICP-MS. The sample was analyzed after the linear fitting parameter of the elemental standard solution reached 0.999. The recoveries of Cd, Fe, Zn, Cu and Mn in the plant standard reference materials were all in the range of 90.0%-110.0%.

[0052] The translocation coefficient (TF) is calculated using the following formula: Root-Straw Metal Translocation Coefficient (TF) root-straw = Straw metal element concentration / Root metal element concentration; Straw-grain metal translocation coefficient (TF) straw-grain = Grain metal element concentration / Straw metal element concentration; Root-to-above-plant metal translocation coefficient (TF) root-shoot = Aboveground metal element concentration / Root metal element concentration. Root Cd absorption efficiency = Total Cd accumulation per plant / Dry weight of roots per plant.

[0053] 5. Gene expression level determination

[0054] Table 1 Primer Information

[0055]

[0056]

[0057] Cd enters wheat plants via other divalent cation transporters. To investigate whether zinc application affects Cd accumulation in wheat by regulating the expression of genes encoding cation transporters, 10 genes involved in Cd transport were selected for analysis. Primer information is shown in Table 1. TaATPases (Ta54227) was used as an internal control gene. The detection method was real-time quantitative PCR, and the analysis method was relative quantitative PCR. -△△Ct Methods. Total RNA was extracted from wheat roots, first node, and flag leaf during the flowering stage using a polyphenol and polysaccharide plant total RNA extraction kit (Meiji Biotechnology, R4150). II. The 1st Strand cDNA Synthesis Kit (Yisheng Biotechnology, 11119ES60) was used to synthesize cDNA. The quantitative PCR detection steps are as follows:

[0058] (1) Dissolve the Mix at 4°C, gently invert and mix well, and then briefly centrifuge.

[0059] (2) Prepare reaction solution on ice: 5 μL SYBR Green Master Mix, 1 μL Forward Primer (2 μM), 1 μL Reverse Primer (2 μM), 2 μL cDNA and 1 μL RNase-free Water;

[0060] (3) Briefly centrifuge the reaction tube to mix the reaction solution;

[0061] (4) Reaction program: 95℃ pre-denaturation for 5 min, 40 amplification cycles (95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s).

[0062] 6 Experimental Results

[0063] 6.1 Agronomic traits at maturity

[0064] like Figures 1-4 The results of the study on the agronomic traits of NM11 and YM27 at maturity under zinc fertilizer treatment in pots are as follows: grain number per plant represents the number of grains per plant, grain dry weight per plant represents the dry weight of grains per plant, straw dry weight per plant represents the dry weight of straw per plant, and thousand-grain weight represents the weight of a thousand grains.

[0065] like Figure 1 As shown, compared with the control, the application of 20 mg / kg and 40 mg / kg ZnSO4 significantly increased the number of grains per plant in NM11 and YM27.

[0066] like Figure 2 and Figure 3 As shown, compared with the control, the application of 20 mg / kg ZnSO4 and 40 mg / kg ZnSO4 significantly increased the dry weight of grain and straw per plant of NM11 and YM27; the application of 40 mg / kg Zn-EDTA significantly increased the dry weight of straw per plant of YM27.

[0067] like Figure 4 As shown, there was no significant difference in the thousand-grain weight of NM11 and YM27 between different treatments.

[0068] 6.2 Concentration of metal elements in wheat at maturity

[0069] Figures 5-9 The results show the concentrations of metal elements in wheat grains, straw, and roots at maturity under different zinc fertilizer treatments in potted plants. Cd concentration in grain represents the Cd concentration in grains, Cd concentration in straw represents the Cd concentration in straw, and Cd concentration in root represents the Cd concentration in roots. Zn concentration in grains represents the Zn concentration in grains, and Zn concentration in root represents the Zn concentration in roots.

[0070] like Figure 5 As shown, compared with the control, the application of 20 mg / kg and 40 mg / kg Zn-EDTA reduced the Cd concentration in NM11 grains from 0.46 mg / kg to 0.31 mg / kg and 0.24 mg / kg, respectively; and reduced the Cd concentration in YM27 grains from 0.79 mg / kg to 0.51 mg / kg and 0.23 mg / kg, respectively; while the application of 40 mg / kg ZnSO4 reduced the Cd concentration in YM27 grains to 0.55 mg / kg.

[0071] like Figure 6 As shown, compared with the control, the application of 40 mg / kg Zn-EDTA reduced the Cd concentration in YM27 straw from 1.34 mg / kg to 1.11 mg / kg; there was no significant difference in Cd concentration in NM11 straw among different treatments.

[0072] like Figure 7 As shown, compared with the control, the application of 20 mg / kg ZnSO4, 20 mg / kg Zn-EDTA and 40 mg / kg Zn-EDTA reduced the Cd concentration in the roots of NM11 from 2.83 mg / kg to 2.26 mg / kg, 1.56 mg / kg and 1.21 mg / kg, respectively; the application of 40 mg / kg ZnSO4 and Zn-EDTA reduced the Cd concentration in the roots of YM27 from 3.17 mg / kg to 2.47 mg / kg and 1.69 mg / kg, respectively.

[0073] like Figure 8As shown, compared with the control, the application of 20 mg / kg ZnSO4, 40 mg / kg ZnSO4, 20 mg / kg Zn-EDTA and 40 mg / kg Zn-EDTA increased the Zn concentration in NM11 grains from 25.1 mg / kg to 31.7 mg / kg, 43.8 mg / kg, 37.8 mg / kg and 37.4 mg / kg, respectively, and increased the Zn concentration in YM27 grains from 30.2 mg / kg to 49.2 mg / kg, 54.6 mg / kg, 73.8 mg / kg and 58.3 mg / kg, respectively.

[0074] like Figure 9 As shown, compared with the control, the application of 40 mg / kg ZnSO4, 20 mg / kg Zn-EDTA and 40 mg / kg Zn-EDTA increased the Zn concentration in the roots of NM11 from 7.93 mg / kg to 11.4 mg / kg, 16.9 mg / kg and 20.3 mg / kg, respectively; the application of 20 mg / kg and 40 mg / kg Zn-EDTA increased the Zn concentration in the roots of YM27 from 8.93 mg / kg to 32.6 mg / kg and 40.1 mg / kg, respectively.

[0075] 6.3 pH and available metal content of rhizosphere and non-rhizosphere soils at maturity

[0076] Figures 10-11 The results show the pH values ​​of rhizosphere and non-rhizosphere soils at maturity for two wheat varieties grown in pots. Rhizosphere soil pH represents the pH of rhizosphere soil, and non-rhizosphere soil pH represents the pH of non-rhizosphere soil.

[0077] like Figure 10 As shown, compared with the control, the application of 20 mg / kg ZnSO4, 20 mg / kg Zn-EDTA, and 40 mg / kg Zn-EDTA reduced the rhizosphere soil pH of NM11 from 7.93 to 7.76, 7.77, and 7.67, respectively. Figure 11 As shown, compared with the control, the application of 40 mg / kg Zn-EDTA reduced the pH of the non-rhizosphere soil of YM27 from 7.35 to 7.06.

[0078] Figures 12-13 To detect the available metal content in rhizosphere and non-rhizosphere soils during the mature stage of potted plants, DTPA-Zn and DTPA-Cd tables represent available Zn and available Cd content, respectively.

[0079] like Figure 12As shown, compared with the control, the application of 20 mg / kg and 40 mg / kg Zn-EDTA increased the concentration of available Cd in the rhizosphere soil of NM11 from 0.44 mg / kg to 0.73 mg / kg and 0.97 mg / kg, respectively; and increased the concentration of available Cd in the non-rhizosphere soil of NM11 from 0.47 mg / kg to 0.69 mg / kg and 0.85 mg / kg, respectively. For YM27, the application increased the concentration of available Cd in the rhizosphere soil from 0.41 mg / kg to 0.59 mg / kg and 0.65 mg / kg, respectively; and increased the concentration of available Cd in the non-rhizosphere soil of YM27 from 0.46 mg / kg to 0.55 mg / kg and 0.60 mg / kg, respectively. Furthermore, the application of ZnSO4 had no significant effect.

[0080] like Figure 13 As shown, compared with the control, the application of ZnSO4 and Zn-EDTA significantly increased the content of available Zn in both rhizosphere and non-rhizosphere soils of the two wheat varieties; the application of 20 mg / kg ZnSO4, 40 mg / kg ZnSO4, 20 mg / kg Zn-EDTA and 40 mg / kg Zn-EDTA significantly increased the content of available Zn in both rhizosphere and non-rhizosphere soils. Zn-EDTA increased the concentration of available Zn in the rhizosphere soil of NM11 from 1.94 mg / kg to 3.74 mg / kg, 5.61 mg / kg, 6.77 mg / kg, and 14.7 mg / kg, respectively; and increased the concentration of available Zn in the rhizosphere soil of YM27 from 1.49 mg / kg to 3.14 mg / kg, 5.13 mg / kg, 5.71 mg / kg, and 10.8 mg / kg, respectively. It also increased the concentration of available Zn in the non-rhizosphere soil of NM11 from 2.77 mg / kg to 6.31 mg / kg, 8.99 mg / kg, 6.65 mg / kg, and 10.5 mg / kg, respectively; and increased the concentration of available Zn in the non-rhizosphere soil of YM27 from 2.51 mg / kg to 5.92 mg / kg, 8.56 mg / kg, 6.57 mg / kg, and 10.3 mg / kg, respectively.

[0081] 6.4 Ratio of available Zn / Cd in rhizosphere soil and non-rhizosphere soil at maturity

[0082] like Figure 14 As shown, the concentration ratio of Zn to Cd in rhizosphere and non-rhizosphere soils of potted mature plants was analyzed. The ratio of DTPA-Zn / Cd represents the concentration ratio of available Zn to available Cd. Compared with the control, the application of 20 mg / kg ZnSO4, 40 mg / kg ZnSO4, 20 mg / kg Zn-EDTA and 40 mg / kg Zn-EDTA significantly increased the available Zn / Cd ratio in rhizosphere and non-rhizosphere soils of both wheat varieties, with increases of 72%, 166%, 109% and 241%, respectively.

[0083] 6.5 Total Accumulation and Translocation Coefficient of Metallic Elements in the Mature Period

[0084] Figures 15-16 The total accumulation and translocation coefficient of metal elements in mature wheat under zinc treatment are given, where total accumulation represents the total accumulation, and TF is the translocation coefficient. strawtograin ofCd represents the straw-grain Cd translocation coefficient, TF fromroottostraw ofCd represents the root-straw Cd translocation coefficient.

[0085] like Figure 15 As shown, compared with the control, the application of 20 mg / kg and 40 mg / kg Zn-EDTA significantly reduced the straw-grain Cd translocation coefficients of NM11 and YM27. Figure 16 As shown, compared with the control, the application of 20 mg / kg and 40 mg / kg Zn-EDTA significantly increased the Cd translocation coefficient of the root-straw of NM11; the application of 40 mg / kg Zn-EDTA significantly increased the Cd translocation coefficient of the root-straw of YM27.

[0086] 6.6 Expression of Cd transporter gene during flowering

[0087] The expression levels of the Cd transport genes TaHMA2, TaZIP3, TaZIP5, TaNRAMP1, TaNRAMP3, and TaIRT1 in wheat roots during the flowering stage were detected under pot treatment with 40 mg / kg ZnSO4 and Zn-EDTA. The results are as follows: Figures 17-24 As shown, relative expression represents the relative expression level. Compared with the control, as... Figure 17 , Figure 18 and Figure 19 As shown, application of 40 mg / kg ZnSO4 significantly reduced the expression levels of TaHMA2, TaZIP5, and TaIRT1 in the roots of YM27 plants; Figures 18-20 The application of 40 mg / kg Zn-EDTA significantly reduced the expression levels of TaZIP5 and TaIRT1 in the roots of NM11 and TaZIP5, TaNRAMP3, and TaIRT1 in the roots of YM27. Furthermore, as shown... Figure 21 As shown, application of 40 mg / kg ZnSO4 significantly increased the expression level of TaZIP3 in the roots of YM27 plants; Figure 22 As shown, the application of 40 mg / kg Zn-EDTA significantly increased the expression level of TaNRAMP1 in the roots of NM11.

[0088] The expression levels of Cd transport genes TaZIP3, TaZIP7, and TaHMA2 in the first node and flag leaf of wheat at flowering stage under pot treatment with 40 mg / kg ZnSO4 and Zn-EDTA were detected. The results are as follows: Figures 23-27 As shown, the same relative expression represents a relative expression level. Figure 23 , Figure 24 and Figure 25 As shown, compared with the control, the application of 40 mg / kg ZnSO4 significantly reduced the expression levels of TaZIP7 in the first node of NM11 and TaZIP7 and TaHMA2 in the flag leaf of YM27; Figure 23 , Figure 24 , Figure 25 , Figure 26 and Figure 27 As shown, the addition of 40 mg / kg Zn-EDTA significantly reduced the expression levels of TaZIP7 and TaHMA2 in the first node of NM11, TaZIP3 and TaZIP7 in the flag leaf, and TaZIP7 and TaHMA2 in the first node and flag leaf of YM27. Furthermore, as... Figure 26 As shown, the application of 40 mg / kg ZnSO4 significantly increased the expression level of TaHMA2 in the first node of YM27.

[0089] 7. Results Analysis:

[0090] 7.1 Impact on wheat yield: such as Figures 1-3 The results showed that application of 20 mg / kg and 40 mg / kg ZnSO4 significantly increased the number of grains per plant, grain dry weight, and straw dry weight of both NM11 and YM27 wheat varieties, indicating that ZnSO4 application has a certain promoting effect on wheat growth. However, neither ZnSO4 nor Zn-EDTA application had a significant effect on the thousand-grain weight of NM11 and YM27 wheat varieties. Figure 4 As shown, this indicates that Zn-EDTA and ZnSO4 have the same effect on promoting wheat yield.

[0091] 7.2 Effect on Cd content in wheat grains: Compared with the control, the application of Zn-EDTA significantly reduced the Cd concentration in NM11 and YM27 grains, and the effect of high concentration Zn-EDTA was more significant than that of low concentration Zn-EDTA; in addition, the application of 40 mg / kg ZnSO4 significantly reduced the Cd concentration in YM27 grains, but the effect of ZnSO4 application was not as significant as that of Zn-EDTA. Figure 5 As shown, under the same concentration conditions, although both Zn-EDTA and ZnSO4 can guarantee grain yield after soil application, Zn-EDTA can more effectively reduce the Cd content in grains.

[0092] Compared with the control, such as Figure 14 As shown, the application of both ZnSO4 and Zn-EDTA significantly increased the ratio of available Zn / Cd in the rhizosphere and non-rhizosphere soils of both wheat varieties. Figure 9 The results also show that the addition of zinc fertilizer activates Zn in the soil more than Cd, resulting in wheat roots absorbing more Zn; Cd 2+ and Zn 2+ There is a competitive interaction between Cd 2+ and Zn 2+ They share a transport system on the plasma membrane of wheat root cells, thus relatively reducing the absorption of Cd by NM11 and YM27 roots, and so on. Figure 7 The results of the Cd concentration detection in the roots are consistent. For example... Figure 12 As shown, although the application of Zn-EDTA significantly increased the available Cd content in both rhizosphere and non-rhizosphere soils, at the same time... Figure 16 The results showed that application of 20 mg / kg and 40 mg / kg Zn-EDTA significantly enhanced the Cd translocation capacity of NM11 roots to straw, while application of 40 mg / kg Zn-EDTA significantly enhanced the Cd translocation capacity of YM27 roots to straw. To mitigate the adverse effects of Cd stress, NM11 and YM27 reduced Cd translocation from straw to grains. Figure 15 The results can be verified, thus achieving the effect of reducing the Cd concentration in the grains.

[0093] like Figure 7 The results showed that the application of 40 mg / kg ZnSO4 and Zn-EDTA significantly reduced the Cd concentration in the roots of YM27, and the application of 40 mg / kg Zn-EDTA significantly reduced the Cd concentration in the roots of NM11. Further analysis was conducted on the expression levels of genes involved in Cd transporter proteins in the roots of the two wheat varieties under the treatment of 40 mg / kg ZnSO4 and Zn-EDTA. Figures 17-21 The results showed that, compared with the control, application of 40 mg / kg ZnSO4 inhibited the expression of TaHMA2, TaZIP5, and TaIRT1 in the roots of YM27, but... Figure 21 This shows that it enhances the expression of TaZIP3. Figures 18-20 The results showed that application of 40 mg / kg Zn-EDTA inhibited the expression of TaZIP5 and TaIRT1 in NM11 roots, and TaZIP5, TaNRAMP3, and TaIRT1 in YM27 roots, but... Figure 22 This showed that it enhanced the expression of TaNRAMP1 in NM11 roots, thereby reducing overall root uptake of Cd. Furthermore, Figure 5 and Figure 6The results showed that application of 40 mg / kg ZnSO4 significantly reduced Cd concentration in YM27 grains, while application of 40 mg / kg Zn-EDTA significantly reduced Cd concentration in both NM11 and YM27 grains, as well as Cd concentration in YM27 straw. Further analysis of the expression levels of Cd transporter-related genes in the first node and flag leaf of both wheat varieties under 40 mg / kg ZnSO4 and Zn-EDTA treatments revealed that application of 40 mg / kg ZnSO4 significantly reduced Cd concentration in the first node and flag leaf of both wheat varieties. Compared with the control, application of 40 mg / kg ZnSO4 inhibited the expression of Cd transporter-related genes in the first node and flag leaf of both wheat varieties. Figure 23 The first node TaZIP7 of NM11 shown, and as shown in the figure Figure 24 and Figure 25 The expression of TaZIP7 and TaHMA2 in the flag leaf of YM27 was inhibited by applying 40 mg / kg Zn-EDTA. Figure 23 and Figure 26 The first node of NM11, TaZIP7 and TaHMA2, are shown below. Figure 22 and Figure 24 The flag blades TaZIP3 and TaZIP7 shown, as well as... Figures 23-26 The expression of YM27 first node and TaZIP7 and TaHMA2 in the flag leaf was reduced, thereby decreasing Cd translocation in the first node and flag leaf of wheat. In summary, application of 40 mg / kg ZnSO4 and Zn-EDTA can reduce Cd uptake or translocation in wheat by inhibiting the expression of Cd translocation genes in the roots, first node, and flag leaf, thus mitigating the harmful effects of Cd stress.

[0094] 7.3. Effect on soil pH: The pH of rhizosphere and non-rhizosphere soils of two wheat varieties at maturity was measured in pots. Figure 10 and Figure 11 The results showed that applying 20 mg / kg ZnSO4, 20 mg / kg Zn-EDTA and 40 mg / kg Zn-EDTA could reduce the pH of both rhizosphere and non-rhizosphere soils, improve the soil to a more neutral pH, and provide a good foundation for sustainable farming in subsequent fields.

[0095] Compared with the control, the application of Zn-EDTA activated Cd and Zn in the rhizosphere and non-rhizosphere soils of NM11 and YM27. Figure 12 and Figure 13 Furthermore, the application of ZnSO4 significantly increased the content of available Zn in both rhizosphere and non-rhizosphere soils of the two wheat varieties, as shown in Figure 1. Figure 13 This indicates that both ZnSO4 and Zn-EDTA application can promote Zn activation in wheat roots. Both ZnSO4 and Zn-EDTA application significantly increased the Zn concentration in NM11 and YM27 grains. Figure 8 This indicates that applying ZnSO4 or Zn-EDTA has a significant effect on zinc biofortification in wheat.

Claims

1. The application of Zn-EDTA in reducing cadmium accumulation in wheat grains, characterized by: The application includes the following steps: Zn-EDTA is applied in three stages. The first stage is before wheat sowing, when a portion of Zn-EDTA is applied as a base fertilizer simultaneously with NPK compound fertilizer. After the base fertilizer is applied, Zn-EDTA is applied again in the second stage, namely the wheat jointing stage, and the third stage, the booting stage. The mass ratio of Zn-EDTA fertilizer applied in the first, second, and third stages is 3:1:

1. Zn-EDTA can increase the Zn / Cd ratio in the rhizosphere soil and reduce the expression of Cd transporter-related genes.

2. The application according to claim 1, characterized in that: The wheat variety in question is a low-Cd accumulation variety.

3. The application according to claim 2, characterized in that: The low Cd accumulation variety is Ningmai 11.

4. The application according to claim 1, characterized in that: The zinc addition amount is calculated per kg of dry soil, and the zinc addition amount of the Zn-EDTA fertilizer is 20~40 mg / kg.

5. The application according to claim 1, characterized in that: The amount of nitrogen, phosphorus, and potassium compound fertilizer added is calculated per kg of dry soil, and the amount of nitrogen, phosphorus, and potassium compound fertilizer added is 1.2 g / kg.

6. The application according to claim 5, characterized in that: The mass ratio of N:P2O5:K2O in the nitrogen-phosphorus-potassium compound fertilizer is 15:15:

15.

7. The application according to claim 1, characterized in that: Apply urea to each pot during the jointing and booting stage to supplement nitrogen.

8. The application according to claim 7, characterized in that: The amount of urea added is calculated per kg of dry soil, and the amount added is 0.2 g / kg.

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