Seed pretreatment method for enhancing drought resistance of wheat in seedling stage and preparation of initiator

Abstract

The invention provides a seed pretreatment method for enhancing drought resistance of wheat in a seedling stage and preparation of an initiator. The method comprises the following steps: soaking wheat seeds at regular time by using the specially-made initiator before sowing; the initiator is composed of wood vinegar and ultrapure water; the pyroligneous liquor is prepared from moso bamboo serving as a raw material. The method comprises the following steps: preparing wood vinegar, preparing an initiator, pretreating wheat seeds, initiating the seeds, germinating the seeds, and transplanting the seeds into a matrix after 7 days. A seed drought resistance response mechanism is activated by accurately controlling a pyroligneous liquor preparation process and an initiator ratio. According to the method, moso bamboos which are widely planted in the southern region of China and are low in price and pruned branches of the moso bamboos are used as raw materials, the refined pyroligneous liquor is prepared through optimized stepped pyrolysis and an optimized purification process, and the drought resistance of the wheat in the seedling stage is remarkably improved by adopting a specific initiator proportion and soaking wheat seeds.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural planting, and particularly relates to a seed pretreatment method for enhancing drought resistance of wheat at the seedling stage and the preparation of an initiator. Background Art

[0002] During their growth, crops face pressure from changes in the natural environment. All environmental stresses affect their growth, development, and yield, thereby impacting agricultural production. Among the many natural adversities, drought, a common natural disaster, is considered the most destructive environmental stress. Its occurrence reduces surface runoff and soil moisture, causing a series of changes in plants at the physiological, biochemical, and molecular levels.

[0003] Wheat is one of my country's major grain crops, boasting the highest total wheat production and consumption in the world. However, northern my country, the primary wheat-producing region, is mostly arid or semi-arid, making its growth susceptible to drought stress, especially during the seedling stage. Drought can lead to reduced emergence rates and slowed seedling growth, which in turn affects final yield. Water is the primary ecological factor determining wheat growth and development, and an adequate water supply is crucial for ensuring high and high-quality wheat yields. As the climate warms, the frequency and intensity of droughts are increasing, further exacerbating the impact of drought on high and stable wheat yields. Therefore, in addition to traditional drought-resistance measures for different wheat growth stages, pre-treating wheat seeds to enhance their drought resistance during the seedling stage has become an effective approach to promoting wheat growth.

[0004] Drought-resistant seed dressings are already used in wheat seed pretreatment, such as Hankefeng No. 1 wheat seed dressing. These agents form a protective film on the surface of wheat seeds to reduce water evaporation and improve their drought resistance. Furthermore, the dressings contain pest attractants, fungicides, and various trace elements, offering benefits such as drought resistance, yield increase, insect resistance, and disease and pest resistance. However, their high production costs increase their cost of use; specialized application techniques and equipment are difficult for ordinary farmers to master; and there are risks associated with environmental residues.

[0005] Wood vinegar, a liquid product produced during biomass pyrolysis, is rich in a variety of organic compounds and bioactive components, including phenols, organic acids, ketones, aldehydes, alcohols, esters, and ethers. It promotes plant growth, inhibits bacteria, and enhances stress resistance. Leveraging these properties, a drought-resistant wheat seed pretreatment method based on a specific wood vinegar preparation process has been developed, which has practical application value and is of great significance, aligning with the development of green agriculture. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention aims to provide a seed pretreatment method and initiator preparation for enhancing drought resistance in wheat seedlings, activating the seed drought resistance response mechanism by precisely controlling the wood vinegar preparation process and initiator ratio. The present invention utilizes bamboo and its pruned branches, which are widely grown and relatively inexpensive in southern my country, as raw materials, optimizes the step-wise pyrolysis and purification processes to prepare refined wood vinegar, adopts a specific initiator ratio, and significantly improves the drought resistance of wheat seedlings by soaking the wheat seeds.

[0007] The present invention discloses a seed pretreatment method for enhancing drought resistance of wheat seedlings and the preparation of an initiator. The method comprises soaking wheat seeds regularly with a special initiator before sowing; the initiator comprises wood vinegar and ultrapure water; and the wood vinegar is prepared using bamboo as a raw material. The method comprises the following steps: Preparation of wood vinegar: a. Raw material processing: The bamboo or bamboo pruned branches are crushed and mixed evenly, dried at a temperature of 60 ℃ for 48 hours, and then the dried bamboo raw materials are high-pressure molded using a biomass compression molding machine; b. Stepwise pyrolysis: The high-pressure formed bamboo raw material obtained in step a was placed in a dry distillation reactor and heated to 300 ℃ for pyrolysis for 3 hours; then heated to 500 ℃ for 2 hours; finally heated to 550 ℃ and calcined for 40 minutes; after cooling, the liquid fraction was collected to obtain crude wood vinegar; c. Purification: The crude wood vinegar obtained in step b was collected into a dark container and placed in a dark space for 14 days, the middle layer of wood vinegar was absorbed, 5% of the crude wood vinegar mass was added to the activated carbon, the liquid was shaken in a oscillator for 2 hours and then allowed to stand for 24 hours; d. The supernatant was drawn and filtered through a 0.22 μm membrane filter to obtain a refined wood vinegar solution having a pH value of 2.5-3.5; Preparation of initiator: e. The refined wood vinegar obtained in step d was mixed with ultrapure water in a volume ratio of 1 mL:500 mL, and stirred to obtain an initiator; Pretreatment of wheat seeds: f. Select wheat seeds with plump, healthy grains and no disease spots. First, disinfect the seeds by soaking them in 75% ethanol for 1 ± 0.5 minutes, and then rinse them with distilled water three times. Seed Priming: g. Soak the wheat seeds treated in step f in the initiator obtained in step e at a ratio of 1:5 (g / mL) of seed weight to initiator volume, and soak at a constant temperature of 20° C. in a dark environment for 8 hours; Seed germination: h. Take out the seeds soaked in step g, rinse them with clean water, germinate them at a constant temperature of 20°C for 7 days, and then transplant them into the substrate.

[0008] Compared with the prior art, the present invention has the following beneficial effects: 1. The wheat seed pretreatment method of the present invention has a simple operation process.

[0009] 2. The wood vinegar used is safe and pollution-free, and is an environmentally friendly seed treatment technology.

[0010] 3. It effectively improves the germination rate and germination potential of wheat seeds, and can quickly and effectively increase the relative water content and chlorophyll of wheat leaves in the seedling stage, reduce the proline content inside the leaves, and promote the growth of wheat seedlings under drought conditions.

[0011] 4. Bamboo is a renewable resource, activated carbon is recyclable, the overall cost is low, and it can be promoted on a large scale.

[0012] 5. It can be prepared from agricultural waste (pruned bamboo branches) to reduce environmental pollution.

[0013] 6. It has both ecological and economic benefits, can be produced on a large scale, and is suitable for promotion in arid and semi-arid areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The phenotypic diagram and statistical data of the length of the aboveground and underground parts and fresh weight of wheat seedlings under drought stress after treatment with water and wood vinegar. Among them: Figure A is the growth phenotype diagram of wheat seedling stage; Figure B is a statistical diagram of the length of the aboveground part of wheat at the seedling stage; Figure C is a statistical graph of root length data at the wheat seedling stage; Figure D is a statistical chart of fresh weight of aboveground parts of wheat at the seedling stage. Figure E is a statistical chart of fresh weight data of underground parts of wheat at the seedling stage.

[0015] Figure 2 The relative water content of wheat leaves at the seedling stage under different degrees of drought stress after treatment with water and wood vinegar.

[0016] Figure 3 The chlorophyll a, chlorophyll b and total chlorophyll contents in wheat seedling leaves under different drought stress levels after treatment with water and wood vinegar.

[0017] Figure 4 Proline content in wheat seedling leaves under different drought stress levels after treatment with water and wood vinegar. DETAILED DESCRIPTION

[0018] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0019] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0020] The technical solutions adopted by the seed pretreatment method and the preparation of the initiator for enhancing drought resistance of wheat seedlings are as follows: wheat seeds are regularly soaked with a special initiator before sowing; the initiator consists of wood vinegar and ultrapure water; and the wood vinegar is preferably prepared using bamboo as a raw material. Example

[0021] Preparation of wood vinegar: a. Raw material processing: The bamboo or bamboo pruning branches are crushed and mixed evenly, dried at a temperature of 60 ° C for 48 hours, and then the dried bamboo raw materials are high-pressure molded using a biomass compression molding machine to improve pyrolysis efficiency; b. Stepwise pyrolysis: The high-pressure formed bamboo raw material obtained in step a is placed in a dry distillation reactor and heated to 300°C for pyrolysis for 3 hours; then heated to 500°C for a further 2 hours; and finally heated to 550°C for calcination for 40 minutes. After cooling, the liquid fraction is collected to obtain crude wood vinegar. The stepwise pyrolysis process optimizes the yield of active ingredients (such as acetic acid and phenols) in the wood vinegar while reducing harmful substances such as tar. c. Purification: The crude wood vinegar obtained in step b was collected into a dark container and placed in a dark space for 14 days, the middle layer of wood vinegar was absorbed, 5% of the crude wood vinegar mass was added to the activated carbon, the liquid was shaken in a oscillator for 2 hours and then allowed to stand for 24 hours; d. The supernatant was drawn and filtered through a 0.22 μm membrane filter to obtain a refined wood vinegar solution having a pH value of 2.5-3.5; Preparation of initiator: e. The refined wood vinegar obtained in step d was mixed with ultrapure water in a volume ratio of 1 mL:500 mL, and stirred to obtain an initiator; Pretreatment of wheat seeds: f. Select wheat seeds with plump, healthy grains and no disease spots. First, disinfect the seeds by soaking them in 75% ethanol for 1 ± 0.5 minutes, and then rinse them with distilled water three times. Seed Priming: g. Soak the wheat seeds treated in step f in the initiator obtained in step e at a ratio of 1:5 (g / mL) of seed weight to initiator volume, and soak at a constant temperature of 20° C. in a dark environment for 8 hours; Seed germination: h. Take out the seeds soaked in step g, rinse them with clean water, germinate them at a constant temperature of 20°C for 7 days, and then transplant them into the substrate.

[0022] The composition of wood vinegar was determined by gas chromatography-mass spectrometry (7890B-7000C GC-MS, Agilent Technologies, Inc., USA). Pure helium was used as the carrier gas at a constant flow rate of 1.2 mL / min. A 30.00 m × 0.25 mm × 0.25 μm DB-WAX capillary column was used for separation. The injection port temperature was 220°C. The injection volume was 1 μL, and split injection was used with a split ratio of 80:1. Helium was used as the carrier gas at a constant flow rate of 1.00 mL / min. The column temperature was maintained at 60°C for 3 min, then increased to 240°C at a heating rate of 5°C / min and maintained for 10 min. The mass spectrometry conditions were an electron energy of 70 eV, an ion source temperature of 230°C, and a mass spectrometry scan range of 35-400 mz. -1 The compounds were identified by comparison of retention time and mass spectrum with mass spectral library data (NIST14), and the content of the compounds was determined by relative peak area.

[0023] Table 1 Main components of wood vinegar

[0024] As shown in Table 1, a total of 35 organic substances were detected in the wood vinegar made from bamboo raw materials; among them, the relative content of phenols was the largest at 72.83%, followed by ketones at 9.78%, organic acids at 6.31%, nitrogen compounds and glucose at 4.78%, lipids at 2.60%, furan derivatives at 1.90%, and alcohols at 1.80%. Example

[0025] Seven-day-old seedlings were transplanted into containers containing a substrate. After seven days of normal growth, drought stress treatment was initiated. Drought conditions were simulated by limiting water supply to evaluate the effects of wood vinegar priming on wheat drought resistance. Three drought levels were selected: mild, moderate, and severe drought, with soil moisture contents of 55–65%, 35–45%, and 15–25% of field capacity, respectively. After 14 days, wheat seedling phenotypes were observed, and the length and fresh weight of the aboveground and underground parts of the wheat were calculated. Figure 1The figures show the phenotype, aboveground and underground length, and fresh weight of wheat under drought stress in the control and priming treatments. The results show that priming with wood vinegar significantly improved the phenotype and biomass of wheat under drought stress, indicating that wood vinegar can enhance the drought tolerance of wheat seedlings. Example

[0026] The moisture content was determined based on Example 2: Experimental materials: wheat leaves subjected to drought stress obtained after treatment using the experimental method in Example 2.

[0027] The relative water content (RWC) of the leaves was weighed after harvest. The fresh weight (FW) of the leaves was weighed after harvest and then immersed in a test tube filled with distilled water at 4°C in the dark for 24 hours. The samples were then blotted dry and weighed immediately (to determine the expanded weight, TW), then dried at 70°C for 24 hours and weighed again to determine the dry weight (DW). The relative water content (RWC) of 10 leaves was determined using the formula: Relative water content (RWC) (%) = ((FW-DW) / (TW-DW)) × 100%. Among them, FW stands for fresh weight, DW stands for dry weight, and TW stands for expanded weight; Measurement results Figure 2 As shown in the results, the relative water content of wheat leaves treated with wood vinegar under drought stress was significantly increased, from 72.65% to 80.82% under mild drought, from 64.30% to 69.55% under moderate drought, and from 52.74% to 58.71% under severe drought compared with the control group. Example

[0028] Chlorophyll was determined based on Example 2 Experimental materials: Wheat leaves after stress treatment obtained after treatment using the experimental method in Example 2.

[0029] Weigh 0.1 g of wheat leaves after stress treatment, cut them into small pieces, and place them in a 15 mL centrifuge tube. Add 10 mL of a 1:1 volume ratio of anhydrous ethanol and acetone mixture to the centrifuge tube and place it in the dark for 24 h to fully extract chlorophyll. Use a microplate reader to measure the absorbance of the extract at 470 nm, 649 nm, and 665 nm. Measurement results Figure 3 As shown in the results, compared with the control group priming with pure water, the wheat primed with the primer can effectively increase the chlorophyll a, chlorophyll b and total chlorophyll contents under different degrees of drought stress, indicating that wood vinegar priming can improve the drought resistance of wheat by increasing the chlorophyll content. Example

[0030] Determination of proline based on Example 2 Experimental materials: Wheat leaves after stress treatment obtained after treatment using the experimental method in Example 2.

[0031] Proline (PRO) content was determined using a proline assay kit (A107-1-1, Nanjing Jiancheng Biological Co., Ltd.). Proline reacts with ninhydrin to form a red complex with a characteristic absorption peak at 515 nm, so the proline content in leaves can be calculated. Samples were added according to the kit instructions, and absorbance was measured at a wavelength of 515 nm and a light path of 1 cm. Measurement results Figure 4 As shown in the results, wheat induced by the initiator can effectively reduce the proline content in leaves under different degrees of drought stress, indicating that wood vinegar induces the regulation of leaf osmotic pressure, enhances the osmotic balance of plants, alleviates stress, and promotes their survival under drought conditions.

[0032] The seed pretreatment method for enhancing drought resistance of wheat seedlings and the preparation technology of the initiator of the present invention can also be applied to the preparation of initiators for wheat seeds using the straw of gramineous crops such as corn and sorghum; the method of the present invention can also be applied to the priming of seeds of other crops to enhance the drought resistance of their seedlings.

[0033] The above is a specific embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A seed pretreatment method for enhancing drought resistance of wheat seedlings and preparation of an initiator, characterized by: Before sowing, wheat seeds are soaked regularly with a special initiator; the initiator is composed of wood vinegar and ultrapure water; the wood vinegar is prepared from bamboo as a raw material; the method comprises the following steps: Preparation of wood vinegar: a. Raw material processing: The bamboo or bamboo pruned branches are crushed and mixed evenly, dried at a temperature of 60 ℃ for 48 hours, and then the dried bamboo raw materials are high-pressure molded using a biomass compression molding machine; b. Stepwise pyrolysis: The high-pressure formed bamboo raw material obtained in step a was placed in a dry distillation reactor and heated to 300 ℃ for pyrolysis for 3 hours; then heated to 500 ℃ for 2 hours; finally heated to 550 ℃ and calcined for 40 minutes; after cooling, the liquid fraction was collected to obtain crude wood vinegar; c. Purification: The crude wood vinegar obtained in step b was collected into a dark container and placed in a dark space for 14 days, the middle layer of wood vinegar was absorbed, 5% of the crude wood vinegar mass was added to the activated carbon, the liquid was shaken in a oscillator for 2 hours and then allowed to stand for 24 hours; d. The supernatant was drawn and filtered through a 0.22 μm membrane filter to obtain a refined wood vinegar solution having a pH value of 2.5-3.5; Preparation of initiator: e. The refined wood vinegar obtained in step d was mixed with ultrapure water in a volume ratio of 1 mL:500 mL, and stirred to obtain an initiator; Pretreatment of wheat seeds: f. Select wheat seeds with plump, healthy grains and no disease spots. First, disinfect the seeds by soaking them in 75% ethanol for 1 ± 0.5 minutes, and then rinse them with distilled water three times. Seed Priming: g. Soak the wheat seeds treated in step f in the initiator obtained in step e at a ratio of 1:5 (g / mL) of seed weight to initiator volume, and soak at a constant temperature of 20° C. in a dark environment for 8 hours; Seed germination: h. Take out the seeds soaked in step g, rinse them with clean water, germinate them at a constant temperature of 20°C for 7 days, and then transplant them into the substrate.

Images