Application of liquid nitrogen fertilizer LNF in improving cold resistance of Brassica campestris

Abstract

The application of liquid nitrogen fertilizer LNF in improving the cold resistance of Brassica napus is disclosed. The liquid nitrogen fertilizer LNF is urea nitrammonium aqueous solution. The application research finds that the rhizosphere irrigation treatment of liquid nitrogen fertilizer significantly relieves the phenomena of leaf water loss, green fading, yellowing, plant wilting and slow growth of Brassica napus seedlings caused by low temperature stress. Under low temperature conditions, appropriate application of liquid nitrogen fertilizer improves the low temperature tolerance of Brassica napus seedlings through various physiological mechanisms, including improving leaf water retention, enhancing antioxidant regulation, promoting osmotic regulation, improving photosynthetic capacity and enhancing active oxygen scavenging capacity. Therefore, the liquid nitrogen fertilizer can effectively improve the low temperature tolerance of Brassica napus seedlings, promote the root development and enhance the seedling growth vigor, and provides an efficient and economical solution for agricultural production under cold climate conditions.

Description

Technical Field

[0001] This invention belongs to the field of agricultural planting technology, and more specifically, this invention relates to the application of liquid nitrogen fertilizer in improving the cold resistance of broccoli. Background Technology

[0002] Broccoli is a major cultivated variety of greenhouse vegetables. It originated in temperate climate zones and is a warm-season vegetable, extremely sensitive to low temperatures. Its suitable growth temperature is 15-25℃. When the ambient temperature is below 10℃, the plant is susceptible to chilling injury, leading to stunted growth; when the temperature drops below 5℃, growth essentially stops.

[0003] Low-temperature chilling injury has become a major abiotic stress for broccoli production in autumn, winter, and spring, especially during the seedling and head formation stages. Low temperatures significantly hinder normal growth, manifesting as slow growth, yellowing leaves, increased spots, and even tissue necrosis. Sustained low-temperature stress can also lead to flower drop and head deformities, severely impacting commercial yield and quality, weakening market competitiveness, and causing significant economic losses for growers. Since low-temperature chilling injury not only causes large-scale yield reductions but also results in severe economic losses, there is an urgent need to develop highly effective chilling-resistant agents and chilling-resistant cultivation techniques to mitigate the negative impact of low-temperature stress on broccoli seedling growth and morphological development, enhance its low-temperature tolerance, and stabilize broccoli yield and quality. Summary of the Invention

[0004] In a first aspect, the present invention provides the application of liquid nitrogen fertilizer (LNF) as a single cold-resistant component in improving the cold resistance of broccoli; the liquid nitrogen fertilizer (LNF) is a urea ammonium nitrate aqueous solution with a mass concentration of 30-35%.

[0005] The liquid nitrogen fertilizer (LNF) is a urea-ammonium nitrate aqueous solution containing water-soluble nitrogen sources such as urea nitrogen, ammonium nitrogen, and nitrate nitrogen. Through a complex nitrogen supply mechanism, it can meet the nutritional needs of crops at different stages and promote crop growth and metabolism.

[0006] Existing research (CN121420750A) indicates that liquid nitrogen fertilizer (LNF) can be used in fertilization methods for head lettuce to improve its cold resistance. However, in this method, the main cold-resistant active ingredient is peptide-based microbial fertilizer, and in addition to liquid nitrogen fertilizer, a high-potassium water-soluble fertilizer is added as an auxiliary agent. That is, peptide-based microbial fertilizer is the main component, supplemented by liquid nitrogen fertilizer and high-potassium water-soluble fertilizer, and the cold resistance of head lettuce is improved through the synergistic effect of multiple fertilizers. This technical solution does not disclose or teach that liquid nitrogen fertilizer can be used independently as a cold-resistant active ingredient without peptide-based microbial fertilizer and high-potassium water-soluble fertilizer, nor can the independent contribution of liquid nitrogen fertilizer to cold resistance be directly determined from its compound system.

[0007] Furthermore, head lettuce and broccoli exhibit significant differences in plant classification, marketable organs, growth and development patterns, and low-temperature response metabolism. Head lettuce, belonging to the genus *Lactuca* of the Asteraceae family, is a leafy vegetable whose primary production goal is to form a compact head; while broccoli, belonging to the genus *Brassica* of the Brassicaceae family, is a flowering vegetable whose main marketable organs are unopened inflorescences and flower buds, and possesses secondary metabolic characteristics such as glucosinolates unique to Brassicaceae crops. The two differ in root-crop growth coordination, water retention, head or inflorescence formation, and sensitivity to chilling injury; therefore, the cold resistance experience gained from compound fertilization systems for head lettuce cannot be directly extrapolated to broccoli seedlings.

[0008] From the perspective of mechanism of action, the liquid nitrogen fertilizer in CN121420750A mainly plays a supporting role in nitrogen supplementation and synergistic action with peptide-based microbial fertilizer and high-potassium water-soluble fertilizer in the head lettuce program, and its cold resistance effect depends on the combined action of multiple fertilizers. However, this invention proves that, without the addition of peptide-based microbial fertilizer, high-potassium water-soluble fertilizer or other cold-resistant agents, using only urea ammonium nitrate liquid nitrogen fertilizer as a single cold-resistant component can improve the low-temperature tolerance of broccoli seedlings through multiple mechanisms, such as promoting root development and increasing the root-to-shoot ratio, improving leaf moisture status, enhancing the activity of antioxidant enzymes such as SOD, CAT, and APX, promoting proline accumulation, maintaining chlorophyll synthesis and reducing H2O2 accumulation. In particular, the 0.5 g / L LNF treatment increased the comprehensive index D value of low-temperature tolerance of broccoli seedlings from 0.158 to 0.527, the root-to-shoot ratio increased by 75% compared with the control group, the proline content increased by 157%, and the APX activity increased by 139%, showing a clear and significant cold-resistant enhancement effect. Therefore, even if those skilled in the art know that the liquid nitrogen fertilizer in CN121420750A can be used as an auxiliary component in a compound system for head lettuce, they would hardly imagine that urea ammonium nitrate liquid nitrogen fertilizer could achieve the above-mentioned significant cold resistance effect in broccoli seedlings as a single cold-resistant component. Thus, the present invention has achieved unexpected technical effects.

[0009] In the application of improving the cold resistance of broccoli, the concentration of the liquid nitrogen fertilizer is 0.1 g / L-0.75 g / L, preferably 0.1 g / L-0.5 g / L, and more preferably 0.25 g / L-0.5 g / L. Experiments show that the rational application of liquid nitrogen fertilizer can significantly improve the cold resistance of broccoli.

[0010] Secondly, the present invention provides a method for improving the cold resistance of broccoli, comprising the following steps: applying a liquid nitrogen fertilizer (LNF) with a single cold-resistant component to the rhizosphere of broccoli seedlings; wherein the liquid nitrogen fertilizer (LNF) is an aqueous solution of urea ammonium nitrate.

[0011] In the method, the application concentration of the liquid nitrogen fertilizer is 0.1 g / L-0.75 g / L, preferably 0.1 g / L-0.5 g / L, and more preferably 0.25 g / L-0.5 g / L.

[0012] In the method, the timing of the rhizosphere irrigation is as follows: 12 days and 6 days before the onset of low temperature stress, respectively, an exogenous application is performed; the amount of rhizosphere irrigation is as follows: 45-55 mL per plant each time.

[0013] In the method, the broccoli seedling is a broccoli plant that has grown to 4-5 true leaves.

[0014] Compared with the prior art, the beneficial effects achieved by the present invention are: This invention significantly alleviated the adverse effects of low-temperature stress on broccoli seedling growth, including leaf dehydration, chlorosis, yellowing, wilting, and slow growth, through rhizosphere irrigation with LNF. Further analysis of physiological and biochemical indicators closely related to low-temperature stress revealed the following improvements in LNF-treated broccoli seedlings: significantly increased activity of antioxidant enzymes such as SOD, CAT, and APX in leaves; increased proline and chlorophyll a content; improved hydroxyl radical scavenging rate; significantly reduced H2O2 accumulation; decreased free water content in leaves; increased bound water content and bound water / free water ratio; and significantly increased root fresh weight, root-shoot ratio, and overall low-temperature tolerance. Under low-temperature stress, the control group broccoli seedlings exhibited a low-temperature tolerance index (D value) of only 0.158, while the optimal LNF concentration significantly increased the D value to 0.527, an increase of 233%, demonstrating that LNF significantly enhanced the low-temperature tolerance of broccoli seedlings. Therefore, LNF can be an effective option for preventing low-temperature damage during the seedling stage of broccoli, not only enhancing its cold resistance but also significantly reducing the risk of yield reduction caused by low temperatures.

[0015] Meanwhile, LNF's cold-resistant regulation technology is simple and easy to implement, suitable for integrated water and fertilizer drip irrigation systems, and suitable for large-scale promotion and application. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0019] Experimental Methods: Effects of LNF on physiological and biochemical indicators related to low-temperature tolerance in broccoli 1. Experimental materials: Broccoli: The main cultivated variety in the Beijing-Tianjin-Hebei region; Liquid nitrogen fertilizer (LNF): urea ammonium nitrate aqueous solution with a mass concentration of 30-35%.

[0020] LNF solutions: Prepare LNF solutions of different concentrations: 0.1 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, and 1 g / L.

[0021] 2. Experimental methods: (1) Broccoli in a light incubator (DGX-260E) (light intensity 400 µmol / m 2 / s, 12 hours of light / 12 hours of darkness, normal temperature control (day and night temperatures 25°C / 18°C), low temperature stress (day and night temperatures 15°C / 5°C), broccoli was grown in substrate cultivation. After the plants grew to 4-5 true leaves, plants of uniform size and growth were selected for experimental treatment.

[0022] (2) 12 days and 6 days before the onset of low temperature stress, an exogenous application was carried out, and a specified concentration of LNF solution was applied to the rhizosphere to ensure that the solution was evenly distributed in the rhizosphere area of ​​the broccoli plants. The control group was treated with distilled water rhizosphere irrigation. Each application was 50 mL / plant.

[0023] (3) On the morning of the 7th day, the light incubator was set to low temperature stress treatment for 3 days, and then the incubator was adjusted to room temperature to restore growth for 2 days.

[0024] (4) Observe the growth of broccoli plants in each group and analyze and detect the physiological and biochemical indicators closely related to low temperature tolerance.

[0025] 3. Experimental Results (1) SPAD value and chlorophyll a content of broccoli seedling leaves SPAD values ​​and chlorophyll a content reflect the level of photosynthetic pigments in plant leaves and directly affect photosynthetic efficiency. Higher SPAD values ​​indicate stronger light energy utilization, enhanced stress resistance, and better growth.

[0026] In this study, the effects of 0.1–1 g / L LNF on chlorophyll synthesis in broccoli under low-temperature stress (5℃) are shown in Table 1. The results showed that 0.1 g / L LNF pretreatment had no significant effect on the SPAD value and chlorophyll a content of broccoli seedling leaves under low-temperature stress, while 0.25–0.5 g / L LNF pretreatment increased both of these chlorophyll synthesis parameters. The increase from 0.5 g / L LNF pretreatment was statistically significant (p<0.05), demonstrating a significant promoting effect on chlorophyll synthesis. However, when the LNF concentration further increased to over 0.5 g / L, chlorophyll synthesis capacity significantly decreased, returning to near the control level. This indicates that appropriate amounts of LNF (0.25–0.5 g / L) can effectively promote chlorophyll synthesis in broccoli leaves under low-temperature stress, while excessively high concentrations of LNF may inhibit chlorophyll synthesis.

[0027] Table 1. Effects of root drenching with LNF on SPAD value and chlorophyll a content in leaves of broccoli seedlings under low temperature stress.

[0028] (2) Root-to-shoot ratio of broccoli seedlings The root-to-shoot ratio (RS) is a key indicator of the coordination between root absorption capacity and aboveground growth in plants. A higher RS ​​indicates that the plant has stronger root absorption capacity, which is crucial for improving the plant's adaptability to low-temperature stress.

[0029] In this study, the root survival rate (RS) was calculated as the ratio of root fresh weight to aboveground fresh weight (see Table 2). The results showed that, compared with the control group, irrigation with 0.1–0.5 g / L LNF significantly increased RS, with the increase in concentration increasing. The 0.5 g / L LNF treatment achieved the highest RS value (0.328), a 75% increase compared to the control group (p<0.05), demonstrating optimal absorption capacity and growth balance. However, when the LNF concentration increased to 0.75–1 g / L, RS significantly decreased, falling to 0.197 and 0.165 respectively, close to or lower than the control group levels (p<0.05), indicating that high concentrations of LNF may inhibit the coordinated growth of roots and aboveground parts. In summary, LNF significantly improved the RS of broccoli seedlings within a suitable concentration range, with 0.5 g / L LNF showing the best regulatory effect, fully demonstrating its role in enhancing root absorption capacity and optimizing resource allocation. This provides a reliable scientific basis for optimizing cold resistance regulation techniques in broccoli and also emphasizes the importance of controlling the application concentration.

[0030] Table 2 Effects of rhizosphere irrigation with LNF on root-shoot ratio of broccoli seedlings under low temperature stress

[0031] (3) Fresh weight of broccoli seedling roots and fresh weight of aboveground parts Root fresh weight and aboveground fresh weight are important indicators for measuring a plant's absorption capacity and aboveground growth status, reflecting the plant's resource allocation and growth adaptation capabilities under adverse conditions. Root fresh weight characterizes the root system's ability to absorb water and nutrients, while aboveground fresh weight reflects the overall level of photosynthesis and nutrient distribution in the plant.

[0032] In this study, root fresh weight and aboveground fresh weight were obtained by fresh weight determination (see Table 3). The results showed that, compared with the control group, irrigation with 0.5 g / L LNF significantly increased the root fresh weight of broccoli seedlings, with an increase of 74% (from 0.273 g / plant to 0.476 g / plant, p<0.05). However, the aboveground fresh weight did not show a significant change after LNF irrigation (1.440 g / plant, not significantly different from the control group's 1.473 g / plant, p>0.05). This indicates that LNF mainly improves plant adaptability by enhancing root absorption capacity, while its direct effect on aboveground fresh weight is limited. In summary, 0.5 g / L LNF significantly increased root fresh weight, improving the plant's resource absorption and utilization capacity, and providing effective support for alleviating low-temperature stress. This result further validates the potential of appropriate concentrations of LNF in enhancing root function and optimizing cold resistance regulation.

[0033] Table 3. Effects of rhizosphere drenching with LNF on root and aboveground fresh weight of broccoli seedlings under low temperature stress.

[0034] (4) Electrical conductivity (REC), hydrogen peroxide (H2O2) content and hydroxyl radical scavenging rate (HRSR) of broccoli seedling leaves In this study, relative conductivity (REC) is one of the important indicators for assessing plant cell membrane integrity and stress resistance. Under low-temperature stress, cell membranes are easily damaged, leading to electrolyte leakage and an increase in relative conductivity (REC). Therefore, REC can directly reflect the plant's tolerance to low-temperature stress. Hydrogen peroxide (H2O2) is one of the common reactive oxygen species (ROS) in plants, and its accumulation is an important marker of oxidative stress. Excessive H2O2 can cause oxidative damage to the cell membrane system and cellular components, further exacerbating the harm of low-temperature stress. Hydroxyl radical scavenging rate (HRSR) characterizes the activity of the plant's antioxidant system. A higher HRSR indicates that the plant can effectively scavenge hydroxyl radicals, thereby mitigating the effects of oxidative stress and enhancing low-temperature resistance. In this study, REC was measured using a conductivity meter to directly quantify the degree of cell membrane damage. H2O2 content was determined using visible spectrophotometry. The kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. to ensure the accuracy and ease of operation of the measurement. HRSR was calculated by the amount of hydroxyl radicals scavenged in the reaction system and was used to quantify the plant's ability to scavenge ROS. The relevant experimental results are detailed in Table 4. The comprehensive analysis of the above indicators provides a reliable basis for a comprehensive assessment of the physiological response and tolerance of broccoli seedlings under low temperature stress, and also lays the data support for in-depth research on the low temperature regulation mechanism.

[0035] The results showed that pretreatment with a predetermined concentration of LNF solution significantly reduced the accumulation of H2O2 in broccoli seedling leaves by 27% (p<0.05), while simultaneously increasing HRSR by 5.5% (p<0.05), indicating that LNF plays a significant role in enhancing the scavenging capacity of the antioxidant system. Furthermore, REC decreased by 4.3%, but the decrease was not statistically significant (p>0.05), suggesting that the effect of LNF solution in reducing electrolyte leakage has certain limitations. Overall, LNF solution pretreatment can effectively inhibit the excessive accumulation of reactive oxygen species and enhance the function of the antioxidant system, thus alleviating oxidative damage to plant cells caused by low-temperature stress and providing a good regulatory effect for improving the low-temperature tolerance of broccoli seedlings.

[0036] Table 4. Effects of rhizosphere drenching with LNF on leaf electrical conductivity (REC), hydrogen peroxide (H2O2) content, and hydroxyl radical scavenging rate (HRSR) of broccoli seedlings under low temperature stress.

[0037] (5) Content of free water (FW) and bound water (BW) in broccoli seedling leaves and the ratio of bound water to free water (BF). Free water (FW), bound water (BW), and the bound water / free water ratio (BF) are important physiological indicators reflecting the water status of plant leaves and their stress resistance. Under low-temperature stress, an increase in free water content in cells leads to exacerbated water loss, while a higher bound water content helps maintain cellular metabolic activity and structural stability. The bound water / free water ratio (BF) is a key parameter for measuring the balance of intracellular water distribution; a higher BF value indicates that the plant has a stronger water retention capacity, which is beneficial for resisting damage caused by low-temperature stress. In this study, free water content (FW) was determined by gravimetric method, while bound water (BW) and the bound water / free water ratio (BF) were calculated based on water distribution characteristics, which can accurately quantify changes in the water status of plant leaves. The relevant measurement results are detailed in Table 5.

[0038] The results showed that rhizosphere application of 0.5 g / L LNF significantly reduced the free water (FW) content in broccoli seedling leaves by 22.46% (p<0.05), while significantly increasing the bound water (BW) content by 7.08% (p<0.05). Furthermore, the bound water to free water ratio (BF) significantly increased by 36.72% (p<0.05). These results indicate that LNF treatment effectively optimizes leaf water status, reducing the proportion of free water and increasing the proportion of bound water, thereby enhancing water retention capacity. Overall, LNF improves cellular water status by regulating the ratio of free to bound water in leaves, helping to maintain normal cellular metabolic function and providing effective support for broccoli seedlings to cope with low-temperature stress.

[0039] Table 5. Effects of rhizosphere irrigation with LNF on the content of free water (FW), bound water (BW), and bound water / free water (BF) in the leaves of broccoli seedlings under low-temperature stress.

[0040] (6) Content of soluble sugar (SS) and proline (Pro) in broccoli seedling leaves Soluble sugars (SS) and proline (Pro) are important indicators for measuring the osmotic regulation capacity of plants under low-temperature stress. Soluble sugars play a role in resisting cold by maintaining cell osmotic balance and protecting membrane structure, while proline can stabilize proteins and membrane structure, scavenge free radicals, and alleviate oxidative stress. Increased levels of SS are usually associated with enhanced stress resistance.

[0041] In this study, soluble sugars were determined by enzyme-linked immunosorbent assay (ELISA), and proline was quantified using the acidic naphthol method. The relevant results are detailed in Table 6. The results showed that irrigation with 0.5 g / L LNF had no significant effect on soluble sugar (SS) content (p>0.05), but significantly increased proline (Pro) content by 157% (p<0.05), from 139 μg / g FW to 357 μg / g FW. This indicates that LNF can significantly promote proline accumulation and enhance cold resistance. Overall, LNF mainly enhances the osmotic regulation capacity of broccoli seedlings by increasing proline content, protecting cell structure and mitigating oxidative damage under low-temperature stress. The stability of soluble sugars reflects the selectivity of LNF regulation, highlighting the crucial role of proline in cold resistance regulation.

[0042] Table 6. Effects of rhizosphere irrigation with LNF on the content of soluble sugar (SS) and proline (Pro) in the leaves of broccoli seedlings under low temperature stress.

[0043] (7) SOD, POD, CAT and APX enzyme activities in broccoli seedling leaves Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) are core enzymes in the plant antioxidant system, responsible for scavenging reactive oxygen species (ROS) and protecting cells from oxidative damage. SOD converts superoxide anions (O2) into oxygen. - ) is converted into hydrogen peroxide (H2O2), while POD, CAT and APX decompose H2O2, further alleviating oxidative stress.

[0044] This study measured SOD activity using the NBT method, while POD, CAT, and APX activities were quantified by substrate degradation rates. Detailed results are shown in Table 7. The results showed that rhizosphere application of 0.5 g / L LNF significantly increased the activity of antioxidant enzymes in broccoli seedling leaves (p<0.05). SOD activity significantly increased by 37.6%; POD activity increased by 10.4%; and CAT activity increased by 43.6%. APX activity showed the most significant increase, reaching 139%. These changes indicate that LNF treatment effectively enhanced the activity of multiple key antioxidant enzymes, significantly improving the plant's antioxidant capacity. This helps alleviate oxidative damage caused by low-temperature stress and provides reliable support for improving the low-temperature tolerance of broccoli seedlings.

[0045] Table 7. Effects of rhizosphere drenching with LNF on the activities of SOD, POD, CAT, and APX enzymes in the leaves of broccoli seedlings under low temperature stress.

[0046] (8) Content of total phenols (TP), flavonoids (FLA) and anthocyanins (ANT) in broccoli seedling leaves Total phenols (TP), flavonoids (FLA), and anthocyanins (ANT) are important antioxidants in plants to cope with low-temperature stress. TP and FLA can protect cells from oxidative damage by scavenging reactive oxygen species, while ANT, as a natural pigment, can enhance plant resistance and reduce stress damage.

[0047] In this study, the contents of total phenols (TP) and flavonoids (FLA) were determined spectrophotometrically, and the contents of anthocyanins (ANT) were quantified using the pH difference method. The results are shown in Table 8. Irrigation with 0.5 g / L LNF showed a significant increasing trend in total phenol content, with an increase of 5.4%, but this was not statistically significant (p>0.05). The contents of flavonoids and anthocyanins did not change significantly (p>0.05), showing a slight increase and remaining basically stable, respectively. This indicates that LNF has a certain promoting effect on the accumulation of total phenols, which may enhance the antioxidant capacity of plants, while the stability of flavonoids and anthocyanins shows that their antioxidant functions were not significantly affected. Overall, the antioxidant regulation of LNF irrigation is mainly reflected in its positive effect on total phenols, which may alleviate oxidative damage caused by low-temperature stress, but its role in antioxidant defense still needs further investigation.

[0048] Table 8 Effects of rhizosphere irrigation with LNF on the content of total phenols (TP), flavonoids (FLA), and anthocyanins (ANT) in the leaves of broccoli seedlings under low temperature stress.

[0049] (9) Comprehensive index of low temperature tolerance of broccoli seedlings (D value) The low-temperature tolerance index (D value) is an important indicator for comprehensively measuring the growth performance and tolerance of plants under low-temperature stress. A higher D value usually indicates that the plant can better adapt to low-temperature environments and exhibits stronger cold resistance.

[0050] In this study, the D-value was calculated by comprehensively analyzing the physiological growth data of broccoli seedlings under low-temperature stress, and the results are detailed in Table 9. After irrigation with 0.5 g / L LNF, the D-value significantly increased from 0.158 in the control group to 0.527, an increase of 233%. This result indicates that LNF treatment significantly enhanced the low-temperature tolerance of broccoli seedlings, demonstrating a stronger ability to adapt to low-temperature stress and better growth. Overall, LNF showed significant effects in improving the comprehensive indicators of low-temperature tolerance, further validating its regulatory potential in alleviating low-temperature stress and providing an important basis for cold-resistant management of crops during the cold season.

[0051] Table 9. Effects of rhizosphere drenching with LNF on the comprehensive index (D value) of low-temperature tolerance in broccoli seedlings under low-temperature stress.

[0052] (10) Content of glucosinolates (GSLs) in broccoli seedlings Glucosinolates (GSLs) and their components are unique secondary metabolites found in cruciferous plants such as broccoli, and are closely related to plant stress resistance and health functions. The metabolic dynamics of GSLs can reflect the plant's adaptation strategies to environmental stresses, including its response to low-temperature stress.

[0053] This study used high-performance liquid chromatography (HPLC) to determine the content of total glucosinolates (GSLs) and their main components (IBE, NAP, 4OH, ERU, NAS, and NEO). The results are detailed in Table 10. After irrigation with 0.5 g / L LNF, the total glucosinolate content decreased slightly by 5.1%, but this decrease was not statistically significant (p>0.05). Among the glucosinolate components, the NAP content significantly increased by 68.2% (p<0.05), while the 4OH content decreased slightly (p>0.05). Furthermore, the ERU and NAS contents increased by 6.0% and 8.5%, respectively, with ERU showing a statistically significant increase (p<0.05). The NEO content decreased slightly, but the difference was not significant (p>0.05). In conclusion, LNF treatment has a selective regulatory effect on glucosinolate metabolism in broccoli seedlings, and the significant increase in NAP and ERU, in particular, may play a role in stress resistance under low-temperature stress. This result indicates that LNF irrigation can enhance the low-temperature resistance of broccoli seedlings by optimizing the distribution of glucosinolate components, but its effect on the total glucosinolate content is relatively limited, providing data support for further research on its role in plant stress resistance.

[0054] Table 10 Effects of rhizosphere drenching with LNF on glucosinolate content and types in broccoli seedlings under low temperature stress.

[0055] This study demonstrates that low-temperature nutrient oxidase (LNF) is significantly effective in regulating chilling injury in broccoli. Rhizosphere application of 0.1–0.5 g / L LNF significantly increased the root-to-shoot ratio (RS) of broccoli seedlings, with the 0.5 g / L LNF treatment achieving an RS of 0.328, a 75% increase compared to the control group (p<0.05), exhibiting optimal balance between root absorption and resource allocation. However, RS significantly decreased at LNF concentrations of 0.75 g / L and above, indicating that high concentrations may inhibit the coordinated growth of roots and shoots. At the optimal concentration, LNF effectively alleviated oxidative damage and maintained cell membrane integrity by increasing the root-to-shoot ratio, reducing H2O2 accumulation, and enhancing the activity of antioxidant enzymes (such as SOD, CAT, and APX). Furthermore, LNF regulates leaf water metabolism, increases the proportion of bound water, enhances water retention capacity, promotes proline accumulation and optimizes photosynthetic parameters, thus improving plant growth. In terms of application potential, LNF possesses advantages such as readily available raw materials, low cost, mature processing technology, and environmental friendliness. Rhizosphere irrigation is a simple and efficient method suitable for protecting broccoli from cold damage during cold seasons and for crops under low-temperature stress, providing an effective cold-resistance strategy for agricultural production. In summary, LNF enhances broccoli's low-temperature tolerance through multiple mechanisms, offering a practical solution for agricultural production in cold regions, and may have potential applications in regulating low-temperature resistance in other crops.

[0056] The embodiments and technical features described in this invention are all based on the application of liquid nitrogen fertilizer (LNF) as a urea-ammonium nitrate aqueous solution for rhizosphere irrigation, combined with experimental data and effect analysis of different application concentrations, application periods, and application rates. For simplicity, not all possible concentrations, application rates, application periods, and combinations thereof are described in detail. However, as long as the combination and adjustment of technical features do not contradict each other, they should be considered within the scope of this specification and fall under the protection of this invention.

[0057] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of liquid nitrogen fertilizer as a single cold-resistant component in improving the cold resistance of broccoli; the liquid nitrogen fertilizer is an aqueous solution of urea ammonium nitrate.

2. The application according to claim 1, characterized in that, The concentration of the liquid nitrogen fertilizer used is 0.1 g / L-0.75 g / L.

3. The application according to claim 1, characterized in that, The concentration of the liquid nitrogen fertilizer used is 0.1 g / L-0.5 g / L.

4. The application according to claim 1, characterized in that, The concentration of the liquid nitrogen fertilizer used is 0.25 g / L-0.5 g / L.

5. A method for improving the cold resistance of broccoli, characterized in that, The process includes the following steps: applying a liquid nitrogen fertilizer with a single cold-resistant component to the root zone of broccoli seedlings; the liquid nitrogen fertilizer is an aqueous solution of urea and ammonium nitrate.

6. The method according to claim 5, characterized in that, The application concentration of the liquid nitrogen fertilizer is 0.1 g / L-0.75 g / L.

7. The method according to claim 5, characterized in that, The application concentration of the liquid nitrogen fertilizer is 0.1 g / L-0.5 g / L.

8. The method according to claim 5, characterized in that, The application concentration of the liquid nitrogen fertilizer is 0.25 g / L-0.5 g / L.

9. The method according to claim 5, characterized in that, The timing of the rhizosphere irrigation is as follows: one application of exogenous material is carried out 12 days and 6 days before the onset of low temperature stress. The amount of water applied to the rhizosphere is 45-55 mL per plant each time.

10. The method according to claim 5, characterized in that, The broccoli seedlings are broccoli plants that have grown to 4-5 true leaves.

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