Compound root stimulator and its application in tobacco seedling floating culture

Abstract

The present application belongs to the technical field of modern agricultural planting and plant growth regulator, and relates to a compound root promoter and application thereof in floating seedling of flue-cured tobacco. The compound root promoter is composed of carboxymethyl chitosan, gamma-aminobutyric acid and gamma-polyglutamic acid. Flue-cured tobacco seeds are sowed in a seedling tray filled with seedling substrate in a floating seedling mode, the seedling tray is placed in a transfer box filled with nutrient solution for seedling, and the compound root promoter is applied to the nutrient solution at one time in the early stage of seedling. The compound root promoter significantly improves total root surface area and root tip number through "fine root proliferation", and significantly improves leaf SPAD value, root activity and nitrate reductase activity, cooperatively drives carbon assimilation of the aboveground part and nitrogen assimilation of the root system, optimizes source-sink relationship and comprehensively improves tobacco seedling quality, and successfully avoids the risk of osmotic stress and growth inhibition caused by high concentration or middle and late application.

Description

Technical Field

[0001] This invention belongs to the field of modern agricultural planting and plant growth regulator technology, and relates to a compound root-promoting agent and its application in floating seedling raising of flue-cured tobacco. Background Technology

[0002] Seedling raising is the primary step in the cultivation and production of flue-cured tobacco (Nicotiana tabacum L.), and cultivating robust seedlings is the foundation for high-quality tobacco leaf production. Floating seedling raising technology is currently the most common method for intensive flue-cured tobacco seedling raising. While its development has matured since its initial promotion and widespread adoption, the rate of robust seedlings often falls short of the demands of tobacco production. In particular, underdeveloped root systems and a limited number of effective roots lead to low transplant survival rates, slow seedling recovery, and weak root vitality. This results in a mismatch between the supply and demand of nutrients such as nitrogen during the early stages of tobacco seedling growth, which has become a serious limiting factor for later yield formation and quality improvement. Therefore, optimizing root structure and enhancing root physiological activity through exogenous regulation is one of the key issues that urgently needs to be addressed in the cultivation of robust flue-cured tobacco seedlings.

[0003] Applying root-promoting agents is an effective way to regulate root morphology and enhance plant stress resistance. However, traditional synthetic hormone-based root-promoting substances easily interfere with the balance and coordination of endogenous plant hormones, and have a certain degree of persistence and bioaccumulation in soil and water, which may pose potential residual risks to the ecological environment. Their single mechanism of action is no longer sufficient to meet the needs of modern ecological agriculture. In recent years, novel root-promoting agents, represented by biostimulants and functional amino acids, have shown great application potential. However, existing research on the application of root-promoting agents in flue-cured tobacco seedling cultivation mainly focuses on the growth-promoting effects of single growth regulators or single substances, with relatively few studies on multi-substance compound root-promoting agents. In particular, how these compound root-promoting agents affect the root morphology, photosynthetic characteristics, and physiological metabolic mechanisms of flue-cured tobacco seedlings under different application periods and concentration ratios remains unclear. Summary of the Invention

[0004] To address the problems of underdeveloped root systems, low number of effective roots, and low transplant survival rates in existing flue-cured tobacco floating seedling cultivation, as well as the environmental risks associated with traditional hormone-based root-promoting agents, this invention provides a novel, non-toxic, and environmentally friendly compound root-promoting agent and its precise application method in flue-cured tobacco seedling cultivation. The compound root-promoting agent provided by this invention can regulate root architecture and physiological metabolic processes, thereby targeting and solving the problem of underdeveloped root systems, synergistically promoting seedling growth, and effectively improving the rate of robust flue-cured tobacco seedlings.

[0005] Invention Concept: Carboxymethyl chitosan, as a water-soluble derivative of the biostimulant chitosan, possesses both excellent biocompatibility and chelation effects, effectively promoting the development of flue-cured tobacco seedlings. Gamma-aminobutyric acid (GABA), a non-protein amino acid, can act as a key signaling molecule, directly regulating the activity of aluminum-activated malate transporter (ALMT), effectively modulating root growth and plant responses to stresses such as acid-base imbalance and aluminum stress. Meanwhile, γ-polyglutamic acid, as an environmentally friendly macromolecular biostimulant, effectively promotes the formation of soil aggregates, improves soil permeability, and enhances water retention capacity. In soilless cultivation systems, its abundant functional groups also exert excellent nutrient chelation and osmotic regulation effects, creating a favorable rhizosphere microenvironment for the roots. The scientific combination of these three substances is expected to create a synergistic and complementary effect from two levels: "rhizosphere environment optimization" and "physiological signal transduction." Specifically, γ-polyglutamic acid and carboxymethyl chitosan synergistically improve the rhizosphere microenvironment, while γ-aminobutyric acid, as a signaling molecule, stimulates the biological potential for root growth.

[0006] This invention systematically evaluated the application effects of a novel compound root-promoting agent composed of carboxymethyl chitosan, γ-aminobutyric acid, and γ-polyglutamic acid at different application stages (early seedling stage and 30 days after sowing) and concentration gradients, exploring the intrinsic relationships between this compound root-promoting agent and the agronomic traits, root architecture, photosynthetic characteristics, and physiological metabolic indicators of flue-cured tobacco. The aim is to clarify its optimal application strategy, reveal its physiological mechanism of growth promotion, and provide a theoretical basis for cultivating robust flue-cured tobacco seedlings and developing novel eco-friendly root-promoting agents.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] This invention discloses a compound root-promoting agent, which is composed of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid; wherein the mass ratio of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid is (8.0~12.0):(45.0~55.0):(0.5~1.5).

[0009] In some embodiments, the mass ratio of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid is 10.0:50.0:1.0.

[0010] The application of the aforementioned compound root-promoting agent in floating seedling cultivation of flue-cured tobacco is also within the scope of protection of this invention.

[0011] In some embodiments, a floating seedling raising method is adopted, in which flue-cured tobacco seeds are sown in seedling trays containing seedling substrate, and the seedling trays are placed in a transport box containing nutrient solution for seedling raising; on the first day after sowing, a 1 / 2 concentration nutrient solution is applied once, and on the 30th day after sowing, the nutrient solution is replaced and a full concentration nutrient solution is applied once; wherein, in the early stage of seedling raising, the above-mentioned compound root-promoting agent is applied to the nutrient solution once.

[0012] In some embodiments, the above-mentioned compound root-promoting agent is applied to the nutrient solution once in the early stage of seedling cultivation, and the seedlings absorb it through the floating seedling system.

[0013] In some embodiments, the seedling substrate is prepared by mixing a base substrate, perlite and vermiculite in a mass ratio of (5.0~7.0):(1.0~2.0):(2.0~3.0).

[0014] In some embodiments, the seedling substrate is prepared from a base substrate, perlite, and vermiculite in a mass ratio of 6.0:1.5:2.5.

[0015] In some embodiments, the base substrate is not specifically limited, and any general-purpose base substrate is applicable to this invention.

[0016] In some embodiments, the nutrient solution is prepared from seedling fertilizer and deionized water; the concentration of seedling fertilizer in the 1 / 2 concentration nutrient solution is 230 mg / L to 250 mg / L; and the concentration of seedling fertilizer in the full concentration nutrient solution is 460 mg / L to 500 mg / L.

[0017] The concentration of seedling fertilizer in the full-concentration nutrient solution is twice the concentration of seedling fertilizer in the half-concentration nutrient solution.

[0018] In some embodiments, the nutrient solution is prepared from seedling fertilizer and deionized water; the concentration of seedling fertilizer in the 1 / 2 concentration nutrient solution is 240 mg / L; and the concentration of seedling fertilizer in the full concentration nutrient solution is 480 mg / L.

[0019] In some embodiments, the seedling fertilizer is not specifically limited and may conform to the DB52 / T 661-2010 standard "Technical Specification for Floating Seedling Raising of Flue-cured Tobacco in Guizhou Province".

[0020] In some embodiments, optionally, the seedling fertilizer has a nitrogen, phosphorus, and potassium mass ratio of 15:10:18, a total nutrient content of ≥43%, a nitrate nitrogen content of ≥55%, and a trace element mass content of: Mg 0.04%, B 0.02%, Zn 0.02%, Mo 0.005%, Mn 0.02%, and Fe 0.04%.

[0021] In some embodiments, the initial seedling stage is the first to third day after the tobacco seeds are sown.

[0022] In some embodiments, the initial seedling stage is the first day after the tobacco seeds are sown.

[0023] In some embodiments, the compound root-promoting agent is composed of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid; wherein the mass ratio of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid is (8.0~12.0):(45.0~55.0):(0.5~1.5).

[0024] In some embodiments, the mass ratio of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid is 10.0:50.0:1.0.

[0025] In some embodiments, the application amount of the compound root-promoting agent is calculated per tobacco seedling: 1 mg / plant to 5 mg / plant of carboxymethyl chitosan, 5 mg / plant to 25 mg / plant of γ-aminobutyric acid, and 0.1 mg / plant to 0.5 mg / plant of γ-polyglutamic acid.

[0026] In some embodiments, the application amount of the compound root-promoting agent is calculated per single flue-cured tobacco seedling: 1 mg / plant carboxymethyl chitosan, 5 mg / plant γ-aminobutyric acid, and 0.1 mg / plant γ-polyglutamic acid.

[0027] In some embodiments, the temperature is controlled at 25℃~28℃ and the relative humidity is 60%~70% during the seedling stage. Natural light is supplemented by artificial light sources, and the photocycle is 12 hours of light and 12 hours of darkness.

[0028] In some embodiments, the compound root-promoting agent can regulate the growth of flue-cured tobacco seedlings raised in floating nurseries, reshape the root system, and promote physiological metabolism.

[0029] In some embodiments, the compound root-promoting agent can increase the plant height, stem diameter, maximum leaf length, maximum leaf width, total root surface area, number of root tips, and overall plant biomass of flue-cured tobacco seedlings.

[0030] In some embodiments, the compound root-promoting agent can increase the SPAD value of flue-cured tobacco seedling leaves, root activity, and nitrate reductase activity.

[0031] Beneficial effects:

[0032] (1) The experimental research of this invention shows that the exogenous addition of low concentrations of compound root-promoting agents (carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid) in the early stage of flue-cured tobacco seedling cultivation can significantly promote the morphological development and biomass accumulation of root system in flue-cured tobacco seedlings. This provides a scientific basis for the promotion and application of this novel compound root-promoting agent in future flue-cured tobacco floating seedling cultivation. Developed seedling roots not only mean stronger physiological metabolic activity and stress resistance, but may also provide more favorable conditions for promoting the mutually beneficial symbiosis between beneficial microorganisms and tobacco roots by improving the rhizosphere microenvironment. This virtuous cycle can not only effectively improve the transplant survival rate and agronomic traits of tobacco seedlings, but also, this novel green growth-promoting strategy based on rhizosphere regulation is expected to replace traditional chemical hormones from the source, significantly reducing agricultural non-point source pollution and environmental risks while ensuring high-quality and high-yield tobacco leaves.

[0033] (2) The active ingredient of the compound root-promoting agent disclosed in this invention is a scientific compound of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid. By clarifying that the compound root-promoting agent has a significant "low-promoting and high-inhibiting" effect and time effect in flue-cured tobacco floating seedling raising, the optimal application strategy was established: at the early stage of seedling raising (the first day after sowing), a low concentration of the formula (single plant dosage: 1 mg / plant of carboxymethyl chitosan, 5 mg / plant of γ-aminobutyric acid, and 0.1 mg / plant of γ-polyglutamic acid) was precisely applied to the nutrient solution. This treatment can reshape the root system structure of seedlings, significantly increase the total root surface area and root tip number through "fine root proliferation", and significantly increase the leaf SPAD value, root activity and nitrate reductase activity. It synergistically drives the aboveground carbon assimilation and root nitrogen assimilation, realizes the optimization of the source-sink relationship and the overall improvement of tobacco seedling quality, and successfully avoids the risk of osmotic stress and growth inhibition caused by high concentration or mid-to-late stage application.

[0034] (3) Significantly reshaping root morphology and promoting "fine root proliferation": The compound root-promoting agent of this invention, when applied at low concentrations, can significantly optimize the root structure of tobacco seedlings. The experimental results show that the total root surface area and root tip number of flue-cured tobacco under the optimal treatment are significantly increased by 97.0% and 125% respectively compared with the control. This fine root promotion strategy effectively expands the contact area between the root system and nutrients.

[0035] (4) Significantly improves biomass and agronomic traits, and coordinates source-sink relationships: When the compound root-promoting agent of this invention is applied at low concentrations, it can achieve a simultaneous and significant increase in the biomass of the aboveground parts and roots. Among them, the fresh weight and dry weight of the aboveground parts are significantly increased by 90.9% and 42.0% respectively compared with the control, the fresh weight and dry weight of the roots are significantly increased by 25.9% and 82.8% respectively compared with the control, while the plant height and stem diameter are significantly increased by 15.6% and 24.7% respectively compared with the control, and the maximum leaf length and leaf width are increased by 21.4% and 9.2% respectively compared with the control, realizing the overall optimization of the source-sink relationship of flue-cured tobacco seedlings.

[0036] (5) Activate key physiological functions and promote a virtuous cycle of carbon and nitrogen metabolism: This invention, through the synergistic effect of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid, when applied at low concentrations, can significantly increase the SPAD value of tobacco seedling leaves (significantly increased by 67.7% compared to the control), root activity (significantly increased by 2.92 times compared to the control treatment) and nitrate reductase (NR) activity (significantly increased by 54.1% compared to the control), while promoting photosynthetic carbon assimilation in the aboveground parts, and synergistically enhancing the nitrogen assimilation capacity of the roots.

[0037] (6) Clarified the appropriate time and concentration conditions for "low-promoting and high-inhibiting": This invention establishes the key process conditions for precise application of "low concentration" (single plant dosage: 1 mg / plant carboxymethyl chitosan, 5 mg / plant γ-aminobutyric acid, 0.1 mg / plant γ-polyglutamic acid) at the "early seedling stage" (one-time application of root promoter on the first day after sowing). This invention confirms that delaying the application to the middle stage of seedling stage (30 days after sowing) or increasing the application concentration will not only weaken or eliminate the growth-promoting effect, but the high concentration of components will also trigger a hyperosmolar microenvironment and osmotic stress, forcing the roots to passively synthesize soluble proteins for regulation, thereby inhibiting root and whole plant growth. This discovery provides a scientific basis for optimizing the application strategy of root promoters in production practice and avoiding the negative effects caused by unreasonable application. Attached Figure Description

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0039] Figure 1 The graph shows the effect of different application times and concentrations of compound root-promoting agents on the biomass of flue-cured tobacco seedlings.

[0040] Figure 2 The graph shows the effect of different application times and concentrations of compound root-promoting agents on the phenotype of flue-cured tobacco seedlings.

[0041] Figure 3 Figure 1 shows the effect of different application times and concentrations of compound root-promoting agents on the aboveground agronomic traits of flue-cured tobacco seedlings.

[0042] Figure 4 The figure shows the effects of different application times and concentrations of root-promoting agents on the photosynthetic capacity of leaves and the physiological metabolism of roots in flue-cured tobacco seedlings.

[0043] Figure 5 Spearman correlation analysis of various agronomic traits, root morphology and physiological metabolic indicators during the seedling stage of flue-cured tobacco. Detailed Implementation

[0044] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0046] Unless otherwise specified, the markings in the accompanying drawings in the instruction manual have the following meanings: CK: Control (no root-promoting agent applied); H: High concentration treatment in the early stage of seedling growth; M: Medium concentration treatment in the early stage of seedling growth; L: Low concentration treatment in the early stage of seedling growth; H30: High concentration treatment in the middle stage of seedling growth; M30: Medium concentration treatment in the middle stage of seedling growth; L30: Low concentration treatment in the middle stage of seedling growth.

[0047] The base substrate used in the seedling substrate in the following examples is a general-purpose base substrate (purchased from Pins, Denmark). The basic physicochemical properties of the base substrate are as follows: pH=6.14; total nitrogen 8.13 g / kg; total phosphorus 0.50 g / kg; total potassium 4.10 g / kg; available phosphorus 85.8 mg / kg; available potassium 952.5 mg / kg; available nitrogen 343.9 mg / kg; organic matter 576.9 g / kg.

[0048] Example 1:

[0049] 1. Materials and Methods

[0050] 1.1 Test Materials

[0051] The tested flue-cured tobacco variety was Guiyan 20, and the seeds were provided by the Qianxinan Prefecture Branch of Guizhou Tobacco Company. The seedling substrate was prepared by mixing basic substrate, perlite, and vermiculite in a mass ratio of 6.0:1.5:2.5. The root-promoting agent mainly consisted of carboxymethyl chitosan, γ-aminobutyric acid, and γ-polyglutamic acid. Seedling fertilizer was prepared according to the standard DB52 / T 661-2010 "Technical Regulations for Floating Seedling Raising of Flue-cured Tobacco in Guizhou Province," using a special seedling fertilizer with a nitrogen, phosphorus, and potassium mass ratio of 15:10:18, total nutrients ≥43%, nitrate nitrogen ≥55%, and trace element content as follows: Mg 0.04% (magnesium sulfate), B 0.02% (sodium tetraborate decahydrate), Zn 0.02% (zinc sulfate), Mo 0.005% (ammonium molybdate), Mn 0.02% (manganese sulfate), and Fe 0.04% (ferrous sulfate). All reagents used in the experiment were of analytical grade.

[0052] 1.2 Experimental Design

[0053] The experiment was conducted in the constant-temperature incubation chamber of the Nanjing Institute of Soil Science, Chinese Academy of Sciences. A single-factor randomized block design was adopted, with a total of 7 treatments: control (CK, no root-promoting agent applied), high concentration treatment in the early seedling stage (H), medium concentration treatment in the early seedling stage (M), low concentration treatment in the early seedling stage (L), high concentration treatment in the middle seedling stage (H30), medium concentration treatment in the middle seedling stage (M30), and low concentration treatment in the middle seedling stage (L30). Each treatment was replicated in 3s.

[0054] The root-promoting agent concentration settings are as follows: High concentration: 10 mg / plant of carboxymethyl chitosan, 50 mg / plant of γ-aminobutyric acid, and 1 mg / plant of γ-polyglutamic acid; Medium concentration is a 1-fold dilution of the high concentration: 5 mg / plant of carboxymethyl chitosan, 25 mg / plant of γ-aminobutyric acid, and 0.5 mg / plant of γ-polyglutamic acid; Low concentration is a 10-fold dilution of the high concentration: 1 mg / plant of carboxymethyl chitosan, 5 mg / plant of γ-aminobutyric acid, and 0.1 mg / plant of γ-polyglutamic acid. The application time settings are as follows: for the initial seedling treatment, apply the root-promoting agent once on the first day after sowing; for the mid-seedling treatment, apply the root-promoting agent once on the 30th day after sowing.

[0055] 1.3 Test Methods

[0056] 1.3.1 Seedling Management

[0057] A floating seedling raising method was adopted, using 60-cell seedling trays, with one coated seed sown per cell. The seedling trays were placed in transport boxes containing 9 L of nutrient solution (seedling-specific fertilizer) per box. A half-concentration seedling-specific fertilizer was applied on the first day after sowing, and the nutrient solution was replaced and a full-concentration seedling-specific fertilizer was applied on the 30th day after sowing. The final volume of the nutrient solution in each box was 9 L, prepared using seedling-specific fertilizer and deionized water. The full concentration was defined as 4.32 g of seedling-specific fertilizer per box, equivalent to a concentration of 480 mg / L, while the half-concentration seedling-specific fertilizer concentration was 240 mg / L. During the seedling period, the temperature was controlled at 25℃~28℃, the relative humidity at 60%~70%, and natural light was supplemented with artificial light, with a photoperiod of 12h / 12h (light / dark). Except for the concentration and timing of the root-promoting agent application, all other management measures were consistent across treatments. Samples were taken and measured 65 days after sowing.

[0058] 1.3.2 Application method of root-promoting agent

[0059] Control (CK): No root-promoting agent was applied. On the first day after sowing, an equal amount of deionized water was added to the nutrient solution. All other seedling management measures were exactly the same as those of the treatment group.

[0060] High-concentration treatment in the early stage of seedling growth (H): Single plant dosage: 10 mg / plant of carboxymethyl chitosan, 50 mg / plant of γ-aminobutyric acid, and 1 mg / plant of γ-polyglutamic acid; apply the root-promoting agent once on the first day after sowing.

[0061] Medium concentration treatment (M) in the early stage of seedling cultivation: Single plant dosage: 5 mg / plant carboxymethyl chitosan, 25 mg / plant γ-aminobutyric acid, 0.5 mg / plant γ-polyglutamic acid; apply the root-promoting agent once on the first day after sowing.

[0062] Low-concentration treatment (L) in the early stage of seedling cultivation: Single plant dosage: 1 mg / plant of carboxymethyl chitosan, 5 mg / plant of γ-aminobutyric acid, and 0.1 mg / plant of γ-polyglutamic acid; apply the root-promoting agent once on the first day after sowing.

[0063] High-concentration treatment during mid-seedling stage (H30): Single plant dosage: 10 mg / plant carboxymethyl chitosan, 50 mg / plant γ-aminobutyric acid, 1 mg / plant γ-polyglutamic acid; apply the root-promoting agent once on the 30th day after sowing.

[0064] Mid-stage seedling treatment (M30): Single plant dosage: 5 mg / plant carboxymethyl chitosan, 25 mg / plant γ-aminobutyric acid, 0.5 mg / plant γ-polyglutamic acid; apply the root-promoting agent once on the 30th day after sowing.

[0065] Low-concentration treatment during mid-seedling stage (L30): Single plant dosage: 1 mg / plant carboxymethyl chitosan, 5 mg / plant γ-aminobutyric acid, 0.1 mg / plant γ-polyglutamic acid; apply the root-promoting agent once on the 30th day after sowing.

[0066] According to the experimental design, the prepared compound root-promoting agent was added to the nutrient solution under the seedling tray at the specified time and stirred well. The control treatment was treated with an equal amount of deionized water.

[0067] 1.3.3. Measurement Items and Methods

[0068] (1) Determination of agronomic traits

[0069] Aboveground parameters: Plant height (from stem base to shoot tip meristem) was measured with a ruler; stem diameter was measured with calipers; leaf number was counted based on fully expanded leaves; maximum leaf length and maximum leaf width were both measured with a ruler. Aboveground and root biomass determination: The fresh weight of the aboveground parts and roots was weighed separately using an electronic balance, followed by blanching at 105℃ for 30 min, drying at 75℃ to constant weight, and weighing the dry weight (i.e., dry biomass) of the aboveground parts and roots; root morphological parameters, including total root length, root surface area, root volume, average diameter, and number of root tips, were analyzed using a root scanner (WinRhizo-LA1600, Regent Instruments Inc., Quebec, QC, Canada).

[0070] (2) Measurement of physiological indicators

[0071] The determination of plant physiological indicators was based on existing techniques (Li Hesheng. Principles and Techniques of Plant Physiological and Biochemical Experiments [M]. Beijing: Higher Education Press, 2000): Chlorophyll content was determined using a SPAD-502 chlorophyll meter, with the midrib of the leaf removed during the measurement; root activity was determined using the triphenyltetrazolium chloride (TTC) method; soluble protein content was determined using the Coomassie brilliant blue G-250 staining method; and nitrate reductase (NR) activity was determined using the sulfonamide colorimetric method.

[0072] 1.4 Data Processing

[0073] Experimental data were processed using Microsoft Excel 2019. One-way ANOVA and Tukey multiple comparisons (P < 0.05) were performed using SPSS 25.0 statistical software, and graphs were plotted using Origin 2021. The Spearman correlation coefficient method was used to assess the degree of association between the indicators. The correlation matrix was plotted using the corrplot package (v0.94) in R (v4.4.1), with color intensity and gradients representing the magnitude and direction of the correlation coefficient; significance markers were added (* P < 0.05, ** P < 0.01, *** P < 0.001).

[0074] 2. Results and Analysis

[0075] 2.1 Effects of different application periods and concentrations of compound root-promoting agents on aboveground and root biomass of flue-cured tobacco seedlings

[0076] Figure 1The figure shows the effect of different application times and concentrations of compound root-promoting agents on the biomass of flue-cured tobacco seedlings; in the figure, a is the fresh weight of the aboveground parts; b is the dry weight of the aboveground parts; c is the fresh weight of the roots; d is the dry weight of the roots; e is the root-to-shoot ratio; different lowercase letters above the column indicate significant differences between treatments, the same below (Tukey multiple comparisons, P < 0.05).

[0077] Figure 2 The graph shows the effect of different application times and concentrations of compound root-promoting agents on the phenotype of flue-cured tobacco seedlings.

[0078] from Figure 1 and Figure 2 It can be seen that the application of compound root-promoting agents has a significant impact on the growth of flue-cured tobacco plants. Regarding aboveground growth, low-concentration treatments had a significant promoting effect. Among them, the L treatment showed the best effect, with aboveground fresh weight and dry weight significantly increasing by 90.9% and 42.0% respectively compared to the control (CK). The L30 treatment also showed a certain growth-promoting effect, with fresh weight and dry weight increasing by 9.6% and 31.0% respectively compared to the CK. High-concentration treatments inhibited aboveground growth. Among them, the H30 treatment showed the strongest inhibition on fresh weight, decreasing by 52.9% compared to the CK treatment; while the H treatment showed the most significant inhibition on aboveground dry matter accumulation, decreasing by 41.4% compared to the CK.

[0079] The response trend of root biomass to the compound root-promoting agent was basically consistent with that of the aboveground parts. Root development was optimal under the L treatment, with root fresh weight and dry weight significantly increased by 25.9% and 82.8% compared to the control (CK), respectively. Although the root fresh weight of the L30 treatment was not significantly different from that of the CK, its root dry weight was significantly increased by 47.5%. In the high-concentration treatment groups, the root fresh weight of the H30 treatment was significantly reduced by 77.4% compared to the CK, while the root dry weight of the H treatment was significantly reduced by 56.6% compared to the CK.

[0080] From the root-to-shoot ratio results, the L and M treatments showed the most significant improvement, increasing the ratio by 28.7% and 27.3% respectively compared to the control (CK). The L30 treatment followed, increasing the ratio by 12.6%, but the difference was not statistically significant. Conversely, the H, M30, and H30 treatments significantly inhibited the root-to-shoot ratio, decreasing it by 25.8%, 26.1%, and 29.3% respectively compared to the CK. Among all treatments, the H30 treatment showed the most significant decrease in root-to-shoot ratio, decreasing it by 45.0% and 44.4% compared to the L and M treatments, respectively.

[0081] 2.2 Effects of different application times and concentrations of compound root-promoting agents on the aboveground phenotypes of flue-cured tobacco seedlings

[0082] Figure 3The figure shows the effect of different application times and concentrations of compound root-promoting agents on the aboveground agronomic traits of flue-cured tobacco seedlings; in the figure, a is plant height; b is stem diameter; c is maximum leaf length; and d is maximum leaf width (Tukey multiple comparisons, P < 0.05).

[0083] from Figure 3 It was found that the application of compound root-promoting agents had a significant regulatory effect on the aboveground growth of flue-cured tobacco seedlings, and this effect was influenced by both the application time and concentration. Overall, application in the early seedling stage was more effective than in the middle seedling stage; regarding concentration effects, low concentrations promoted growth, while medium and high concentrations showed inhibitory effects. In terms of plant height and stem diameter, the L treatment showed the best growth-promoting effect, with plant height and stem diameter significantly increasing by 15.6% and 24.7% compared to the control (CK), respectively. If application was delayed until the middle seedling stage, the growth-promoting effect of the L30 treatment essentially disappeared. Medium and high concentration treatments inhibited both plant height and stem diameter, with H30 showing the strongest inhibitory effect, significantly reducing plant height and stem diameter by 40.7% and 34.6% compared to the control (CK), respectively. Under the condition of application in the early seedling stage, the inhibitory effects of the M and H treatments were relatively weak, with plant height decreasing by 9.4% and 12.2% compared to the control (CK), and stem diameter decreasing by 11.0% and 17.9%, respectively.

[0084] The compound root-promoting agent also significantly regulated the maximum leaf length and width of flue-cured tobacco seedlings, and its trend was consistent with that of plant height and stem diameter. The L treatment effectively promoted leaf development, increasing maximum leaf length and width by 21.4% and 9.2% respectively compared to the control (CK), with a significant effect on maximum leaf length. The L30 treatment, however, showed no effect on either maximum leaf length or width. Medium and high concentrations also significantly inhibited leaf development. The H30 treatment significantly reduced maximum leaf length and width by 42.6% and 18.3% respectively compared to the CK; the M and H treatments reduced maximum leaf length and width by 16%–18% and 12%–14% respectively compared to the CK.

[0085] 2.3 Effects of different application times and concentrations of compound root-promoting agents on root development in flue-cured tobacco seedlings

[0086] Table 1 shows that the application of compound root-promoting agents significantly affected the root development of flue-cured tobacco, but there were significant differences among the different treatments. Specifically, the L treatment showed the best promoting effect on root development, with its total root surface area and root tip number significantly increasing by 97.0% and 125% compared to the CK, respectively. The promoting effect of the L30 treatment was significantly weaker than that of the L treatment. Compared with the L treatment, the total root length of the M and H treatments decreased significantly by 48.2% and 59.9%, respectively; in terms of total root volume, compared with the L treatment, the M and H treatments decreased by 35.1% and 77.6%, respectively; while the root tip number of the H and H30 treatments decreased significantly by 48.1% and 49.1% compared to the control treatment. When applied in the mid-seedling stage, the H30 treatment showed a strong inhibitory effect, with its total root volume and average diameter significantly decreasing by 86.4% and 79.7% compared to the CK, respectively.

[0087] Table 1. Effects of different application times and concentrations of root-promoting agents on root morphology in flue-cured tobacco seedlings.

[0088]

[0089] Note: Different lowercase letters in the same column of the table indicate significant differences between different treatments (P < 0.05).

[0090] 2.4 Effects of different application periods and concentrations of compound root-promoting agents on the photosynthetic capacity of leaves and root physiological metabolism in flue-cured tobacco seedlings.

[0091] Figure 4 The effects of different application times and concentrations of root-promoting agents on the photosynthetic capacity of leaves and the physiological metabolism of roots in flue-cured tobacco seedlings are shown in the figure. In the figure, a is the SPAD value of leaves; b is the root activity; c is the nitrate reductase (NR) activity; and d is the soluble protein content (Tukey multiple comparisons, P < 0.05).

[0092] from Figure 4 It can be seen that, in terms of leaf photosynthetic performance, low-concentration treatments have a strong effect on improving the SPAD value of flue-cured tobacco. Specifically, the L treatment significantly increased the SPAD value by 67.7% compared to the control (CK); the L30 treatment also showed a significant promoting effect, increasing the SPAD value by 43.4% compared to the CK, but the difference was not statistically significant. In contrast, the H and H30 treatments failed to improve the photosynthetic capacity of flue-cured tobacco.

[0093] Regarding root physiological metabolism, the responses of various indicators to the compound root-promoting agents varied significantly. Root activity indicators showed that low-concentration treatments had the strongest promoting effect, with L and L30 treatments significantly increasing root activity by 2.92 and 2.34 times compared to the control (CK) treatment, respectively. Medium-concentration treatments also showed significant promoting effects, with M and M30 treatments significantly increasing root activity by 1.16 and 2.05 times compared to the CK treatment, respectively. However, high-concentration treatments showed no significant difference in root activity compared to the CK. Furthermore, nitrate reductase (NR) activity results indicated that L and L30 treatments performed best, significantly increasing activity by 54.1% and 45.5% compared to the CK treatment, respectively. Application of the compound root-promoting agents significantly increased the soluble protein content of flue-cured tobacco roots, with high-concentration treatments (H and H30) showing the most significant improvement, increasing by 108.3% and 91.4% compared to the CK treatment, respectively. The L treatment significantly increased soluble protein content by 86.0% compared to the CK treatment.

[0094] 2.5 Correlation analysis of different application periods and concentrations of compound root-promoting agents on agronomic traits and physiological metabolism in flue-cured tobacco seedlings.

[0095] Figure 5 Spearman correlation analysis plots for various agronomic traits, root morphology, and physiological metabolic indicators during the seedling stage of flue-cured tobacco are shown. In the plot, red squares represent positive correlations, and blue squares represent negative correlations. The intensity of the color reflects the magnitude of the correlation coefficient. An asterisk inside a square indicates that the correlation is significant (* P < 0.05, ** P < 0.01, *** P < 0.001).

[0096] To further elucidate the intrinsic relationship between compound root-promoting agents and the regulation of growth and physiological metabolism in flue-cured tobacco seedlings, Spearman correlation analysis was conducted on the aboveground agronomic traits, root morphology, and physiological and biochemical indicators of flue-cured tobacco seedlings. Figure 5 It was found that root development and aboveground growth in flue-cured tobacco exhibit a high degree of phenotypic synergy. Root surface area and root tip number showed highly significant positive correlations with aboveground phenotypic parameters (plant height, stem diameter, maximum leaf length, maximum leaf width) and total plant biomass. However, the average root diameter showed negative or weak correlations with root surface area, root tip number, and aboveground phenotypic indicators. Physiological function indicators also showed strong correlations with aboveground biomass. Leaf SPAD value, root activity, and NR activity were all significantly positively correlated with the dry matter accumulation of tobacco seedlings. In contrast to the above indicators, root soluble protein content showed a completely different response. Correlation analysis results showed that soluble protein showed highly significant negative correlations with aboveground and root biomass, agronomic traits (plant height, stem diameter, maximum leaf length, maximum leaf width), and root tip number.

[0097] 3. Discussion of Results

[0098] 3.1 The regulatory mechanism of compound root-promoting agents on seedling phenotype and source-sink relationship in flue-cured tobacco.

[0099] The effect of compound root-promoting agents on the phenotype of flue-cured tobacco seedlings varies depending on the application concentration and timing. This invention demonstrates that a novel compound root-promoting agent composed of carboxymethyl chitosan, γ-aminobutyric acid, and γ-polyglutamic acid exhibits a significant "low-promoting, high-inhibiting" effect on the development of both the aboveground parts and roots of flue-cured tobacco seedlings. Applying a low concentration of the root-promoting agent (L treatment) in the early seedling stage significantly promotes plant height, stem diameter, and leaf expansion. Figure 3 This is mainly attributed to the fact that the early seedling stage is a critical and sensitive period for active division and morphogenesis of root meristems. At this time, low concentrations of carboxymethyl chitosan and γ-aminobutyric acid (GABA) act as signaling molecules, effectively stimulating cell elongation. Simultaneously, γ-polyglutamic acid synergistically promotes the growth of flue-cured tobacco seedlings by improving nutrient availability in the root growth environment. However, when the concentration of root-promoting agents is too high or application is delayed until the middle of the seedling stage, the growth-promoting effect is significantly weakened or even turns into an inhibitory effect. This inhibitory effect caused by high concentrations may be due to excessive exogenous substances interfering with the dynamic balance of endogenous hormones in the plant, leading to receptor saturation; and high concentrations of macromolecules easily form physical barriers on the root surface or induce sudden changes in osmotic potential in the growth environment, thereby limiting the absorption of nutrients and water by the roots.

[0100] Roots are the main organs for nutrient absorption in crops, and a well-developed root system is the foundation for high-quality and high-yield flue-cured tobacco. This study found that the average root diameter was highest in the CK treatment (2.26 mm), while it decreased to 1.64 mm in the L treatment (Table 1). This indicates that the L treatment mainly exhibited "fine root proliferation" rather than root thickening. The root-promoting strategy of the L treatment significantly increased the root surface area of ​​tobacco seedlings, and this optimized root architecture effectively expanded the contact area between roots and nutrients. Regarding biomass accumulation, the L treatment promoted a synchronous increase in aboveground and root biomass, indicating that low-concentration compound root-promoting agents can effectively coordinate carbon and nitrogen metabolism, achieving overall optimization of the source-sink relationship in flue-cured tobacco seedlings. This study shows that exogenous active substances mainly increase the total root length and root volume by promoting root elongation or increasing the number of roots, thereby promoting aboveground development. Furthermore, the total root length of the tobacco plant is significantly positively correlated with the dry matter accumulation of tobacco leaves. Notably, the M treatment showed the most significant improvement in the root-to-shoot ratio. Figure 1e) This reflects an adaptive resource allocation strategy that tobacco seedlings may adopt when faced with moderate concentrations of exogenous stimulants: that is, plants actively adjust carbon flow, transferring more dry matter to the roots to strengthen root development, and coping with environmental changes by enhancing nutrient acquisition for organ development; while the stimulant concentration under the L treatment is within the optimal range for synergistic aboveground and belowground growth. In contrast, the H30 treatment significantly inhibited phenotypic indicators such as total root length, total root volume, and average diameter (Table 1), possibly because the tobacco seedling roots have already acquired a complete morphology in the mid-stage of seedling growth, and their plasticity has significantly decreased; high concentrations of root-promoting agents lead to an increase in osmotic pressure around the roots, thereby damaging root cells and inhibiting the root's growth-promoting response.

[0101] 3.2 Analysis of the potential synergistic mechanism of compound root-promoting agents in the regulation of tobacco seedling growth

[0102] In this study, both L and L30 treatments effectively increased the SPAD value of tobacco seedling leaves (the difference between the L treatment and the CK treatment was significant) and root NR activity ( Figure 4 These results indicate that low-concentration compound root-promoting agents synergistically enhance root nitrogen assimilation capacity while promoting aboveground carbon assimilation (photosynthesis), thus driving a virtuous cycle of carbon and nitrogen metabolism. Furthermore, the significant increase in root activity further confirms the synergistic effect of this compound root-promoting agent in activating root physiological functions. This suggests that biostimulants not only promote carbon assimilation by enhancing photosynthesis but also strengthen nitrogen assimilation by promoting root morphogenesis and nutrient absorption. Correlation analysis results show that leaf SPAD values, root activity, and NR activity are all significantly positively correlated with the dry matter accumulation of tobacco seedlings. Figure 5 The study also revealed the synergistic driving effect of root-promoting agents on aboveground carbon assimilation (photosynthesis), root nitrogen assimilation (NR activity), and nutrient absorption kinetics (root vitality).

[0103] However, both H and H30 treatments showed significant inhibitory effects on the biomass and phenotype of the aboveground parts and roots of flue-cured tobacco, but the content of soluble protein in the roots was significantly higher than that of the control treatment. Figure 4(d) This phenomenon can be attributed to the passive defense mechanism of tobacco seedling roots in the face of a high-osmotic environment. High concentrations of γ-aminobutyric acid (GABA), γ-polyglutamic acid (PGA), and carboxymethyl chitosan (MCCH) create a high-osmotic microenvironment, inducing mild osmotic stress. High concentrations of exogenous plant growth regulators can exert a stress effect on plants, inhibiting their growth; however, when plants are exposed to adversity, their cells spontaneously accumulate osmotic regulators such as soluble proteins, enhancing their water absorption capacity to maintain reasonable water content and normal physiological activities. Therefore, to maintain the water content of tobacco roots, the roots synthesize soluble proteins for osmotic regulation. Furthermore, the application of γ-polyglutamic acid significantly promotes the accumulation of osmotic regulators such as soluble proteins in the plant, thereby effectively improving the water-holding capacity of the roots and maintaining their water metabolism balance during osmotic regulation. Therefore, to maintain cell turgor pressure and water homeostasis, tobacco roots under high-concentration treatment adopt a passive defense mechanism of forced synthesis and accumulation of soluble proteins for osmotic regulation. Figure 5 The correlation analysis results showed that soluble protein exhibited a highly significant negative correlation with aboveground and root biomass, agronomic traits (plant height, stem diameter, maximum leaf length, maximum leaf width), and root tip number in tobacco seedlings. This significant negative effect also suggests that the abnormal accumulation of soluble protein did not translate into a positive driving force for tobacco seedling growth, but rather was a stress-induced passive accumulation when overall plant growth and root architecture were suppressed.

[0104] In conclusion, the precise application of low-concentration compound root-promoting agents in the early stages of flue-cured tobacco seedling cultivation can comprehensively improve the quality of tobacco seedlings from multiple levels, including phenotypic remodeling, physiological metabolism, and improvement of the rhizosphere microenvironment. This provides a scientific basis for the cultivation of high-quality and robust flue-cured tobacco seedlings and the research and development of green and ecological biostimulants in modern ecological agriculture.

[0105] This invention provides a compound root-promoting agent and its application in floating seedling cultivation of flue-cured tobacco, along with its concept and method. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A compound root-promoting agent, characterized in that, The compound root-promoting agent is composed of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid; wherein the mass ratio of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid is (8.0~12.0):(45.0~55.0):(0.5~1.5).

2. The compound root-promoting agent according to claim 1, characterized in that, The mass ratio of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid is 10.0:50.0:1.

0.

3. The application of the compound root-promoting agent according to claim 1 or 2 in floating seedling cultivation of flue-cured tobacco.

4. The application according to claim 3, characterized in that, The floating seedling method is adopted. Flue-cured tobacco seeds are sown in seedling trays containing seedling substrate, and the seedling trays are placed in a transfer box containing nutrient solution for seedling cultivation. On the first day after sowing, a 1 / 2 concentration nutrient solution is applied once, and on the 30th day after sowing, the nutrient solution is replaced and a full concentration nutrient solution is applied once. Among them, the compound root-promoting agent described in claim 1 or 2 is applied to the nutrient solution once in the early stage of seedling cultivation.

5. The application according to claim 4, characterized in that, The seedling substrate is prepared by mixing basic substrate, perlite and vermiculite in a mass ratio of (5.0~7.0):(1.0~2.0):(2.0~3.0).

6. The application according to claim 4, characterized in that, The nutrient solution is prepared from seedling fertilizer and deionized water; the concentration of seedling fertilizer in the 1 / 2 concentration nutrient solution is 230 mg / L to 250 mg / L; and the concentration of seedling fertilizer in the full concentration nutrient solution is 460 mg / L to 500 mg / L.

7. The application according to claim 4, characterized in that, The initial stage of seedling cultivation refers to the first 1 to 3 days after the tobacco seeds are sown.

8. The application according to claim 4, characterized in that, The compound root-promoting agent is composed of carboxymethyl chitosan, γ-aminobutyric acid, and γ-polyglutamic acid; wherein the mass ratio of carboxymethyl chitosan, γ-aminobutyric acid, and γ-polyglutamic acid is (8.0~12.0):(45.0~55.0):(0.5~1.5). Optionally, the mass ratio of carboxymethyl chitosan, γ-aminobutyric acid and γ-polyglutamic acid is 10.0:50.0:1.

0.

9. The application according to claim 4, characterized in that, The application amount of the compound root-promoting agent is calculated per single flue-cured tobacco seedling: 1 mg / plant to 5 mg / plant of carboxymethyl chitosan, 5 mg / plant to 25 mg / plant of γ-aminobutyric acid, and 0.1 mg / plant to 0.5 mg / plant of γ-polyglutamic acid.

10. The application according to claim 4, characterized in that, The application amount of the compound root-promoting agent is calculated per single flue-cured tobacco seedling: 1 mg / plant carboxymethyl chitosan, 5 mg / plant γ-aminobutyric acid, and 0.1 mg / plant γ-polyglutamic acid.

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