A complex plant growth regulating composition, its preparation and use

Abstract

The application belongs to the technical field of plant growth regulators, and discloses a compound plant growth regulator composition, a preparation method and application.The composition is composed of estragole and neoberalozulene in a mass ratio of (45:55) to (55:45). The composition is composed of estragole and neoberalozulene in a mass ratio of 50.43:49.57.The raw materials are low in price, easy to obtain, and convenient for repeated preparation, quality control of raw materials and standardization of subsequent products.

Description

Technical Field

[0001] This invention belongs to the field of plant growth regulator technology, and particularly relates to a compound plant growth regulator composition, its preparation method and application. Background Technology

[0002] Plant volatile oils are composed of various terpenoids, phenylpropanoids, and other volatile components. Current research mainly focuses on their applications in antibacterial, insecticidal, herbicidal, and seed treatment applications. Their effects on plant growth exhibit a clear dose-dependent pattern: higher concentrations typically inhibit seed germination or seedling growth, while at suitable lower concentrations, some volatile oils or volatile monomers can promote root elongation or biomass accumulation. Therefore, the use of well-defined volatile components for low-dose plant growth regulation holds further development value.

[0003] Artemisia dracunculus is a perennial aromatic plant belonging to the genus Artemisia in the family Asteraceae. The composition of its volatile oil is influenced by factors such as germplasm, geographical origin, growth conditions, and organ origin. Current research largely focuses on the chemical composition, antibacterial activity, and inhibitory phytochemical effects of volatile oils from the aboveground parts or individual organs of Artemisia dracunculus. There is a lack of parallel comparisons of the low-concentration growth-regulating effects of volatile oils from roots, stems, and leaves of the same source. The main components and proportions differ among different organs, and directly using the complex composition of whole oils is not conducive to the standardization of active ingredients and stable formulations.

[0004] Existing research mostly focuses on single volatile oils or monomeric components. While there are reports of artemisia argyi promoting root growth in Arabidopsis thaliana, publicly available research on neo-ocimene primarily concerns natural sources and biosynthesis, lacking direct verification of its plant growth-regulating applications. Summary of the Invention

[0005] To overcome the problems existing in related technologies, this invention discloses a compound plant growth regulator composition, its preparation method, and its application, specifically relating to a compound plant growth regulator composition and its application in promoting root growth through seed treatment. This invention extracts and analyzes the volatile oils from the roots, stems, and leaves of the same batch of Artemisia annua using GC-MS, and conducts activity screening at concentrations of 10–2000 μg / mL in lettuce, amaranth, and wheat. The maximum root-promoting effect of the volatile oils from the three organs on the tested plants occurred in the range of 20–100 μg / mL, with a 67.49% increase in wheat root length after treatment with 50 μg / mL of root volatile oil. The volatile oils from the stems and leaves contain high levels of artemisinin and neo-ocimene; therefore, a binary composition based on these two components was further constructed, and its application was verified in petri dishes and wheat pots.

[0006] The technical solution is as follows: A compound plant growth regulator composition comprising two active ingredients, estragole (CAS 140-67-0) and neo-allo-ocimene (CAS 7216-56-0), wherein the mass ratio of estragole to neo-allo-ocimene is (45:55) to (55:45) based on the mass of the active ingredients, and is used to promote plant root growth.

[0007] Furthermore, the composition, based on the mass ratio of the active ingredients, is artemisia argyi: neo-ocimene = 50.43: 49.57.

[0008] Furthermore, the purity of artemisia argyi was 99.77%, the purity of neo-ocimene was 95%, and the actual weighing ratio of artemisia argyi to neo-ocimene was 49.20:50.80.

[0009] Another object of the present invention is to provide a method for preparing a compound plant growth regulator composition, the method being used to prepare the compound plant growth regulator composition, characterized in that the preparation method includes the following steps:

[0010] S1. Perform mass correction based on the purity of the standard. Weigh out the 99.77% purity artemisia argyi standard and the 95.00% purity neo-ocimene standard so that the actual effective component mass ratio of the two compounds reaches (45:55) to (55:45).

[0011] S2. Weigh out artemisia argyi and neo-ocimene, dissolve them separately in acetone, and prepare monomer stock solutions.

[0012] S3. After complete dissolution, the two stock solutions are mixed and ultrasonically treated to ensure uniform dispersion, thereby obtaining a binary mixed mother liquor of artemisia annua and neo-ocimene.

[0013] Furthermore, based on the target treatment concentration requirements, the obtained binary mixed mother liquor was diluted stepwise with distilled water to prepare a treatment solution containing the plant growth regulator composition of artemisia annua and neo-ocimene at the required concentration; wherein, the volume fraction of acetone in the final treatment solution was 0.5%.

[0014] Another object of the present invention is to provide the application of the compound plant growth regulator composition in promoting root growth through plant seed treatment.

[0015] Furthermore, in the application of the compound plant growth regulator composition in promoting root growth through plant seed treatment, the maximum root length promotion effect is 20 μg / mL to 100 μg / mL.

[0016] Furthermore, the plant includes wheat; in the application of the compound plant growth regulator composition in wheat seed treatment to promote root growth, the maximum root length promoting effect is 50 μg / mL.

[0017] Furthermore, in the application of the compound plant growth regulator composition in wheat seed treatment to promote root growth, wheat seeds are soaked for 8 hours.

[0018] Furthermore, the compound plant growth regulator composition is used as a root growth regulator in the growth of lettuce and amaranth.

[0019] Combining all the above technical solutions, the beneficial effects of this invention are as follows:

[0020] This invention utilizes two main components from the volatile oils of Artemisia annua stems and leaves—artemisinin and neo-ocimene—as active ingredients to prepare a binary composition for promoting plant root growth. Compared to directly using the complex whole oil of Artemisia annua, this invention clearly defines the active ingredients, their mass ratios, effective concentrations, and application methods, facilitating repeated formulations, raw material quality control, and subsequent product standardization.

[0021] In petri dish experiments, artemisia annua, neo-ocimene, and their binary combination all exhibited concentration-dependent effects. Artemisia annua showed maximum root growth promotion rates of 27.95%, 30.15%, and 9.77% for lettuce, amaranth, and wheat, respectively; neo-ocimene showed rates of 21.30%, 33.38%, and 16.60%; and the binary combination reached 41.89%, 36.33%, and 43.97%, respectively. The maximum root growth promotion of the binary combination in all three species was higher than the individual maximum values ​​of the two monomers, indicating that the combination produces a stronger low-concentration growth response. However, only one specific ratio has been experimentally verified so far.

[0022] In the wheat pot seed soaking experiment, the average root length of the 50 μg / mL treatment increased from 34.31 cm in the control group (CK) to 42.25 cm, and the average fresh weight per plant increased from 0.622 g to 0.903 g, representing increases of 23.1% and 45.2% respectively compared to the CK, with statistically significant differences; seedling height showed no significant change. The 100 μg / mL treatment increased the fresh weight per plant by 27.8% compared to the CK, with a statistically significant difference, but the increase in root length did not reach a statistically significant level. The results from petri dishes and pots corroborate each other, indicating that 50 μg / mL is a relatively stable candidate concentration under the conditions of this experiment.

[0023] This invention utilizes two volatile components with clearly defined compositions, providing the proportion range, optimal implementation examples, purity calculation methods, low-dose ranges, and soaking steps. This approach facilitates batch-to-batch consistency compared to directly using whole oil from Artemisia annua. Treatment with 50 μg / mL resulted in an increase in IAA and chlorophyll content in wheat, while ABA and SA showed a decrease. Attached Figure Description

[0024] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the disclosure of this invention and, together with the description, serve to explain the principles of this disclosure;

[0025] Figure 1 This is a flowchart of the preparation method of the compound plant growth regulator composition provided in the embodiments of the present invention;

[0026] Figure 2 This invention provides a diagram showing the effect of volatile oils from the roots, stems, and leaves of Artemisia annua on the root length of three tested plants. (A) shows the effect on the root length of lettuce (Lactuca sativa L.); (B) shows the effect on the root length of amaranth (Amaranthus tricolor L.); and (C) shows the effect on the root length of wheat (Triticum aestivum L.).

[0027] Figure 3 This is a graph showing the effect of the present invention on the root length of three tested plants, including artemisia argyi, neo-ocimene and their fixed-ratio binary mixtures; where (A) is the effect on the root length of lettuce (Lactuca sativa L.); (B) is the effect on the root length of amaranth (Amaranthus tricolor L.); and (C) is the effect on the root length of wheat (Triticum aestivum L.).

[0028] Figure 4 This is a representative phenotypic diagram of potted wheat after soaking in a binary mixture of artemisia annua and neo-ocimene provided by the present invention.

[0029] Figure 5 This is a graph showing the effect of soaking seeds with a fixed-ratio binary mixture of artemisia annua and neo-ocimene on the growth of wheat pot plants. Among them, (A) shows the change in wheat root length after treatment with different concentrations of artemisia annua and neo-ocimene mixture; (B) shows the change in wheat seedling height after treatment with different concentrations of artemisia annua and neo-ocimene mixture; and (C) shows the change in fresh weight of single wheat plants after treatment with different concentrations of artemisia annua and neo-ocimene mixture.

[0030] Figure 6 This is a graph showing the content of 50 μg / mL artemisia annua-neoproximate binary mixture in the tender stems and leaves of wheat after soaking; where (A) is the content of IAA, (B) is the content of ABA, and (C) is the content of SA.

[0031] Figure 7 This is a graph showing the chlorophyll a, chlorophyll b, and total chlorophyll content in wheat leaves after soaking in a 50 μg / mL arugula-neoprene binary mixture provided by the present invention. Detailed Implementation

[0032] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] The innovation of this invention lies in the following: Based on the analysis of the active components of Artemisia annua volatile oil, this invention screens out the main active components with plant growth regulation potential, estragole (CAS 140-67-0) and neo-allo-ocimene (CAS 7216-56-0), from complex natural volatile components. These components are then compounded in specific mass ratios to construct a plant-derived growth-regulating composition with a well-defined and controllable proportion. This composition differs from the traditional direct utilization of complex plant extracts, realizing the transformation of natural active resources into a form with well-defined components and controllable quality. It can be used for plant seed treatment to promote root growth, providing a new technical solution for the high-value utilization of aromatic plant resources and the development of plant-derived growth-regulating materials.

[0034] Example 1.

[0035] The compound plant growth regulator composition provided in this embodiment of the invention includes two active ingredients: estragole (CAS140-67-0) and neo-allo-ocimene (CAS 7216-56-0). The mass ratio of estragole to neo-allo-ocimene is (45:55) to (55:45) based on the mass of the active ingredients, and is used to promote plant root growth.

[0036] Preferably, the composition has an active ingredient mass ratio of artemisia argyi: neo-ocimene = 50.43: 49.57.

[0037] Artemisia argyi has a purity of 99.77%, and neo-ocimene has a purity of 95%. The actual weighing ratio of artemisia argyi to neo-ocimene is 49.20:50.80.

[0038] It is known that this invention combines artemisia argyi and neo-ocimene in a near-equal mass ratio and uses them for low-dose root growth regulation; it also provides repeatable ratios, effective concentrations and soaking conditions, and provides support data for petri dishes and potted plants.

[0039] In petri dish experiments, artemisia argyi, neo-ocimene, and their binary combination all exhibited concentration-dependent effects. Artemisia argyi showed maximum root growth promotion rates of 27.95%, 30.15%, and 9.77% for lettuce, amaranth, and wheat, respectively; neo-ocimene showed rates of 21.30%, 33.38%, and 16.60%; and the binary combination reached 41.89%, 36.33%, and 43.97%, respectively. The maximum root growth promotion of the binary combination in all three species was higher than the individual maximum values ​​of the two monomers, indicating that the combination produced a stronger low-concentration growth response.

[0040] In the wheat pot seed soaking experiment, the average root length of the 50 μg / mL treatment increased from 34.31 cm in the control group (CK) to 42.25 cm, and the average fresh weight per plant increased from 0.622 g to 0.903 g, representing increases of 23.1% and 45.2% respectively compared to the CK, with statistically significant differences; seedling height showed no significant change. The 100 μg / mL treatment increased the fresh weight per plant by 27.8% compared to the CK, with a statistically significant difference, but the increase in root length did not reach a statistically significant level. The results from petri dishes and pots corroborate each other, indicating that 50 μg / mL is a relatively stable candidate concentration under the conditions of this experiment.

[0041] This invention employs two volatile components with clearly defined compositions, providing the proportion range, optimal embodiment, purity calculation method, low-dose range, and soaking steps. This approach facilitates batch-to-batch consistency compared to directly using whole oil from Artemisia annua. Treatment with 50 μg / mL resulted in an increase in IAA and chlorophyll content in wheat, while ABA and SA showed a decrease, but none of the differences were statistically significant.

[0042] This invention provides a compound plant growth regulator composition derived from plant volatile components. The composition uses artemisinin and neo-ocimene as the main active ingredients, and features clearly defined components, controllable proportions, and ease of standardized preparation, making it suitable for further development as a plant-derived growth regulator. Compared to the complex composition of natural volatile oils, this technical solution facilitates the identification of active ingredients and product quality control, providing a new technical pathway for the high-value utilization of aromatic plant resources. In the future, based on further field trials and product development results, it can be applied to fields such as crop seed treatment, root promotion, and plant growth regulation.

[0043] Currently, there are no reports on the use of a specific ratio of artemisinin and neo-ocimene to promote plant root growth. This invention screens, combines, and verifies the main active ingredients in the volatile oil of Artemisia annua, establishing a plant growth regulation application scheme based on a clearly defined combination of volatile components, providing a new research direction for the development of plant-derived growth regulators.

[0044] This invention does not address major technical problems that have remained unsolved for a long time. Instead, it provides a plant-derived compound growth regulation scheme with a clear source and controllable composition, which addresses the problems of complex active ingredients, single source, or insufficient utilization efficiency of natural plant resources in existing plant growth regulators.

[0045] There is no evidence to suggest that this invention overcomes the explicit technical biases in existing technologies. However, this technical solution breaks through the traditional approach of using only complex plant extracts or single growth regulators. By screening and combining the main components of natural volatile oils, a compound plant growth regulation scheme composed of clearly defined compounds has been established.

[0046] Example 2.

[0047] The compound plant growth regulator composition provided in this embodiment of the invention is a binary mixture prepared according to a mass ratio of 50.43:49.57 (preferred) of acetamiprid and neo-ocimene.

[0048] like Figure 1 As shown, the preparation method of the compound plant growth regulator composition provided in this embodiment of the invention includes:

[0049] S1. Perform mass correction based on the purity of the standard. Weigh out the 99.77% purity artemisia argyi standard and the 95.00% purity neo-ocimene standard so that the actual effective component mass ratio of the two compounds reaches (45:55) to (55:45).

[0050] Preferably, the actual effective component mass ratio of the two compounds reaches 50.43:49.57, corresponding to an actual weighed mass ratio of 49.20:50.80.

[0051] S2. Weigh out artemisia argyi and neo-ocimene, dissolve them separately in acetone, and prepare monomer stock solutions.

[0052] S3. After complete dissolution, the two stock solutions are mixed and ultrasonically treated to ensure uniform dispersion, thereby obtaining a binary mixed mother liquor of artemisia annua and neo-ocimene.

[0053] Based on the target treatment concentration requirements, the obtained binary mixed mother liquor was gradually diluted with distilled water to prepare a treatment solution containing the required concentration of artemisia annua-neoprolide plant growth regulator composition. The acetone volume fraction in the final treatment solution was controlled at 0.5% (v / v) to ensure sufficient dissolution of the active ingredients. An aqueous solution containing an equal volume of acetone was also provided as a control treatment.

[0054] Example 3.

[0055] Application of a compound plant growth regulator composition in promoting root growth through seed treatment.

[0056] In the application of the compound plant growth regulator composition in promoting root growth through seed treatment, the maximum root length promotion effect is 20–100 μg / mL.

[0057] The plant mentioned includes wheat.

[0058] In the application of the compound plant growth regulator composition in wheat seed treatment to promote root growth, the maximum root length promotion effect is 50 μg / mL.

[0059] In the application of the compound plant growth regulator composition in wheat seed treatment to promote root growth, wheat seeds are soaked for 8 hours.

[0060] The compound plant growth regulator composition is used as a root growth regulator in the growth of lettuce and amaranth.

[0061] Example 4.

[0062] The compound plant growth regulator composition provided in this embodiment of the invention includes two active ingredients, artemisia argyi and neo-ocimene. The mass ratio of artemisia argyi to neo-ocimene is 45:55 based on the mass of the active ingredients, and it is used to promote plant root growth.

[0063] The method for preparing the compound plant growth regulator composition provided in this embodiment of the invention includes:

[0064] S1. Based on the purity of the two standards, a mass correction was performed by weighing appropriate amounts of artemisia argyi standard (purity 99.77%) and neo-ocimene standard (purity 95.00%) to make the actual effective component mass ratio of the two compounds reach 45:55.

[0065] S2, after weighing, artemisia argyi and neo-ocimene were dissolved in an appropriate amount of acetone to prepare monomer stock solutions;

[0066] S3, after being fully dissolved, the two stock solutions are mixed according to the above mass ratio, and ultrasonic treatment is used to make them fully and evenly dispersed to obtain a binary mixed mother liquor of artemisia argyi and neo-ocimene.

[0067] In this process, the resulting binary mixed mother liquor is gradually diluted with distilled water according to the target treatment concentration requirements to prepare the treatment solution of the arbutin-neoprolol plant growth regulator composition at the desired concentration. The acetone volume fraction in the final treatment solution is controlled at 0.5% (v / v) to ensure sufficient dissolution of the active ingredients. An aqueous solution containing an equal volume of acetone is also provided as a control treatment.

[0068] Example 5.

[0069] The compound plant growth regulator composition provided in this embodiment of the invention includes two active ingredients, artemisia argyi and neo-ocimene. The mass ratio of artemisia argyi to neo-ocimene is 55:45 based on the mass of the active ingredients, and it is used to promote plant root growth.

[0070] The method for preparing the compound plant growth regulator composition provided in this embodiment of the invention includes:

[0071] S1. Based on the purity of the two standards, a mass correction was performed by weighing appropriate amounts of artemisia argyi standard (purity 99.77%) and neo-ocimene standard (purity 95.00%) to make the actual effective component mass ratio of the two compounds reach 55:45.

[0072] S2, after weighing, artemisia argyi and neo-ocimene were dissolved in an appropriate amount of acetone to prepare monomer stock solutions;

[0073] S3, after being fully dissolved, the two stock solutions are mixed according to the above mass ratio, and ultrasonic treatment is used to make them fully and evenly dispersed to obtain a binary mixed mother liquor of artemisia argyi and neo-ocimene.

[0074] In this process, the resulting binary mixed mother liquor is gradually diluted with distilled water according to the target treatment concentration requirements to prepare the treatment solution of the arbutin-neoprolol plant growth regulator composition at the desired concentration. The acetone volume fraction in the final treatment solution is controlled at 0.5% (v / v) to ensure sufficient dissolution of the active ingredients. An aqueous solution containing an equal volume of acetone is also provided as a control treatment.

[0075] Example 6.

[0076] The compound plant growth regulator composition provided in this embodiment of the invention includes two active ingredients, artemisia argyi and neo-ocimene. The mass ratio of artemisia argyi to neo-ocimene is 50:50 based on the mass of the active ingredients, and it is used to promote plant root growth.

[0077] The method for preparing the compound plant growth regulator composition provided in this embodiment of the invention includes:

[0078] S1. Based on the purity of the two standards, perform mass correction by weighing appropriate amounts of artemisia argyi standard (purity 99.77%) and neo-ocimene standard (purity 95.00%) to make the actual effective component mass ratio of the two compounds reach 50:50.

[0079] S2, after weighing, artemisia argyi and neo-ocimene were dissolved in an appropriate amount of acetone to prepare monomer stock solutions;

[0080] S3, after being fully dissolved, the two stock solutions are mixed according to the above mass ratio, and ultrasonic treatment is used to make them fully and evenly dispersed to obtain a binary mixed mother liquor of artemisia argyi and neo-ocimene.

[0081] In this process, the resulting binary mixed mother liquor is gradually diluted with distilled water according to the target treatment concentration requirements to prepare the treatment solution of the arbutin-neoprolol plant growth regulator composition at the desired concentration. The acetone volume fraction in the final treatment solution is controlled at 0.5% (v / v) to ensure sufficient dissolution of the active ingredients. An aqueous solution containing an equal volume of acetone is also provided as a control treatment.

[0082] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0083] To further illustrate the effects of the embodiments of the present invention, the following experiments were conducted.

[0084] 1.1 Materials and reagents.

[0085] 1.1.1 Plant collection.

[0086] Mature *Artemisia annua* plants were collected in August 2024 from a natural wild population in a town in a county of a city in an autonomous region (approximately 43°N, 87°E, altitude 1669m). Healthy plants without obvious pests or diseases were selected. After removing surface soil, the roots, stems, and leaves were separated and allowed to air dry naturally in a cool, well-ventilated place. The tested plants were wheat (*Triticum aestivum*), lettuce (*Lactucasativa*), and amaranth (*Amaranthus tricolor*). The purity of artemisia annua was 99.77% (CAS 140-67-0), and the purity of neo-ocimene was 95% (CAS 7216-56-0).

[0087] 1.1.2 Extraction and preparation of volatile oils;

[0088] 120g each of the shade-dried roots, stems, and leaves of *Artemisia annua* were weighed, chopped, and placed in a Clevenger apparatus for water distillation for 3 hours. The distillate oil was dried with anhydrous sodium sulfate and transferred to brown sample bottles, then stored at 4°C in the dark. 2.5, 1.5, and 1.5 mL of volatile oil were obtained from the roots, stems, and leaves, respectively, with corresponding yields of 2.08%, 1.25%, and 1.25% (v / w, dry weight).

[0089] 1.1.3 Chemical composition analysis of volatile oils;

[0090] The volatile oils were analyzed using an Agilent 7890A gas chromatography-mass spectrometry system with a DB-5MS capillary column. Chemical components were matched against an EI-MS library and compared with experimental retention indices calculated from n-alkanes under the same chromatographic conditions. A total of 52 volatile components were obtained that showed good agreement between EI-MS and RI information. 36, 30, and 14 components were detected in the root, stem, and leaf oils, respectively, totaling 6 components across the three organs. The relative peak areas of the listed components in the roots, stems, and leaves were 90.90%, 96.62%, and 96.61%, respectively.

[0091] The volatile oil from the roots was mainly composed of capillary ene and 5-phenyl-1,3-pentadiyne, with relative peak areas of 58.10% and 11.15%, respectively. In the volatile oil from the stems, neo-ocimene and artemisinin accounted for 30.64% and 25.36%, respectively. In the volatile oil from the leaves, artemisinin and neo-ocimene accounted for 36.11% and 29.79%, respectively. The volatile oil from the roots was mainly composed of polyacetylene compounds, while the volatile oils from the stems and leaves were mainly composed of monoterpenes and phenylpropanoids, showing significant differences between the organs.

[0092] 1.1.4 Petri dish experiments on volatile oils from different organs;

[0093] The volatile oils from the roots, stems, and leaves were prepared into stock solutions using acetone, and then diluted with distilled water to concentrations of 10, 20, 50, 100, 200, 500, 1000, and 2000 μg / mL. The final acetone concentration in all treatment solutions was 0.5% (v / v), and the control (CK) was an aqueous solution of acetone at the same concentration. Each 9cm diameter petri dish was lined with filter paper and 10 seeds were placed on it. 5mL of the corresponding treatment solution was added, and the dish was sealed. Different treatments were cultured separately to minimize cross-influence between volatile components. The culture temperature was 25±2℃ and the environment was kept dark. The taproot length and seedling length of lettuce and amaranth were measured on day 5, and that of wheat on day 7. Each treatment was replicated in three petri dishes, with 10 seedlings per dish, for a total of 30 seedlings measured.

[0094] The maximum root length promotion rate of volatile oils from all three organs occurred within the range of 20–100 μg / mL. The maximum promotion rate of root volatile oils was 24.82% (20 μg / mL) for lettuce, and 62.74% and 67.49% (both 50 μg / mL) for amaranth and wheat, respectively. At 100 μg / mL, the maximum promotion rates of stem volatile oils for the three plants were 56.65%, 53.78%, and 55.12%, respectively; and at 50 μg / mL, the maximum promotion rates of leaf volatile oils were 20.90%, 46.57%, and 37.30%, respectively. With further increases in concentration, the promotion rate gradually decreased, and some treatments at 1000–2000 μg / mL turned into inhibitory effects.

[0095] in, Figure 2 The effects of volatile oils from the roots, stems, and leaves of Artemisia annua on the root length of three tested plants were investigated. Figure 2 Figure (A) shows the effect of root length on lettuce (Lactuca sativa); Figure 2 Figure (B) shows the effect of root length on amaranth (Amaranthus tricolor); Figure 2 Figure C shows the effect of different concentrations on root length in wheat (Triticum aestivum). The control (CK) was a 0.5% (v / v) aqueous solution of acetone, with treatment concentrations ranging from 10 to 2000 μg / mL. All values ​​are the mean ± standard error of three biological replicates (n=30). Different lowercase letters within the volatile oil of the same species and the same organ indicate significant differences in concentration between treatments (Tukey HSD test after one-way ANOVA, P<0.05).

[0096] 1.1.5 Tests on artemisia argyi, neo-ocimene, and binary mixtures;

[0097] Artemisia argyi, neo-ocimene, and the binary composition were cultured using the same concentration gradient and method as for organ volatile oils. The mass ratio of artemisia argyi to neo-ocimene active ingredients in the binary composition was set to (45:55) to (55:45); this example used the validated ratio of 50.43:49.57. After purity correction according to the commercial standard used in this experiment, the actual weighing ratio was 49.20:50.80. The concentration of the mixture was calculated as the sum of the masses of the two active ingredients, and the final concentration of acetone in the treatment solution was maintained at 0.5% (v / v).

[0098] The maximum growth-promoting rates of artemisia argyi on the root length of lettuce, amaranth, and wheat were 27.95%, 30.15%, and 9.77%, respectively, at concentrations of 100, 50, and 20 μg / mL. The maximum growth-promoting rates of neo-ocimene were 21.30%, 33.38%, and 16.60%, respectively, at concentrations of 100, 50, and 100 μg / mL. The maximum growth-promoting rates of the binary composition on the root length of lettuce and wheat were 41.89% and 43.97%, respectively, both occurring at 50 μg / mL; the maximum growth-promoting rate on amaranth was 36.33%, occurring at 20 μg / mL. The maximum growth-promoting rates of the binary composition in all three species were higher than the individual maximum values ​​of the two monomers, but the results of a single formulation cannot be used to determine synergistic, additive, or antagonistic relationships.

[0099] Figure 3 The effects of artemisia argyi, neo-ocimene, and their fixed-ratio binary mixtures on the root length of three tested plants. Figure 3 Figure (A) shows the effect of root length on lettuce (Lactuca sativa); Figure 3 Figure (B) shows the effect of root length on amaranth (Amaranthus tricolor); Figure 3 Figure C shows the effect on wheat (Triticum aestivum) root length. Mixture is a mixture of artemisia annua and neo-ocimene; CK is a 0.5% (v / v) aqueous solution of acetone, with treatment concentrations ranging from 10 to 2000 μg / mL. All values ​​are the mean ± standard error of three biological replicates (n=30). Different lowercase letters within the same species and treatment type indicate significant differences in concentration between treatments (Tukey HSD test after one-way ANOVA, P<0.05).

[0100] 1.1.6 Pot experiment on wheat seeds soaked in a mixture of artemisia annua and neo-ocimene;

[0101] Based on the petri dish results, a binary mixture was selected for a wheat pot seed soaking experiment. Four treatments were set up: CK, 20, 50, and 100 μg / mL. CK was distilled water containing 0.5% (v / v) acetone. After surface disinfection, wheat seeds were soaked for 8 hours and then sown in a substrate of sterilized nutrient soil, vermiculite, and sand in a 3:1:1 (v / v / v) mixture. The pots were 15 cm in diameter and 20 cm in height. The plants were cultivated in a greenhouse at approximately 25℃ with a 16-hour photoperiod. Each treatment had 3 independent pots, with 10 seedlings per pot. After 25 days of cultivation, the roots, seedling height, and fresh weight of 30 wheat plants were measured.

[0102] The average root lengths of the CK, 20, 50, and 100 μg / mL treatments were 34.31, 36.02, 42.25, and 38.80 cm, respectively; the average seedling heights were 33.26, 35.13, 33.88, and 32.47 cm, respectively; and the average fresh weight per plant was 0.622, 0.742, 0.903, and 0.795 g, respectively. The 50 μg / mL treatment significantly increased root length by 23.1% and fresh weight by 45.2% compared to the CK treatment. The 100 μg / mL treatment significantly increased fresh weight by 27.8% compared to the CK treatment, but the increase in root length was not significant. There were no significant differences in seedling height among the treatments.

[0103] Figure 4 Representative phenotypes of potted wheat after seed soaking treatment with a binary mixture of artemisia annua and neo-ocimene. Wheat seeds were soaked for 8 h in CK, 20, 50, and 100 μg / mL solutions, respectively. CK was distilled water containing 0.5% (v / v) acetone. The images, from left to right, show the CK, 20, 50, and 100 μg / mL treatments, with a 50 cm ruler on the left.

[0104] Figure 5 The effect of soaking seeds in a fixed-ratio binary mixture of artemisia annua and neo-ocimene on the growth of wheat in pots. Figure 5Figure (A) shows the changes in wheat root length after treatment with different concentrations of artemisia annua-neoproside mixture; Figure 5 Figure (B) shows the changes in wheat seedling height after treatment with different concentrations of artemisia annua-neoproside mixture; Figure 5 Figure (C) shows the change in fresh weight of individual wheat plants after treatment with different concentrations of artemisia annua-neoproside mixture. Mixture is a binary mixture of artemisia annua-neoproside in a fixed ratio; CK is a 0.5% (v / v) aqueous solution of acetone. Different lowercase letters indicate significant differences between different treatments for the same indicator (Tukey HSD test after one-way ANOVA, P < 0.05). All values ​​are the mean ± standard error of three biological replicates.

[0105] Wheat seeds were soaked for 8 hours. All values ​​are the mean ± standard error of three biological replicates (n=30). Different lowercase letters within the same trait indicate significant differences between treatments (Tukey HSD test after one-way ANOVA, P<0.05).

[0106] 1.1.7 Determination of plant hormones and chlorophyll;

[0107] Based on the potted plant phenotypic results, the contents of IAA, ABA, SA, and chlorophyll in young wheat stems and leaves were determined using the control (CK) and a 50 μg / mL treatment. Each treatment was repeated in triplicate. IAA, ABA, and SA were determined using HPLC with external standard method, while chlorophyll was determined using 95% ethanol extraction-spectrophotometry.

[0108] After treatment with 50 μg / mL, the average IAA content increased from 3.737 μg / g to 4.902 μg / g, an increase of 31.2%; ABA decreased from 0.320 μg / g to 0.141 μg / g, a decrease of 56.0%; and SA decreased from 0.223 μg / g to 0.175 μg / g, a decrease of 21.6%. The average contents of chlorophyll a, chlorophyll b, and total chlorophyll increased from 0.214, 0.144, and 0.358 mg / g fresh weight to 0.310, 0.216, and 0.525 mg / g fresh weight, respectively, representing increases of 44.7%, 49.5%, and 46.6%.

[0109] Figure 6 The figure shows the content of 50 μg / mL artemisia annua-neoproside binary mixture in the tender stems and leaves of wheat after seed soaking. Figure 6 Figure (A) shows the content of IAA. Figure 6 Figure (B) shows the contents of ABA (B) and SA (C). All values ​​are the mean ± standard error of three independent biological replicates (n=3). The same lowercase letter indicates that there is no significant difference between CK and the 50 μg / mL treatment (Tukey HSD test after one-way ANOVA, P>0.05).

[0110] Figure 7 The values ​​represent the chlorophyll a, chlorophyll b, and total chlorophyll contents in wheat leaves after seed soaking in a 50 μg / mL mixture of artemisia annua and neobasene. All values ​​are the mean ± standard error of three independent biological replicates (n=3). The same lowercase letter indicates no significant difference between the control (CK) and the 50 μg / mL treatment (Tukey HSD test after one-way ANOVA, P>0.05).

[0111] 1.1.8 Comparative examples and comparison results.

[0112] Comparative Example 1 (Artemisia argyi monomer).

[0113] Except for the use of only artemisinin as the active ingredient, the concentration gradient, solvent system, culture conditions, and assay methods were the same as those for the binary composition petri dish test. Within the tested concentration range, artemisinin achieved maximum root growth promotion rates of 27.95%, 30.15%, and 9.77% for lettuce, amaranth, and wheat, respectively.

[0114] Comparative Example 2 (Neo-Ocimethylene Monomer);

[0115] Except for the use of neo-ocimene as the only active ingredient, the other conditions were the same as those for the binary composition. The maximum root growth promotion rates of neo-ocimene on lettuce, amaranth, and wheat were 21.30%, 33.38%, and 16.60%, respectively.

[0116] Example 4 (Binary Composition);

[0117] A validated formulation using artemisia argyi:neo-ocimene in a ratio of 50.43:49.57 (mass ratio of active ingredients) was employed. This composition achieved maximum root growth promotion rates of 41.89%, 36.33%, and 43.97% for lettuce, amaranth, and wheat, respectively, all exceeding the maximum values ​​of the two monomers within the same test concentration range. Therefore, artemisia argyi monomer and neo-ocimene monomer can be used as comparative examples to illustrate the experimental effects of the binary composition relative to the single-component formulation.

[0118] Comparative Example 3 (Pot-plant solvent control and non-optimal concentration);

[0119] In potted wheat, the average root lengths of the CK, 20, 50, and 100 μg / mL treatments were 34.31, 36.02, 42.25, and 38.80 cm, respectively, and the average fresh weight per plant was 0.622, 0.742, 0.903, and 0.795 g, respectively. The root length and fresh weight of the 50 μg / mL treatment increased by 23.1% and 45.2% compared to the CK, respectively, indicating that this concentration maintained a significant growth-promoting effect under substrate cultivation conditions. The increase in root length decreased at 100 μg / mL, suggesting that the present invention has a suitable dosage window.

[0120] 1.1.9 Data processing.

[0121] All data were analyzed using IBM SPSS Statistics 26.0, and results are expressed as mean ± standard error. Two-way ANOVA was used for the volatile oil experiments in different organs, as well as for the experiments involving artemisinin, neo-ocimene, and binary compositions, to test treatment type, concentration, and their interactions. The control (CK) was not included in the two-way model. One-way ANOVA was used to analyze the differences between different concentrations within each treatment type and the CK, as well as potted plant growth, plant hormones, and chlorophyll indices. Tukey's HSD test was used for multiple comparisons when overall differences were significant. Normality and homogeneity of variance were tested before analysis, with a significance level set at P < 0.05.

[0122] This invention constructs a binary composition with a well-defined structure using artemisia argyi and neo-ocimene. Based on the normalized ratio of the two main components in the volatile oils of the stems and leaves, the effective component mass ratio of artemisia argyi to neo-ocimene can be expressed as (45:55) to (55:45); the experimentally verified example shows a ratio of 50.43:49.57. When using raw materials of different purities, the ratio can be converted according to the effective component content. The actual weighing ratio of 99.77% artemisia argyi and 95% neo-ocimene used in this experiment is 49.20:50.80. Compared with directly using whole oil from Artemisia annua, this method is more convenient for standardized formulation and batch quality control.

[0123] Petri dish experiments showed that the binary composition promoted root growth in lettuce, amaranth, and wheat at low concentrations. The maximum growth promotion rate for all three species was higher than the individual maximum values ​​of artemisia annua and neo-ocimene monomers. Specifically, the 50 μg / mL treatment promoted wheat root length by 43.97%. Pot seed soaking experiments further showed that the 50 μg / mL treatment increased wheat root length and whole plant fresh weight by 23.1% and 45.2%, respectively, with significant differences, indicating that this effect could be carried over from filter paper culture conditions to substrate culture conditions.

[0124] This invention provides a specific method for soaking seeds in a low-concentration binary composition for 8 hours, and clarifies the suitable concentration range. 50 μg / mL is a candidate concentration that showed relatively stable performance under the conditions of this experiment. The formulation is simple in composition and has a clear ratio, facilitating further development into a plant-derived root growth regulator product. Existing data primarily support seedling root elongation and early fresh weight accumulation; further validation in subsequent greenhouse and field trials is needed for a wider range of formulations, other crops, yield, dry matter accumulation, residues, safety, and long-term effects.

[0125] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any person skilled in the art within the scope of the technology disclosed in the present invention, within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A compound plant growth regulator composition, characterized in that, The composition includes two active ingredients, artemisia argyi and neo-ocimene. The mass ratio of artemisia argyi to neo-ocimene is (45:55) to (55:45) based on the mass of the active ingredients, and it is used to promote plant root growth.

2. The compound plant growth regulator composition according to claim 1, characterized in that, The composition, by mass ratio of active ingredients, is artemisia argyi: neo-ocimene = 50.43: 49.

57.

3. The compound plant growth regulator composition according to claim 1, characterized in that, The purity of artemisia argyi was 99.77%, and the purity of neo-ocimene was 95%. The actual weighing ratio of artemisia argyi to neo-ocimene was 49.20:50.

80.

4. A method for preparing a compound plant growth regulator composition, wherein the method is used to prepare the compound plant growth regulator composition according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: S1. Perform mass correction based on the purity of the standard. Weigh out the 99.77% purity artemisia argyi standard and the 95.00% purity neo-ocimene standard so that the actual effective component mass ratio of the two compounds reaches (45:55) to (55:45). S2. Weigh out artemisia argyi and neo-ocimene, dissolve them separately in acetone, and prepare monomer stock solutions. S3. After complete dissolution, the two stock solutions are mixed and ultrasonically treated to ensure uniform dispersion, thereby obtaining a binary mixed mother liquor of artemisia annua and neo-ocimene.

5. The method for preparing the compound plant growth regulator composition according to claim 4, characterized in that, According to the target treatment concentration requirement, the obtained binary mixed mother liquor was diluted stepwise with distilled water to prepare a treatment solution containing the plant growth regulator composition of artemisia annua and neo-ocimene at the required concentration; wherein, the volume fraction of acetone in the final treatment solution was 0.5%.

6. The use of a compound plant growth regulator composition as described in any one of claims 1-3 in promoting root growth through plant seed treatment.

7. The application according to claim 6, characterized in that, In the application of the compound plant growth regulator composition in promoting root growth through seed treatment, the maximum root length promotion effect is 20 μg / mL to 100 μg / mL.

8. The application according to claim 6, characterized in that, The plant includes wheat; in the application of the compound plant growth regulator composition in wheat seed treatment to promote root growth, the maximum root length promoting effect is 50 μg / mL.

9. The application according to claim 8, characterized in that, In the application of the compound plant growth regulator composition in wheat seed treatment to promote root growth, wheat seeds are soaked for 8 hours.

10. The application according to claim 6, characterized in that, The compound plant growth regulator composition is used as a root growth regulator in the growth of lettuce and amaranth.

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