Method for promoting flower fragrance release of dendrobium violaceum by using exogenous melatonin

Abstract

The invention discloses a method for promoting flower fragrance release of dendrobium violaceum by utilizing exogenous melatonin, and belongs to the technical field of plant fragrance regulation and plant growth regulation. According to the method, melatonin is externally applied to regulate and control the physiological process of the flowering phase of the dendrobium purple petals, accumulation and volatilization of floral substances are enhanced, and the concentration and durability of fragrance are improved; specifically, according to the method, when flower buds of the dendrobium violaceum are about 2 cm long in the flower bud stage, melatonin solution is applied in a spraying and root flushing mode, the concentration is preferably (500 + / -100) micromol / L, preferably, the melatonin solution is applied at 10: 00 in the morning, the temperature is controlled to be 25-30 DEG C, the humidity is controlled to be 60%-75%, and the illumination period of 12-hour illumination and 12-hour darkness is set for cultivation; release of flower fragrance substances of the purple petal dendrobium is effectively promoted, the variety and content of fragrance components are increased, the lasting time of the flower fragrance is prolonged, meanwhile, the stress resistance and the ornamental value of plants are enhanced, and the method has important application prospects in improvement of the ornamental value of dendrobium plants and research and development of products.

Description

Technical Field

[0001] This invention specifically relates to a method for promoting the release of fragrance from Dendrobium nobile flowers using exogenous melatonin, belonging to the technical fields of plant aroma regulation and plant growth regulation. Background Technology

[0002] Dendrobium purpureum ( Dendrobium parishii Dendrobium purpureum is a common Dendrobium species, a perennial epiphytic herb belonging to the genus Dendrobium in the Orchidaceae family. It possesses high ornamental, medicinal, and economic value, with vibrant flowers, a long flowering period, and a delicate fragrance, making it an important ornamental orchid resource. Floral fragrance is an important secondary metabolic characteristic of plants, composed of various low-boiling-point volatile organic compounds (VOCs), and its quality is one of the most important factors determining consumer sensory experience and market value. The main fragrance components of Dendrobium purpureum are fatty acid-derived ketones (such as 2-pentadecanone, 2-trigecanone, and cyclopentadecanone). The content and release intensity of these compounds not only directly determine the quality of the fragrance but also attract pollinators and resist external biotic stress, playing an important ecological role in plant defense. Therefore, enhancing the ornamental and economic value of Dendrobium purpureum is of great significance. However, the synthesis and release of floral fragrance substances are regulated by multiple factors, including environmental conditions, developmental stage, and endogenous hormone signals, resulting in a complex mechanism. Currently, relevant regulatory methods are still very limited.

[0003] Traditional methods for controlling floral fragrance mainly include regulating light, temperature, and nutrients. For example, in the cultivation of Dendrobium nobile, maintaining the greenhouse temperature at 25±2°C can prevent the decline in fragrance caused by high-temperature heat stress. Zinc treatment (ZnSO4·7H2O) on the plant leaves can significantly increase the content of terpenes and their derivatives in the petals of Dendrobium nobile, especially α-terpenes. pinene, β Key aroma components such as pinene; in addition, JA is induced by methyl jasmonate (MeJA). Signaling and enriching carbon dioxide to create a microenvironment can also promote the synthesis of floral fragrance substances. However, these methods often suffer from unstable regulatory effects and pose potential environmental pollution risks.

[0004] Melatonin (MT), also known as pineal hormone, is an essential indoleamine compound. As an endogenous hormone in plants, it has potential regulatory effects on hormone balance, plant metabolism, photosynthesis, stress resistance, and aroma synthesis. However, there is currently no technical solution to utilize melatonin to enhance the release of fragrance compounds in orchids. Summary of the Invention

[0005] This invention aims to provide a method for promoting the release of fragrance from Dendrobium nobile flowers using melatonin. By applying exogenous melatonin to promote the release of fragrance substances from Dendrobium nobile flowers, the physiological and metabolic processes at different stages of flower development are precisely regulated, significantly promoting the synthesis and volatilization of fragrance substances and increasing the concentration and intensity of the aroma. This method has the advantages of being simple to operate, low in cost, and environmentally friendly, making it suitable for greenhouse cultivation and large-scale production. By controlling the concentration and timing of melatonin application, green and precise regulation of fragrance release can be achieved, providing a new technical path for the industrial production of Dendrobium nobile fragrance. In addition, this invention can also provide a reference for the aroma regulation of other flowers or aromatic crops, and has broad application prospects.

[0006] This invention provides the following technical solution: This invention provides a method for promoting the release of fragrance from Dendrobium nobile flowers using exogenous melatonin. By applying exogenous melatonin to Dendrobium nobile, the method effectively promotes the early release of fragrance substances from Dendrobium nobile flowers and increases the types and content of volatile aroma components in Dendrobium nobile flowers, thereby prolonging the duration of the fragrance.

[0007] Furthermore, the exogenous melatonin was applied to Dendrobium nobile in solution form.

[0008] Furthermore, the purple-petaled Dendrobium refers to the purple-petaled Dendrobium in its bud stage.

[0009] The method for promoting the release of fragrance from Dendrobium nobile flowers using exogenous melatonin, provided by this invention, includes the following specific steps: S1. Select exogenous melatonin as a growth regulator and prepare a melatonin solution; S2. Select Dendrobium purpureum in the bud stage as the treatment target, and apply the melatonin solution to Dendrobium purpureum in the bud stage. The application methods include spraying and / or root irrigation. S3. Then, the purple-petaled Dendrobium in the bud stage is cultivated to the initial flowering stage, the early flowering stage, or the late flowering stage of the purple-petaled Dendrobium, and the synthesis and volatilization of the floral fragrance substances of the obtained purple-petaled Dendrobium are effectively promoted.

[0010] The method provided by this invention can effectively regulate the types and contents of floral fragrance substances in cultivated and harvested Dendrobium purpureus (early flowering stage, early flowering stage, or late flowering stage) by changing the concentration and / or amount of exogenous melatonin solution. This can improve the synthesis and volatilization of floral fragrance substances in Dendrobium purpureus at different flowering stages and extend the duration of floral fragrance, thereby significantly enhancing its market competitiveness in the fragrance industry.

[0011] Furthermore, the melatonin concentration of the melatonin solution in step S1 is preferably no more than 1000 μmol / L; more preferably, the concentration of the melatonin solution is 500 μmol / L, which can effectively promote the release of fragrance substances from Dendrobium nobile flowers.

[0012] Furthermore, in step S2, the melatonin solution is applied by spraying and root irrigation; the preferred application amount is 300 mL / plant; preferably, the melatonin solution is applied to the leaves and stems of Dendrobium nobile and the soil; more preferably, the melatonin solution can be sprayed directly onto the leaves of Dendrobium nobile first, and then absorbed through the roots by root irrigation after the leaves are fully wetted.

[0013] Further, in step S2, the melatonin solution is applied when the longitudinal diameter of the flower bud of Dendrobium nobile is (2.00±0.03) cm during the bud stage, preferably when the length is 2 cm; the melatonin solution can be applied 1 to 2 times, preferably 2 times, with an interval of 7 to 10 days between the two applications.

[0014] Furthermore, in step S2, the melatonin solution is applied between 9:00 AM and 11:00 AM, preferably at 10:00 AM, when the fragrance release of Dendrobium nobile is at its highest, which is beneficial to improving the absorption efficiency of melatonin.

[0015] Furthermore, the specific cultivation conditions described in step S3 include: controlling the ambient temperature at 25℃~30℃ and the humidity at 60%~75% under natural light conditions.

[0016] In the cultivation of Dendrobium purpureus, reasonable control of the light cycle and light intensity helps the synthesis and function of melatonin in the plant. The present invention preferably sets a light cycle of 12 hours of light and 12 hours of darkness to simulate the natural light conditions.

[0017] The present invention has the following beneficial effects: 1. This invention provides a method for promoting the release of floral fragrance from Dendrobium nobile using exogenous melatonin. The exogenous melatonin is prepared as a solution and applied by spraying and / or root irrigation when the flower buds of Dendrobium nobile are approximately 2 cm long during the bud stage. By adjusting the concentration and / or amount of the melatonin solution, the physiological and metabolic processes of Dendrobium nobile at different flowering stages can be precisely controlled, thereby effectively promoting the synthesis and volatilization of floral fragrance substances at different flowering stages, and improving the concentration and persistence of the fragrance. This method utilizes the plant's natural background to synthesize a large amount of floral fragrance substances, is green and safe, simple to operate, and environmentally friendly. It not only improves the quality and stability of floral fragrance but also enhances the plant's antioxidant and ecological defense capabilities, possessing significant ecological and industrial application value.

[0018] 2. This invention effectively optimizes the composition and content structure of volatiles in Dendrobium nobile flowers and enhances the accumulation of fatty acid-derived ketone compounds, while significantly improving the intensity and characteristic aroma of the fragrance. The results show that when the melatonin solution concentration is 500 μmol / L and the application rate is 300 mL / plant, the effect on improving the types and total content of fragrance substances is the best. The total amount of fragrance substances is about 1.4 to 4.0 times higher than that of other concentration control groups, the aroma spectrum is richer, the types of aroma components increase by about 25% in the early flowering stage, and although they decrease in the full flowering stage, the proportion of key components increases several times, which is significantly better than that of other concentration control groups. The study found that the enhancement of aroma mainly comes from ketone compounds in fatty acid derivatives, among which 2-pentadecanone accumulates most significantly and is the key component that determines the aroma concentration and characteristic aroma.

[0019] 3. The method of promoting the release of fragrance from Dendrobium purpureum flowers by using exogenous melatonin provided by the present invention also significantly enhances the ornamental value of Dendrobium purpureum flowers, including making the flower shape fuller and the color more vibrant. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the different developmental stages of Dendrobium nobile flowers.

[0021] Figure 2 It is the content of the main fragrance substances in the early flowering stage (Ck) of Dendrobium purpureum.

[0022] Figure 3 It is the content of the main fragrance substances in the early flowering stage (Sa) of Dendrobium nobile flowers.

[0023] Figure 4 It is the content of the main fragrance substances in the late blooming stage (Sp) of Dendrobium purpureum flowers. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available; and all methods used in the following examples are conventional methods.

[0026] This invention provides a method for promoting the release of fragrance from Dendrobium purpureum flowers using exogenous melatonin. The exogenous melatonin is prepared into a melatonin solution and applied to Dendrobium purpureum flowers, thereby effectively promoting the early release of fragrance substances from Dendrobium purpureum flowers and increasing the types and contents of volatile aroma components in Dendrobium purpureum flowers, thus prolonging the duration of the fragrance. Melatonin, with the molecular formula C, is mentioned above. 13 H16 N2O2, with a molecular weight of 232.28 and a purity of ≥95%; The preparation steps of the melatonin solution are as follows: Weigh 1161.4 mg of melatonin and place it in a clean test tube or beaker. Add deionized water in several portions while stirring and shaking until the volume is brought to 500 mL. Heat the solution in a 50°C water bath for about 4 hours to aid dissolution. Once the melatonin is completely dissolved, a melatonin stock solution with a concentration of 10 mmol / L is obtained. After preparation, transfer the stock solution to a light-proof, sealed container and store at 4°C to prevent photodegradation. When using, dilute the stock solution according to the experimental design to prepare melatonin solutions of different concentrations.

[0027] The method for promoting the release of fragrance from Dendrobium nobile flowers using exogenous melatonin in this invention includes the following specific steps: The prepared melatonin solution is applied to the leaves, stems, and soil of Dendrobium purpureum during the bud stage by spraying and / or fertigation; then the Dendrobium purpureum during the bud stage is cultivated until the initial flowering stage, the early flowering stage, or the late flowering stage of Dendrobium purpureum.

[0028] In some preferred embodiments, the melatonin solution is applied when the longitudinal diameter of the Dendrobium nobile flower bud is (2.00±0.03) cm, preferably 2 cm. The application is performed 1 to 2 times, preferably 2 times. When the application is performed 2 times, the interval between the two applications is 7 to 10 days, more preferably 7 days. The first application is also performed when the longitudinal diameter of the flower bud is (2.00±0.03) cm, which is a key time point in flower development. The second application is a reinforcement of the first application, which is generally performed during the peak flowering period of Dendrobium nobile, thus enhancing the effect of the first application.

[0029] In some preferred embodiments, the melatonin solution is applied between 9:00 AM and 11:00 AM. When the application concentration does not exceed 1000 μmol / L, the melatonin solution effectively promotes the release of fragrance substances from *Dendrobium nobile* flowers. Preferably, an application concentration of 500 μmol / L achieves the best effect in promoting the release of fragrance substances, and a concentration within the range of (500±100) μmol / L also achieves a similar effect. The application rate is 200-500 mL / plant, preferably 300 mL / plant. This application rate ensures that the soil layer at a depth of 40 cm is thoroughly moistened.

[0030] In some preferred embodiments, after applying the prepared melatonin solution to the leaves, stems, and soil of Dendrobium nobile, it is also preferred to manage the Dendrobium nobile plants, which can be done using conventional management methods in the art.

[0031] In some preferred embodiments, the specific cultivation conditions include: controlling the ambient temperature at 25°C to 30°C and the humidity at 60% to 75% under natural light conditions; more preferably, the natural light conditions are set to a light cycle of 12 hours of light and 12 hours of darkness per day.

[0032] In the following examples, floral fragrance substances were collected from *Dendrobium purpureus* at the following stages: early flowering stage (Ck, sepals and petals half-open, not fully extended, sepal diameter of the lower two petals approximately (2.02±0.1) cm), early full bloom stage (Sa, bright flower color, sepal diameter of the lower two petals approximately (3.97±0.5) cm), and late full bloom stage (Sq, flower fully open, sepals, petals, and lip fade to pale pink, sepal diameter of the lower two petals approximately (5.57±0.61) cm). These samples were collected daily from 9:00 AM to 11:00 AM for the determination of the types and contents of floral fragrance substances. The collected floral fragrance substance samples were analyzed for volatile organic compounds (VOCs) using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS).

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0034] Example 1 In this embodiment, *Dendrobium purpureum* was selected as the treatment target, and a 1000 μmol / L melatonin solution was used as a growth regulator. When the longitudinal diameter of the flower buds of *Dendrobium purpureum* was 2 cm, the melatonin solution was sprayed onto the flowers, with an average application of 300 mL per plant. The ambient temperature was then controlled within the range of 25℃ to 30℃, and the humidity was controlled within the range of 60% to 75%. The plant was cultivated under natural light conditions (12 hours of light and 12 hours of darkness per day). Seven days after the first application of the melatonin solution, the same steps and conditions were followed for another application of the melatonin solution, and cultivation continued.

[0035] Example 2 In this embodiment, Dendrobium nobile in the bud stage was selected as the treatment object. Following the same steps as in Example 1, Dendrobium nobile in the bud stage with a bud diameter of 2 cm was treated and cultivated. The difference from Example 1 is that in this embodiment, a 500 μmol / L melatonin solution was used as a growth regulator.

[0036] Example 3 In this embodiment, Dendrobium nobile in the bud stage was selected as the treatment object. Following the same steps as in Example 1, Dendrobium nobile in the bud stage with a bud diameter of 2 cm was treated and cultivated. The difference from Example 1 is that pure water (i.e., melatonin concentration of 0 μmol / L) was used as the functional control group for the growth regulator in this embodiment.

[0037] Test results Figure 1 This diagram illustrates the different developmental stages of *Dendrobium purpureum* (bud stage, initial flowering stage, pre-full bloom stage, and post-full bloom stage). In this invention, *Dendrobium purpureum* from the bud stage of the treatment groups in Examples 1-3 were sequentially cultivated to the initial flowering stage, pre-full bloom stage, and post-full bloom stage. During this period, samples of floral fragrance substances from *Dendrobium purpureum* at different flowering stages were collected for component and content analysis. The results are as follows: (1) Initial flowering period Flowers of *Dendrobium purpureus* at the initial flowering stage were collected for the analysis of floral fragrance compounds in each embodiment, with three replicates for each group. As shown in Table 1, compared with 0 μmol / L, the 500 μmol / L group showed a 25% increase in the types of floral fragrance components and a 1.41-fold increase in the total content; the 1000 μmol / L group showed a 53.125% increase in the types of floral fragrance components and a 1.31-fold increase in the total content. The 500 μmol / L treatment group increased the overall volatile matter content (approximately 1.4 times that of 0 μmol / L), while the 1000 μmol / L treatment, although still higher than 0 μmol / L, was lower than 500 μmol / L.

[0038] Ketone compounds in fatty acid derivatives (especially C11-C17 2-alkylketones and cyclic ketones) are major aroma-enhancing components, with typical examples including 2-tetanetone, 2-pentadecanone, 2-heptadecanone, 2-undecanetone, and cyclopentadecanone. For example... Figure 2 As shown, the 500 μmol / L treatment significantly increased the average content of the above ketones, with 2-pentadecanone showing the largest accumulation (1.49 times higher than 0 μmol / L), which made a decisive contribution to the improvement of the overall aroma intensity.

[0039] Table 1. Comparison of volatile compounds (Ck) in Dendrobium purpureus flowers at the initial flowering stage.

[0040] (2) Pre-peak flowering period Flowers of *Dendrobium purpureus* in the early flowering stage of each embodiment were collected for fragrance substance analysis, with each group sampled three times. As shown in Table 2, compared with 0 μmol / L, the 500 μmol / L group showed a 31.48% reduction in the types of fragrance components and a 3.88-fold increase in the total content; the 1000 μmol / L group showed a 7.41% reduction in the types of fragrance components and a 2.39-fold increase in the total content. The 500 μmol / L treatment group significantly increased the overall volatile matter content (approximately four times that of 0 μmol / L), while the 1000 μmol / L treatment, although still higher than 0 μmol / L, was lower than 500 μmol / L.

[0041] Ketone compounds in fatty acid derivatives (especially C11-C17 2-alkylketones and cyclic ketones) are major aroma-enhancing components, with typical examples including 2-tetanetone, 2-pentadecanone, 2-heptadecanone, 2-undecanetone, and cyclopentadecanone. For example... Figure 3 As shown, the 500 μmol / L treatment significantly increased the average content of the above ketones, with 2-pentadecanone showing the largest accumulation (5.38 times higher than 0 μmol / L), which made a decisive contribution to the improvement of the overall aroma intensity.

[0042] Table 2 Comparison of Sa volatile compounds in Dendrobium purpureus flowers during the early flowering stage

[0043] (3) Late bloom Flowers of *Dendrobium purpureus* in the late blooming stage of each embodiment were collected for fragrance substance analysis, with each group sampled three times. As shown in Table 3, compared with 0 μmol / L, the 500 μmol / L group showed a 38.46% reduction in the types of fragrance components and a 2.59-fold increase in the total content; the 1000 μmol / L group showed a 17.31% reduction in the types of fragrance components and a 2.03-fold increase in the total content. The 500 μmol / L treatment group significantly increased the overall volatile matter content, while the 1000 μmol / L treatment, although still higher than 0 μmol / L, was lower than 500 μmol / L.

[0044] Ketone compounds in fatty acid derivatives (especially C11-C17 2-alkylketones and cyclic ketones) are major aroma-enhancing components, with typical examples including 2-tetanetone, 2-pentadecanone, 2-heptadecanone, 2-undecanetone, and cyclopentadecanone. For example... Figure 4 As shown, the 500 μmol / L treatment significantly increased the average content of the above ketones, with 2-pentadecanone showing the largest accumulation (1.93 times higher than 0 μmol / L), which made a decisive contribution to the improvement of the overall aroma intensity.

[0045] Table 3. Comparison of Sp volatile compounds in Dendrobium purpureus flowers during the later stages of full bloom.

[0046] As can be seen from the above embodiments, the method for promoting the release of fragrance from Dendrobium purpureum using exogenous melatonin provided by the present invention has significant effects, effectively improving the ornamental quality and market competitiveness of Dendrobium purpureum. At the same time, it also has certain reference value for the quality improvement of other fragrant orchids and the development of industrial fragrance enhancement solutions, and has high practicality and economic value.

[0047] Furthermore, the above embodiments also show that by adjusting the concentration and / or application amount of exogenous melatonin solution, the composition and content of fragrance substances in Dendrobium nobile flowers at different flowering stages can be precisely controlled. Among them, with an application amount of 300 mL / plant, the 500 μmol / L concentration of exogenous melatonin solution has the best effect, which effectively optimizes the composition and content structure of volatiles in Dendrobium nobile flowers. In particular, it significantly improves the fragrance intensity and characteristic aroma by enhancing the accumulation of fatty acid-derived ketone compounds.

[0048] Finally, the above embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for promoting the release of fragrance from Dendrobium nobile flowers using exogenous melatonin, characterized in that, Includes the following steps: S1. Select exogenous melatonin as a growth regulator and prepare a melatonin solution; S2. Select Dendrobium nobile in the bud stage as the treatment object, and apply the melatonin solution to the Dendrobium nobile in the bud stage. The application methods include spraying and / or root irrigation. S3. Then, the purple-petaled Dendrobium in the bud stage is cultivated to the initial flowering stage, the early flowering stage, or the late flowering stage of the purple-petaled Dendrobium, and the release of the floral fragrance of the purple-petaled Dendrobium is effectively promoted.

2. The method for promoting the release of fragrance from *Dendrobium nobile* flowers using exogenous melatonin according to claim 1, characterized in that, The melatonin concentration of the melatonin solution in step S1 shall not exceed 1000 μmol / L.

3. The method for promoting the release of fragrance from *Dendrobium nobile* flowers using exogenous melatonin according to claim 2, characterized in that... In step S2, the melatonin solution is applied to the purple-petaled Dendrobium during the bud stage, and the amount of melatonin solution applied is 300 mL / plant.

4. The method for promoting the release of fragrance from *Dendrobium nobile* flowers using exogenous melatonin according to claim 1, characterized in that, In step S2, the melatonin solution is applied to the Dendrobium purpureum during the bud stage. The application time of the melatonin solution is when the longitudinal diameter of the Dendrobium purpureum bud is (2.00±0.03) cm.

5. The method for promoting the release of fragrance from *Dendrobium nobile* flowers using exogenous melatonin according to claim 4, characterized in that... In step S2, the melatonin solution is applied to the purple-petaled Dendrobium during the bud stage. The melatonin solution is applied 1 to 2 times, and when it is applied 2 times, the interval between the two applications is 7 to 10 days.

6. The method for promoting the release of fragrance from *Dendrobium nobile* flowers using exogenous melatonin according to claim 5, characterized in that, In step S2, the melatonin solution is applied to the Dendrobium purpureum during the bud stage. The application time of the melatonin solution is 10:00 am, when the fragrance release of Dendrobium purpureum is the highest, which is conducive to improving the absorption efficiency of melatonin.

7. The method for promoting the release of fragrance from *Dendrobium nobile* flowers using exogenous melatonin according to claim 1, characterized in that, The specific cultivation conditions described in step S3 include: controlling the ambient temperature at 25℃~30℃ and the humidity at 60%~75% under natural light conditions.

8. The method for promoting the release of fragrance from *Dendrobium nobile* flowers using exogenous melatonin according to claim 7, characterized in that, The specific natural lighting conditions are: 12 hours of light and 12 hours of darkness per day.

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