A method to promote the release of fragrance during the flowering period of Phalaenopsis purpurea.

Abstract

This invention provides a method for promoting the release of fragrance during the flowering period of Phalaenopsis 'Purple Diamond' orchids. It compares the number of flowering days during peak bloom and the effects of different light intensities and temperatures on fragrance release, exploring the light and temperature adaptability of potted Phalaenopsis orchids during their peak bloom. This provides a foundational basis for breeding aromatic Phalaenopsis orchid varieties and for studying the mechanism of fragrance in important economic ornamental flowers. Furthermore, this invention further investigates and explores the optimal culture medium formulation for each stage of the rapid tissue culture propagation of Phalaenopsis 'Purple Diamond' orchids, enabling its industrial-scale development and application.

Description

Technical Field

[0001] This application relates to the field of agricultural cultivation technology, and in particular to a method for promoting the release of fragrance during the flowering period of Phalaenopsis 'Purple Diamond' flowers. Background Technology

[0002] The Purple Diamond Phalaenopsis orchid exhibits vigorous growth, with fragrant small flowers, beautiful flower shape, purple stripes, a long flowering period, and stronger resistance to adverse conditions. However, it is difficult to propagate the Purple Diamond Phalaenopsis orchid through conventional asexual reproduction. Current technologies lack a well-established and perfected tissue culture rapid propagation technique for the Purple Diamond Phalaenopsis orchid. There is an urgent need for in-depth research and exploration to screen the optimal culture medium formula for each stage of the rapid tissue culture propagation of the Purple Diamond Phalaenopsis orchid, enabling its industrial-scale development and application.

[0003] Meanwhile, fragrance is one of the most important traits of Phalaenopsis orchids. However, the impact of different cultivation conditions on fragrance release during the flowering period remains unclear. Therefore, this study selected the potted variety "Purple Diamond" as the model plant. First, the composition of the fragrance components during the peak flowering period was analyzed; then, the effects of the number of flowering days during the peak flowering period, as well as different light intensities and temperatures, on fragrance release were compared. Exploring the light and temperature adaptability of the fragrance during the peak flowering period of potted Phalaenopsis orchids can provide a foundation for the breeding of aromatic Phalaenopsis orchid varieties and the study of the mechanism of fragrance in important economic ornamental flowers. Summary of the Invention

[0004] To address the aforementioned technical problems, this application provides a method for promoting the release of fragrance during the flowering period of the Phalaenopsis 'Purple Diamond' orchid, comprising the following steps: applying fragrance to the Phalaenopsis 'Purple Diamond' orchid... Phalaenopsis'Purple Diamond' Starting from the first flower, the light and temperature treatment is carried out from day 5 to day 10, maintaining a constant light intensity of 2000 Lux for 24 hours and a constant temperature of 30-38℃ for 3-6 days.

[0005] Furthermore, prior to the light and temperature treatment during the flowering period of the Purple Diamond Phalaenopsis, the following steps are also included:

[0006] (1) Material preparation: Use healthy, disease-free Phalaenopsis Purple Diamond orchids with dormant buds that have passed their peak flowering period. (Phalaenopsis 'Purple Diamond' When collecting flower stalks, cut them from the bottom of the plant as material.

[0007] (2) Disinfection method for explants: Cut mature flower stalks of Phalaenopsis purpurea into segments with one bud, leaving 1.5-2cm above and below the bud. Soak in laundry detergent or dish soap water for 10 minutes, shaking continuously during the process. After cleaning, rinse with tap water for 30 minutes. Use sterile filter paper to absorb the water droplets on the flower stalks. Wipe the surface of the flower stalks with 75% alcohol cotton balls. Then soak in 0.1% mercuric chloride or 10% NaClO for 15-20 minutes, shaking continuously during the process. Wash with sterile water 2-3 times.

[0008] (3) Induction of differentiation and proliferation

[0009] (3.1) Induction of lateral buds on flower stalks

[0010] Remove parts of both ends of the lateral bud segment on the flower stalk, and inoculate it in MS medium containing 1.5 mg / L 6-BA, 1.0 g / L peptone, 30 mg / L banana puree, 30 g / L sucrose, 6.0 g / L agar powder, and pH 5.8 in the normal growth direction to induce vegetative shoots. Culture environment: 25±2℃, light intensity 2500-3000 Lux, light duration 12h.

[0011] (3.2) Differentiation and proliferation of clustered buds

[0012] The induced 1.5cm long vegetative shoots were cut from the base of the flower stalk segment for cluster shoot differentiation. The differentiation medium was MS + 6-BA 4.5mg / L + NAA 0.5mg / L + coconut juice 10% + banana puree 30mg / L, sucrose 30g / L, agar powder 6.0g / L, pH 5.6-5.8. The culture environment was 25±2℃, light intensity 2500-3000Lux, and light duration 12h.

[0013] (4) Strong seedlings take root

[0014] Cut buds longer than 1.5 cm from the clustered buds of Phalaenopsis orchids and carry out seedling rooting culture. The culture medium is: 1 / 2 MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + activated carbon 2.0 g / L, sucrose 30 g / L, agar powder 6.0 g / L, pH 5.6-5.8. The culture environment is: 25±2℃, light intensity 2500-3000 Lux, light duration 12h.

[0015] (5) Planting of tissue culture seedlings after removal from bottle

[0016] (5.1) Requirements for removing tissue culture seedlings from the bottle

[0017] Phalaenopsis tissue culture seedlings are pollution-free. When the seedlings have developed 3-4 leaves and 3-5 roots, with leaves 3-4cm long and roots 3-5cm long, they can be removed from the bottle and planted.

[0018] (5.2) Seedling hardening

[0019] When the tissue culture seedlings meet the requirements for transplanting, they are moved into the greenhouse prepared for planting for acclimatization. The acclimatization temperature is 25-30℃, the light intensity is 10000 Lux, and the environment is kept clean and well-ventilated for 3-4 weeks.

[0020] (5.3) Material preparation

[0021] The cultivation substrate is high-quality sphagnum moss. Before transplanting, the sphagnum moss is soaked in a 2000-fold potassium permanganate solution for 12 hours. The sphagnum moss is then scooped up and squeezed until no water comes out. Seedlings are planted in transparent seed trays.

[0022] (5.4) Transplanting

[0023] When transplanting, open the sealing film, take out the seedling, wash the root culture medium to prevent root rot after planting, soak the seedling in 0.2% potassium permanganate solution for 3-5 minutes to disinfect the seedling, take it out, then loosen the sphagnum moss, take a small amount of sphagnum moss under the roots, and at the same time wrap the roots and single-axis stem of the seedling with sphagnum moss, leaving the heart leaves exposed, plant the seedling upright in the center of the seedling tray, and squeeze the seedling tray with your hand to make it firm and elastic. The temperature should be controlled in the range of 22-28℃ when it is first taken out of the bottle, which is conducive to the recovery of the Phalaenopsis orchid seedling and the rejuvenation of the root system. After 1 month, gradually adjust it to the temperature range of 20-30℃ for the vegetative growth of Phalaenopsis orchid, the relative humidity should be above 80%, the light intensity should be below 10000 Lux, and the environment should be clean and well-ventilated.

[0024] (6) Seedling cultivation

[0025] When the distance between the two leaves of the plant reaches more than 12cm, the seedlings can be repotted. After repotting, spray the seedlings with a 2000-fold dilution of carbendazim fungicide once every two weeks, for a total of three applications. Newly transplanted seedlings should be treated on the first... Do not water during the first week to encourage early root development and prevent soft rot. Provide moisture to the seedlings via misting, spraying twice daily, once in the morning and once in the afternoon. Watering can begin in the second week. Water thoroughly when the surface of the seedling is dry and there are no water droplets or mist at the bottom of the transparent seed tray. Begin applying a balanced fertilizer (nitrogen, phosphorus, and potassium in a 20:20:20 ratio) on the 21st day after repotting. Fertilize and water alternately, using a 3000-5000 times dilution. Start with a lower concentration and gradually increase it, but never too high. Adhere to the principle of frequent, light fertilization, aiming for an EC value between 0.6 and 0.8. Initially, use a 5000 times dilution, gradually increasing to 3000 times, but never exceeding the maximum EC value of 0.8.

[0026] (7) Fertilizing during flowering period

[0027] Before Phalaenopsis orchids bloom, reduce the amount of nitrogen fertilizer used. Specifically, during the flowering period, apply water-soluble fertilizer with a nitrogen, phosphorus, and potassium ratio of 9:45:15. When the flower stalk reaches 25cm or more, apply water-soluble fertilizer with a nitrogen, phosphorus, and potassium ratio of 10:30:20. Apply once a week or every two weeks at a dilution of 3000-5000 times. Do not fertilize during the flowering period.

[0028] Furthermore, this application also protects Phalaenopsis purpureus orchids whose fragrance release is enhanced by the two methods described above.

[0029] Furthermore, the formulation of the lateral bud induction medium for flower stalks was: MS + 6-BA 1.5 mg / L + peptone 1.0 g / L + banana puree 30 mg / L, sucrose 30 g / L, agar powder 6.0 g / L, and light intensity 3000 Lux.

[0030] Furthermore, the formula for the bud differentiation and proliferation medium was: MS + 6-BA 4.5 mg / L + NAA 0.5 mg / L + coconut juice 10% + banana puree 30 mg / L, sucrose 30 g / L, agar powder 6.0 g / L, and light intensity 3000 Lux.

[0031] Furthermore, the formula for the seedling rooting induction medium is: 1 / 2 MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + activated carbon 2.0 g / L, sucrose 30 g / L, agar powder 6.0 g / L, and light intensity 2500 Lux.

[0032] Furthermore, the hardening temperature was 30℃ and the light intensity was 10000 Lux.

[0033] Furthermore, for transplanting, the temperature should be controlled within the range of 28℃ immediately after transplanting to facilitate the recovery of the Phalaenopsis seedlings and the rejuvenation of the root system. After one month, the temperature should be gradually adjusted to the range of 25℃ for the vegetative growth of Phalaenopsis, with a relative humidity of over 80%, a light intensity of 8000 Lux, and a clean and well-ventilated environment.

[0034] Furthermore, the initial flowering period was calculated from the first bloom of the Purple Diamond Phalaenopsis orchid. On the 5th day, light and temperature treatment was carried out, maintaining a constant light intensity of 2000 Lux for 24 hours and a constant temperature of 34℃ for 3 days.

[0035] Furthermore, this application also protects the application of the above method in any of the following:

[0036] (1) Application in improving the ratio of induced vegetative buds on the orchid stem of Phalaenopsis purpurea;

[0037] (2) Application in improving the ratio of induced cluster shoots in Phalaenopsis purpurea;

[0038] (3) Application in improving the rooting rate of vigorous seedlings of Phalaenopsis purpurea;

[0039] (4) Application in promoting the release of fragrance during the flowering period of Phalaenopsis purpurea.

[0040] Beneficial effects:

[0041] 1. Phalaenopsis 'Purple Diamond' is difficult to propagate asexually using conventional methods. This application establishes and improves the tissue culture rapid propagation technology for Phalaenopsis 'Purple Diamond', which is a key step in the propagation of Phalaenopsis. Through in-depth research and discussion, the optimal culture medium formula for each step of the rapid tissue culture propagation of Phalaenopsis 'Purple Diamond' has been selected.

[0042] 2. This study found that light is the key factor affecting the release rate of volatile organic compounds (VOCs) from the Phalaenopsis orchid, specifically the Purple Diamond variety, and its influence exceeds that of temperature. Under light-free conditions, the release rate of VOCs increased slightly with increasing temperature, but the effect was limited. However, under light conditions, the release rate of VOCs remained high at higher temperatures, indicating a more significant effect of light. Among all tested treatments, the L2T2 treatment (34℃, 2000 Lux light) showed the highest total VOC release rate after 3 days, indicating that this phototemperature condition is favorable for the release of the Phalaenopsis orchid's Purple Diamond fragrance.

[0043] 3. Dimethyl phthalate (DMT) is one of the main components of the fragrance in Phalaenopsis orchids, and its release rate under light conditions is significantly higher than under no light conditions. The highest release rate, exceeding 40 ng / g / h, was observed after 3 days of treatment at L2T2 (34℃, 2000 Lux light). The effect of light on the fragrance release of Phalaenopsis orchids may be related to photosynthesis; light provides energy and promotes the synthesis and release of volatile organic compounds. This finding is significant for regulating the fragrance release of Phalaenopsis orchids, as fragrance release can be enhanced by adjusting light conditions.

[0044] 4. The effect of temperature on floral fragrance release may be related to enzyme activity; suitable temperatures can increase enzyme activity, thereby promoting the synthesis of floral fragrance compounds. However, excessively high or low temperatures may inhibit enzyme activity, leading to a reduction in floral fragrance compound synthesis. In this study, the effects of light and temperature on the release rates of total fragrance, 2,4-di-tert-butylphenol, and dimethyl phthalate were particularly significant, possibly because these compounds dominate the fragrance of Phalaenopsis Purple Diamond and are more sensitive to changes in environmental conditions.

[0045] 5. The findings of this study have guiding significance for the cultivation and management of Phalaenopsis orchids. By adjusting light and temperature conditions, the fragrance quality of Phalaenopsis orchids can be optimized, thereby improving their ornamental value and market competitiveness.

[0046] 6. This application also investigated the effects of different tissue culture conditions on the growth and fragrance of *Phalaenopsis purpurea*. We found that the optimal combination formula for achieving promoted fragrance release in *Phalaenopsis purpurea* was Example 1. Comparing Example 2, reducing light intensity during the induction of lateral buds on flower stalks, the induction of clustered buds, and the hardening-off period, as well as lowering the temperature after removal from the container, were all detrimental to the fragrance release of *Phalaenopsis purpurea* during its peak bloom. Simultaneously, to promote root induction in *Phalaenopsis purpurea*, light intensity needed to be reduced during the strong seedling rooting period. By modifying the culture medium for *Phalaenopsis purpurea* during the induction of lateral buds on flower stalks, the induction of clustered buds, and the strong seedling rooting period in Examples 1-2, 3-4, and 5-6, we found that the induction rates obtained were significantly lower than those of the formula in Example 1. Comparative fragrance promotion experiments in Examples 7-8 revealed that changing the ratio of nitrogen, phosphorus, and potassium in the water-soluble fertilizer during the catalytic period, as well as the start time of light and temperature treatments, significantly affected the total fragrance release. Attached Figure Description

[0047] Figure 1. Image of the tested Phalaenopsis orchid, Purple Diamond;

[0048] Figure 2. Changes in the total fragrance release rate of Phalaenopsis orchids under different light and temperature conditions;

[0049] Figure 3. Changes in the release rate of dimethyl phthalate;

[0050] Figure 4. Changes in the release rate of 2,4-di-tert-butylphenol. Detailed Implementation

[0051] To make the objectives, solutions, and advantages of the technical solutions of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Unless otherwise stated, the terms used herein have their ordinary meanings in the art. The same reference numerals in the drawings represent the same parts.

[0052] Example 1

[0053] A method for promoting the fragrance release of Phalaenopsis purpurea during its flowering period includes the following steps:

[0054] (1) Material preparation: Use healthy, disease-free Phalaenopsis Purple Diamond orchids with dormant buds that have passed their peak flowering period. (Phalaenopsis 'Purple Diamond' When collecting flower stalks, the plant is used as the material, and the stalks are cut from the bottom.

[0055] (2) Method for disinfecting explants: Cut mature flower stalks of Phalaenopsis purpurea into segments with one bud, leaving 1.5 cm above and below the bud. Soak in water with laundry detergent or dish soap for 10 minutes, shaking continuously during the process. After cleaning, rinse with tap water for 30 minutes. Use sterile filter paper to absorb the water droplets on the flower stalks. Wipe the surface of the flower stalks with cotton balls soaked in 75% alcohol. Then soak in 0.1% mercuric chloride or 10% NaClO for 20 minutes, shaking continuously during the process. Wash 3 times with sterile water.

[0056] (3) Induction of differentiation and proliferation

[0057] (3.1) Induction of lateral buds on flower stalks

[0058] Remove parts of both ends of the lateral bud segment on the flower stalk, and inoculate it in MS medium containing 1.5 mg / L 6-BA, 1.0 g / L peptone, 30 mg / L banana puree, 30 g / L sucrose, 6.0 g / L agar powder, and pH 5.8 in the normal growth direction to induce vegetative shoots. Culture environment: 25±2℃, light intensity 3000 Lux, light duration 12h.

[0059] (3.2) Differentiation and proliferation of clustered buds

[0060] The induced 1.5cm long vegetative shoots were cut from the base of the flower stalk segment for cluster shoot differentiation. The differentiation medium was MS + 6-BA 4.5mg / L + NAA 0.5mg / L + coconut juice 10% + banana puree 30mg / L, sucrose 30g / L, agar powder 6.0g / L, pH 5.6. The culture environment was 25±2℃, light intensity 3000Lux, and light exposure for 12h.

[0061] (4) Strong seedlings take root

[0062] Cut buds longer than 1.5 cm from the clustered buds of Phalaenopsis orchids and carry out seedling rooting culture. The culture medium is: 1 / 2 MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + activated carbon 2.0 g / L, sucrose 30 g / L, agar powder 6.0 g / L, pH 5.6. The culture environment is: 25±2℃, light intensity 2500 Lux, light duration 12h.

[0063] (5) Planting of tissue culture seedlings after removal from bottle

[0064] (5.1) Requirements for removing tissue culture seedlings from the bottle

[0065] Phalaenopsis tissue culture seedlings are pollution-free. When the seedlings develop to 3 leaves and 4 roots, with leaves 3cm long and roots 5cm long, they are removed from the bottle and planted.

[0066] (5.2) Seedling hardening

[0067] When the tissue culture seedlings reach the requirements for transplanting, they are moved into the greenhouse prepared for planting for acclimatization. The acclimatization temperature is 30℃, the light intensity is 10000 Lux, and the environment is kept clean and well-ventilated. Acclimatization takes 4 weeks. During the acclimatization period, do not remove the sealing film of the bottle mouth to prevent contamination inside the bottle and avoid root rot due to miscellaneous bacteria.

[0068] (5.3) Material preparation

[0069] The cultivation substrate is high-quality sphagnum moss. Before transplanting, the sphagnum moss is soaked in a 2000-fold potassium permanganate solution for 12 hours. The sphagnum moss is then scooped up and squeezed until no water comes out. Seedlings are planted in 100-square transparent seedling trays.

[0070] (5.4) Transplanting

[0071] When transplanting, open the sealing film, take out the seedling, wash the root culture medium to prevent root rot after planting, soak the seedling in 0.2% potassium permanganate solution for 5 minutes to disinfect the seedling, take it out, then loosen the sphagnum moss, take a small amount of sphagnum moss under the roots, and at the same time wrap the roots and single-axis stem of the seedling with sphagnum moss, leaving the heart leaves exposed, plant the seedling upright in the center of the seedling tray, and squeeze the seedling tray with your hand to make it firm and elastic. The temperature should be controlled within 28℃ when it is first taken out of the bottle, which is conducive to the recovery of the Phalaenopsis orchid seedling and the rejuvenation of the root system. After 1 month, gradually adjust it to the temperature range of 25℃ for the vegetative growth of Phalaenopsis orchid, the relative humidity should be above 80%, the light intensity should be 8000 Lux, and the environment should be clean and well-ventilated.

[0072] (6) Seedling cultivation

[0073] When the distance between the two leaves of the plant reaches more than 12cm, the seedlings can be repotted. After repotting, spray the seedlings with a 2000-fold dilution of carbendazim fungicide once every two weeks for three consecutive times. Do not water the newly transplanted seedlings during the first week to allow them to root early and avoid soft rot. Provide water to the seedlings by spraying twice a day, once in the morning and once in the afternoon. Watering can begin after the second week. The principle of watering is to water thoroughly when the surface of the seedlings is dry and there are no water droplets or mists at the bottom of the transparent seed tray. Begin applying a balanced fertilizer (nitrogen, phosphorus, and potassium ratio of 20:20:20) on the 21st day after repotting, with an interval of 1 fertilization followed by 1 watering, using a 5000-fold dilution.

[0074] (7) Fertilizing during flowering period

[0075] Before Phalaenopsis orchids bloom, reduce the amount of nitrogen fertilizer used. Specifically, during the flowering period, apply water-soluble fertilizer with a nitrogen, phosphorus, and potassium ratio of 9:45:15. When the flower stalk is more than 25cm long, apply water-soluble fertilizer with a nitrogen, phosphorus, and potassium ratio of 10:30:20. Apply once a week or every half month with a 3000-fold dilution. Do not fertilize during the flowering period.

[0076] (8) Regulation of light intensity and temperature during flowering period

[0077] The initial flowering period of Phalaenopsis orchids is calculated from the first flower. On the 5th day, light and temperature treatment is carried out, maintaining a constant light intensity of 2000 Lux for 24 hours and a constant temperature of 34℃ for 3 days to obtain Phalaenopsis orchids with enhanced fragrance release.

[0078] (9) Leaving the nursery

[0079] Release the nursery stock as needed by the market.

[0080] Example 2

[0081] A method for promoting the fragrance release of Phalaenopsis purpurea during its flowering period includes the following steps:

[0082] (1) Material preparation: Use healthy, disease-free Phalaenopsis 'Purple Diamond' orchids that have passed their peak flowering period and have dormant buds. Phalaenopsis 'Purple Diamond' When collecting flower stalks, cut them from the bottom of the plant as material.

[0083] (2) Method for disinfecting explants: Cut mature flower stalks of Phalaenopsis purpurea into segments with one bud, leaving 2cm above and below the bud. Soak in water with laundry detergent or dish soap for 10 minutes, shaking continuously during the process. After cleaning, rinse with tap water for 30 minutes. Use sterile filter paper to absorb the water droplets on the flower stalks. Wipe the surface of the flower stalks with 75% alcohol cotton balls. Then soak in 0.1% mercuric chloride or 10% NaClO for 20 minutes, shaking continuously during the process. Wash 3 times with sterile water.

[0084] (3) Induction of differentiation and proliferation

[0085] (3.1) Induction of lateral buds on flower stalks

[0086] Remove parts of both ends of the lateral bud segment of the flower stalk, and inoculate it in MS medium containing 1.5 mg / L 6-BA, 1.0 g / L peptone, 30 mg / L banana puree, 30 g / L sucrose, 6.0 g / L agar powder, and pH 5.8 in the normal growth direction to induce vegetative shoots. Culture environment: 25±2℃, light intensity 2500 Lux, light duration 12h.

[0087] (3.2) Differentiation and proliferation of clustered buds

[0088] The induced 1.5cm long vegetative shoots were cut from the base of the flower stalk segment for cluster shoot differentiation. The differentiation medium was MS + 6-BA 4.5mg / L + NAA 0.5mg / L + coconut juice 10% + banana puree 30mg / L, sucrose 30g / L, agar powder 6.0g / L, pH 5.6. The culture environment was 25±2℃, light intensity 2500Lux, and light duration 12h.

[0089] (4) Strong seedlings take root

[0090] Cut buds longer than 1.5 cm from the clustered buds of Phalaenopsis orchids and carry out seedling rooting culture. The culture medium is: 1 / 2 MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + activated carbon 2.0 g / L, sucrose 30 g / L, agar powder 6.0 g / L, pH 5.6. The culture environment is: 25±2℃, light intensity 3000 Lux, light duration 12h.

[0091] (5) Planting of tissue culture seedlings after removal from bottle

[0092] (5.1) Requirements for removing tissue culture seedlings from the bottle

[0093] Phalaenopsis tissue culture seedlings are pollution-free. When the seedlings develop to 3 leaves and 4 roots, with leaves 3cm long and roots 5cm long, they are removed from the bottle and planted.

[0094] (5.2) Seedling hardening

[0095] When the tissue culture seedlings reach the requirements for transplanting, they are moved into the greenhouse prepared for planting for acclimatization. The acclimatization temperature is 25℃, the light intensity is within 8000 Lux, and the environment is kept clean and well-ventilated. Acclimatization takes 4 weeks. During the acclimatization period, do not remove the sealing film of the bottle mouth to prevent contamination inside the bottle and avoid root rot due to miscellaneous bacteria.

[0096] (5.3) Material preparation

[0097] The cultivation substrate is high-quality sphagnum moss. Before transplanting, the sphagnum moss is soaked in a 2000-fold potassium permanganate solution for 12 hours. The sphagnum moss is then scooped up and squeezed until no water comes out. Seedlings are planted in 100-square transparent seedling trays.

[0098] (5.4) Transplanting

[0099] When transplanting, open the sealing film, take out the seedling, wash the root culture medium to prevent root rot after planting, soak the seedling in 0.2% potassium permanganate solution for 5 minutes to disinfect the seedling, take it out, then loosen the sphagnum moss, take a small amount of sphagnum moss under the roots, and at the same time wrap the roots and single-axis stem of the seedling with sphagnum moss, leaving the heart leaves exposed, plant the seedling upright in the center of the seedling tray, and squeeze the seedling tray with your hand to make it firm and elastic. The temperature should be controlled within 25℃ when it is first taken out of the bottle, which is conducive to the recovery of the Phalaenopsis orchid seedling and the rejuvenation of the root system. After 1 month, gradually adjust it to the temperature range of 25℃ for the vegetative growth of Phalaenopsis orchid, the relative humidity should be above 80%, the light intensity should be 8000 Lux, and the environment should be clean and well-ventilated.

[0100] (6) Seedling cultivation

[0101] When the distance between the two leaves of the plant reaches more than 12cm, the seedlings can be repotted. After repotting, spray the seedlings with a 2000-fold dilution of carbendazim fungicide once every two weeks for three consecutive times. Do not water the newly transplanted seedlings during the first week to allow them to root early and avoid soft rot. Provide water to the seedlings by spraying twice a day, once in the morning and once in the afternoon. Watering can begin after the second week. The principle of watering is to water thoroughly when the surface of the seedlings is dry and there are no water droplets or mists at the bottom of the transparent seed tray. Begin applying a balanced fertilizer (nitrogen, phosphorus, and potassium ratio of 20:20:20) on the 21st day after repotting, with an interval of 1 fertilization followed by 1 watering, using a 5000-fold dilution.

[0102] (7) Fertilizing during flowering period

[0103] Before Phalaenopsis orchids bloom, reduce the amount of nitrogen fertilizer used. Specifically, during the flowering period, apply water-soluble fertilizer with a nitrogen, phosphorus, and potassium ratio of 9:45:15. When the flower stalk is more than 25cm long, apply water-soluble fertilizer with a nitrogen, phosphorus, and potassium ratio of 10:30:20. Apply once a week or every half month with a 3000-fold dilution. Do not fertilize during the flowering period.

[0104] (8) Regulation of light intensity and temperature during flowering period

[0105] The initial flowering period of Phalaenopsis orchids is calculated from the first flower. On the 5th day, light and temperature treatment is carried out, maintaining a constant light intensity of 2000 Lux for 24 hours and a constant temperature of 34℃ for 3 days to obtain Phalaenopsis orchids with enhanced fragrance release.

[0106] (9) Leaving the nursery

[0107] Release the nursery stock as needed by the market.

[0108] Experiment 1: Effects of light and temperature treatment on the fragrance release during the flowering period of Phalaenopsis 'Purple Diamond' flowers

[0109] Experimental Methods: The experimental material was the Phalaenopsis cultivar Purple Diamond, located at the Xiongxi Germplasm Resource Nursery of the Zhejiang Subtropical Crops Research Institute (118˚21'~120˚30'E, 29˚11'~30˚33'N). Purple Diamond varieties with uniform growth were selected and placed in an artificial climate chamber (PERCIVALE-41H02, USA) for different light and temperature treatments. The treatment of the experimental material followed the guidelines in Example 1, ensuring good flower growth. First, the main fragrance components of the Phalaenopsis orchids were measured under natural conditions on the 5th day of flowering. Then, a light and temperature treatment experiment was conducted: the light condition was set to a constant 24-hour light intensity. The specific experimental treatment settings are as follows: (The light and temperature adjustments are shown in the example 1). The treatment started 5 days after the first flower bud opened following the completion of the Phalaenopsis orchid's stem elongation, and continued until the peak flowering period ended. Flowers at the same growth position were collected on the 30th day of peak flowering under 3-day and 6-day treatment conditions for fragrance collection and component analysis. Each treatment was repeated 3 times, with 5 pots per replicate. The Phalaenopsis orchid fragrance volatiles were collected using a dynamic headspace method. Whole flowers from potted Phalaenopsis orchids were randomly selected and placed in a glass container (30 cm high, 10 cm diameter at the mouth). The container was supplied with 0.7 min / L activated carbon to purify the air. After equilibration for 30 min, the container was connected to an adsorption column packed with Porpark Q (Waters Corporation, USA) silica gel. The column was eluted with 200 μL of dichloromethane (chromatographic grade, Shanghai Maclean Biotechnology Co., Ltd.) containing 0.001% nonyl acetate (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.) as an internal standard before injection for analysis. A blank headspace collection bottle was set up as a control group. All samples were collected over a period of 4 hours, and the fresh weight of whole Phalaenopsis orchid flowers was then weighed. The floral fragrance volatiles were analyzed and identified using high-resolution gas chromatography-mass spectrometry (Q Exactive™ GC Orbitrap™ GC-MS / MS). Helium gas was injected at a flow rate of 1 min / L onto an HP-5MS column (30 m × 0.25 mm × 0.25 μm). The floral fragrance splitless mode was set, the injection port temperature was 260 °C, and the gradient temperature program started at 40 °C, then increased to 250 °C at a rate of 5 °C / min and held for 20 min. MS conditions: ion source temperature 230 °C, ionization energy 70 eV, and mass spectrometry scan range 40–500 amu. Retention indices were calculated based on the retention times of the volatiles and compared with reference values ​​in the NIST17 database for qualitative analysis. The formula for calculating the release rate of floral fragrances is: Release rate (ng / g / h) = (peak area of ​​sample volatiles / peak area of ​​internal standard volatiles) × content of internal standard / (headspace collection time × fresh weight of each sample). Excel 2023 software was used to calculate the release rates of various floral fragrance volatiles, and GraphPad Prism 9 software was used to compare and analyze the release amounts of volatiles from different varieties.

[0110] Table 1 Experimental treatment settings

[0111] Processing Number Lighting Processing temperature L1T1 dark 38℃ L1T2 dark 34℃ L1T3 dark 30℃ L2T1 2000Lux 38℃ L2T2 2000Lux 34℃ L2T3 2000Lux 30℃

[0112] Table 2. Main components of the fragrance of Phalaenopsis orchid's purple jewel pattern.

[0113] serial number Ingredient name Retention time (min) Release rate (ng / g / h) 1 Toluene 5.244 47.45548727 2 β-Osimerene 14.908 24.32113519 3 p-xylene 8.926 16.15027651 4 5-Methyl-1-heptanol 14.576 7.423290944 5 benzene 9.789 6.56451277 6 Linaloyl acetate 16.538 6.047101582 7 3-carene 11.181 6.033041227 8 α-Brassene 27.626 1.691690244 9 Dimethyl phthalate 24.912 10.90306376 10 2,4-Di-tert-butylphenol 25.865 8.4730503

[0114] Experimental results: The main fragrance components of the Purple Diamond cultivar are dimethyl phthalate, 2,4-di-tert-butylphenol, monoterpenes (β-ocimene), and sesquiterpenes (α-brassene). The changes in the total fragrance release rate of Phalaenopsis orchids under different light and temperature conditions are shown in the following figures. Figure 2 As shown, under dark conditions (L1T1, L1T2, L1T3), VOC emission rates decreased with decreasing temperature. L1T1 had the highest emission rate, and L1T3 had the lowest. In the 3-day and 6-day treatments, VOC emission rates under illumination conditions (L2T1, L2T2, L2T3) were generally higher than those under dark conditions (L1T1, L1T2, L1T3). This indicates that illumination is a significant factor promoting VOC emissions. Under 2000 Lux illumination, the VOC release rates in the 6-day treatment were lower than those in the 3-day treatment, but still remained at a high level. Under 2000 Lux illumination, the highest VOC emission rate was observed at 34℃ (L2T2), which may indicate that this temperature is the optimal condition for VOC synthesis. The relatively low emission rates at 38℃ (L2T1) and 30℃ (L2T3) may be due to the adverse effects of excessively high or low temperatures on VOC synthesis.

[0115] from Figure 3 As can be seen, the highest dimethyl phthalate (DME) release rate, exceeding 40 ng / g / h, was observed after 3 days of treatment with L2T2 (34℃, 2000 Lux illumination). Under dark conditions (L1T1, L1T2, L1T3), the DME release rate decreased with decreasing temperature, and almost no release was observed after 6 days of treatment with L1T3 (30℃, dark). At the same temperature, the DME release rate was generally higher after 3 days under 2000 Lux illumination (L2T1, L2T2, L2T3) compared to the dark treatment (L1T1, L1T2, L1T3). Under the 2000 Lux illumination conditions (L2T1, L2T2, L2T3), a temperature of 34℃ resulted in the highest DME release rate; excessively high or low temperatures decreased the DME release rate. Overall, the release rate of dimethyl phthalate (DMT) follows the same trend as the total release rate.

[0116] from Figure 4As can be seen, the highest release rate of 2,4-di-tert-butylphenol (2,4-di-tert-butylphenol) was observed after 3 days of treatment with L2T2 (34℃, 2000 Lux illumination), reaching 37.96 ng / g / h. When the treatment time was 6 days, under dark conditions (L1T1, L1T2, L1T3), the release rate of 2,4-di-tert-butylphenol showed an increasing trend with decreasing temperature, reaching its highest point in L1T3 (30℃, dark treatment). At the same temperature, the release rate of 2,4-di-tert-butylphenol was generally higher after 3 days under 2000 Lux illumination treatment (L2T1, L2T2, L2T3) compared to the dark treatment (L1T1, L1T2, L1T3). Under 2000 Lux illumination (L2T1, L2T2, L2T3) conditions, the temperature of 34℃ resulted in the highest release rate of 2,4-di-tert-butylphenol. Excessively high or low temperatures decreased the release rate of 2,4-di-tert-butylphenol. Overall, the release rate of 2,4-di-tert-butylphenol showed a consistent trend with the total release rate.

[0117] Experiment 2: Effects of different tissue culture conditions on the growth and fragrance of Phalaenopsis purpurea.

[0118] Experimental conditions: The experimental material was the Phalaenopsis cultivar Purple Diamond, and the experimental site was the Xiongxi Germplasm Resource Nursery of the Zhejiang Subtropical Crops Research Institute (118˚21'~120˚30'E, 29˚11'~30˚33'N). Material treatments were as described in Example 1, and the treatment groups are listed in Control Examples 1-8 below. Lateral bud induction was recorded at 90 days post-inoculation, with the ratio of lateral buds inducing vegetative shoots recorded. Cluster bud induction was recorded at 60 days post-inoculation, with the rooting rate of vigorous seedlings recorded at 70 days post-inoculation. The fragrance promotion experiment was the same as in Experiment 1, with only some parameters replaced. Flowers with consistent growth positions were collected on the 30th day of full bloom for fragrance collection and component analysis. Each treatment consisted of 30 bottles, with 3 replicates.

[0119] Comparative Example 1: Same as Example 1, except that the lateral bud induction medium was replaced: MS + 6-BA 5mg / L + peptone 1.0g / L + banana puree 30mg / L, sucrose 30g, and agar powder 6.0g.

[0120] Comparative Example 2: Same as Example 1, except that the lateral bud induction medium was replaced with MS + NAA 1.5 mg / L + peptone 1.0 g / L + banana puree 30 mg / L, sucrose 30 g, and agar powder 6.0 g.

[0121] Comparative Example 3: Same as Example 1, except that the bud differentiation and proliferation medium was replaced: MS + 6-BA 1.5 mg / L + NAA 1.5 mg / L + coconut juice 10% + banana puree 30 mg / L, sucrose 30 g, agar powder 6.0 g.

[0122] Comparative Example 4: Same as Example 1, except that the bud differentiation and proliferation medium was replaced: MS + KT 4.5mg / L + NAA 0.5mg / L + coconut juice 10% + banana puree 30 mg / L, sucrose 30 g, agar powder 6.0 g.

[0123] Comparative Example 5: Same as Example 1, except that the seedling rooting medium was replaced: 1 / 2 MS + 6-BA 1.5 mg / L + NAA 0.5 mg / L + activated carbon 2.0 g / L, sucrose 30 g, and agar powder 6.0 g.

[0124] Comparative Example 6: Same as Example 1, except that the seedling rooting medium was replaced: 1 / 2MS + 6-BA 0.5 mg / L + KT 3 mg / L + activated carbon 2.0 g / L, sucrose 30 g, and agar powder 6.0 g.

[0125] Comparative Example 7: Same as Example 1, except that the water-soluble fertilizer applied during the flowering period was replaced with a nitrogen, phosphorus and potassium ratio of 10:30:20, and the water-soluble fertilizer applied when the flower stalk reached 25cm or more was replaced with a nitrogen, phosphorus and potassium ratio of 9:45:15.

[0126] Comparative Example 8: Same as Example 1, except that the light and temperature treatment was replaced on the 10th day of flowering.

[0127] Table 3 Comparison of growth and fragrance characteristics of Phalaenopsis Purple Diamond orchid

[0128] serial number Flower stalk induced vegetative bud ratio % % of clustered buds Seedling rooting rate % VOCng / g / h Example 1 90 58 88 484.54 Example 2 86 43 71 246.34 Comparative Example 1 73 - - - Comparative Example 2 56 - - - Comparative Example 3 - 47 - - Comparative Example 4 - 23 - - Comparative Example 5 - - 67 - Comparative Example 6 - - 32 - Comparative Example 7 - - - 320.75 Comparative Example 8 - - - 316.31

[0129] Experimental Results: The results of the experiment on the effects of different tissue culture conditions on the growth and fragrance of Phalaenopsis 'Purple Diamond' showed that the optimal combination formula for obtaining Phalaenopsis 'Purple Diamond' with promoted fragrance release was Example 1. Comparing Example 2, reducing light intensity and lowering the temperature after removal from the culture vessel during the induction of lateral buds on the flower stalk, the induction of clustered buds, and the hardening-off period were all detrimental to the fragrance release of Phalaenopsis 'Purple Diamond' during its peak flowering period. Simultaneously, to promote root induction, light intensity needed to be reduced during the strong seedling rooting induction period. By comparing the culture media of Phalaenopsis 'Purple Diamond' at the three stages of lateral bud induction, clustered bud induction, and strong seedling rooting induction in Examples 1-2, 3-4, and 5-6, respectively, we found that the induction rates obtained were significantly lower than those of the formula in Example 1. Comparing the fragrance promotion experiments of Examples 7-8, we found that changing the ratio of nitrogen, phosphorus, and potassium in the water-soluble fertilizer during the catalytic period and the start time of light and temperature treatments significantly affected the total fragrance release.

[0130] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for promoting the release of fragrance during the flowering period of Phalaenopsis purpurea, characterized in that, Includes the following steps: Starting from the first bloom of the Purple Diamond Phalaenopsis orchid, the light and temperature treatment was carried out on the 5th day, maintaining a constant light intensity of 2000 Lux for 24 hours and a constant temperature of 34℃ for 3 days; the fragrance release refers to the release of 2,4-di-tert-butylphenol and dimethyl phthalate from the petals of the Purple Diamond Phalaenopsis orchid. Specifically, the light intensity was increased to 3000 Lux, 3000 Lux, and 10000 Lux during the induction of lateral buds on flower stalks, the induction of clustered buds, and the hardening-off period, respectively. Meanwhile, in order to promote the induction of roots in Phalaenopsis purpureus, the light intensity was reduced to 2500 Lux during the strong seedling induction and rooting period; and the temperature after removal from the bottle was increased to 28℃. Before flowering, reduce the amount of nitrogen fertilizer used for Phalaenopsis Purple Diamond. Specifically, during the flowering period, apply a water-soluble fertilizer with a nitrogen, phosphorus, and potassium ratio of 9:45:

15. When the flower stalk reaches more than 25cm, apply a water-soluble fertilizer with a nitrogen, phosphorus, and potassium ratio of 10:30:

20. Apply once a week or every half month, each time with a 3000-5000 times dilution. Do not fertilize during the flowering period.

2. The method as described in claim 1, characterized in that, The culture medium formula for inducing lateral buds on flower stalks is: MS + 6-BA 1.5 mg / L + peptone 1.0 g / L + banana puree 30 mg / L, sucrose 30 g / L, agar powder 6.0 g / L, and light intensity 3000 Lux.

3. The method as described in claim 1, characterized in that, The culture medium formula for inducing shoot clusters is: MS + 6-BA 4.5 mg / L + NAA 0.5 mg / L + coconut juice 10% + banana puree 30 mg / L, sucrose 30 g / L, agar powder 6.0 g / L, and light intensity 3000 Lux.

4. The method as described in claim 1, characterized in that, The culture medium formula for the seedling induction and rooting period is as follows: 1 / 2 MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + activated carbon 2.0 g / L, sucrose 30 g / L, agar powder 6.0 g / L, and light intensity 2500 Lux.

5. The application of the method as described in any one of claims 1-4 in any of the following: (1) Application in improving the ratio of induced vegetative buds on the orchid stem of Phalaenopsis purpurea; (2) Application in improving the ratio of induced cluster shoots in Phalaenopsis purpurea; (3) Application in improving the rooting rate of vigorous seedlings of Phalaenopsis purpurea; (4) Application in promoting the release of fragrance during the flowering period of Phalaenopsis purpurea.

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