Method for promoting release of fragrance in orchid period of phalaenopsis splendens

Abstract

According to the method for promoting the fragrance release of the Phalaenopsis amabilis in the flowering stage, the influence of flowering days, different light intensities and different temperatures on the fragrance release in the full-bloom stage is compared, and the light and temperature adaptability of the fragrance of potted Phalaenopsis amabilis in the full-bloom stage is proved; and a basic basis can be provided for breeding aromatic phalaenopsis varieties and researching the action mechanism of the flower fragrance of important economic ornamental flowers. Meanwhile, the optimal culture medium formula of each link of tissue culture and rapid propagation of the phalaenopsis purpurascens is further deeply researched, discussed and screened, so that the phalaenopsis purpurascens can be developed and applied on an industrial scale.

Description

Technical Field

[0001] The present application relates to the field of agricultural cultivation technology, and in particular to a method for promoting the release of fragrance of purple phalaenopsis flowers during the flowering period. Background Art

[0002] Purple Diamond Phalaenopsis grows vigorously, with small fragrant flowers, beautiful flower shape, purple stripes, long flowering period, and stronger resistance. However, Purple Diamond Phalaenopsis is difficult to carry out conventional asexual reproduction, and there is no established and perfect Purple Diamond Phalaenopsis tissue culture rapid propagation technology in the existing technology. It is urgent to screen out the best culture medium formula for each link of Purple Diamond Phalaenopsis tissue culture rapid propagation through in-depth research and discussion, so that it can be developed and applied on an industrial scale.

[0003] At the same time, the fragrance of flowers is one of the traits of Phalaenopsis that cannot be ignored. However, during the flowering period of Phalaenopsis, the effects of different cultivation conditions on the release of fragrance are still unclear. Therefore, this study selected the potted variety Purple Diamond as a model plant, first analyzing the composition of the fragrance components during the flowering period; then, comparing the number of days of flowering during the flowering period, the effects of different light intensities and temperatures on the release of fragrance. Finding out the light and temperature adaptability of the fragrance of potted Phalaenopsis during the flowering period can provide a basic basis for the selection of aromatic Phalaenopsis varieties and the study of the mechanism of the fragrance of important economic ornamental flowers. Summary of the invention

[0004] In order to solve the above-mentioned technical problems to be solved, the present application provides a method for promoting the release of fragrance during the flowering period of purple diamond phalaenopsis, comprising the following steps: Phalaenopsis 'Purple Diamond' ) Starting from the first flower opening, light and temperature treatment was carried out on the 5th to 10th day, maintaining a constant light intensity of 2000 Lux for 24 hours and a constant temperature of 30-38℃ for 3-6 days.

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

[0006] (1) Material preparation: healthy and disease-free Phalaenopsis Purple Diamond with dormant buds that have passed their peak flowering period (Phalaenopsis 'Purple Diamond' ) plants as materials, when collecting the flower stalks, cut them from the bottom;

[0007] (2) Explant disinfection method: Cut the mature pedicels of Phalaenopsis purpurogenum into segments with one bud, leaving 1.5-2 cm length above and below the bud, soak them in washing powder or detergent water for 10 minutes, shaking continuously during the period, rinse with tap water for 30 minutes after cleaning, use sterile filter paper to absorb the water droplets on the pedicel, wipe the surface of the pedicel with 75% alcohol cotton ball, then soak them in 0.1% mercuric chloride or 10% NaClO for 15-20 minutes, shaking continuously, and wash them with sterile water 2-3 times;

[0008] (3) Inducing differentiation and proliferation

[0009] (3.1) Induction of lateral buds on pedicels

[0010] Remove the upper and lower ends of the lateral buds of the pedicel, and inoculate them in the normal growth direction into a medium containing 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, pH 5.8 to induce nutrient buds. The culture environment is: 25±2℃, light intensity 2500-3000 Lux, and light for 12 hours.

[0011] (3.2) Cluster bud differentiation and proliferation

[0012] The induced 1.5 cm long nutrient buds were cut from the base of the pedicel segment for cluster bud differentiation. The differentiation 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, pH 5.6-5.8, culture environment: 25±2℃, light intensity 2500-3000 Lux, light for 12 h;

[0013] (4) Strong seedlings and rooting

[0014] Cut off the buds over 1.5 cm in the Phalaenopsis clusters and culture them for seedling rooting. The culture medium is 1 / 2MS + 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 2500-3000Lux, and light for 12h.

[0015] (5) Planting of tissue culture seedlings out of bottles

[0016] (5.1) Requirements for tissue culture seedlings to be taken out of bottles

[0017] Phalaenopsis tissue culture seedlings are pollution-free. When the seedlings grow to 3-4 leaves, 3-5 roots, with leaves 3-4cm long and roots 3-5cm long, they are taken out of the bottle for planting.

[0018] (5.2) Tempering Seedlings

[0019] When the tissue culture bottle seedlings meet the requirements for bottle release, they are moved to the greenhouse for acclimatization. The temperature for seedling hardening is 25-30℃, the light intensity is 10000Lux, and the environment is kept clean and ventilated. The seedling hardening lasts for 3-4 weeks.

[0020] (5.3) Preparation of materials

[0021] The cultivation medium is high-quality sphagnum moss. Before transplanting, the sphagnum moss is soaked in 2000 times potassium permanganate solution for 12 hours. The sphagnum moss is picked up and squeezed hard until no water comes out. The seedlings are planted in a transparent plug tray.

[0022] (5.4) Transplanting

[0023] When transplanting, open the sealing film, clip out the seedlings, wash the root culture medium to prevent root rot after planting, soak the seedlings in 0.2% potassium permanganate solution for 3-5 minutes, disinfect the seedlings, fish them out, shake loose the sphagnum moss, put a small amount of sphagnum moss under the root system, and wrap the roots and monoaxial stems of the seedlings with sphagnum moss to expose the heart leaves, plant the seedlings vertically in the center of the plug tray, and press the plug tray with your hands to make it firm and elastic. The temperature just out of the bottle is controlled within the range of 22-28℃, which is conducive to the slow growth of Phalaenopsis seedlings and the rejuvenation of the root system. After one month, gradually adjust to the temperature range of 20-30℃ for the nutritional growth of Phalaenopsis, the relative humidity is above 80%, the light intensity is below 10000Lux, and the environment is clean and 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 and cultivated. After the seedlings are repotted, spray them uniformly with 2000 times carbendazim fungicide once every 2 weeks for 3 times in a row. No watering within a week can make the seedlings take root early and avoid soft rot diseases. Supply water to the seedlings by spraying, and keep spraying twice a day in the morning and afternoon to increase humidity; watering can be done after the second week. The principle of watering is that the surface of the water plants of the seedlings is dry and there is no water droplets or mist in the lower part of the transparent plug tray, then water it thoroughly. On the 21st day after the seedlings are repotted, even fertilizer (nitrogen, phosphorus, and potassium ratio is 20:20:20) is applied. The fertilization interval is 1 fertilizer and 1 water. The nitrogen, phosphorus, and potassium ratio of even fertilizer is 20:20:20. The fertilization interval is 1 fertilizer and 1 water, 3000-5000 times liquid; the concentration of fertilizer is low at the beginning, and the fertilizer concentration is gradually increased later, but not too high. Pay attention to the principle of thin fertilizer and frequent application. The EC value of fertilizer and water is required to be between 0.6-0.8. The fertilizer concentration is 5000 times liquid at the beginning, and the concentration is appropriately increased to 3000 times liquid later, but it should not exceed the EC maximum value of 0.8;

[0026] (7) Fertilization during flowering period

[0027] When fertilizing before Phalaenopsis blooms, the amount of nitrogen fertilizer should be reduced. Specifically, water-soluble fertilizer should be applied during the flowering period: the ratio of nitrogen, phosphorus and potassium should be 9:45:15. Water-soluble fertilizer should be applied from the pedicel to over 25 cm: the ratio of nitrogen, phosphorus and potassium should be 10:30:20. Apply once a week or half a month, 3000-5000 times the liquid. No fertilizer should be applied during the flowering period.

[0028] Furthermore, the present application also protects the purple diamond Phalaenopsis orchid with enhanced fragrance release obtained by the above two methods.

[0029] Furthermore, the formula of the pedicel lateral bud induction medium 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.

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

[0031] Furthermore, the formula of the seedling rooting induction medium is: 1 / 2MS + 6-BA 0.5 mg / L + NAA0.3 mg / L+ activated carbon 2.0 g / L, sucrose 30 g / L, agar powder 6.0g / L, and light intensity 2500Lux.

[0032] Furthermore, the seedling hardening temperature is 30°C and the light intensity is 10000Lux.

[0033] Furthermore, for transplanting and planting, the temperature just out of the bottle is controlled within the range of 28℃, which is conducive to the growth of Phalaenopsis seedlings and the rejuvenation of the root system. After one month, it is gradually adjusted to the temperature range of Phalaenopsis' nutritional growth of 25℃, the relative humidity is above 80%, the light intensity is 8000Lux, and the environment is clean and ventilated.

[0034] Furthermore, the initial flowering period was calculated from the first flower of the Purple Diamond Phalaenopsis, and light and temperature treatment was carried out on the 5th day, maintaining a constant light intensity of 2000 Lux for 24 hours, and the temperature was maintained at a constant temperature of 34°C for 3 days.

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

[0036] (1) Application in improving the ratio of nutrient bud induction of Phalaenopsis purpurogenum orchid stalks;

[0037] (2) Application in improving the ratio of vegetative buds to induce clustered buds in Phalaenopsis purpurogenum;

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

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

[0040] Beneficial effects:

[0041] 1. Purple Diamond Phalaenopsis is difficult to propagate asexually in a conventional manner. This application establishes and improves the tissue culture rapid propagation technology of Purple Diamond Phalaenopsis, which is a key link in the reproduction of Phalaenopsis. Through in-depth research and discussion, the best culture medium formula for each link of the tissue culture rapid propagation of Purple Diamond Phalaenopsis is selected.

[0042] 2. This study found that light is a key factor affecting the release rate of Phalaenopsis Purple Diamond's floral fragrance volatiles, and its influence exceeds that of temperature. Under no-light conditions, as the temperature rises, the release rate of floral fragrance volatiles (VOCs) increases, but the impact is limited. Under light conditions, the release rate of floral fragrance volatiles (VOCs) remains at a high level at higher temperatures, and the effect of light is more significant. Among all the treatments tested, the total VOC release rate of L2T2 (34°C, 2000Lux light) for 3 days was the highest, indicating that this light and temperature condition is conducive to the release of Phalaenopsis Purple Diamond's floral fragrance.

[0043] 3. Dimethyl phthalate is one of the main components of the Phalaenopsis Purple Diamond flower fragrance. The release rate under light conditions is also significantly higher than that under no light conditions. The release rate is the highest at L2T2 (34℃, light 2000Lux) for 3 days, exceeding 40 ng / g / h. The effect of light on the release of Phalaenopsis Purple Diamond flower fragrance may be related to photosynthesis. Light provides energy and promotes the synthesis and release of volatile organic compounds. This discovery is of great significance for regulating the release of Phalaenopsis flower fragrance. The release of flower fragrance can be enhanced by adjusting light conditions.

[0044] 4. The effect of temperature on the release of floral fragrance may be related to enzyme activity. Appropriate temperature can increase enzyme activity and thus promote the synthesis of floral fragrance compounds. However, too high or too low temperature may inhibit enzyme activity, resulting in reduced synthesis of floral fragrance compounds. In this study, the effects of light and temperature on the release rate of total fragrance, 2,4-di-tert-butylphenol, and dimethyl phthalate were particularly significant. This may be because these compounds dominate the floral 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. By adjusting the light and temperature conditions, the floral fragrance quality of Phalaenopsis can be optimized, thereby improving its ornamental value and market competitiveness.

[0046] 6. The present application also studies the effects of different tissue culture conditions on the growth and fragrance of purple diamond Phalaenopsis. We find that the optimal combination formula for obtaining purple diamond Phalaenopsis with promoted fragrance release is Example 1. By comparing with Example 2, namely, reducing the light intensity during the pedicel lateral bud induction, cluster bud induction, and seedling hardening period, and reducing the temperature after the bottle is not conducive to the release of the flower fragrance of purple diamond Phalaenopsis during the flowering period. At the same time, in order to promote the induction of rooting of purple diamond Phalaenopsis, the light intensity should be reduced during the seedling induction rooting period. By changing the culture medium culture of purple diamond Phalaenopsis in the three periods of pedicel lateral bud induction, cluster bud induction, and seedling induction rooting in Comparative Examples 1-2, 3-4, and 5-6, we found that the induction rate obtained was significantly lower than the formula of Example 1. The comparison of the fragrance promotion test of Comparative Examples 7-8 found that changing the ratio of nitrogen, phosphorus, and potassium in the catalytic period water-soluble fertilizer and the start time of light and temperature treatment would significantly affect the release of total fragrance. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1: Image of Phalaenopsis purple diamond;

[0048] Fig. 2 Changes in the release rate of total floral fragrance of Phalaenopsis at different light and temperature;

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

[0050] Fig. 4 Changes in the release rate of 2,4-di-tert-butylphenol. DETAILED DESCRIPTION

[0051] In order to make the purpose, scheme and advantages of the technical solution of the present application clearer, the technical solution of the embodiment of the present application will be clearly and completely described in conjunction with the drawings of the specific embodiments of the present application. Unless otherwise specified, the terms used herein have the usual meanings in the art. The same reference numerals in the drawings represent the same components.

[0052] Example 1

[0053] A method for promoting the release of fragrance of purple butterfly flowers during the blooming period, comprising the following steps:

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

[0055] (2) Explant disinfection method: Cut the mature pedicels of Phalaenopsis purpurogenum into segments with one bud, leaving 1.5 cm length above and below the bud, soak them in washing powder or detergent water for 10 minutes, shaking continuously during the period, rinse with tap water for 30 minutes after cleaning, use sterile filter paper to absorb the water droplets on the pedicel, wipe the surface of the pedicel with 75% alcohol cotton ball, then soak them in 0.1% mercuric chloride or 10% NaClO for 20 minutes, shaking continuously, and wash them with sterile water 3 times;

[0056] (3) Inducing differentiation and proliferation

[0057] (3.1) Induction of lateral buds on pedicels

[0058] Remove the upper and lower ends of the lateral buds of the pedicel, and inoculate them in the normal growth direction into a medium containing 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, pH 5.8 to induce vegetative buds. The culture environment is: 25±2℃, light intensity 3000Lux, light for 12h;

[0059] (3.2) Cluster bud differentiation and proliferation

[0060] The induced 1.5 cm long nutrient buds were cut from the base of the pedicel segment for cluster bud differentiation. The differentiation 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, pH 5.6, culture environment: 25 ± 2 ° C, light intensity 3000 Lux, light for 12 h;

[0061] (4) Strong seedlings and rooting

[0062] Cut off the buds of Phalaenopsis that are more than 1.5 cm in diameter and culture them for rooting. The culture medium is 1 / 2MS + 6-BA 0.5 mg / L + NAA0.3 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 2500Lux, and light for 12h.

[0063] (5) Planting of tissue culture seedlings out of bottles

[0064] (5.1) Requirements for tissue culture seedlings to be taken out of bottles

[0065] Phalaenopsis tissue culture seedlings are pollution-free. When the seedlings grow to 3 leaves and 4 roots, with leaves 3 cm long and roots 5 cm long, they are taken out of the bottle for planting.

[0066] (5.2) Tempering Seedlings

[0067] When the tissue culture bottle seedlings meet the requirements for bottle release, they are moved to the greenhouse for acclimatization. The temperature for acclimatization is 30℃, the light intensity is 10000Lux, and the environment is kept clean and ventilated. The acclimatization can last for 4 weeks. During the acclimatization period, the sealing film of the bottle mouth should not be removed to prevent contamination in the bottle and root rot caused by bacteria.

[0068] (5.3) Preparation of materials

[0069] The cultivation medium is high-quality sphagnum moss. Before transplanting, soak the sphagnum moss in 2000 times potassium permanganate solution for 12 hours. Pick up the sphagnum moss and squeeze it hard until no water comes out. Use a 100-grid square transparent plug tray to plant the seedlings.

[0070] (5.4) Transplanting

[0071] When transplanting, open the sealing film, clip out the seedlings, wash the root culture medium to prevent root rot after planting, soak the seedlings in 0.2% potassium permanganate solution for 5 minutes, disinfect the seedlings, fish them out, shake loose the sphagnum moss, put a small amount of sphagnum moss under the root system, and wrap the roots and monoaxial stems of the seedlings with sphagnum moss to expose the heart leaves, plant the seedlings vertically in the center of the plug tray, and press the plug tray with your hands to make it firm and elastic. The temperature just out of the bottle is controlled within the range of 28℃, which is conducive to the slow growth of Phalaenopsis seedlings and the rejuvenation of the root system. After one month, gradually adjust to the temperature range of 25℃ for the nutritional growth of Phalaenopsis, the relative humidity is above 80%, the light intensity is 8000Lux, and the environment is clean and 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 and cultivated. After the seedlings are repotted, they are uniformly sprayed with 2000 times carbendazim fungicide, once every 2 weeks, and used continuously for 3 times; No watering in the first week of the newly transplanted seedlings can make the seedlings take root early and avoid soft rot diseases. Water the seedlings by spraying, and keep spraying twice a day in the morning and afternoon to increase humidity; Watering can be done after the second week. The principle of watering is that the surface of the water plants of the seedlings is dry and there is no water droplets or mist in the lower part of the transparent plug tray, then water it thoroughly. Start to apply even fertilizer (nitrogen, phosphorus, potassium ratio is 20:20:20) on the 21st day after the seedlings are repotted. The fertilization interval is 1 fertilizer and 1 water, 5000 times liquid;

[0074] (7) Fertilization during flowering period

[0075] Before Phalaenopsis blooms, the amount of nitrogen fertilizer should be reduced. Specifically, water-soluble fertilizer should be applied during the flowering period: the ratio of nitrogen, phosphorus and potassium should be 9:45:15. Water-soluble fertilizer should be applied from the peduncle to more than 25 cm: the ratio of nitrogen, phosphorus and potassium should be 10:30:20. Apply once a week or half a month, 3000 times the liquid, and no fertilizer should be applied during the flowering period.

[0076] (8) Light intensity and temperature regulation during flowering period

[0077] The initial flowering period starts from the first flower of Phalaenopsis. On the fifth 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°C for 3 days to obtain a purple Phalaenopsis with enhanced fragrance release.

[0078] (9) Leave the field

[0079] Plant out in time according to market demand.

[0080] Example 2

[0081] A method for promoting the release of fragrance of purple butterfly flowers during the blooming period, comprising the following steps:

[0082] (1) Material preparation: healthy and disease-free Phalaenopsis Purple Diamond with dormant buds that have passed their peak flowering period ( Phalaenopsis 'Purple Diamond' ) plants as materials, when collecting the flower stalks, cut them from the bottom;

[0083] (2) Explant disinfection method: Cut the mature pedicels of Phalaenopsis purpurogenum into segments with one bud, leaving 2 cm of length above and below the bud, soak them in washing powder or detergent water for 10 minutes, shaking them continuously during the period, rinse them with tap water for 30 minutes after cleaning, use sterile filter paper to absorb the water droplets on the pedicels, wipe the surface of the pedicels with 75% alcohol cotton balls, and then soak them in 0.1% mercuric chloride or 10% NaClO for 20 minutes, shaking them continuously, and wash them with sterile water three times;

[0084] (3) Inducing differentiation and proliferation

[0085] (3.1) Induction of lateral buds on pedicels

[0086] Remove the upper and lower ends of the lateral buds of the pedicel, and inoculate them in the normal growth direction into a medium containing MS + 6-BA 1.5 mg / L + peptone 1.0 g / L + banana puree 30 mg / L, sucrose 30g / L, agar powder 6.0g / L, pH 5.8 to induce vegetative buds. The culture environment is: 25±2℃, light intensity 2500Lux, light for 12h;

[0087] (3.2) Cluster bud differentiation and proliferation

[0088] The induced 1.5 cm long nutrient buds were cut from the base of the pedicel segment for cluster bud differentiation. The differentiation 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, pH 5.6, culture environment: 25 ± 2 ° C, light intensity 2500 Lux, light for 12 h;

[0089] (4) Strong seedlings and rooting

[0090] Cut off the buds of Phalaenopsis over 1.5 cm in diameter and culture them for rooting. The culture medium is 1 / 2MS + 6-BA 0.5 mg / L + NAA0.3 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, and light for 12 hours.

[0091] (5) Planting of tissue culture seedlings out of bottles

[0092] (5.1) Requirements for tissue culture seedlings to be taken out of bottles

[0093] Phalaenopsis tissue culture seedlings are pollution-free. When the seedlings grow to 3 leaves and 4 roots, with leaves 3 cm long and roots 5 cm long, they are taken out of the bottle for planting.

[0094] (5.2) Tempering Seedlings

[0095] When the tissue culture bottle seedlings meet the requirements for bottle release, they are moved to the greenhouse for acclimatization. The temperature for acclimatization is 25℃, the light intensity is within 8000Lux, and the environment is kept clean and ventilated. The acclimatization can last for 4 weeks. During the acclimatization period, the sealing film of the bottle mouth should not be removed to prevent contamination in the bottle and root rot caused by bacteria.

[0096] (5.3) Preparation of materials

[0097] The cultivation medium is high-quality sphagnum moss. Before transplanting, soak the sphagnum moss in 2000 times potassium permanganate solution for 12 hours. Pick up the sphagnum moss and squeeze it hard until no water comes out. Use a 100-grid square transparent plug tray to plant the seedlings.

[0098] (5.4) Transplanting

[0099] When transplanting, open the sealing film, clip out the seedlings, wash the root culture medium to prevent root rot after planting, soak the seedlings in 0.2% potassium permanganate solution for 5 minutes, disinfect the seedlings, fish them out, shake loose the sphagnum moss, put a small amount of sphagnum moss under the root system, and wrap the roots and monoaxial stems of the seedlings with sphagnum moss to expose the heart leaves, plant the seedlings vertically in the center of the plug tray, and press the plug tray with your hands to make it firm and elastic. The temperature just out of the bottle is controlled within the range of 25℃, which is conducive to the slow growth of Phalaenopsis seedlings and the rejuvenation of the root system. After one month, gradually adjust to the temperature range of 25℃ for the nutritional growth of Phalaenopsis, the relative humidity is above 80%, the light intensity is 8000 Lux, and the environment is clean and 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 and cultivated. After the seedlings are repotted, they are uniformly sprayed with 2000 times carbendazim fungicide, once every 2 weeks, and used continuously for 3 times; No watering in the first week of the newly transplanted seedlings can make the seedlings take root early and avoid soft rot diseases. Water the seedlings by spraying, and keep spraying twice a day in the morning and afternoon to increase humidity; Watering can be done after the second week. The principle of watering is that the surface of the water plants of the seedlings is dry and there is no water droplets or mist in the lower part of the transparent plug tray, then water it thoroughly. Start to apply even fertilizer (nitrogen, phosphorus, potassium ratio is 20:20:20) on the 21st day after the seedlings are repotted. The fertilization interval is 1 fertilizer and 1 water, 5000 times liquid;

[0102] (7) Fertilization during flowering period

[0103] Before Phalaenopsis blooms, the amount of nitrogen fertilizer should be reduced. Specifically, water-soluble fertilizer should be applied during the flowering period: the ratio of nitrogen, phosphorus and potassium should be 9:45:15. Water-soluble fertilizer should be applied from the peduncle to more than 25 cm: the ratio of nitrogen, phosphorus and potassium should be 10:30:20. Apply once a week or half a month, 3000 times the liquid, and no fertilizer should be applied during the flowering period.

[0104] (8) Light intensity and temperature regulation during flowering period

[0105] The initial flowering period starts from the first flower of Phalaenopsis. On the fifth 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°C for 3 days to obtain a purple Phalaenopsis with enhanced fragrance release.

[0106] (9) Leave the field

[0107] Plant out in time according to market demand.

[0108] Experiment 1: Effects of light and temperature treatment on the fragrance release of purple phalaenopsis during the blooming period

[0109] Test method: The test material is Phalaenopsis horticultural variety Purple Diamond, located in Xiongxi Germplasm Resource Nursery of Zhejiang Subtropical Crops Research Institute (118˚21'~120˚30'E, 29˚11'~30˚33'N). Select Purple Diamond with consistent growth and place it in an artificial climate box (USA, PERCIVALE-41H02) for different light and temperature treatments. The test material treatment refers to Example 1, and the flowers are guaranteed to grow well. First, the main floral fragrance components of Phalaenopsis on the 5th day of flowering were determined under natural conditions, and then the light and temperature treatment test was carried out: the light condition was set to 24h constant light intensity, and the specific test treatment settings were as follows (The parameters of Example 1 are replaced by the corresponding light and temperature regulation). The treatment start time is based on the opening of the first flower bud after the Phalaenopsis orchid completes the extraction of stems. The light and temperature treatment is carried out on the 5th day, and then the flowering period turns into the decay period. Under the conditions of 3 days and 6 days of treatment respectively, the flowers with the same growth position are collected on the 30th day of flowering in the flowering period for flower fragrance collection and component analysis. Each treatment is repeated 3 times, and each repetition is 5 pots. The dynamic headspace collection method is used to collect the volatiles of Phalaenopsis orchid fragrance. The whole flower of potted Phalaenopsis orchid is randomly selected and placed in a glass container (30 cm high and 10 cm in diameter), and 0.7 min / L activated carbon purified air is supplied to the bottle. After balancing for 30 minutes, the adsorption column filled with Porpark Q (Waters Technology Co., Ltd., USA) adsorption silica gel is connected, and 200 μL of dichloromethane (chromatographically pure, Shanghai McLean Biochemical Technology Co., Ltd.) containing 0.001% volume fraction of nonyl acetate (chromatographically pure, Sigma Aldrich Shanghai Trading Co., Ltd.) as the internal standard is eluted and then sampled and measured. A blank headspace collection bottle was set up as a control group. All samples were collected for 4 hours, and then the fresh weight of the Phalaenopsis orchid flower samples was weighed. High-resolution gas chromatography-mass spectrometry (Q Exactive™GC Orbitrap™ GC-MS / MS) was used to analyze and identify the volatile components of floral fragrance. Helium was separated on an HP-5MS column (30 m×0.25 mm×0.25 μm) at a flow rate of 1 min / L. The floral fragrance splitless mode was set, the inlet temperature was 260 ℃, the gradient temperature program started at 40 ℃, then increased to 250 ℃ at a rate of 5 ℃ / min, and maintained for 20 min. MS conditions: ion source temperature was 230 ℃, ionization energy was 70 eV, and mass spectrometry scanning range was 40-500 amu. The retention index was calculated according to the retention time of the volatiles, and the qualitative comparison was made with the reference value of the compound retrieved in the database NIST17. The formula for calculating the floral fragrance release rate is: release rate (ng / g / h) = (sample volatile peak area / internal standard volatile peak area) × internal standard content / (headspace collection time × fresh weight of each sample group). The release rates of various floral volatiles were calculated using Excel 2023 software, and the volatile release amounts of different varieties were compared and analyzed using GraphPad prism 9 software.

[0110] Table 1 Experimental treatment settings

[0111] Processing number Dealing with 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 Phalaenopsis Purple Diamond fragrance

[0113] serial number Ingredient name Retention time (min) Release rate (ng / g / h) 1 Toluene 5.244 47.45548727 2 β-Ocimene 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 Linalyl acetate 16.538 6.047101582 7 3-Carene 11.181 6.033041227 8 α-Bulletin 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 components of the floral fragrance of the purple diamond variety are dimethyl phthalate, 2,4-di-tert-butylphenol, monoterpene (β-ocimene), and sesquiterpene (α-buhlerene). The changes in the release rate of the total floral fragrance of Phalaenopsis under different light and temperature are shown in Figure 2. Under dark conditions (L1T1, L1T2, L1T3), the VOC emission rate shows a downward trend as the temperature decreases. The emission rate of L1T1 is the highest and that of L1T3 is the lowest. In the 3d and 6d treatments, the VOC emission rate under light conditions (L2T1, L2T2, L2T3) is generally higher than that under dark conditions (L1T1, L1T2, L1T3). This shows that light is an important factor in promoting VOC emissions. Under the condition of 2000Lux light, the VOC release rate of the 6d treatment is lower than that of the 3d treatment, but still maintains a high level. Under the condition of 2000 Lux, the VOC emission rate was the highest at 34 °C (L2T2), which may indicate that this temperature is the optimal condition for VOC synthesis. The emission rates at 38 °C (L2T1) and 30 °C (L2T3) were relatively low, which may be due to the adverse effects of too high or too low temperatures on VOC synthesis.

[0115] As can be seen from Figure 3, the release rate of dimethyl phthalate was the highest when L2T2 (34℃, 2000Lux) was treated for 3 days, exceeding 40 ng / g / h. Under dark conditions (L1T1, L1T2, L1T3), the release rate of dimethyl phthalate also decreased with decreasing temperature, and there was almost no release after 6 days of treatment with L1T3 (30℃, dark). At the same temperature, the release rate of dimethyl phthalate was generally higher at 3 days in the treatment of 2000Lux light (L2T1, L2T2, L2T3) than in the dark treatment (L1T1, L1T2, L1T3). The release rate of dimethyl phthalate was the highest at 34℃ under the conditions of 2000Lux light treatment (L2T1, L2T2, L2T3), and too high or too low temperature would reduce the release rate of dimethyl phthalate. Overall, the release rate of dimethyl phthalate is consistent with the total release rate.

[0116] As can be seen from Figure 4, the release rate of 2,4-di-tert-butylphenol was the highest at 37.96 ng / g / h when L2T2 (34°C, 2000 Lux) was treated for 3 days. 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, and was the highest when treated with L1T3 (30°C, dark). At the same temperature, the release rate of 2,4-di-tert-butylphenol was generally higher at 3 days in the treatments of 2000 Lux (L2T1, L2T2, L2T3) than in the dark treatments (L1T1, L1T2, L1T3). The release rate of 2,4-di-tert-butylphenol was the highest at 34°C under the conditions of 2000 Lux illumination (L2T1, L2T2, L2T3). Too high or too low a temperature would reduce the release rate of 2,4-di-tert-butylphenol. Overall, the release rate of 2,4-di-tert-butylphenol was consistent with the trend of the total release rate.

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

[0118] Experimental conditions: The experimental material is Phalaenopsis horticultural variety Purple Diamond, located in Xiongxi Germplasm Resources Garden of Zhejiang Subtropical Crops Research Institute (118˚21'~120˚30'E, 29˚11'~30˚33'N). Material treatment refers to Example 1, and the treatment groups are shown in the following control examples 1-8. The pedicel lateral bud induction is to record the ratio of pedicel lateral bud induction vegetative buds 90 days after inoculation, the cluster bud induction is to record the ratio of vegetative bud induction cluster buds 60 days after inoculation, and the seedling rooting induction is to record the seedling rooting rate 70 days after inoculation. The fragrance promotion test is the same as the first test, only some parameters are replaced, and the flowers with the same growth position are collected on the 30th day of flowering during the peak flowering period for flower fragrance collection and component analysis. 30 bottles are treated for 3 times.

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

[0120] Comparative Example 2: Same as Example 1, except that the pedicel 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 culture medium for clustered bud differentiation and proliferation was replaced with: MS + 6-BA 1.5 mg / L + NAA 1.5 mg / L + coconut juice 10% + banana puree 30 mg / L, sucrose 30 g, and agar powder 6.0 g.

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

[0123] Comparative Example 5: Same as Example 1, except that the culture medium for rooting seedlings was replaced with: 1 / 2MS + 6-BA 1.5 mg / L + NAA0.5 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 culture medium for rooting seedlings was replaced with: 1 / 2MS + 0.5 mg / L 6-BA + 3 mg / L KT + 2.0 g / L activated carbon, 30 g sucrose and 6.0 g agar powder.

[0125] Comparative Example 7: The same as Example 1, except that water-soluble fertilizer was applied during the flowering period: the ratio of nitrogen, phosphorus and potassium was 10:30:20, and water-soluble fertilizer was applied from the pedicel to more than 25 cm: the ratio of nitrogen, phosphorus and potassium was 9:45:15.

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

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

[0128] serial number The ratio of pedicel-induced vegetative buds % Cluster bud ratio% Rooting rate of strong seedlings % 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] Test results: The effects of different tissue culture conditions on the growth and fragrance of purple diamond Phalaenopsis. We can see that the optimal combination formula for purple diamond Phalaenopsis with promoted fragrance release is Example 1. By comparing with Example 2, namely, reducing the light intensity during the pedicel lateral bud induction, cluster bud induction, and seedling hardening period, and reducing the temperature after the bottle is not conducive to the release of the flower fragrance of purple diamond Phalaenopsis during the flowering period. At the same time, in order to promote the induction of rooting of purple diamond Phalaenopsis, the light intensity should be reduced during the seedling induction rooting period. By changing the culture medium culture of purple diamond Phalaenopsis in the three periods of pedicel lateral bud induction, cluster bud induction, and seedling induction rooting in Examples 1-2, 3-4, and 5-6, we found that the induction rate obtained was significantly lower than the formula of Example 1. The comparison of the flower fragrance promotion test of Example 7-8 found that changing the ratio of nitrogen, phosphorus, and potassium in the catalytic period water-soluble fertilizer and the start time of light and temperature treatment would significantly affect the release of total fragrance.

[0130] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for promoting the release of fragrance during the blooming period of purple phalaenopsis, characterized in that: The following steps are involved: In Purple Diamond Phalaenopsis ( Phalaenopsis 'Purple Diamond' ) Starting from the first flower opening, light and temperature treatment was carried out on the 5th to 10th day, maintaining a constant light intensity of 2000 Lux for 24 hours and a constant temperature of 30-38℃ for 3-6 days.

2. The method according to claim 1, characterized in that Starting from the time the first flower of the Purple Diamond Phalaenopsis blooms, 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°C for 3 days.

3. The method according to claim 1, characterized in that Before the light and temperature treatment of the purple diamond Phalaenopsis during its blooming period, the following steps are also included: (1) Material preparation: healthy and disease-free Phalaenopsis Purple Diamond with dormant buds that have passed their peak flowering period ( Phalaenopsis 'Purple Diamond' ) plants as materials, when collecting the flower stalks, cut them from the bottom; (2) Explant disinfection method: Cut the mature pedicels of Phalaenopsis purpurogenum into segments with one bud, leaving 1.5-2 cm length above and below the bud, soak them in washing powder or detergent water for 10 minutes, shaking continuously during the period, rinse with tap water for 30 minutes after cleaning, use sterile filter paper to absorb the water droplets on the pedicel, wipe the surface of the pedicel with 75% alcohol cotton ball, and then soak them in 0.1% mercuric chloride or 10% NaClO for 15-20 minutes, shaking continuously during the period, and wash them with sterile water 2-3 times; (3) Inducing differentiation and proliferation (3.1) Induction of lateral buds on pedicels Remove the upper and lower ends of the lateral buds of the pedicel, and inoculate them in the normal growth direction into a medium containing 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, pH 5.8 to induce nutrient buds. The culture environment is: 25±2℃, light intensity 2500-3000 Lux, and light for 12 hours. (3.2) Cluster bud differentiation and proliferation The induced 1.5 cm long nutrient buds were cut from the base of the pedicel segment for cluster bud differentiation. The differentiation 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, pH 5.6-5.8, culture environment: 25±2℃, light intensity 2500-3000 Lux, light for 12 hours; (4) Strong seedlings and rooting Cut off the buds over 1.5 cm in the Phalaenopsis clusters and culture them for seedling rooting. The culture medium is 1 / 2MS + 6-BA 0.5 mg / L + NAA0.3 mg / L + activated carbon 2.0 g / L, sucrose 30g / L, agar powder 6.0g / L, pH 5.6-5.

8. The culture environment is 25±2℃, light intensity 2500-3000Lux, and light for 12h. (5) Planting of tissue culture seedlings out of bottles (5.1) Requirements for tissue culture seedlings to be taken out of bottles Phalaenopsis tissue culture seedlings are pollution-free. When the seedlings grow to 3-4 leaves, 3-5 roots, with leaves 3-4cm long and roots 3-5cm long, they are taken out of the bottle for planting. (5.2) Tempering Seedlings When the tissue culture bottle seedlings meet the requirements for bottle release, they are moved to the greenhouse for acclimatization. The temperature for seedling hardening is 25-30℃, the light intensity is 10000Lux, and the environment is kept clean and ventilated. The seedling hardening lasts for 3-4 weeks. (5.3) Preparation of materials The cultivation medium is high-quality sphagnum moss. Before transplanting, the sphagnum moss is soaked in 2000 times potassium permanganate solution for 12 hours. The sphagnum moss is picked up and squeezed hard until no water comes out. The seedlings are planted in a transparent plug tray. (5.4) Transplanting When transplanting, open the sealing film, clip out the seedlings, wash the root culture medium to prevent root rot after planting, soak the seedlings in 0.2% potassium permanganate solution for 3-5 minutes, disinfect the seedlings, fish them out, shake loose the sphagnum moss, put a small amount of sphagnum moss under the root system, and wrap the roots and monoaxial stems of the seedlings with sphagnum moss, exposing the heart leaves, and plant the seedlings vertically in the center of the plug tray. The temperature just out of the bottle should be controlled within the range of 22-28℃, which is conducive to the slow growth of Phalaenopsis seedlings and the rejuvenation of the root system. After one month, gradually adjust the temperature to the range of 20-30℃ for the nutritional growth of Phalaenopsis, the relative humidity should be above 80%, the light intensity should be below 10000Lux, and the environment should be clean and ventilated; (6) Seedling cultivation When the distance between the two leaves of the plant reaches more than 12cm, the seedlings are repotted and cultivated. After the seedlings are repotted, they are uniformly sprayed with 2000 times of carbendazim fungicide, once every 2 weeks, and used continuously for 3 times; the newly transplanted seedlings are not watered in the first week, and the seedlings are supplied with water by spraying, and the humidification is maintained twice a day in the morning and afternoon; watering is applied after the second week. The principle of watering is to water thoroughly when the surface of the water plants of the seedlings is dry and there is no water droplet or mist in the lower part of the transparent plug tray. On the 21st day after the seedlings are repotted, equal fertilizer is applied. The ratio of nitrogen, phosphorus and potassium in the equal fertilizer is 20:20:20, and the fertilization interval is 1 fertilizer and 1 water, each time 3000-5000 times liquid; (7) Fertilization during flowering period When fertilizing before Phalaenopsis blooms, the amount of nitrogen fertilizer should be reduced. Specifically, during the flowering period, water-soluble fertilizer with nitrogen, phosphorus and potassium ratio of 9:45:15 should be applied. From the pedicel to over 25 cm, water-soluble fertilizer with nitrogen, phosphorus and potassium ratio of 10:30:20 should be applied. Apply once a week or half a month, 3000-5000 times the liquid each time. No fertilizer should be applied during the flowering period.

4. The method according to claim 3, characterized in that Formula of pedicel lateral bud induction medium: 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, light intensity 3000 Lux.

5. The method according to claim 3, characterized in that Formula of cluster bud differentiation and proliferation medium: MS + 6-BA4.5mg / L+ NAA 0.5mg / L + coconut juice 10% + banana puree 30 mg / L, sucrose 30g / L, agar powder 6.0g / L, light intensity 3000Lux.

6. The method according to claim 3, characterized in that Culture medium formula: Seedling rooting induction culture medium formula: 1 / 2MS + 6-BA 0.5 mg / L + NAA0.3 mg / L + activated carbon 2.0 g / L, sucrose 30g / L, agar powder 6.0g / L, light intensity 2500Lux.

7. The method according to claim 3, characterized in that The seedling hardening temperature is 30℃ and the light intensity is 10000Lux.

8. The method according to claim 3, characterized in that For transplanting and planting, the temperature should be controlled within 28℃ just out of the bottle, which is conducive to the growth of Phalaenopsis seedlings and the rejuvenation of the root system. After one month, the temperature should be gradually adjusted to 25℃, the range of Phalaenopsis vegetative growth, relative humidity should be above 80%, light intensity should be 8000Lux, and the environment should be clean and ventilated.

9. Purple Diamond Phalaenopsis orchid with enhanced fragrance release obtained by the method of claim 1 or 3.

10. Use of the method according to claim 1 or 3 in any of the following: (1) Application in improving the ratio of nutrient bud induction of Phalaenopsis purpurogenum orchid stalks; (2) Application in improving the ratio of vegetative buds to induce clustered buds in Phalaenopsis purpurogenum; (3) Application in improving the rooting rate of purple diamond Phalaenopsis seedlings; (4) Application in promoting the release of fragrance from Phalaenopsis purpurogenum during the flowering period.

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