Method for improving survival rate of bottle-out transplanting of phalaenopsis tissue culture seedlings

Abstract

The method comprises the following steps: using an intelligent positive-pressure sterile greenhouse as a seedling hardening place, putting bottle seedlings into the positive-pressure sterile greenhouse, carrying out seedling hardening, adopting a gradient seedling hardening mode, carrying out seedling hardening for 10 days, and carrying out transplanting operation; the culture medium sphagna is soaked in cold water and then dehydrated; after dehydration, performing high-pressure steam sterilization for 25-30 minutes; and after transplanting, carrying out post-transplanting management in a greenhouse environment. The method solves the problems of high incidence of plant diseases and insect pests, low survival rate, poor environmental adaptability and the like in the hardening and transplanting process of the butterfly orchid tissue culture seedlings, and compared with a traditional method, the survival rate is increased by 20% or above, the production cycle of commercial seedlings is shortened by 15-20 days, the production efficiency and economic benefits of butterfly orchid are remarkably improved, and the method is suitable for industrial production. And a technical support is provided for high-quality development of the phalaenopsis industry.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for reducing the contamination rate of bottle seedlings during the seedling hardening period and improving the survival rate of Phalaenopsis tissue culture seedlings after being taken out of bottles and transplanted. Background Art

[0002] Existing Phalaenopsis orchids are usually cultivated through tissue culture bottle seedlings. Tissue culture bottle seedlings are grown in a sealed, sterile environment with sufficient nutrition supply, suitable light, constant temperature and high humidity. However, after the tissue culture seedlings have fully taken root, they need to be removed from the culture bottle and transplanted into a natural environment. In order to smoothly realize the process of normal growth of tissue culture seedlings from culture bottles to natural environments, the conventional method is to pre-treat the bottle seedlings before transplanting: seedling hardening. Seedling hardening refers to the process of moving the bottle seedlings from a clean, constant temperature and low light culture room to a greenhouse environment before taking them out of the bottle for transplanting, and gradually changing the temperature, humidity, light and other conditions of the environment surrounding the tissue culture bottle seedlings so that they gradually adapt to the natural environment of the greenhouse. The main purpose of seedling hardening is to improve the survival rate of the bottle seedlings after transplanting.

[0003] However, the survival rate of transplanting Phalaenopsis tissue culture seedlings after hardening is still low, usually less than 70%. The main reasons include the following:

[0004] 1. High pest and disease infection rates: Fungal diseases (such as Fusarium root rot) can have infection rates exceeding 30%. Two main reasons contribute to this high infection rate: First, poor cleanliness in the seedling hardening area, which can easily lead to contamination of the culture medium during the uncapped hardening phase; and second, the growing medium (such as sphagnum moss) harbors pathogens, insect eggs, and weed seeds. Traditional sphagnum moss treatment involves two 30-minute soaks in cold water, or a 30-minute soak in 80°C hot water followed by another 30-minute soak in cold water. However, these methods do not completely kill pathogen spores and weed seeds, making them susceptible to contamination after transplanting.

[0005] 2. Inappropriate seedling hardening methods: Conventional seedling hardening involves simply opening the bottle cap for 7-10 days. This direct opening of the bottle cap can cause a sudden change in the environment inside and outside the bottle, which can cause physiological shock in the tissue culture seedlings. Because the seedlings fail to undergo a gradual adaptation process to the changing external environment, it is difficult for them to successfully transition from a sterile culture medium to a sphagnum moss medium.

[0006] 3. Improper environmental regulation and management during the initial transplanting phase: During the initial transplanting phase, there are often inadequate regulation and management of water, fertilizer, light, temperature, and ventilation conditions. For example, unbalanced water and fertilizer supply, excessive or insufficient light, large temperature fluctuations, and poor ventilation can all hinder the growth of tissue culture seedlings or even lead to their death.

[0007] In view of the above problems, it is necessary to further optimize the seedling hardening environment, improve the seedling hardening method and strengthen the environmental regulation and management in the early stage of transplanting to improve the transplanting survival rate of Phalaenopsis tissue culture seedlings. Summary of the Invention

[0008] The present invention aims to reduce the contamination rate of Phalaenopsis orchid tissue culture seedlings during the hardening period and significantly improve their survival rate after transplantation. To this end, the present invention proposes novel technical solutions for the seedling acclimation process, optimized pretreatment of the transplanting medium, water and fertilizer management, and environmental regulation.

[0009] The technical solution adopted in the present invention is:

[0010] A method for improving the survival rate of Phalaenopsis tissue culture seedlings after being taken out of bottles and transplanted, the method comprising the following steps:

[0011] (1) Prepare a site for hardening seedlings:

[0012] Use intelligent positive pressure sterile greenhouse as the seedling hardening place, and the fresh air entering the positive pressure sterile greenhouse has an air purification level of ISO5;

[0013] Furthermore, the positive pressure sterile greenhouse is equipped with an air purification processor, and its filtration system complies with the requirements of ISO 14644-1 standard, so that the purification level reaches ISO5, ensuring that the air entering the positive pressure sterile greenhouse is sterile air.

[0014] Furthermore, the positive pressure sterile greenhouse is equipped with a positive pressure buffer room. Personnel and materials first enter the positive pressure buffer room for 75% alcohol disinfection before entering the positive pressure sterile greenhouse. The absolute positive pressure difference of the positive pressure sterile greenhouse can be adjusted to 10-15 Pa, which reduces bacterial contamination caused by gas flow.

[0015] The positive pressure sterile greenhouse has the function of intelligently controlling temperature and humidity, light intensity, carbon dioxide concentration, etc., thus ensuring the environmental conditions for hardening seedlings.

[0016] (2) Planting substrate treatment:

[0017] The cultivation medium sphagnum moss is first soaked in cold water and then dehydrated; after dehydration, it is sterilized with high-pressure steam for 25 to 30 minutes;

[0018] Cold water soaking generally involves soaking the sphagnum moss in cold water 2 to 3 times, each time for 30 to 40 minutes.

[0019] The dehydration standard is: pinch the sphagnum moss tightly with your hands and feel that water seems to be dripping out.

[0020] High-pressure steam sterilization can completely kill pathogens, spores, insect eggs, weed seeds, etc. in sphagnum moss.

[0021] The conditions for high-pressure steam sterilization are: high-pressure steam temperature of 120-125° C., sterilization for 25-30 minutes, preferably high-pressure steam temperature of 121° C., sterilization for 25 minutes.

[0022] (3) The bottled seedlings are placed in a positive pressure sterile greenhouse for hardening:

[0023] Three hours before the bottle seedlings enter the positive pressure sterile greenhouse, the greenhouse is first irradiated with ultraviolet light for 30 minutes, then disinfected with ozone for 30 minutes, and then the ventilation system is turned on, and the temperature, humidity and light are adjusted in advance to the environmental conditions required for seedling hardening;

[0024] The seedlings are hardened in a gradient manner, as follows:

[0025] Day 1-2: Do not open the bottle cap, and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a day-night temperature difference of 2-3℃; adjust the light intensity to between 3000-5000 lux, the humidity to between 60%-70%, and the positive pressure difference to between 10-15Pa;

[0026] Day 3-4: Do not open the bottle caps. Adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night. Adjust the light intensity to 5000-7000 lux, humidity to 60%-70%, and positive pressure difference to 10-15 Pa.

[0027] Day 5-6: Open the lid completely, adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to 5000-7000 lux, humidity to 80%-90%, and positive pressure difference to 10-15 Pa;

[0028] Day 7-8: Open the lid completely and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to 5000-7000 lux, humidity to 70%-80%, and positive pressure difference to 10-15 Pa;

[0029] Day 9-10: Open the lid completely and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 7000-9000 lux, the humidity to between 60%-70%, and the positive pressure difference to between 10-15Pa;

[0030] Generally speaking, when the tissue culture seedlings grow to a height of 3-5 cm, 3-5 leaves, 2-4 roots, and a root length of 2.5-5.0 cm, they are hardened. After hardening, they can be removed from the bottle and transplanted.

[0031] (4) Harden the seedlings for 10 days and then transplant them:

[0032] Select healthy seedlings with normal leaf color, 3-5 leaves, and more than 2 aerial roots; use high-pressure steam sterilized sphagnum moss as the substrate, plant the hardened plants in 1.5-inch soft pots, and then place them in a greenhouse environment for management;

[0033] The specific operation method is: use clips to pick up the seedlings from the bottle, and use clean water to wash away the culture medium remaining on the roots;

[0034] Planting method: After high-pressure steam sterilization, cool the sphagnum moss to room temperature; loosen the moss with your hands, place the roots of the plant in the middle of the moss, then wrap the roots with the loosened moss and plant in a 1.5-inch soft pot;

[0035] Furthermore, when wrapping the plant with sphagnum moss, make sure that the roots are completely covered with sphagnum moss, which can provide good moisturizing and support for the roots.

[0036] When planting, place the plant wrapped in sphagnum moss in a 1.5-inch soft pot. The soft pot usually has three ring lines. When placing the plant in the soft pot, make sure that the roots of the plant and the wrapped sphagnum moss are below the three ring lines of the soft pot. This step is to ensure that the plant can grow stably in the pot and prevent the roots from being exposed to the air.

[0037] After placing the plant in the soft pot, use your thumb to gently press the sphagnum moss around the pot. The purpose of compaction is to ensure that the sphagnum moss is in close contact with the roots and provide stable support for the plant.

[0038] During the compaction process, be careful to use moderate force to ensure that the sphagnum moss is in close contact with the roots, but avoid pressing too tightly to prevent affecting the breathing and growth of the roots.

[0039] The final effect is that the sphagnum moss below the roots is slightly loose so that the roots can grow freely; while the sphagnum moss around the soft pot should be compacted to provide stable support.

[0040] If the seedlings in the bottle are too small, they can be discarded or planted in a 128-grid hole tray. Larger seedlings can be directly planted in a 1.5-inch soft pot and then placed in a greenhouse environment for management.

[0041] (5) Management after transplanting:

[0042] Greenhouse environment control is as follows:

[0043] Light intensity: Control the light intensity at 5000-7000 lux in the first 4 weeks, and increase it to 7000-10000 lux in the 5th to 8th weeks; after 8 weeks, when new roots begin to grow, increase the light intensity to 12000-15000 lux;

[0044] Temperature: 26±2℃ during the day and 23±1℃ at night. Control the temperature difference between day and night to be ≥3℃ to promote rooting.

[0045] Humidity: Use intermittent spraying to control RH 70-75%;

[0046] Water and fertilizer management: The water quality standard for preparing water and fertilizer is an EC content of less than 0.38mS / cm, preferably less than 0.10mS / cm;

[0047] After transplanting, spray fungicide to prevent pathogens. Leave it for 4 to 7 days after spraying, then apply water and fertilizer. Use fertilizer with a N:P:K ratio of 10:30:20, and add water to adjust the EC value to between 0.6-0.7mS / cm as water and fertilizer; fungicides can be selected from carbendazim diluted 800-1000 times and kasugamycin diluted 800-1000 times, prepare and spray them separately, one day apart.

[0048] Generally speaking, 1 to 2 months after transplanting, more than 95% of the tissue culture seedlings have grown new roots, and the fertilizer should be changed to a balanced fertilizer with an N:P:K ratio of 20:20:20, and the EC value of water and fertilizer should be increased to 0.8-1.0mS / cm.

[0049] Generally speaking, water and fertilizer should be applied every 6 to 12 days. The frequency of watering and fertilizer application can be adjusted according to the temperature and humidity, as well as the dryness of the sphagnum moss. For example, water and fertilizer should be applied every 10 to 12 days in winter and every 6 to 7 days in summer.

[0050] Furthermore, when the sphagnum moss in the pot is dry and feels light but still heavy when picked up, it is the best time to water and fertilize.

[0051] Do not water or fertilize on rainy days to avoid oxygen deficiency in the roots;

[0052] Pest and disease control: Insecticides and fungicides need to be sprayed once every half a month.

[0053] Furthermore, the fungicide can be selected from carbendazim diluted 800-1000 times and kasugamycin diluted 800-1000 times, which can be prepared and sprayed separately; the insecticide is thiamethoxam diluted 800-1000 times and abamectin diluted 800-1000 times, which can be sprayed separately or mixed in a 1:1 ratio.

[0054] The present invention establishes a three-in-one management system of "zero pest and disease source contact - gradient adaptation - intelligent environmental control". Through systematic technological innovation, it improves the survival rate of Phalaenopsis tissue culture seedlings during hardening and transplanting. The beneficial effects of the present invention are:

[0055] 1. Significantly reduce the infection rate of pests and diseases

[0056] By using an intelligent positive pressure sterile greenhouse as a seedling hardening site, strictly controlling the entry and exit of personnel and materials, and combining air purification processors and disinfection measures, the entry of pests and diseases during the seedling hardening process is eliminated from the source. At the same time, the cultivation medium is sterilized with high-pressure steam to thoroughly kill pathogens, insect eggs, and weed seeds in the sphagnum moss, fundamentally solving the problem of high incidence of pests and diseases during traditional seedling hardening. The disease incidence rate of the present invention dropped from 27.6% in the control group to 4.3%, significantly reducing seedling mortality caused by pests and diseases and improving transplant survival rate.

[0057] 2. Improve transplant survival rate

[0058] The present invention adopts a gradient seedling hardening method, gradually adjusting environmental conditions such as temperature, humidity, and light, so that tissue culture seedlings can smoothly transition from the sterile, high-humidity culture bottle environment to the natural environment. This gradual acclimatization process avoids the physiological shock caused by traditional direct hardening of seedlings with the lid opened, ensuring that the tissue culture seedlings can better adapt to the external environment. The transplant survival rate of Phalaenopsis tissue culture seedlings increased from 73.5% in the control group to over 90%, significantly improving production efficiency and reducing economic losses caused by transplant failure.

[0059] 3. Optimize environmental control and improve growth quality

[0060] The intelligent positive-pressure sterile greenhouse is equipped with an advanced environmental control system that monitors and automatically adjusts key parameters such as temperature, humidity, light intensity, and carbon dioxide concentration in real time, ensuring optimal environmental conditions during the hardening and transplanting processes. Furthermore, intermittent spraying and precise water and fertilizer management further optimize the plant's growth environment. Phalaenopsis tissue culture seedlings experience accelerated growth after transplanting, with new roots emerging earlier and more robustly. Root tips are visible 15 days after transplanting, 5-10 days earlier than usual. The overall plant growth quality is also significantly improved.

[0061] 4. Improve production efficiency and economic benefits

[0062] The method of the present invention improves the transplant survival rate, shortens the production cycle of Phalaenopsis, and improves production efficiency and economic benefits.

[0063] The method of the present invention effectively solves the problems of high incidence of diseases and pests, low survival rate, and poor environmental adaptability in the process of hardening and transplanting Phalaenopsis tissue culture seedlings. Compared with traditional methods, the survival rate is increased by more than 20%, and the production cycle of commercial seedlings is shortened by 15-20 days, which significantly improves the production efficiency and economic benefits of Phalaenopsis, and provides strong technical support for the high-quality development of the Phalaenopsis industry. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0065] Example 1:

[0066] The method of the present invention was applied to cultivate the "Big Pepper" variety of Phalaenopsis in a tissue culture seedling production base in Sanya City.

[0067] When the tissue culture seedlings grow to 3-5cm in height, 3-5 leaves, 2-4 roots, and 2.5-5.0cm in length, they can be hardened. After hardening, they can be taken out of the bottle and transplanted.

[0068] (1) Prepare a site for hardening seedlings:

[0069] An intelligent positive pressure sterile greenhouse is used as the seedling hardening place, equipped with an air purification processor. Its filtration system complies with the requirements of ISO14644-1 standard, so that the purification level of the fresh air entering the positive pressure sterile greenhouse reaches ISO5, ensuring that the air entering the positive pressure sterile greenhouse is sterile air.

[0070] Three hours before the bottle seedlings enter the positive pressure sterile greenhouse, the greenhouse is first irradiated with ultraviolet light for 30 minutes, then disinfected with ozone for 30 minutes, and then the ventilation system is turned on, and the temperature, humidity and light are adjusted in advance to the environmental conditions required for seedling hardening;

[0071] Furthermore, the positive pressure sterile greenhouse is equipped with a positive pressure buffer room. Personnel and materials first enter the positive pressure buffer room for 75% alcohol disinfection before entering the positive pressure sterile greenhouse. The absolute positive pressure difference of the positive pressure sterile greenhouse can be adjusted to 10-15 Pa, which reduces bacterial contamination caused by gas flow.

[0072] The positive pressure sterile greenhouse has the functions of intelligently controlling temperature and humidity, light intensity, carbon dioxide concentration, etc., which ensures the environmental conditions for hardening seedlings;

[0073] (2) Planting substrate treatment:

[0074] The cultivation medium is sphagnum moss, which is first soaked in cold water twice, 30 minutes each time, and then dehydrated; after dehydration, sterilize with high-pressure steam for 25 to 30 minutes;

[0075] The dehydration standard is: pinch the sphagnum moss tightly with your hands and feel that water seems to be dripping out.

[0076] The high-pressure steam temperature is 121°C and the sterilization time is 25 minutes.

[0077] High-pressure steam sterilization can completely kill pathogens, spores, insect eggs, weed seeds, etc. in sphagnum moss.

[0078] (3) The bottled seedlings are placed in a positive pressure sterile greenhouse for hardening:

[0079] The seedlings are hardened in a gradient manner, as follows:

[0080] Day 1-2: Do not open the bottle cap, and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a day-night temperature difference of 2-3℃; adjust the light intensity to between 3000-5000 lux, the humidity to between 60%-70%, and the positive pressure difference to between 10-15Pa;

[0081] Day 3-4: Do not open the bottle cap, and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 5000-7000 lux, the humidity to between 60%-70%, and the positive pressure difference to between 10-15Pa;

[0082] Day 5-6: Open the lid completely, adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to 5000-7000 lux, the humidity to 80%-90%, and the positive pressure difference to 10-15 Pa;

[0083] Day 7-8: Open the lid completely and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to 5000-7000 lux, humidity to 70%-80%, and positive pressure difference to 10-15 Pa;

[0084] Day 9-10: Open the lid completely and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 7000-9000 lux, the humidity to between 60%-70%, and the positive pressure difference to between 10-15 Pa;

[0085] (4) After 10 days of hardening, transplanting is carried out:

[0086] Seedling selection criteria: Select healthy seedlings with normal leaf color, 3-5 leaves, and more than 2 aerial roots;

[0087] Operation method: Use clips to remove the seedlings from the bottle and wash away the culture medium remaining on the roots with clean water;

[0088] Planting method: Leave the sterilized sphagnum moss overnight and use it after the temperature drops to room temperature; loosen the sphagnum moss with your hands, place the roots of the plant in the middle of the sphagnum moss, then wrap the roots with the loosened sphagnum moss and plant it in a 1.5-inch soft pot; then place it in a greenhouse environment for management.

[0089] When wrapping the plant with sphagnum moss, make sure the roots are completely covered with sphagnum moss, which can provide good moisture retention and support for the roots.

[0090] When planting, place the plant wrapped in sphagnum moss in a 1.5-inch soft pot. The soft pot usually has three ring lines. When placing the plant in the soft pot, make sure that the roots of the plant and the wrapped sphagnum moss are below the three ring lines of the soft pot. This step is to ensure that the plant can grow stably in the pot and prevent the roots from being exposed to the air.

[0091] After placing the plant in the soft pot, use your thumb to gently press the sphagnum moss around the pot. The purpose of compaction is to ensure that the sphagnum moss is in close contact with the roots and provide stable support for the plant.

[0092] During the compaction process, be careful to use moderate force to ensure that the sphagnum moss is in close contact with the roots, but avoid pressing too tightly to prevent affecting the breathing and growth of the roots.

[0093] The final effect is that the sphagnum moss below the roots is slightly loose so that the roots can grow freely; while the sphagnum moss around the soft pot should be compacted to provide stable support.

[0094] If the seedlings in the bottle are too small, they can be discarded or planted in a 128-cell hole tray. Larger seedlings in the bottle can be planted directly in a 1.5-inch soft pot.

[0095] (5) Management after transplanting:

[0096] After transplanting, place the plants in a greenhouse and manage them under the following environmental control:

[0097] Light intensity: Control the light intensity at 5000-7000 lux in the first 4 weeks, and increase it to 7000-10000 lux in the 5th to 8th weeks; after 8 weeks, when new roots begin to grow, increase the light intensity to 12000-15000 lux;

[0098] Temperature: 26±2℃ during the day and 23±1℃ at night. Control the temperature difference between day and night to be ≥3℃ to promote rooting.

[0099] Humidity: Use intermittent spraying to control RH 70-75%;

[0100] Water and fertilizer management: The water quality standard for preparing water and fertilizer is an EC content of less than 0.38mS / cm, preferably less than 0.10mS / cm;

[0101] After transplanting, spray carbendazim diluted 1000 times and carbendazim-kasugamycin diluted 1000 times as fungicides, one day apart to prevent pathogens. Leave the spray for 4-7 days after spraying before applying water and fertilizer. Use fertilizer with an NPK (nitrogen-phosphorus-potassium) ratio of 10-30-20 and adjust the EC value to between 0.6-0.7mS / cm. When preparing water and fertilizer, use filtered water with an EC value below 0.1mS / cm.

[0102] 1-2 months after transplanting, when new roots have grown, increase the EC value of water and fertilizer to 0.8-1.0mS / cm, and use a balanced fertilizer with an NPK ratio of 20-20-20;

[0103] Furthermore, when the sphagnum moss in the pot is dry and feels light but still heavy when picked up, it is the best time to water and fertilize.

[0104] Generally speaking, water and fertilizer should be applied every 6 to 12 days. The frequency of watering and fertilizer application can be adjusted according to the temperature and humidity, as well as the dryness of the sphagnum moss. For example, water and fertilizer should be applied every 10 to 12 days in winter and every 6 to 7 days in summer.

[0105] Do not water or fertilize on rainy days to avoid oxygen deficiency in the roots;

[0106] Pest control: Spray insecticides and fungicides once every two weeks. The insecticide is a 1000-fold diluted buprofezin solution and a 1000-fold diluted abamectin solution, which can be sprayed separately or mixed. The fungicide is a 1000-fold diluted carbendazim solution and a 1000-fold diluted kasugamycin solution, which can be prepared and sprayed separately.

[0107] Ten days after transplanting, the plants were checked daily for growth, disease incidence, and the date of new root growth. Thirty days later, the survival rate was calculated, and the results showed that the transplanted survival rate of the "Big Pepper" variety reached 93.2%.

[0108] Root tips can be seen 15 days after transplanting, and new roots have grown, which is 5 days earlier than the normal time for new roots to grow (about 20 days).

[0109] The disease incidence rate dropped to 4.3%.

[0110] Conventional technology was used to harden and transplant the seedlings for comparison.

[0111] Comparative Example 1

[0112] During the same time period, the same batch of "Big Pepper" variety Phalaenopsis orchids were cultivated.

[0113] The difference from Example 1 is:

[0114] Instead of using a positive pressure sterile greenhouse, the conventional seedling hardening method is:

[0115] Transport the bottle seedlings to the glass greenhouse for hardening. The specific operations are as follows:

[0116] Day 1-2: Do not open the bottle cap, control the temperature of the glass greenhouse between 26±1℃ during the day and 24±1℃ at night, and ensure that the temperature difference between day and night is between 2-3℃; adjust the light intensity to between 3000-5000lux.

[0117] Day 3-4: Do not open the bottle caps, control the temperature of the glass greenhouse between 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 5000-7000lux.

[0118] Days 5-6: Open the lid for 2 hours during the daytime (8-10 AM, 12-2 PM, and 4-6 PM), and keep it covered the rest of the time. Adjust the glass greenhouse temperature to 26 ± 1°C during the day and 24 ± 1°C at night, ensuring a 2-3°C difference between day and night. Adjust the light intensity to between 5,000 and 7,000 lux, and use intermittent misting to maintain humidity between 80% and 90%.

[0119] Day 7-8: Open the lid completely, adjust the temperature of the glass greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 5000-7000lux, and use intermittent spray control to control the humidity between 70%-80%.

[0120] Day 9-10: Open the lid completely, adjust the temperature of the glass greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 7000-9000 lux, and use intermittent spray control to control the humidity between 60%-70%.

[0121] The transplanting steps and post-transplanting management are the same as steps (4) and (5) of Example 1.

[0122] The results showed that since the glass greenhouse is not a sterile environment, the seedlings are easily contaminated during hardening, resulting in a lower transplant survival rate of only 83.5%.

[0123] Comparative Example 2

[0124] During the same time period, the same batch of "Big Pepper" variety Phalaenopsis orchids were cultivated.

[0125] The difference from Example 1 is:

[0126] In the transplanting step (2), the sphagnum moss is treated using conventional methods:

[0127] Soak in 80℃ hot water for 30 minutes, then soak in cold water for 30 minutes, then dehydrate. The dehydration standard is: pinch the moss tightly with your hands and feel water dripping out.

[0128] Other management methods after hardening and transplanting are the same as in Example 1.

[0129] The results showed that the transplant survival rate was 82.0%. Because the sphagnum moss was not sterilized with high-pressure steam, the pathogens, insect eggs, and weed seeds in the sphagnum moss were not completely killed, and the incidence of pests and diseases was as high as 17.8%.

[0130] Comparative Example 3

[0131] During the same time period, the same batch of "Big Pepper" variety Phalaenopsis orchids were cultivated.

[0132] The difference from Example 1 is:

[0133] Step (5) Post-transplant management adopts conventional methods:

[0134] Environmental controls are as follows:

[0135] For the first 4 weeks, the light intensity is controlled at 7000-12000 lux, the temperature is 26±2℃ during the day and 23±1℃ at night, and the temperature difference between day and night is controlled at about 3℃ to promote rooting. Every day (except rainy days), spray clean water once at 9:00 and 11:00 in the morning and 13:00 and 15:00 in the afternoon to prevent leaf dehydration. From the 5th to the 8th week, the light intensity is controlled at 7000-12000 lux, the temperature is 26±2℃ during the day and 23±1℃ at night, and the temperature difference between day and night is controlled at ≥3℃ to promote rooting. After about 2 months, when more than 95% of the plants have grown new roots, the light intensity is increased to 12000-15000 lux and the normal management stage is entered.

[0136] Water and Fertilizer Management: After transplanting, spray a 1000-fold diluted solution of carbendazim and a 1000-fold diluted solution of kasugamycin to prevent pathogens. For the 20 days before transplanting, water only. Apply the first watering and fertilizing 20 days after transplanting. For the first two months, follow a watering and fertilizing cycle, watering once every two months. Use a fertilizer with an NPK (nitrogen-phosphorus-potassium) ratio of 10-30-20, with an EC value between 0.6-0.7 mS / cm. When mixing water and fertilizer, use filtered water with an EC value below 0.1 mS / cm.

[0137] Two months after transplanting, when new roots have grown, increase the EC value of water and fertilizer to 0.8-1.0mS / cm, and switch to a balanced fertilizer with an NPK ratio of 20-20-20;

[0138] When the sphagnum moss in the pot is dry and feels light but still heavy when you pick it up, it is the best time to water and fertilize.

[0139] Do not water or fertilize on rainy days to avoid oxygen deficiency in the roots;

[0140] Pest control: Spray insecticides and fungicides every two weeks. The insecticides are 1000-fold diluted buprofezin and 1000-fold diluted abamectin, and the fungicides are 1000-fold diluted carbendazim and 1000-fold diluted kasugamycin, which are sprayed separately.

[0141] Ten days after transplanting, the plants were checked daily for growth, disease incidence, and the date of new root growth. Thirty days later, the survival rate was calculated, and the results showed that the transplant survival rate of the "Big Pepper" variety was 86.5%.

[0142] Compared with Example 1, new root tips were visible 24 days after transplanting, and the occurrence of new roots was delayed by 9 days compared with Example 1.

[0143] Comparative Example 4

[0144] During the same time period, the same batch of "Big Pepper" variety Phalaenopsis orchids were cultivated using conventional methods.

[0145] (1) Using a positive pressure sterile greenhouse, the conventional seedling hardening method is:

[0146] Transport the bottle seedlings to the glass greenhouse for hardening. The specific operations are as follows:

[0147] Day 1-2: Do not open the bottle cap, control the temperature of the glass greenhouse between 26±1℃ during the day and 24±1℃ at night, and ensure that the temperature difference between day and night is between 2-3℃; adjust the light intensity to between 3000-5000lux.

[0148] Day 3-4: Do not open the bottle caps, control the temperature of the glass greenhouse between 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 5000-7000lux.

[0149] Days 5-6: Open the lid for 2 hours during the daytime (8-10 AM, 12-2 PM, and 4-6 PM), and keep it covered the rest of the time. Adjust the glass greenhouse temperature to 26 ± 1°C during the day and 24 ± 1°C at night, ensuring a 2-3°C difference between day and night. Adjust the light intensity to between 5,000 and 7,000 lux, and use intermittent misting to maintain humidity between 80% and 90%.

[0150] Day 7-8: Open the lid completely, adjust the temperature of the glass greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 5000-7000lux, and use intermittent spray control to control the humidity between 70%-80%.

[0151] Day 9-10: Open the lid completely, adjust the temperature of the glass greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 7000-9000 lux, and use intermittent spray control to control the humidity between 60%-70%.

[0152] (2) Planting substrate treatment:

[0153] The cultivation medium is sphagnum moss. Soak it in 80℃ hot water for 30 minutes and then in cold water for 30 minutes. Then dehydrate it. The dehydration standard is: squeeze the sphagnum moss tightly with your hands and feel water dripping out.

[0154] (3) After 10 days of hardening, transplanting is carried out:

[0155] Seedling selection criteria: Select healthy seedlings with normal leaf color, 3-5 leaves, and more than 2 aerial roots;

[0156] Operation method: Use clips to remove the seedlings from the bottle and wash away the culture medium remaining on the roots with clean water;

[0157] Planting method: Loosen the sphagnum moss with your hands, place the roots of the plant in the middle of the sphagnum moss, then wrap the roots with the loosened sphagnum moss and plant them in a 1.5-inch soft pot; then place it in a greenhouse environment for management.

[0158] When wrapping the plant with sphagnum moss, make sure the roots are completely covered with sphagnum moss, which can provide good moisture retention and support for the roots.

[0159] When planting, place the plant wrapped in sphagnum moss in a 1.5-inch soft pot. The soft pot usually has three ring lines. When placing the plant in the soft pot, make sure that the roots of the plant and the wrapped sphagnum moss are below the three ring lines of the soft pot. This step is to ensure that the plant can grow stably in the pot and prevent the roots from being exposed to the air.

[0160] After placing the plant in the soft pot, use your thumb to gently press the sphagnum moss around the pot. The purpose of compaction is to ensure that the sphagnum moss is in close contact with the roots and provide stable support for the plant.

[0161] During the compaction process, be careful to use moderate force to ensure that the sphagnum moss is in close contact with the roots, but avoid pressing too tightly to prevent affecting the breathing and growth of the roots.

[0162] The final effect is that the sphagnum moss below the roots is slightly loose so that the roots can grow freely; while the sphagnum moss around the soft pot should be compacted to provide stable support.

[0163] If the seedlings in the bottle are too small, they can be discarded or planted in a 128-cell hole tray. Larger seedlings in the bottle can be planted directly in a 1.5-inch soft pot.

[0164] (4) Management after transplanting: Environmental control is as follows:

[0165] For the first four weeks, control the light intensity at 7000-12000 lux, the temperature at 26±2℃ during the day and 23±1℃ at night, and control the temperature difference between day and night to about 3℃ to promote rooting. Every day (except rainy days), spray the leaves with clean water once at 9:00 and 11:00 in the morning and 13:00 and 15:00 in the afternoon to prevent dehydration. From the 5th to the 8th week, control the light intensity at 7000-12000 lux, the temperature at 26±2℃ during the day and 23±1℃ at night, and control the temperature difference between day and night to ≥3℃ to promote rooting. After about 2 months, when more than 95% of the plants have grown new roots, increase the light intensity to 12000-15000 lux and enter the normal management stage.

[0166] Water and Fertilizer Management: After transplanting, spray a 1000-fold diluted solution of carbendazim and a 1000-fold diluted solution of kasugamycin every other day to prevent pathogens. For the 20 days before transplanting, water only. Apply the first watering and fertilizing 20 days after transplanting. For the first two months, follow a watering and fertilizing cycle, watering once every two months. Use a fertilizer with an NPK (nitrogen-phosphorus-potassium) ratio of 10-30-20, with an EC value between 0.6-0.7 mS / cm. When mixing water and fertilizer, use filtered water with an EC value below 0.1 mS / cm.

[0167] Two months after transplanting, when more than 95% of the plants have grown new roots, the EC value of water and fertilizer is increased to 0.8-1.0mS / cm, and the fertilizer is changed to a balanced fertilizer with an NPK ratio of 20-20-20;

[0168] When the sphagnum moss in the pot is dry and feels light but still heavy when you pick it up, it is the best time to water and fertilize.

[0169] Do not water or fertilize on rainy days to avoid oxygen deficiency in the roots;

[0170] Pest control: Spray insecticides and fungicides every two weeks. The insecticides are 1000-fold diluted buprofezin and 1000-fold diluted abamectin, and the fungicides are 1000-fold diluted carbendazim and 1000-fold diluted kasugamycin, which are sprayed separately.

[0171] Ten days after transplanting, check the growth status of the plants every day, observe the incidence of diseases, and record the date of new root growth.

[0172] After 30 days, survival rates were calculated, revealing a 73.5% survival rate for the "Big Pepper" variety, while the incidence of pests and diseases reached 27.6%. Because conventional methods don't utilize a sterile greenhouse, tissue culture seedlings are susceptible to contamination during hardening. Furthermore, the sphagnum moss is not sterilized with high-pressure steam, which can lead to pests and diseases. Consequently, the transplant survival rate dropped significantly.

Claims

1. A method for improving the survival rate of Phalaenopsis tissue culture seedlings after transplanting out of bottles, characterized in that The method comprises the following steps: (1) Prepare a site for hardening seedlings: Use intelligent positive pressure sterile greenhouse as the seedling hardening place, and the fresh air entering the positive pressure sterile greenhouse has an air purification level of ISO5; (2) Planting substrate treatment: The cultivation medium sphagnum moss is first soaked in cold water and then dehydrated; after dehydration, it is sterilized with high-pressure steam for 25 to 30 minutes; (3) The bottled seedlings are placed in a positive pressure sterile greenhouse for hardening: The seedlings are hardened in a gradient manner, as follows: Day 1-2: Do not open the bottle cap, and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a day-night temperature difference of 2-3℃; adjust the light intensity to between 3000-5000 lux, the humidity to between 60%-70%, and the positive pressure difference to between 10-15Pa; Day 3-4: Do not open the bottle caps. Adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night. Adjust the light intensity to 5000-7000 lux, humidity to 60%-70%, and positive pressure difference to 10-15 Pa. Day 5-6: Open the lid completely, adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to 5000-7000 lux, humidity to 80%-90%, and positive pressure difference to 10-15 Pa; Day 7-8: Open the lid completely and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to 5000-7000 lux, humidity to 70%-80%, and positive pressure difference to 10-15 Pa; Day 9-10: Open the lid completely and adjust the temperature of the positive pressure sterile greenhouse to 26±1℃ during the day and 24±1℃ at night, ensuring a temperature difference of 2-3℃ between day and night; adjust the light intensity to between 7000-9000 lux, the humidity to between 60%-70%, and the positive pressure difference to between 10-15Pa; (4) Harden the seedlings for 10 days and then transplant them: Select healthy seedlings with normal leaf color, 3-5 leaves, and more than 2 aerial roots; use high-pressure steam sterilized sphagnum moss as the substrate, transplant the hardened plants into 1.5-inch soft pots, and then place them in a greenhouse environment for management; (5) Management after transplanting: Greenhouse environment control is as follows: Light intensity: Control the light intensity at 5000-7000 lux in the first 4 weeks, and increase it to 7000-10000 lux in the 5th to 8th weeks; after 8 weeks, when new roots begin to grow, increase the light intensity to 12000-15000 lux; Temperature: 26±2℃ during the day / 23±1℃ at night, control the temperature difference between day and night to be ≥3℃; Humidity: Use intermittent spraying to control RH 70-75%; Water and fertilizer management: The water quality standard for preparing water and fertilizer is EC content below 0.38mS / cm; After transplanting, spray the plants with fungicide, leave them for 4-7 days, and then apply water and fertilizer. Use fertilizer with a N:P:K ratio of 10:30:20, and adjust the EC value of water to between 0.6-0.7mS / cm as water and fertilizer. 1-2 months after transplanting, change the fertilizer to a balanced fertilizer with an N:P:K ratio of 20:20:20, and increase the EC value of water and fertilizer to 0.8-1.0mS / cm.

2. The method according to claim 1, wherein In step (3), 3 hours before the bottle seedlings enter the positive pressure sterile greenhouse, the greenhouse is first irradiated with ultraviolet light for 30 minutes, then disinfected with ozone for 30 minutes, and then the ventilation system is turned on, and the temperature, humidity and light are adjusted in advance to the environmental conditions required for seedling hardening.

3. The method according to claim 1, wherein The positive pressure sterile greenhouse is equipped with a positive pressure buffer room. Personnel and materials first enter the positive pressure buffer room for 75% alcohol disinfection and then enter the positive pressure sterile greenhouse. The absolute positive pressure difference of the positive pressure sterile greenhouse is adjusted to 10-15Pa.

4. The method according to claim 1, wherein In the step (2), the cold water soaking is to soak the sphagnum moss in cold water for 2 to 3 times, each time for 30 to 40 minutes.

5. The method according to claim 1, wherein In step (2), the conditions for high-pressure steam sterilization are: high-pressure steam temperature is 120-125°C, and sterilization takes 25-30 minutes.

6. The method according to claim 1, wherein In step (4), the transplanting operation method is: Use clips to remove the seedlings from the bottle and wash away the culture medium remaining on the roots with clean water; After high-pressure steam sterilization, cool the sphagnum moss to room temperature; loosen the sphagnum moss with your hands, place the roots of the plant in the middle of the sphagnum moss, then wrap the roots with the loosened sphagnum moss and plant them in a 1.5-inch soft pot.

7. The method according to claim 1, wherein In step (5), the water quality standard for preparing the water fertilizer is an EC content of less than 0.1mS / cm.

8. The method according to claim 1, wherein In the step (5), when the sphagnum moss is dry, water and fertilizer are applied, with a frequency of once every 6 to 12 days; no water and fertilizer are applied on rainy days.

9. The method according to claim 1, wherein In the step (5), the insecticide and fungicide are sprayed once every half a month.

10. The method according to claim 1, wherein In step (5), 1-2 months after transplanting, more than 95% of the tissue culture seedlings grow new roots, and the fertilizer is changed to a balanced fertilizer with an N:P:K ratio of 20:20:20.