A high-efficiency seedling raising method for improving survival rate of trichosanthes kirilowii tissue culture seedlings
Patent Information
- Application Number
- CN202611141120.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-30
- Publication Date
- 2026-10-30
AI Technical Summary
[0026]本发明通过科学筛选最佳苗龄、精准优化基质配方以及集成化设施管理，使栝楼组培苗移栽成活率最高达91%、有效苗率达86%，操作简便、成本可控，适合规模化推广。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of Trichosanthes kirilowii seedling cultivation, specifically relating to an efficient seedling cultivation method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings. Background Technology
[0002] Trichosanthes kirilowii Maxim., commonly known as hanging gourd, is a perennial herbaceous vine belonging to the genus Trichosanthes in the Cucurbitaceae family. Its fruit, seeds, and rhizomes can all be used medicinally, possessing properties such as clearing heat and resolving phlegm, relieving chest congestion, and promoting bowel movements. Meanwhile, Trichosanthes seeds (hanging gourd seeds) are popular as a high-end snack due to their crisp texture and rich nutrition, with demand increasing year by year. With the continuous expansion of the Trichosanthes industry, the limitations of traditional seedling cultivation methods are becoming increasingly apparent. Currently, Trichosanthes propagation mainly relies on sowing and root division. Sowing and seedling raising presents challenges due to the thick and hard seed coat of Trichosanthes kirilowii seeds, resulting in extremely uneven germination and the inability to distinguish between male and female plants before flowering (improper matching of male and female plants severely affects yield; currently, a male-to-female plant ratio of 15:1 is commonly used in planting). Furthermore, the seedling raising cycle is long, and the offspring of seedlings exhibit significant variation, making it difficult to guarantee varietal consistency and the stable inheritance of superior commercial traits. While root division can maintain varietal characteristics, it has an extremely low propagation coefficient. Long-term asexual reproduction can also easily lead to virus accumulation and seed degeneration, and consumes a large amount of rhizome raw materials, making it difficult to meet the urgent demand for large-scale, standardized planting of high-quality seedlings.
[0003] Plant tissue culture technology has gradually become an important method for the propagation of Trichosanthes kirilowii seedlings due to its significant advantages, such as rapid propagation, virus-free and rejuvenating capabilities, high preservation of the superior traits of the parent plant, and year-round industrialized production without seasonal restrictions. However, in the industrial application of Trichosanthes kirilowii tissue culture, the process of transplanting tissue-cultured seedlings from a sterile, constant-temperature, and high-humidity culture environment to the external natural environment has become a serious technical bottleneck restricting its widespread application. Tissue culture seedlings of Trichosanthes kirilowii generally face the following prominent problems during the transplanting period: On the one hand, tissue culture seedlings are in a heterotrophic or commensal state for a long time, resulting in incomplete development of the cuticle and wax layer of the leaf epidermis, poor stomatal regulation ability, and easy rapid water loss and wilting after transplanting, making them weak in adapting to the external environment; on the other hand, the roots induced during the rooting stage of tissue culture seedlings are often not vigorous, with sparse or absent root hairs, poor nutrient absorption capacity, long recovery period after transplanting, and difficulty in the development of new roots; in addition, if the seedling conditions such as transplanting substrate, water and fertilizer management, light and humidity control are not properly controlled, it is easy to cause diseases such as root rot and stem base rot, leading to a large number of tissue culture seedling deaths.
[0004] Currently, research on improving the survival rate of transplanted plant tissue culture seedlings mainly focuses on optimizing single aspects, such as substrate formulation or single hormone treatment. There is a lack of systematic seedling cultivation methods that address the biological characteristics of *Trichosanthes kirilowii* and the synergistic regulation of multiple factors during transplanting. In particular, existing technologies lack scientific basis for selecting the transplanting age, resulting in significant differences in survival and effective seedling rates among tissue culture seedlings of different ages. Furthermore, the mechanism by which the precise formulation of the transplanting substrate (especially the ratio of organic fertilizer to calcium magnesium phosphate fertilizer) affects the survival rate of *Trichosanthes kirilowii* tissue culture seedlings remains unclear; excessive fertilization can severely inhibit seedling growth. In addition, the synergistic optimization of the rhizosphere microenvironment and aboveground growth environment after transplanting (such as temperature and humidity control, light management, and the configuration of automatic sprinkler systems) also lacks systematic research and practical verification specifically for *Trichosanthes kirilowii* tissue culture seedlings.
[0005] Given the critical bottleneck of generally low survival rates of Trichosanthes kirilowii tissue culture seedlings after transplanting, developing a highly efficient seedling cultivation method that can systematically cover the optimal seedling age selection before transplanting, precise substrate formulation, and synergistic optimization of the rhizosphere microenvironment and aboveground growth environment after transplanting is of great practical significance for overcoming obstacles to the industrial application of Trichosanthes kirilowii tissue culture seedlings, ensuring an efficient supply of high-quality seedlings, and promoting the sustainable and healthy development of the Trichosanthes kirilowii industry. Summary of the Invention
[0006] The purpose of this invention is to provide an efficient seedling raising method to improve the survival rate of Trichosanthes kirilowii tissue culture seedlings.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0008] A highly efficient seedling cultivation method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings includes the following steps:
[0009] (1) Seedling selection: Select Trichosanthes kirilowii tissue culture seedlings that have been rooted for 45-60 days, and remove weak seedlings, diseased seedlings and individuals with poor growth;
[0010] (2) Substrate preparation and disinfection: Mix the seedling substrate, bio-organic fertilizer and calcium magnesium phosphate fertilizer in a mass ratio of 24:4:0~4 and sterilize them.
[0011] (3) Determining the seedling time: Seedling raising operations shall be carried out from late February to early March;
[0012] (4) Seedling facility construction and environmental control: A seedling facility system is built in the greenhouse. The seedling facility system includes a bottom insulation and heating system, seedling containers and small arched sheds, shading system, supplemental lighting system and spray system to precisely control the temperature, light and humidity during the seedling process.
[0013] In step (1), the Trichosanthes kirilowii tissue culture seedlings are obtained by the following method: using healthy, disease-free stem segments of Trichosanthes kirilowii from the current year as explants, after disinfection, they are inoculated into a basal culture medium for primary culture to obtain sterile initiating buds; the obtained sterile initiating buds are cut into single-bud stem segments and inoculated into a proliferation culture medium for proliferation culture to obtain cluster buds; the obtained cluster buds are cut into single buds and inoculated into a rooting culture medium for rooting culture to obtain the Trichosanthes kirilowii tissue culture seedlings.
[0014] In step (1), the optimal seedling age of the Trichosanthes kirilowii tissue culture seedlings is 45 days after rooting culture.
[0015] In step (2), the optimal mass ratio of the seedling substrate, bio-organic fertilizer and calcium magnesium phosphate fertilizer is 24:4:1.
[0016] In step (4), the method for constructing the bottom insulation and heating system is as follows: a black mulch film is laid on the greenhouse floor to divide the seedling beds; two layers of geotextile are laid on the seedling bed surface, and heating wires are laid between the two layers of geotextile. The heating wires are arranged back and forth at 15cm intervals, and isolation materials are set between adjacent heating wires; a temperature control device with a temperature probe is installed at each seedling bed head, and the start and stop temperature threshold of the temperature control device is set to 10℃.
[0017] In step (4), the method of using the seedling container is as follows: place seedling trays on the seedling bed and arrange black nutrient bags in the seedling trays; fill the black nutrient bags with the sterilized and cooled substrate, and fill the substrate to a height of 2-5 mm from the bag opening; when transplanting, make a hole in the center of the substrate, place the roots of the tissue culture seedling in the hole, backfill the substrate and compact it, and the transplanting depth is such that the base of the tissue culture seedling roots is level with the surface of the substrate. After transplanting, water thoroughly to settle the roots.
[0018] In step (4), the method for building and managing the small arched shed is as follows: fiber rods are laid out at intervals on the seedbed and bent to form an arch frame, and a transparent heat-insulating film is covered on the outside of the arch frame to form a small arched shed;
[0019] Two weeks before seedling cultivation, open the insulation film on both sides of the small arched shed for ventilation at 9:00 am every day. After ventilation for 1 hour, cover the insulation film back.
[0020] Two weeks after the seedlings are established, open the insulation film on both sides of the small arched greenhouse at 9:00 a.m. every day for ventilation, while keeping the top insulation film closed. At 4:00 p.m., decide whether to close the insulation film tightly based on the forecast of the lowest temperature at night: if the forecast of the lowest temperature at night is not lower than 15°C, keep the ventilation on both sides open; if the forecast of the temperature at night is lower than 15°C, close the insulation film tightly.
[0021] In step (4), the management method of the shading, supplemental lighting and sprinkler system is as follows:
[0022] Shading management: Use shade nets with a shading rate of 60%; cover the shade nets all day for the first three weeks of seedling cultivation; from the fourth week of seedling cultivation, cover the shade nets from 10:00 am to 4:00 pm on sunny days, and leave them open during the rest of the time; do not cover the shade nets at all during cloudy or rainy weather; remove the shade nets after the sixth week of seedling cultivation; the shade nets will automatically close when the temperature inside the greenhouse exceeds 30℃.
[0023] Supplemental lighting management: per 8m 2 A standard, evenly spaced array of 100W full-spectrum LED supplementary lights is installed and turned on when natural light is insufficient due to continuous cloudy or rainy weather. The supplementary lighting period is from 6:00 AM to 6:00 PM daily.
[0024] Spraying management: Spray 5 times a day at even intervals during the first week of seedling cultivation. From the second week onwards, spray 3 times a day at even intervals, completing the spraying during the day. The standard for stopping a single spraying is when the substrate moisture reaches 70%. In cloudy or rainy weather, when the air humidity reaches 85% or above, spray only once a day at 6 pm, also with the standard of when the substrate moisture reaches 70%.
[0025] The significant advantages of this invention are:
[0026] This invention achieves a survival rate of up to 91% and an effective seedling rate of 86% for tissue culture seedlings of Trichosanthes kirilowii through scientific selection of the optimal seedling age, precise optimization of substrate formula, and integrated facility management. It is easy to operate, cost-effective, and suitable for large-scale promotion. Attached Figure Description
[0027] Figure 1 Example 1 describes the initial seedling condition during transplanting.
[0028] Figure 2 Seedling condition 30 days after transplanting using the method described in Example 1. Detailed Implementation
[0029] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.
[0030] The seedling substrate used in this invention is a mixture of peat moss and perlite in a volume ratio of 4:1.
[0031] The registration certificate number of the bio-organic fertilizer used in this invention is Microbial Fertilizer (2018) Approval No. (6271).
[0032] The effective component content of the calcium magnesium phosphate fertilizer used in this invention is: P2O5≥12%, CaO≥20%, MgO≥4%.
[0033] The Trichosanthes kirilowii tissue culture seedlings used in this invention were cultivated according to the methods disclosed in the prior art (Rao Baorong, Chen Yonghe, Liu Zhonghui, et al. Research on rapid propagation system of Trichosanthes kirilowii tissue culture [J]. Tropical Agricultural Science, 2020, 40(3): 55-60.), and the steps are as follows:
[0034] (1) Explant collection, disinfection and primary culture
[0035] Healthy, disease-free stem segments of *Trichosanthes kirilowii* (from the current year's growth) were collected. First, they were rinsed with running water, then soaked in a suitable laundry detergent solution for 30 minutes, rinsed again, and transferred to a clean bench. Leaves were removed, and the segments were trimmed into 3-5 cm segments with nodes. The segments were first disinfected by soaking in 75% alcohol for 15 seconds, rinsed twice with sterile water, then disinfected with 0.1% mercuric chloride solution for 8 minutes, and rinsed 6 times with sterile water. Both ends were removed, and the segments were cut into 1-1.5 cm segments with nodes. These segments were then inoculated onto induction medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sugar + 7.8 g / L carrageenan, pH 5.8) for primary culture to obtain sterile initiating shoots. The primary culture conditions were: temperature 25±1℃, light intensity 2000 lx, photoperiod 12 h / d, and culture time 20 days.
[0036] (2) Proliferation culture
[0037] The aseptic initiating buds obtained in step (1) were cut into single-bud stem segments of approximately 1 cm in length and inoculated into proliferation medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L white sugar + 7.8 g / L carrageenan, pH 5.8) for proliferation culture to obtain cluster buds. The proliferation culture conditions were: temperature 25 ± 1℃, light intensity 2000 lx, photoperiod 12 h / d, and culture time 40 days.
[0038] (3) Rooting culture
[0039] Select the clustered shoots obtained from the proliferation culture in step (2), cut off single shoots about 1 cm long, and inoculate them into rooting medium (MS + 0.6 mg / L NAA + 0.6 mg / L IBA + 20 g / L white sugar + 7.8 g / L carrageenan, pH 5.8) for rooting culture to obtain Trichosanthes kirilowii tissue culture seedlings. The rooting culture conditions are: temperature 25±1℃, light intensity 2500 lx, light duration 12 h / d, and culture time 45~60 days.
[0040] Example 1:
[0041] A highly efficient seedling cultivation method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings includes the following steps:
[0042] (1) Seedling selection
[0043] Trichosanthes tissue culture seedlings that have undergone rooting culture for 45 days were selected as seedling materials. Before transplanting, all tissue culture seedlings need to be inspected one by one. Priority should be given to selecting healthy individuals that are free from pests and diseases, have dark green leaves, thick stems, and well-developed root systems. Weak seedlings, diseased seedlings, and individuals with poor growth should be removed to ensure that the seedlings entering the next stage are of uniform quality and vigorous.
[0044] (2) Matrix preparation and sterilization
[0045] Add organic fertilizer and calcium magnesium phosphate fertilizer in a mass ratio of 24:4:1 (seedling substrate, bio-organic fertilizer, and calcium magnesium phosphate fertilizer), and mix thoroughly. After preparation, sterilize the substrate with high-temperature steam to completely kill any pathogens and insect eggs it may carry. Allow the substrate to cool naturally to room temperature before use.
[0046] (3) Determining the seedling raising time
[0047] The seedling cultivation operation will be carried out in Nanping City, Fujian Province, from late February to early March 2026. During this period, the temperature will gradually rise but will not yet reach the high-temperature period, which will help the tissue culture seedlings slowly adapt to the external environment after transplanting, reduce the physiological stress caused by drastic temperature fluctuations, and thus improve the survival rate.
[0048] (4) Construction of seedling facilities and environmental control
[0049] To achieve precise control of key environmental factors such as temperature, light, and humidity, an integrated seedling cultivation facility system is built inside the greenhouse, which includes the following steps.
[0050] ① Bottom layer insulation and heating system
[0051] First, a layer of black mulch is laid on the ground inside the greenhouse to retain heat and moisture and inhibit weed growth. Then, seedling beds are divided according to the mulch coverage area, with a bed width of 185cm and a length between 20-30m, which can be adjusted flexibly according to the actual space in the greenhouse. Two layers of geotextile are laid on the bed surface, and heating wires are laid between the two layers of geotextile. The heating wires are arranged back and forth along the fixed poles at the head of the bed at 15cm intervals to ensure uniform temperature distribution across the entire bed surface. To prevent wear and leakage caused by contact between the heating wires, 15mm thick wooden strips are embedded between adjacent heating wires as insulation. A temperature control device with a temperature probe is installed at each bed head, and the start / stop threshold is set to 10℃. When the ambient temperature drops below 10℃ at night, the temperature control device automatically starts heating; when the temperature rises back above 10℃, it automatically stops heating, thus keeping the seedbed temperature stable within the suitable range for the growth of Trichosanthes kirilowii tissue culture seedlings.
[0052] ② Seedling containers, substrate filling and transplanting
[0053] Four 43.5cm square black seedling trays are placed side-by-side along the width of each seedling bed. In each tray, a black nutrient bag with a 7cm diameter opening is placed neatly in 7 rows by 7 columns. The purpose of using black nutrient bags is to utilize their excellent light-blocking properties to create a dark environment for root growth, thereby promoting root development.
[0054] Fill the sterilized and cooled substrate into the nutrient bags, with the filling height about 2-5 mm from the bag opening. After filling, gently compact the substrate to maintain a moderate density, ensuring both air permeability and water retention capacity.
[0055] After filling the substrate, immediately proceed with the transplanting. Use a bamboo skewer or thin stick to vertically poke a small hole, approximately 1-1.5 cm deep, in the center of the substrate in the nutrient bag. Then, carefully place the roots of the selected Trichosanthes kirilowii tissue culture seedlings into the hole, ensuring the roots are spread out and not directly adhered to the substrate over a large area. Next, fill the hole with substrate and gently press around the roots to ensure close contact between the roots and the substrate. The transplanting depth should be such that the base of the original tissue culture seedling is level with the surface of the substrate; it should not be too deep to avoid burying the growth point, nor too shallow to expose the roots. After transplanting, immediately water thoroughly, a process known as "settling-in watering," to ensure full contact between the roots and the substrate, eliminate gaps in the substrate, and lay the foundation for subsequent temperature, light, water, and air regulation.
[0056] ③ Small arched sheds and ventilation management
[0057] In each seedling bed, a fiber pole is inserted every 2 meters, bent to form an arch, and covered with a transparent insulating film to create a small arched shed. This is used for phased, differentiated opening and closing of the shed for ventilation, adapting to the growth needs of the tissue culture seedlings. The first two weeks are a critical period for seedling establishment, focusing on heat preservation and moisture retention. Every morning at 9:00 AM, the insulating film on both sides of the small arched shed is opened for ventilation. After one hour of ventilation, the insulating film is promptly covered back to maintain warmth and prevent seedling dehydration and temperature stress damage. After two weeks of seedling establishment, the insulating film on both sides of the small arched shed is opened for ventilation every morning at 9:00 AM, while the top insulating film remains intact to effectively block rainwater, prevent excessive humidity and disease, and ensure ventilation. At 4:00 PM, the decision to cover the shed tightly for warmth is based on the forecast of the nighttime minimum temperature: if the forecast minimum temperature is not lower than 15℃, there is no need to cover the shed; ventilation on both sides is maintained to continuously strengthen the seedlings' resistance. If the forecast nighttime temperature is lower than 15℃, the insulating film is covered tightly in advance to prevent frost damage to the seedlings.
[0058] ④ Shading, supplemental lighting and sprinkler system
[0059] Shading nets, LED supplemental lighting units, and automatic sprinkler systems are installed 2 meters above each bed, along with light, temperature, and humidity control systems. The shading nets used provide 60% shading. During the first three weeks of seedling cultivation, the tissue culture seedlings are immature and have weak tolerance to strong light. Therefore, they are covered with 60% shading nets all day to effectively block strong light, prevent scorching of tender leaves, and ensure the seedlings recover smoothly.
[0060] Starting from the fourth week of seedling cultivation, the seedlings officially enter the hardening-off stage under light conditions. Intermittent shading helps the tissue-cultured seedlings gradually adapt to the strong natural light environment, preventing wilting and scorching caused by sudden changes in light: on sunny days, 60% shade netting is used during the hottest and most intense light period from 10:00 AM to 4:00 PM, while the seedlings are left uncovered during the rest of the day; on cloudy or rainy days, no shade netting is used, fully utilizing natural diffused light to promote seedling growth. After the sixth week of seedling cultivation, the hardening-off stage is completed, the shade netting is removed, and full-light cultivation is implemented. Simultaneously, temperature-controlled shading is used for auxiliary regulation; when the temperature inside the greenhouse exceeds 30℃, the shade netting automatically closes to assist in cooling and avoid high-temperature stress.
[0061] If two or more consecutive days of overcast or rainy weather result in insufficient natural sunlight to meet the photosynthetic needs of seedlings, promptly turn on the LED supplemental lighting system. Supplemental lighting equipment should be installed at intervals of 8 meters. 2 A standard, evenly spaced 100W full-spectrum LED supplemental light is installed, and the light is turned on from 6 a.m. to 6 p.m. every day to continuously supplement the light and ensure the photosynthetic growth of the seedlings.
[0062] The sprinkler system regulates the humidity and substrate moisture content of the seedling environment. Management guidelines are as follows: Spray 5 times daily at even intervals during the first week of seedling cultivation; from the second week onwards, spray 3 times daily at even intervals, completing all spraying operations during the day. Each spraying session ends when the substrate moisture content reaches 70%. During cloudy or rainy weather, reduce the spraying frequency according to air humidity. When the air humidity reaches 85% or higher during cloudy or rainy weather, spray only once daily at 6 PM, again stopping at 70% substrate moisture content. This avoids excessive spraying that can lead to high substrate moisture, root rot, and disease growth, thus maintaining stable and suitable substrate moisture and environmental conditions for seedling growth.
[0063] Example 2: Effects of different seedling ages on the survival rate and effective seedling rate of Trichosanthes kirilowii tissue culture seedlings
[0064] This embodiment aims to explore the effects of different ages of Trichosanthes kirilowii tissue culture seedlings on seedling survival rate and effective seedling rate, so as to determine the optimal transplanting seedling age.
[0065] (1) Experimental Design
[0066] Four treatment groups were established by selecting Trichosanthes kirilowii tissue culture seedlings that had undergone rooting culture for 45, 50, 55, and 60 days, respectively. Each treatment group consisted of three trays, with 49 seedlings planted in each tray, and the results were replicated three times. All tissue culture seedlings were selected according to the seedling screening criteria described in Example 1, and weak, diseased, and poorly growing seedlings were removed.
[0067] (2) Seedling raising methods
[0068] The seedling cultivation methods for each group of tissue culture seedlings were the same as in Example 1, including substrate preparation and disinfection, seedling time arrangement, seedling facility construction and environmental control, etc., to eliminate interference from other factors on the experimental results.
[0069] (3) Survey indicators
[0070] Fifty days after transplanting, the tissue culture seedlings of each treatment group were surveyed and statistically analyzed, and the following two indicators were recorded:
[0071] Survival rate (%) = (Number of surviving plants / Number of plants planted) × 100%
[0072] Effective seedling rate (%) = (Number of surviving plants with ≥3 leaves and ≥5cm height / Number of plants planted) × 100%
[0073] The effects of different seedling ages on the survival rate and effective seedling rate of Trichosanthes kirilowii tissue culture seedlings are shown in Table 1.
[0074] Table 1. Effects of different seedling ages on seedling survival rate of tissue culture seedlings
[0075]
[0076] Table 1 shows that the survival rate and effective seedling rate both decreased with increasing seedling age. The highest survival rate (91%) and effective seedling rate (86%) were observed in tissue culture seedlings aged 45 days. Conversely, the lowest survival rate (71%) and effective seedling rate (52%) were observed in tissue culture seedlings aged 60 days. Therefore, under the conditions of this experiment, *Trichosanthes kirilowii* tissue culture seedlings that have undergone rooting culture for 45 days are most suitable for transplanting.
[0077] Example 3: Effect of different substrate ratios on the survival rate of Trichosanthes kirilowii tissue culture seedlings
[0078] This embodiment aims to investigate the effects of different addition ratios of organic fertilizer and calcium magnesium phosphate fertilizer in the substrate on the survival rate of Trichosanthes kirilowii tissue culture seedlings, in order to screen the optimal substrate formula.
[0079] (1) Experimental Design
[0080] Trichosanthes kirilowii tissue culture seedlings that had been rooted for 45 days were used as seedling material. Based on this, five different substrate ratio treatment groups were set up. The mass ratios of seedling substrate, bio-organic fertilizer, and calcium magnesium phosphate fertilizer in each treatment group are as follows:
[0081] H0: 24∶4∶0
[0082] H1: 24∶4∶1
[0083] H2: 24:4:2
[0084] H3: 24:4:3
[0085] H4: 24∶4∶4
[0086] Each treatment group consisted of 3 trays, with 49 seedlings planted in each tray, and the results were replicated 3 times. All tissue culture seedlings were selected according to the seedling screening criteria described in Example 1, and weak, diseased, and poorly growing seedlings were removed.
[0087] (2) Seedling raising methods
[0088] The seedling cultivation methods for each group of tissue culture seedlings were the same as in Example 1, including the seedling cultivation time arrangement, seedling facility construction, and environmental control steps, which were kept consistent. The only difference between the treatment groups was the substrate ratio, in order to eliminate the interference of other factors on the experimental results.
[0089] (3) Survey indicators
[0090] Fifty days after transplanting, the survival rate of tissue culture seedlings in each treatment group was investigated and statistically analyzed. Survival rate (%) = (number of surviving seedlings / number of transplanted seedlings) × 100%.
[0091] The effects of different substrate ratios on the survival rate of Trichosanthes kirilowii tissue culture seedlings are shown in Table 2.
[0092] Table 2. Effects of different substrate ratios on the survival rate of Trichosanthes kirilowii tissue culture seedlings
[0093]
[0094] Table 2 shows that different substrate ratios significantly affected the survival rate of *Trichosanthes kirilowii* tissue culture seedlings. The H1 treatment group (seedling substrate: bio-organic fertilizer: calcium magnesium phosphate fertilizer = 24:4:1) had the highest survival rate, reaching 91%, significantly higher than other treatment groups. The H0 treatment group (without calcium magnesium phosphate fertilizer) had a survival rate of 63%, lower than H1 but higher than the other treatment groups. With further increases in the proportion of calcium magnesium phosphate fertilizer, the survival rate dropped sharply, with the survival rates of the H3 and H4 treatment groups being only 10% and 6%, respectively. The results indicate that appropriate addition of calcium magnesium phosphate fertilizer (seedling substrate: bio-organic fertilizer: calcium magnesium phosphate fertilizer = 24:4:1) is beneficial to improving the transplant survival rate of *Trichosanthes kirilowii* tissue culture seedlings, while excessive addition has a significant inhibitory effect.
[0095] Example 4: Effects of different facility management on the survival rate and growth of Trichosanthes kirilowii tissue culture seedlings
[0096] This embodiment aims to explore the impact of different facility management conditions on the survival rate and growth status of Trichosanthes kirilowii tissue culture seedlings, so as to optimize the seedling facility configuration scheme.
[0097] (1) Experimental Design
[0098] Using *Trichosanthes kirilowii* tissue culture seedlings that had undergone rooting culture for 45 days as the seedling material, the substrate formula adopted the optimized ratio from Example 1, namely, a mass ratio of seedling substrate, bio-organic fertilizer, and calcium magnesium phosphate fertilizer of 24:4:1. Based on this, five different facility management treatment groups were set up. The differences between the treatment groups in terms of whether they were equipped with automatic sprinklers, geothermal heating, small arched greenhouse coverings, and supplemental lighting are as follows:
[0099] A1: Equipped with automatic sprinklers, underfloor heating, small arched greenhouse cover, and supplemental lighting.
[0100] A2: No automatic sprinkler system, but with underfloor heating, small arched greenhouse cover, and supplemental lighting.
[0101] A3: Equipped with automatic sprinklers, no underfloor heating, small arched greenhouse cover, and supplemental lighting.
[0102] A4: Equipped with automatic sprinklers, underfloor heating, no small arched greenhouse cover, and supplemental lighting.
[0103] A5: Equipped with automatic sprinklers, underfloor heating, and a small arched greenhouse cover; no supplemental lighting.
[0104] Each treatment group consisted of 3 trays, with 49 seedlings planted in each tray, and the results were replicated 3 times. All tissue culture seedlings were selected according to the seedling screening criteria described in Example 1, and weak, diseased, and poorly growing seedlings were removed.
[0105] (2) Seedling raising methods
[0106] Except for the facility management conditions, which were adjusted as described above, the seedling cultivation methods for each group of tissue culture seedlings were the same as in Example 1, including substrate preparation and disinfection, seedling time arrangement, and transplanting operation, in order to eliminate interference from other factors on the experimental results.
[0107] (3) Survey indicators
[0108] Fifty days after transplanting, the tissue culture seedlings of each treatment group were surveyed and statistically analyzed, and the following two indicators were recorded:
[0109] Survival rate (%) = (Number of surviving plants / Number of plants planted) × 100%
[0110] Seedling growth: Observe and describe the overall growth status of surviving plants, including stem thickness, leaf color, etc.
[0111] The effects of different facility management on the survival rate and growth of Trichosanthes kirilowii tissue culture seedlings are shown in Table 3.
[0112] Table 3. Effects of different facility treatments on the survival rate and growth of Trichosanthes kirilowii seedlings.
[0113]
[0114] Table 3 shows that different facility management conditions significantly affected the survival rate and growth of *Trichosanthes kirilowii* tissue culture seedlings. The A1 treatment group (equipped with all four facilities) had the highest survival rate at 91%, and the seedlings were robust and vibrant green. The survival rates of the A2 treatment group (without automatic sprinkler system) and the A5 treatment group (without supplemental lighting) were 81% and 83%, respectively, slightly lower than A1, but the seedling growth differed: A2 seedlings remained robust and vibrant green, while A5 seedlings were slightly thinner and had paler leaves. The A3 treatment group (without geothermal heating) had a survival rate of 73%, and the seedling growth was acceptable. The A4 treatment group (without a small arched greenhouse covering) had the lowest survival rate at only 55%, indicating that the small arched greenhouse covering played a crucial role in maintaining a suitable temperature and humidity environment and improving the transplant survival rate. In summary, under the conditions of this experiment, the treatment with a complete set of facilities (A1) including automatic sprinklers, geothermal heating, small arched greenhouse coverings, and supplemental lighting showed the best results and can be considered as the recommended facility configuration for efficient seedling cultivation of Trichosanthes kirilowii tissue culture seedlings.
[0115] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.
Claims
1. A highly efficient seedling cultivation method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings, characterized in that: Includes the following steps: (1) Seedling selection: Select Trichosanthes kirilowii tissue culture seedlings that have been rooted for 45-60 days, and remove weak seedlings, diseased seedlings and individuals with poor growth; (2) Substrate preparation and disinfection: Mix the seedling substrate, bio-organic fertilizer and calcium magnesium phosphate fertilizer in a mass ratio of 24:4:0~4 and sterilize them. (3) Determining the seedling time: Seedling raising operations shall be carried out from late February to early March; (4) Seedling facility construction and environmental control: A seedling facility system is built in the greenhouse. The seedling facility system includes a bottom insulation and heating system, seedling containers and small arched sheds, shading system, supplemental lighting system and spray system to precisely control the temperature, light and humidity during the seedling process.
2. The efficient seedling raising method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step (1), the Trichosanthes kirilowii tissue culture seedlings are obtained by the following method: using healthy, disease-free stem segments of Trichosanthes kirilowii from the current year as explants, after disinfection, they are inoculated into a basal culture medium for primary culture to obtain sterile initiating buds; the obtained sterile initiating buds are cut into single-bud stem segments and inoculated into a proliferation culture medium for proliferation culture to obtain cluster buds; the obtained cluster buds are cut into single buds and inoculated into a rooting culture medium for rooting culture to obtain the Trichosanthes kirilowii tissue culture seedlings.
3. The efficient seedling raising method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step (1), the optimal seedling age of the Trichosanthes kirilowii tissue culture seedlings is 45 days after rooting culture.
4. The efficient seedling raising method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step (2), the optimal mass ratio of the seedling substrate, bio-organic fertilizer and calcium magnesium phosphate fertilizer is 24:4:
1.
5. The efficient seedling raising method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step (4), the method for constructing the bottom insulation and heating system is as follows: a black mulch film is laid on the greenhouse floor to divide the seedling beds; two layers of geotextile are laid on the seedling bed surface, and heating wires are laid between the two layers of geotextile. The heating wires are arranged back and forth at 15cm intervals, and isolation materials are set between adjacent heating wires; a temperature control device with a temperature probe is installed at each seedling bed head, and the start and stop temperature threshold of the temperature control device is set to 10℃.
6. The efficient seedling raising method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step (4), the method of using the seedling container is as follows: place seedling trays on the seedling bed and arrange black nutrient bags in the seedling trays; fill the black nutrient bags with the sterilized and cooled substrate, and fill the substrate to a height of 2-5 mm from the bag opening; when transplanting, make a hole in the center of the substrate, place the roots of the tissue culture seedling in the hole, backfill the substrate and compact it, and the transplanting depth is such that the base of the tissue culture seedling roots is level with the surface of the substrate. After transplanting, water thoroughly to settle the roots.
7. The efficient seedling raising method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step (4), the method for building and managing the small arched shed is as follows: fiber rods are laid out at intervals on the seedbed and bent to form an arch frame, and a transparent heat-insulating film is covered on the outside of the arch frame to form a small arched shed; Two weeks before seedling cultivation, open the insulation film on both sides of the small arched shed for ventilation at 9:00 am every day. After ventilation for 1 hour, cover the insulation film back. Two weeks after the seedlings are established, the insulation film on both sides of the small arched shed is opened for ventilation at 9:00 a.m. every day, while the top insulation film is left on. At 4 PM, decide whether to cover the insulation film tightly based on the nighttime low temperature forecast: if the forecast nighttime low temperature is not lower than 15℃, keep both sides ventilated; if the forecast nighttime temperature is lower than 15℃, cover the insulation film tightly.
8. The efficient seedling raising method for improving the survival rate of Trichosanthes kirilowii tissue culture seedlings according to claim 1, characterized in that: In step (4), the management method of the shading, supplemental lighting and sprinkler system is as follows: Shading management: Use shade nets with a shading rate of 60%; cover the shade nets all day for the first three weeks of seedling cultivation; from the fourth week of seedling cultivation, cover the shade nets from 10:00 am to 4:00 pm on sunny days, and leave them open during the rest of the time; do not cover the shade nets at all during cloudy or rainy weather; remove the shade nets after the sixth week of seedling cultivation; the shade nets will automatically close when the temperature inside the greenhouse exceeds 30℃. Supplemental lighting management: per 8m 2 A standard, evenly spaced array of 100W full-spectrum LED supplementary lights is installed and turned on when natural light is insufficient due to continuous cloudy or rainy weather. The supplementary lighting period is from 6:00 AM to 6:00 PM daily. Spraying management: Spray 5 times a day at even intervals during the first week of seedling cultivation. From the second week onwards, spray 3 times a day at even intervals, completing the spraying during the day. The standard for stopping a single spraying is when the substrate moisture reaches 70%. In cloudy or rainy weather, when the air humidity reaches 85% or above, spray only once a day at 6 pm, also with the standard of when the substrate moisture reaches 70%.