A method for cultivating seedlings directly from the germination of *Gynostemma pentaphyllum* seeds in Yunnan.

Abstract

This application discloses a method for directly cultivating seedlings from the germination of *Cymbidium goeringii* seeds in Yunnan. The method includes the following steps: sowing *Cymbidium goeringii* seeds on a modified culture medium, followed by 6 months of constant temperature cultivation at 25℃ to obtain small pseudobulbs suitable for direct transplanting. This method is simple to operate; seeds begin to germinate and turn green 20 days after sowing, begin to grow leaves after 45 days, begin to root after 60 days, and show significant root growth after 90 days. The pseudobulbs also enlarge synchronously. After 6 months, the plants naturally shed their leaves in the tissue culture bottle, and the pseudobulbs can be transplanted to the field for acclimatization and cultivation. This method eliminates the need to change the culture medium, removes the manual transfer step in the tissue culture process, and solves the problems of yellowing, difficult rooting, and high contamination rates after transfer of tissue culture seedlings. Furthermore, the plants naturally shed their leaves in the tissue culture bottle, directly obtaining small pseudobulbs without hardening off, and improving the transplant survival rate, which is beneficial for the large-scale cultivation of *Cymbidium goeringii*.

Description

Technical Field

[0001] This invention belongs to the field of *Orchid of Yunnan* cultivation technology, specifically relating to a cultivation method for directly germinating seedlings from *Orchid of Yunnan* seeds. Background Technology

[0002] Yunnan single garlic orchid ( Pleione. yunnanensis The pseudobulb of *Cremastra appendiculata*, listed as the source plant of "Shan Ci Gu" in the Chinese Pharmacopoeia, is called "Bing Qiu Zi" and possesses various medicinal properties, including clearing heat and detoxifying, resolving phlegm and dissipating nodules, relieving asthma and cough, reducing inflammation, relieving pain, and stopping bleeding. It is used to treat carbuncles, boils, scrofula, lymph node tuberculosis, snake and insect bites, bronchitis, pertussis, asthma, hemoptysis, traumatic injuries, and suppurative osteomyelitis. It is also an important component of over 20 traditional Chinese medicine preparations, such as Toujie Quwen Granules, Longbi Shu Capsules, and Rupiqing Capsules. However, due to excessive resource destruction, all species of the *Cremastra appendiculata* genus were listed as Class II National Key Protected Wild Plants in 2021.

[0003] Yunnan single-bulb orchid has a deciduous habit in winter, and it survives winter dormancy through a typical pseudobulb structure. Its seeds are as fine as dust, lack endosperm, and have incomplete embryo development. Propagation requires natural division of pseudobulbs and tissue culture, but natural division has a low propagation coefficient and slow speed. Tissue culture technology offers faster propagation, but traditional tissue culture seedling cultivation involves numerous steps, requires high operational skills, and results in low survival rates when seedlings are directly planted with leaves. For example, different culture media are typically needed during seed germination and rooting stages, requiring extensive manual transfer. After transfer, tissue culture seedlings are prone to yellowing and contamination, leading to low final seedling efficiency, a long propagation cycle, and high resource consumption, making it costly and unsuitable for large-scale application.

[0004] To overcome the above problems, we need to explore a cultivation method for *Cymbidium goeringii* seeds in Yunnan to directly form pseudobulbs and thus seedlings. This method will simplify the operation process, improve the transplant survival rate, reduce production costs, promote the artificial propagation of *Cymbidium goeringii*, and contribute to the protection of wild resources. Summary of the Invention

[0005] To overcome the problems existing in traditional techniques, this application provides a method for cultivating seedlings directly from the germination of Yunnan single-flower orchid seeds. Taking advantage of the large number of seeds and high germination rate of Yunnan single-flower orchid capsules, seedlings are directly cultivated from the seeds. They grow rapidly in tissue culture bottles, naturally shed their leaves, and form small pseudobulbs. Planting with these pseudobulbs improves the seedling rate, reduces production costs, and is simple to operate and easy to promote, thus facilitating the large-scale production and cultivation of Yunnan single-flower orchid.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] The cultivation method includes the following steps:

[0008] (1) Seed harvesting: Select plump, uniformly sized, disease-free Yunnan single garlic capsules that have been artificially self-pollinated for 150 days for harvesting;

[0009] (2) Preparation of modified culture medium: The modified culture medium consists of MS + 1.5 mg / L NAA + 1.0 mg / L 6-BA + 50 g / L banana + 50 g / L potato + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.6–5.8, sterilized for later use;

[0010] (3) Sowing and cultivation: The harvested capsules are disinfected by washing with detergent, wiping with alcohol, soaking in sodium hypochlorite solution, and washing with sterile water. Then the capsules are taken out and dried, and sown into culture bottles containing improved culture medium. They are cultivated for 6 months until the tissue culture seedlings naturally shed their leaves to obtain small pseudobulbs.

[0011] (4) Transplanting: Take out the small pseudobulbs from the culture bottle, disinfect them, and transplant them into humus soil.

[0012] Further preferred, in step (2), the sterilization temperature is 121°C and the sterilization time is 15 minutes.

[0013] Further preferred, in step (3), the method of rinsing with detergent is as follows: place the capsule in a container, add detergent, and rinse under tap water for 2-3 minutes; the detergent includes soap, dishwashing liquid, laundry powder, etc.

[0014] Further preferred, in step (3), the alcohol wiping method is as follows: wipe the surface of the capsule 2-3 times with degreased cotton soaked in alcohol with a volume ratio of 75%.

[0015] Further preferred, in step (3), the volume fraction of the sodium hypochlorite solution is 1%; and the soaking time is 15 minutes.

[0016] Further preferred, in step (3), the number of times the sterile water is rinsed is 3-5 times.

[0017] Further preferred, in step (3), the drying method is: using the high temperature of the outer flame of an alcohol lamp to instantaneously heat the surface of the capsule to evaporate the moisture for a duration of 2-3 seconds.

[0018] Further preferred, in step (3), the specific operation of sowing seeds into the culture bottle containing the improved culture medium is as follows: cut open the capsule, use tweezers to pick up a small amount of seeds and sow them evenly on the surface of the improved culture medium, sowing 100-120 seeds per bottle.

[0019] Further preferred, in step (3), the specific conditions for the culture period of 6 months until the tissue culture seedlings naturally shed their leaves are: temperature 25±1℃, relative humidity 40%–50%, light intensity 1800–2000 lx, light 12 h / dark 12 h.

[0020] Further preferred, in step (4), the specific operation of the disinfection treatment is as follows: rinse the small pseudobulbs with clean water until all residual culture medium and tissue culture seedling leaves are removed, then place the small pseudobulbs in a carbendazim aqueous solution to soak for 10 minutes, and then rinse with clean water 3-5 times; the volume ratio of carbendazim in the carbendazim aqueous solution is 1 / 1000.

[0021] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application.

[0022] The beneficial effects of this invention are:

[0023] 1. This application provides a method for directly cultivating seedlings from the germination of Yunnan *Cymbidium goeringii* seeds. This method is simple to operate; after seed germination, no artificial transfer is required, and rooted seedlings can be directly formed. The resulting seedlings have well-developed root systems and large pseudobulbs. After 6 months, the leaves naturally fall off in the culture vessel, forming dormant small pseudobulbs, which are easy to transplant and survive. This is because this application screens and sterilizes the explants used, namely the *Cymbidium goeringii* capsules, ensuring a high germination rate and low contamination rate. Simultaneously, this application discloses for the first time an improved culture medium that not only induces seed germination but also induces rooting of *Cymbidium goeringii* tissue culture seedlings without medium conversion. Furthermore, the rooted seedlings grow rapidly and directly form pseudobulbs. The small pseudobulbs formed after leaf fall from the cultured tissue culture seedlings can be directly transplanted, solving the problem of large-scale cultivation of *Cymbidium goeringii*. The overall method is simple to operate and easy to promote.

[0024] 2. This application, through a modified culture medium, simultaneously promotes seed germination, seedling rooting, and pseudobulb growth in *Cymbidium goeringii*, while also allowing for natural leaf drop within the culture vessel, resulting in dormant small pseudobulbs. Typically, *Cymbidium goeringii* tissue culture requires at least two media: germination medium and rooting medium. Tissue-cultured seedlings do not root on germination medium and need to be transferred to rooting medium to promote rooting, allowing for direct planting. However, this application achieves the same effects using only one modified culture medium, directly yielding pseudobulbs for easy planting. This is due to the simultaneous use of specific concentrations of auxin and cytokinin (NAA and 6-BA). NAA, as an auxin, primarily stimulates longitudinal cell growth, especially at the shoot and root tips, by promoting cell elongation and regulating developmental processes, thereby enhancing tissue differentiation. 6-BA is a cytokinin that mainly regulates cell division and delays senescence, promoting cell division and bud differentiation. The two have significant complementary effects in plant physiology. NAA can effectively promote rooting, while 6-BA induces bud differentiation. Furthermore, the addition of bananas and potatoes to the culture medium provides natural organic additives and natural auxin analogs (such as indoleacetic acid precursors) for tissue culture seedlings, which can promote cell differentiation, stimulate adventitious root differentiation and morphogenesis, enabling the tissue culture seedlings of *Ormosia henryi* to develop a well-developed root system, grow vigorously, and promote rapid expansion of their pseudobulbs.

[0025] 3. This application, by controlling the cultivation conditions, enables *Cymbidium goeringii* tissue culture seedlings to begin rooting within 60 days, with pseudobulbs expanding synchronously. This solves the problems of easy yellowing of tissue culture seedlings and the need for artificial transfer. Dormant small pseudobulbs can be obtained for transplanting after 6 months of cultivation, with a survival rate of up to 85%. This is because suitable cultivation conditions promote rooting of *Cymbidium goeringii* tissue culture seedlings, resulting in vigorous plant growth, rapid expansion of pseudobulbs, and rapid natural leaf drop, leading to the acquisition of dormant small pseudobulbs. Transplanting these dormant pseudobulbs can significantly improve the survival rate and shorten the planting cycle. Attached Figure Description

[0026] Figure 1 This is a growth variation diagram of *Cymbidium goeringii* from Yunnan Province, as per this application;

[0027] Figure 2 These are comparative images of tissue culture seedlings after 90 days of inoculation on different culture media according to this application; Note: From left to right: Example 1; Comparative Example 2;

[0028] Figure 3 This is a comparison of pseudobulb diameters after 6 months of sowing on different culture media in this application;

[0029] Figure 4 This is a diagram showing the state of tissue culture seedlings 6 months after sowing, according to an embodiment of this application.

[0030] Figure 5 This is a diagram showing the growth status of *Cymbidium goeringii* after transplanting, according to an embodiment of this application.

[0031] Figure 6 This is a schematic diagram of the growth process of plants in the genus *Gnaphalium*. Note: 1: Dormant pseudobulb; 2: Pseudobulb lateral buds bloom; 3: Flowers wither, leaves form, and new pseudobulbs begin to swell; 4: Old pseudobulbs gradually shrivel, and new pseudobulbs continue to swell; 5: Old pseudobulbs completely shrivel, leaves turn yellow and fall off, and dormant pseudobulbs are formed.

[0032] Figure 7 This is the growth of the tissue culture seedlings with leaves in Comparative Example 3 of this application after 120 days of direct field planting;

[0033] Figure 8 This is the growth of tissue culture seedlings in this application embodiment after natural leaf fall to form pseudobulbs, 120 days after field planting. Detailed Implementation

[0034] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below to facilitate understanding by those skilled in the art.

[0035] This invention provides a method for cultivating *Cymbidium goeringii* seeds directly into seedlings, which simplifies the operation process, shortens the propagation cycle, promotes the enlargement of pseudobulbs in tissue culture seedlings, effectively improves the survival rate of transplanted seedlings, facilitates promotion, and is conducive to the large-scale cultivation of *Cymbidium goeringii*.

[0036] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0037] Example:

[0038] (1) Seed harvesting: Harvest the seeds of Yunnan single-flowered garlic (which blooms in May) P. yunnanensis Artificial self-pollination was carried out, and capsules were obtained after 150 days of cultivation. Full, uniform-sized, disease-free capsules were selected for harvesting.

[0039] (2) Preparation of modified culture medium: The modified culture medium consists of MS + 1.5 mg / L NAA + 1.0 mg / L 6-BA + 50 g / L banana + 50 g / L potato + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.8, sterilized at 121℃ for 15 minutes for later use;

[0040] (3) Sowing and Cultivation: The harvested capsules were surface-sterilized. First, the capsules were placed in a beaker, a small amount of detergent was added, and they were rinsed under running tap water for 2–3 minutes. Then, the capsule surface was wiped twice with cotton soaked in 75% alcohol by volume. Next, the capsules were soaked in 1% sodium hypochlorite solution by volume for 15 minutes and rinsed three times with sterile water. Finally, the capsules were removed with high-temperature sterilized tweezers and quickly baked back and forth in the outer flame of an alcohol lamp for 3 seconds to dry the surface moisture. This completed the sterilization of the capsules of *Gynostemma pentaphyllum*. Then, the capsules were cut open with a knife to scatter the seeds into the culture dish. A small amount of seeds were then picked up with tweezers and evenly sown on the surface of the modified culture medium. 100 seeds were sown in each bottle. The culture medium was maintained at a temperature of 25±1℃, relative humidity of 40%–50%, light intensity of 1800–2000 lx, and a light-dark cycle of 12 h / 12 ​​h. Cultured under h conditions for 6 months until the tissue culture seedlings naturally shed their leaves to obtain small pseudobulbs;

[0041] (4) Transplanting: Take out the small pseudobulbs from the culture bottle, rinse the small pseudobulbs with clean water until all residual culture medium and tissue culture seedling leaves are removed, then soak the small pseudobulbs in a carbendazim solution for 10 minutes, and then rinse them with clean water 3 times. The volume ratio of carbendazim (purchased from Jiangsu Lanfeng Biochemical Co., Ltd., pesticide registration number PD85150-8) in the carbendazim solution is 1 / 1000. After completing the above disinfection treatment, transplant the small pseudobulbs into humus soil for domestication and cultivation. The planting density is 800,000 seedlings / mu. Field water and fertilizer management is carried out in accordance with conventional methods.

[0042] Comparative Example 1:

[0043] Except for replacing the culture medium in step (2) with germination medium, all other steps are the same as in the example. The germination medium consists of: MS + 1.0 mg / L NAA + 100 mL / L coconut juice + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.8.

[0044] Comparative Example 2:

[0045] Except for replacing the culture medium in step (2) with rooting medium, all other steps are the same as in the example. The rooting medium consists of: MS + 1.5 mg / L NAA + 50 g / L banana + 50 g / L potato + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.8.

[0046] Comparative Example 3:

[0047] Except for replacing the culture medium in step (2) with germination medium: MS + 1.0 mg / L NAA + 100 mL / L coconut juice + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.8, after three months of sowing, the seedlings are artificially transferred to rooting medium: MS + 1.5 mg / L NAA + 50 g / L banana + 50 g / L potato + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.8, and then undergoing three months of rooting culture, all other steps are the same as in the example.

[0048] Data Analysis:

[0049] Seed germination media typically contain high concentrations of cytokinins (such as 6-BA) combined with auxins (such as 2,4-D or NAA) to promote seed dedifferentiation and protocorm formation. Some formulations require the addition of coconut juice or casein hydrolysate to enhance germination efficiency. Rooting and seedling strengthening media usually reduce or remove cytokinins, replacing them with low concentrations of auxins (such as NAA 0.2-0.5 mg / L) to induce root development and form complete seedlings suitable for transplanting. However, this application achieves the combined effects of both types of media using only one modified medium. It not only promotes seed germination but also promotes the growth and development of pseudobulbs and roots in *Ormosia rubescens* tissue culture seedlings, directly forming pseudobulbs and improving transplant survival rate.

[0050] from Figure 1 It is known that after adopting the cultivation method of the present application embodiment, the *Cymbidium goeringii* begins to germinate and turn green 20 days after sowing, begins to grow leaves after 45 days, begins to root after 60 days, and the roots grow significantly after 90 days. The pseudobulbs also swell synchronously. After 6 months, the plant naturally loses its leaves in the tissue culture bottle, and the pseudobulbs can be transplanted to the field for domestication and cultivation. After 3 months of planting in the field, the plant grows well.

[0051] According to Tables 1 and 2 Figure 1-5 As shown in points 7 and 8, although the seed germination rates of the examples and the three comparative examples were not significantly different, after six months of sowing, the roots and leaves of the examples were larger and had longer root systems compared to Comparative Example 1. Comparative Example 1 only differentiated leaves but did not develop root systems. The examples allowed the tissue culture seedlings to better absorb nutrients and consume the culture medium, thus forming larger pseudobulbs. The plants quickly and naturally shed their leaves, which could increase the survival rate of subsequent plantings. In contrast, after germination, the seeds of Comparative Example 2 could only form protocorms and could not differentiate leaves and roots, thus failing to grow normally. The protocorms were also small, which was not conducive to subsequent planting survival. Three months after germination, the seeds of Comparative Example 3 required artificial transfer. After transfer, problems such as yellowing and slow growth were likely to occur. After six months, although root and leaf differentiation was completed and pseudobulbs were formed, the small pseudobulbs did not enter a dormant state. Direct transplanting of seedlings resulted in a low survival rate.

[0052] Table 1. Comparison of traits of tissue culture seedlings 6 months after sowing on different culture media.

[0053]

[0054] Table 2. Comparison of traits of *Orchid of the Chinese Garlic* cultivar 120 days after transplanting on different culture media.

[0055]

[0056] Figure 7 To compare the growth of tissue culture seedlings from Example 3 120 days after direct field transplantation, the following data was collected: Figure 6 and Figure 7 It can be seen that, due to the yellowing of tissue culture seedlings in Comparative Example 3 during artificial transfer, the leaves of the tissue culture seedlings did not fall off after 6 months of cultivation after sowing. Even though pseudobulbs were formed, the single garlic orchid did not enter a dormant state. Therefore, its growth was poor and the survival rate was low after direct field transplantation.

[0057] Figure 8 This describes the growth of tissue culture seedlings after 6 months of cultivation, when the leaves naturally fell off and the seedlings were transplanted into the field 120 days later. Figure 1 , 4 -8 shows that when the leaves of the tissue culture seedlings of *Ormosia uniflora* fall off, the pseudobulbs that are formed enter a dormant state. When they are directly transplanted to the field, they grow well. After the pseudobulb lateral buds flower, they can form leaves and new pseudobulbs. The new pseudobulbs continue to swell, while the old pseudobulbs gradually wither, resulting in a high survival rate.

[0058] Therefore, the method provided in this application can directly form dormant pseudobulbs of *Cymbidium goeringii*, which can be directly used for field transplanting, eliminating the need to change the culture medium and harden the seedlings after they are taken out of the culture bottle. This greatly reduces the difficulty of cultivating *Cymbidium goeringii*, shortens the time required for cultivation, and saves manpower and material costs.

[0059] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for cultivating seedlings directly from the germination of *Gynostemma pentaphyllum* seeds in Yunnan, characterized in that... The cultivation method includes the following steps: (1) Seed harvesting: Select plump, uniformly sized, disease-free Yunnan single garlic capsules that have been artificially self-pollinated for 150 days for harvesting; (2) Preparation of modified culture medium: The components of the modified culture medium are: MS + 1.5 mg / L NAA + 1.0 mg / L 6-BA + 50 g / L banana + 50 g / L potato + 30 g / L sucrose + 5 g / L agar + 1 g / L activated carbon, pH 5.6–5.8, sterilized for later use; (3) Sowing and cultivation: The harvested capsules are disinfected by washing with detergent, wiping with alcohol, soaking in sodium hypochlorite solution, and washing with sterile water. Then the capsules are taken out and dried, and sown into culture bottles containing improved culture medium. They are cultivated for 6 months until the tissue culture seedlings naturally shed their leaves, and dormant small pseudobulbs are obtained. (4) Transplanting: Take out the dormant small pseudobulbs from the culture bottle, disinfect them, and transplant them into humus soil.

2. The culture method according to claim 1, characterized by, In step (2), the sterilization temperature is 121°C and the sterilization time is 15 minutes.

3. The culturing method according to claim 1, wherein In step (3), the method of rinsing with detergent is as follows: place the capsule in a container, add detergent, and rinse under tap water for 2-3 minutes; the detergent includes soap, dishwashing liquid, and laundry powder.

4. The culturing method according to claim 1, wherein In step (3), the alcohol wiping method is as follows: wipe the surface of the capsule 2-3 times with degreased cotton soaked in alcohol with a volume ratio of 75%.

5. The culturing method according to claim 1, wherein In step (3), the method of soaking in sodium hypochlorite solution is as follows: soak in a sodium hypochlorite solution with a volume fraction of 1% for 15 minutes.

6. The culturing method according to claim 1, wherein In step (3), the sterile water is rinsed 3-5 times.

7. The culturing method according to claim 1, wherein In step (3), the drying method is to use the outer flame of an alcohol lamp to heat the surface of the capsules for 2-3 seconds to evaporate the moisture.

8. The culturing method according to claim 1, wherein In step (3), the specific operation of sowing seeds into the culture bottle containing the improved culture medium is as follows: cut open the capsule, use tweezers to pick up a small amount of seeds and sow them evenly on the surface of the improved culture medium, sowing 100-120 seeds per bottle.

9. The culturing method according to claim 1, wherein In step (3), the specific conditions for the culture period of 6 months until the tissue culture seedlings naturally shed their leaves are: temperature 25±1℃, relative humidity 40%–50%, light intensity 1800–2000 lx, light 12h / dark 12h.

10. The culturing method according to claim 1, wherein In step (4), the specific operation of the disinfection treatment is as follows: rinse the small pseudobulbs with clean water until all residual culture medium and tissue culture seedling leaves are removed, then place the small pseudobulbs in a carbendazim aqueous solution to soak for 10 minutes, and then rinse with clean water 3-5 times; the volume ratio of carbendazim in the carbendazim aqueous solution is 1 / 1000.

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