Calanthe davidiana seed tissue culture rapid propagation method

Abstract

The invention discloses a calanthe davidiana seed tissue culture rapid propagation method. Belongs to the field of plant seedling breeding. The tissue culture rapid propagation system of calanthe davidiana comprises the steps of seed preparation and harvesting, explant disinfection, seed treatment, protocorm, adventitious bud and adventitious root induction, rooting and strong seedling culture, seedling transplanting and seedling hardening and the like, and the embryo rate of the seeds can be increased to 86.76% on average according to the system; the germination rate of the seeds is increased to 65.74%, macroscopic seeds can start to germinate and form protocorms within 60 days at the soonest, adventitious buds can grow from the protocorms within 100 days, and adventitious roots can grow from the protocorms within 120 days. And carrying out rooting and seedling strengthening culture and acclimatization and transplantation to produce high-quality seedlings for field normalization. The method is an efficient, rapid and environment-friendly propagation technology system, large-scale production of calanthe davidiana can be achieved in a short time, and the method has important practical value in protection and propagation of the rare orchid plant.

Description

Technical Field

[0001] This invention relates to plant tissue culture technology, and more particularly to a tissue culture method for *Cymbidium goeringii*, including steps such as hybridization seed production, explant disinfection, seed treatment, induction of protocorms and adventitious buds and roots, rooting and seedling cultivation, and seedling transplanting and hardening. Background Technology

[0002] Orchids are one of the most diverse groups of angiosperms. *Orchidia macrantha*, belonging to the genus *Orchidia* in the family Orchidaceae, has a narrow distribution and a small population.

[0003] The population of *Cymbidium davidii* is endangered. Conservation sites have been established in its distribution areas, such as Langshan Mountain in Xinning County, Hunan Province, and Jindong Forest Farm in Yongzhou City, Hunan Province, where in-situ and ex-situ conservation measures have been implemented. However, due to the fragility of its ecosystem and the extreme difficulty in natural seed germination in the wild, relying solely on in-situ conservation and natural propagation is insufficient to ensure the continued stability of its population. Only by achieving artificial breeding of *Cymbidium davidii* and combining it with naturalization techniques to promote population recovery to a sufficient base can the sustainable reproduction and propagation of this species in its native habitat be guaranteed. Although there are some reports on the breeding techniques of *Cymbidium davidii*, problems such as long germination time and low germination rate still limit the large-scale breeding process.

[0004] Furthermore, existing tissue culture methods for other orchid species cannot be directly applied and have several problems. For example, patent CN106258997A uses vegetative shoots as explants, which creates wounds on the tuber during acquisition, making it susceptible to pathogen invasion and death of the entire plant, thus hindering the protection of wild resources. It also uses highly toxic mercuric chloride for disinfection. Another example is patent CN106386481A, which requires complex techniques under a stereomicroscope to obtain a thin layer of protocorm cells, making it impractical for widespread application.

[0005] Therefore, this invention discloses a method for improving the germination rate and shortening the seedling cycle of *Cymbidium faberi* seeds by treating the seeds and controlling environmental and biological factors such as nutrition, temperature, humidity, and light intensity, while also overcoming other defects of the prior art. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing methods for germinating *Cymbidium faberi* seeds, which suffer from long germination times and low germination rates, by providing a simple and efficient method for germinating *Cymbidium faberi* seeds into seedlings. This method utilizes unopened mature capsules of *Cymbidium faberi* for sterilization without HgCl2, obtaining a large number of embryogenic seeds as explants. It can rapidly induce protocorms, buds, and roots, achieving excellent germination time and germination rate.

[0007] The objective of this invention is achieved through the following means.

[0008] A method for rapid propagation of *Cymbidium goeringii* by seed tissue culture includes the following steps:

[0009] (1) Seeds were obtained by hybridization of large yellow-flowered shrimp-shaped orchids under sterile conditions after the mature capsules that had not cracked open were disinfected.

[0010] (2) The seed coat is damaged by sterile NaOH or KOH solution treatment;

[0011] (3) Induction of protocorms, adventitious buds, and adventitious roots;

[0012] (4) Seedling rooting and seedling strengthening cultivation;

[0013] (5) Hardening off seedlings and transplanting;

[0014] Step (3) Under aseptic conditions, the seeds obtained in step (2) are spread evenly on the surface of the culture medium. The culture medium consists of 1 / 2 MS medium + 1.0~5.0 mg / L 6-BA + 0.2~0.7 mg / L NAA + 0.2~0.6 mg / L TDZ + 1.0~4.0 g / L activated carbon. After culturing for 60~80 days, protocorms are formed in large quantities; after 100~120 days, adventitious buds are induced from the protocorms; after 120~140 days, the adventitious buds elongate, and adventitious roots are produced at the base, forming seedlings.

[0015] Step (4) Transfer the seedlings obtained in step (3) to a rooting and seedling strengthening culture medium. The culture medium consists of 1 / 2 MS medium + 0.2~0.7 mg / L NAA + 1.0~5.0 g / L activated carbon. After culturing for 30~50 days, the seedlings grow into strong seedlings with a height of 2~4 cm, 2~3 leaves, and 2~5 roots. Then, they are transferred to the hardening stage.

[0016] Furthermore, in step (1), in early April, during the flowering season of *Cymbidium goeringii*, anthers of *Cymbidium goeringii* from different cities are collected for hybridization and pollination; after pollination and fruiting, the capsules develop naturally for 6 to 8 months until the fruit matures, and the capsules are healthy and mature with yellow-green skin, no disease spots, no insect infestation, and no cracks.

[0017] The disinfection process of the capsule in step (1) is as follows: After rinsing the surface of the capsule with pure water, according to the aseptic operation specifications, first soak it in 70-75% alcohol for 30-90 seconds, preferably 40-60 seconds; then rinse it once with sterile water, and then soak it in 6-10% NaClO for 8-12 minutes, preferably 7-8% NaClO for 9-10 minutes; then rinse it three times with sterile water for later use; under aseptic conditions, use sterile filter paper to absorb the moisture on the surface of the capsule after disinfection in step (1), and then cut it open to obtain the seeds inside the pericarp for later use.

[0018] Further, in step (2), the sterile seeds obtained in step (1) are soaked in a sterile NaOH or KOH solution with a concentration of 0.05~0.40 mol / L, preferably 0.20~0.30 mol / L, for a soaking time of 7~12 min, preferably 8~10 min, and then rinsed three times with sterile water for later use.

[0019] Further, in step (3), 1 mL of sterile water is added to the surface of the culture medium to spread the seeds evenly on the surface of the culture medium.

[0020] In steps (3) and (4), the sucrose concentration in the culture medium is 25-35 g / L, the agar concentration is 5-8 g / L, the pH is adjusted to 5.5-6.0, the culture temperature is 23±1℃, the humidity is 70-80%, the light duration is 12-14 h / d, and the light intensity is 2000-3500 lx.

[0021] The preferred culture medium for inducing protocorms, adventitious buds, and adventitious roots in this invention is 1 / 2 MS + 2.0-3.0 mg / L 6-BA + 0.3-0.5 mg / L NAA + 0.4-0.5 mg / L TDZ, with an activated carbon concentration of 2.0 g / L, a sucrose concentration of 30 g / L, and an agar powder concentration of 6 g / L, adjusted to pH 5.8. The preferred culture medium for rooting and seedling cultivation is 1 / 2 MS + 0.3-0.4 mg / L NAA, with an activated carbon concentration of 2.0 g / L, a sucrose concentration of 30 g / L, and an agar powder concentration of 4 g / L, adjusted to pH 5.8.

[0022] Further, in step (5), after hardening off the robust seedlings obtained in step (4), they are transferred to the transplanting substrate, which is a mixture of peat moss, perlite, and vermiculite in a volume ratio of (6~7):(1~2):(2~3). After mixing, the mixture is sterilized at 121℃ for 1~2 hours.

[0023] The preferred transplanting substrate consists of peat moss with a particle size of 1-10 mm, perlite with a particle size of 2-4 mm, and vermiculite with a particle size of 3-6 mm.

[0024] Furthermore, place the tissue culture containers containing strong seedlings under natural light and room temperature conditions for 3-5 days, then open the container cap halfway and culture for 12-24 hours. Remove the seedlings from the container, gently wash off the culture medium attached to the roots in 20-30℃ clean water, and transplant the strong seedlings into a high-temperature sterilized transplanting substrate. Cultivate in a natural light greenhouse with ventilation 2-4 times a day. When the individual plants reach a height of 8-10cm, the longest leaf length reaches 5-8cm, and the longest leaf width reaches 2-4cm, transplant the large yellow-flowered shrimp-shaped orchid seedlings to a suitable wild habitat.

[0025] Beneficial effects of this invention: This invention provides a complete set of techniques for cultivating *Cymbidium faberi* seedlings under controlled conditions using seeds as explants. These techniques have the following advantages:

[0026] (1) Saves time and effort: The explants are taken from the seeds inside the unopened mature capsules of the large yellow flower shrimp orchid, which does not require the use of tubers, making it easier to disinfect and control the contamination rate.

[0027] (2) Simple, efficient, and HgCl2-free: The method involves first soaking the explants containing a large number of seeds in 70-75% alcohol for 40-60 seconds, then soaking them in 7-8% NaClO for 9-10 minutes for stepwise disinfection, and finally rinsing them briefly with sterile water. Since HgCl2 is not used for disinfection, there is no HgCl2 residue, and there is no need for large amounts of sterile water for rinsing, saving water resources and reducing potential environmental hazards. The above sterilizing agents do not need to act directly on the explants. This method has good disinfection effects and can control the seed contamination rate to within 4%.

[0028] (3) Protect wild resources of *Cymbidium faberi*: Explants were taken from unopened mature capsules. Since the seeds of *Cymbidium faberi* are extremely difficult to germinate on their own in the wild, the harvesting of capsules will not affect the growth and reproduction of wild *Cymbidium faberi*.

[0029] (4) High embryo rate of seeds: Using the technical method of the present invention, the embryo rate of seeds can reach 86.76%±3.82%, which is much higher than the average embryo rate of 45.15%±2.68% of the previously published large yellow flower shrimp orchid seeds, showing obvious technical advantages.

[0030] (5) Short seed germination time and high germination efficiency: Using the technical method of this invention, the seed germination time can be shortened to 60-80 days, which is much shorter than the previously reported shortest germination time of 120 days. At the same time, the germination rate of 65.74±3.04% is also much higher than the previously reported highest germination rate of 39.64%±3.34%. It has obvious technological advantages.

[0031] (6) Complete technical system: It provides a complete set of technical methods for the tissue culture of *Cymbidium goeringii*, from the preparation and collection of explants to the germination and rooting culture of protocorms. Each technical link has been fully optimized. Complete seedlings can be obtained in as little as 150 days, which is suitable for the large-scale production of *Cymbidium goeringii* seedlings. Attached Figure Description

[0032] Figure 1 Microscopic observation of hybrid seeds of *Cymbidium goeringii* (0d);

[0033] Figure 2 Seeds of hybrid *Cymbidium goeringii* were damaged after treatment with NaOH solution (10 days).

[0034] Figure 3 Micrographs of the embryonic swelling of the seeds of *Cymbidium macranthum* into protocorms (60 days).

[0035] Figure 4 : The seed embryo of *Cymbidium goeringii* swells into a protocorm (60 days);

[0036] Figure 5 Adventitious buds are produced from the protocorm of *Cymbidium lancifolium* (100 days).

[0037] Figure 6 The adventitious buds of the large yellow-flowered shrimp-shaped orchid elongate and produce adventitious roots at the base (120 days).

[0038] Figure 7 : Cultivation of rooted and robust seedlings of *Cymbidium goeringii* (150 days).

[0039] Figure 8 Transplanting and hardening-off cultivation of *Cymbidium goeringii* (190 days). Detailed Implementation

[0040] The female parent of *Cymbidium faberi* used in the following examples came from the *Cymbidium faberi* distribution protection site in Jindong Forest Farm, Yongzhou City, Hunan Province. The male parent anther material came from the *Cymbidium faberi* protection site in Langshan Scenic Area, Xinning County, Shaoyang City, Hunan Province. The anthers were collected in April 2024 with the assistance of staff from the Xinning County Forestry Bureau. The inventor conducted artificial pollination with the assistance of staff from Jindong Forest Farm to obtain hybrid capsules. The capsules were collected when they were mature and unopened. Cytokinin 6-BA, TDZ, auxin NAA, and NaOH were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0041] Example 1

[0042] In this embodiment, the 1 / 2 MS medium is obtained by halving the macroelements in the MS medium, while the remaining components are the same as the MS medium. Medium A is used for induction of protocorms, adventitious buds, and adventitious roots. It is obtained by adding 2.0 mg / L of 6-BA, 0.3 mg / L of NAA, and 0.4 mg / L of TDZ to the 1 / 2 MS medium, with an activated carbon concentration of 2.0 g / L, a sucrose concentration of 30 g / L, and an agar powder concentration of 6 g / L, and adjusting the pH to 5.8. Medium B is used for rooting and seedling cultivation. It is obtained by adding 0.4 mg / L of NAA, 2.0 g / L of activated carbon, 30 g / L of sucrose, and 4 g / L of agar powder to the 1 / 2 MS medium, and adjusting the pH to 5.8.

[0043] 1. Hybrid seed production

[0044] The standard for hybridization pollination is as follows: after the flowers are fully open during the flowering period, collect the anthers of the male parent and immediately transport them to the location of the female parent. Remove the original anthers of the female parent and remove the outer shell of the male parent's anthers to expose the anthers. Use tweezers to pick them up and place them in the cavity below the stigma of the *Cymbidium goeringii* column to complete pollination. The standard for capsule collection is as follows: after flowering and pollination in early April, *Cymbidium goeringii* undergoes 8 months of development. By the end of December of the same year, the capsules should be healthy and mature, with a surface that is light yellowish-green to yellow, free of disease spots, insect damage, and cracks.

[0045] 2. Disinfection and treatment of explants

[0046] After rinsing the capsule surface with tap water, place it on a sterile dish in a laminar flow hood. Following aseptic procedures, first soak it in 75% alcohol for 1 minute; then rinse it once with sterile water, and then soak it in 8% NaClO for 8 minutes; subsequently rinse it three times with sterile water for later use. Data analysis after 7 days showed that the explant contamination rate was 3%. Under aseptic conditions, the capsule surface moisture was absorbed with sterile filter paper, then it was dissected to obtain the seeds for later use. A portion of the seeds were examined under a microscope, and the embryogenicity rate was calculated. The results of three parallel experiments were 86.76% ± 3.82%. Figure 1 );

[0047] 3. Seed treatment

[0048] The sterile seeds obtained in step 2 were soaked in a sterile NaOH solution with a concentration of 0.20 mol / L for 8 minutes, rinsed three times with sterile water, and set aside. A portion of the seeds were then examined under a microscope to check for any damage to the seed coat. Figure 2 );

[0049] 4. Induction of protocorms, adventitious buds, and adventitious roots

[0050] Under aseptic conditions in a laminar flow hood, the seeds treated in step 3 were placed on the surface of culture medium A using sterile forceps. 1 mL of sterile water was added to ensure even distribution of the seeds. The culture medium consisted of 1 / 2 MS medium + 2.0 mg / L 6-BA + 0.3 mg / L NAA + 0.4 mg / L TDZ + 2.0 g / L activated carbon, with a sucrose concentration of 30 g / L and an agar concentration of 6 g / L. The pH was adjusted to 5.8, the culture temperature was 23 ± 1℃, and the humidity was 70%. Cultured under a photoperiod of 12 h light and 12 h dark, and a light intensity of 2500 lx, protocorms began to form in large quantities after 60 days. Figure 3 , Figure 4 The germination rate was statistically analyzed using microscopic methods, with the embryo swelling into a spherical shape and breaking through the seed coat as the standard. The germination rate in three parallel experiments was 65.74 ± 3.04%; after 100 days, adventitious buds were induced from the original corm. Figure 5); 120 days later, adventitious buds elongate, adventitious roots develop at the base, and seedlings are formed. Figure 6 );

[0051] 5. Cultivation of strong seedlings and rooting

[0052] The seedlings obtained in step 4 were transferred to a rooting and seedling strengthening medium for further rooting and seedling strengthening culture. The seedlings were evenly spread on the surface of the medium, leaving space for their growth. The rooting medium consisted of 1 / 2 MS medium + 0.4 mg / L NAA + 2.0 g / L activated carbon, 30 g / L sucrose, and 4 g / L agar powder, adjusted to pH 5.8. The culture temperature was 23 ± 1℃, and the humidity was 70%. After 30 days of culture under a photoperiod of 12 h light and 12 h dark, and a light intensity of 2500 lx, the seedlings grew to a height of 2-4 cm, with 2-3 leaves and 2-5 roots, achieving a rooting rate of 100%. Figure 7 ), and can then proceed to the seedling hardening stage;

[0053] 6. Transplanting and hardening off seedlings

[0054] Place the tissue culture container containing the strong seedlings under natural light and room temperature conditions and culture for 5 days. Then, open the container cap halfway and culture for 24 hours. Remove the seedlings from the container and gently wash off the culture medium attached to the roots in 25°C water. Transplant the strong seedlings into a high-temperature sterilized transplanting substrate. The transplanting substrate is made by mixing peat moss (particle size 1~10mm), perlite (particle size 2~4mm), and vermiculite (particle size 3~6mm) in a volume ratio of 7:1:2 and sterilizing at 121°C for 2 hours.

[0055] Cultivated in a natural light greenhouse with ventilation three times a day, the survival rate of *Cymbidium goeringii* was 100% after 40 days. Figure 8 Once the individual plants reach a height of 8-10cm, the longest leaf length reaches 5-8cm, and the longest leaf width reaches 2-4cm, the seedlings of *Cymbidium lancifolium* can be transplanted to suitable wild habitats.

[0056] Example 2

[0057] In this embodiment, the 1 / 2 MS medium is obtained by halving the macroelements in the MS medium, while the remaining components are the same as the MS medium. Medium A is used for induction of protocorms, adventitious buds, and adventitious roots. It is obtained by adding 3.0 mg / L of 6-BA, 0.5 mg / L of NAA, and 0.5 mg / L of TDZ to the 1 / 2 MS medium, with an activated carbon concentration of 2.0 g / L, a sucrose concentration of 30 g / L, and an agar powder concentration of 6 g / L, and adjusting the pH to 5.8. Medium B is used for rooting and seedling cultivation. It is obtained by adding 0.3 mg / L of NAA, 2.0 g / L of activated carbon, 30 g / L of sucrose, and 4 g / L of agar powder to the 1 / 2 MS medium, and adjusting the pH to 5.8.

[0058] 1. Hybrid seed production

[0059] The standard for hybridization pollination is as follows: after the flowers are fully open during the flowering period, collect the anthers of the male parent and immediately transport them to the location of the female parent. Remove the original anthers of the female parent and remove the outer shell of the male parent's anthers to expose the anthers. Use tweezers to pick them up and place them in the cavity below the stigma of the *Cymbidium goeringii* column to complete pollination. The standard for capsule collection is as follows: after flowering and pollination in early April, *Cymbidium goeringii* undergoes 8 months of development. By the end of December of the same year, the capsules should be healthy and mature, with a surface that is light yellowish-green to yellow, free of disease spots, insect damage, and cracks.

[0060] 2. Disinfection and treatment of explants

[0061] After rinsing the capsule surface with tap water, place it on a sterile dish in a laminar flow hood. Following aseptic procedures, first soak it in 75% alcohol for 50 seconds; then rinse it once with sterile water, and then soak it in 8% NaClO for 10 minutes; subsequently rinse it three times with sterile water for later use. After 7 days, the data showed that the explant contamination rate was 1%. Under aseptic conditions, the surface moisture of the capsule was absorbed with sterile filter paper, then it was cut open to obtain the seeds for later use. Some seeds were examined under a microscope, and the embryo-bearing rate was found to be 83.24% ± 4.53%.

[0062] 3. Seed treatment

[0063] The sterile seeds obtained in step 2 were soaked in a sterile NaOH solution with a concentration of 0.40 mol / L for 8 minutes, and then rinsed three times with sterile water.

[0064] 4. Induction of protocorms, adventitious buds, and adventitious roots

[0065] Under aseptic conditions in a laminar flow hood, a suitable amount of the seeds treated in step 3 was placed on the surface of culture medium A using sterile forceps. 1 mL of sterile water was added to ensure the seeds were evenly spread across the surface. The culture medium consisted of 1 / 2 MS medium + 3.0 mg / L 6-BA + 0.5 mg / L NAA + 0.5 mg / L NAA. The culture was conducted using TDZ + 2.0 / L activated carbon, sucrose concentration 30 g / L, agar powder concentration 6 g / L, pH adjusted to 5.8, culture temperature 23±1℃, humidity 80% under a photoperiod of 12 h light, 12 h dark, and light intensity of 3000 lx. After 70 days, protocorms began to form in large quantities. The germination rate was statistically analyzed under microscopic methods, with the embryo swelling into a spherical shape and breaking through the seed coat as the standard. The germination rate in three parallel experiments was 49.40±3.03%. At 110 days, adventitious buds were induced from the protocorms; at 130 days, the adventitious buds elongated, and adventitious roots were produced at the base, forming seedlings.

[0066] 5. Cultivation of strong seedlings and rooting

[0067] The seedlings obtained in step 4 were transferred to a rooting and seedling strengthening medium for further rooting and seedling strengthening culture. The seedlings were evenly spread on the surface of the medium, leaving space for seedling growth in the middle. The rooting medium consisted of 1 / 2 MS medium + 0.3 mg / L NAA + 2.0 g / L activated carbon, 30 g / L sucrose and 4 g / L agar powder, adjusted to pH 5.8, cultured at 23±1℃ and 80% humidity. After 40 days of culture under a photoperiod of 12 h light and 12 h dark, and a light intensity of 3000 lx, the seedlings grew into robust seedlings with a height of 2-4 cm, 2-3 leaves and 2-5 roots, with a rooting rate of 100%, and were ready for the hardening-off stage.

[0068] 6. Transplanting and hardening off seedlings

[0069] Place the tissue culture container containing the strong seedlings under natural light and room temperature conditions and culture for 3 days. Then, open the container cap halfway and culture for 12 hours. Remove the seedlings from the container and gently wash off the culture medium attached to the roots in 25°C clean water. Transplant the strong seedlings into a high-temperature sterilized transplanting substrate. The transplanting substrate is made by mixing peat moss (particle size 1~10mm), perlite (particle size 2~4mm), and vermiculite (particle size 3~6mm) in a volume ratio of 6:2:2 and sterilizing at 121°C for 2 hours.

[0070] Cultivated in a natural light greenhouse with ventilation four times a day, the survival rate of *Cymbidium lancifolium* is 95% after 40 days. When the individual plants reach a height of 8-10cm, the longest leaf length reaches 5-8cm, and the longest leaf width reaches 2-4cm, the *Cymbidium lancifolium* seedlings can be transplanted to a suitable wild habitat.

[0071] Comparative Example 1

[0072] Following the method described in the patent "Non-symbiotic Germination Method of Seeds of the Rare and Endangered Plant *Cymbidium faberi*", publication number CN114431149A, *Cymbidium faberi* was cultured. After 180 days, an increase in protocorms was observed, with a seed germination rate of up to 34.85% ± 13.49%. In contrast, in Example 1 of this invention, an increase in protocorms was observed after 60 days, with a germination rate reaching 65.74 ± 3.04%, significantly superior to the patent. The comparative patent requires 360 days from capsule treatment to the formation of robust seedlings (see the comparative patent). Figure 1 In Embodiment 1 of the present invention, it only takes 150 days from capsule treatment to the formation of strong seedlings, which greatly shortens the seedling cycle.

[0073] Comparative Example 2

[0074] According to the method described in the literature: Characteristics and Aseptic Sowing of *Cymbidium goeringii* seeds, Liu Haiping, Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2020, 49(01):29-34. DOI:10.13323 / j.cnki.j.fafu(nat.sci.).2020.01.006, the average embryogenicity of *Cymbidium goeringii* seeds was 45.15%±2.68%. However, using the method of this invention, the embryogenicity can reach 86.76%±3.82%, which is significantly higher than that in the literature. Furthermore, the germination time of seeds using this invention can be shortened to 60-80 days, far less than the shortest germination time of 120 days in the literature. Simultaneously, the germination rate of this invention, 65.74%±3.04%, is also significantly higher than the highest germination rate of 39.64%±3.34% in the literature. It has obvious technological advantages.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of *Cymbidium goeringii* seeds via tissue culture, characterized in that... Includes the following steps: (1) Seeds were obtained by hybridization of large yellow-flowered shrimp-shaped orchids under sterile conditions after the mature capsules that had not cracked open were disinfected. (2) The seed coat is damaged by sterile NaOH or KOH solution treatment; (3) Induction of protocorms, adventitious buds, and adventitious roots; (4) Seedling rooting and seedling strengthening cultivation; (5) Hardening off seedlings and transplanting; Step (3) Under aseptic conditions, the seeds obtained in step (2) are spread evenly on the surface of the culture medium. The culture medium consists of 1 / 2 MS medium + 1.0~5.0 mg / L 6-BA + 0.2~0.7 mg / L NAA + 0.2~0.6 mg / L TDZ + 1.0~4.0 g / L activated carbon. After culturing for 60~80 days, protocorms are formed in large quantities; after 100~120 days, adventitious buds are induced from the protocorms; after 120~140 days, the adventitious buds elongate, and adventitious roots are produced at the base, forming seedlings. Step (4) Transfer the seedlings obtained in step (3) to a rooting and seedling strengthening culture medium. The culture medium consists of 1 / 2 MS medium + 0.2~0.7 mg / L NAA + 1.0~5.0 g / L activated carbon. After culturing for 30~50 days, the seedlings grow into strong seedlings with a height of 2~4 cm, 2~3 leaves, and 2~5 roots. Then, they are transferred to the hardening stage.

2. The method according to claim 1, characterized in that, In step (1), in early April, during the flowering season of *Cymbidium goeringii*, anthers of *Cymbidium goeringii* from different cities were collected for hybridization and pollination. After pollination and fruiting, the capsules naturally develop for 6 to 8 months until they reach maturity, when the pericarp is yellow-green, the surface is free of disease spots, insect infestation, and cracks, and the capsules are healthy and mature.

3. The method according to claim 1, characterized in that, The disinfection process of the capsule in step (1) is as follows: After rinsing the surface of the capsule with pure water, according to the aseptic operation specifications, first soak it in 70-75% alcohol for 30-90 seconds, preferably 40-60 seconds; then rinse it once with sterile water, and then soak it in 6-10% NaClO for 8-12 minutes, preferably 7-8% NaClO for 9-10 minutes; then rinse it three times with sterile water for later use; under aseptic conditions, use sterile filter paper to absorb the moisture on the surface of the capsule after disinfection in step (1), and then cut it open to obtain the seeds inside the pericarp for later use.

4. The method according to claim 1, characterized in that, Step (2) Soak the sterile seeds obtained in step (1) in sterile NaOH or KOH solution with a concentration of 0.05~0.40 mol / L, preferably 0.20~0.30 mol / L, for 7~12 min, preferably 8~10 min, and rinse three times with sterile water for later use.

5. The method according to claim 1, characterized in that, Step (3) Add 1 mL of sterile water to the surface of the culture medium to spread the seeds evenly on the surface of the culture medium.

6. The method according to claim 1, characterized in that, In steps (3) and (4), the sucrose concentration in the culture medium is 25-35 g / L, the agar concentration is 5-8 g / L, the pH is adjusted to 5.5-6.0, the culture temperature is 23±1℃, the humidity is 70-80%, the light duration is 12-14 h / d, and the light intensity is 2000-3500 lx.

7. The method according to claim 1, characterized in that, Step (5) After hardening off the strong seedlings obtained in step (4), they are transferred to the transplanting substrate. The transplanting substrate is a mixture of peat moss, perlite and vermiculite in a volume ratio of (6~7): (1~2): (2~3).

8. The method according to claim 7, characterized in that, Place the tissue culture container containing the strong seedlings under natural light and room temperature conditions for 3-5 days, then open the container cap halfway and incubate for 12-24 hours. Remove the seedlings from the container and gently wash off the culture medium attached to the roots in clean water at 20-30℃. Transplant the strong seedlings into a high-temperature sterilized transplanting substrate. Cultivate in a natural light greenhouse with ventilation 2-4 times a day. When the individual plants are 8-10cm tall, the longest leaf is 5-8cm long, and the longest leaf is 2-4cm wide, transplant the large yellow-flowered shrimp-shaped orchid seedlings to a suitable wild habitat.

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