Tissue culture and rapid propagation method of guizhou germplasm dendrobium devonianum

Abstract

The application provides a Guizhou germplasm Dendrobium devonianum tissue culture rapid propagation method, which comprises the following steps: pre-treatment of explant material, sterile protocorm induction culture, cluster bud subculture proliferation culture, strong seedling rooting induction and seedling culture, and finally obtains Guizhou germplasm Dendrobium devonianum tissue culture seedlings. The application induces protocorms from Guizhou germplasm Dendrobium devonianum seeds, forms cluster buds through subculture proliferation, and obtains in-vitro regenerated plants through strong seedling rooting, seedling and transplanting, and establishes a complete tissue culture rapid propagation system. The method has the characteristics of high success rate, high induction rate, high proliferation coefficient, high genetic stability, good seedling quality, low cost, consistent growth, short seedling cycle and the like, and has universal applicability, and provides reliable technical support for mass breeding, rapid propagation and high-quality germplasm resource protection of Guizhou germplasm Dendrobium devonianum.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a rapid propagation method for Guizhou germplasm of Dendrobium nobile through tissue culture. Background Technology

[0002] Dendrobium purpureum ( Dendrobium parishii ) belongs to the genus Dendrobium in the family Orchidaceae ( Dendrobium This plant, whose fresh or dried stems can be used medicinally, possesses the effects of nourishing the stomach and promoting the production of body fluids, as well as nourishing yin and clearing heat. Purple-petaled Dendrobium not only has medicinal uses but also certain health benefits. In recent years, with national development and the expansion of the traditional Chinese medicine market, purple-petaled Dendrobium, as one of the sources of Dendrobium medicinal materials, has gradually gained attention. All plants in the Dendrobium genus are national second-class protected plants.

[0003] Currently, research on *Dendrobium aurantium* is limited. For rare and protected plants, establishing sound, stable, and reproducible artificial propagation techniques is particularly important. *Dendrobium aurantium* is a valuable plant resource, and how to obtain high-quality, high-yield *Dendrobium aurantium* through artificial propagation has become a primary problem for researchers. This will also provide technical support for the protection of *Dendrobium aurantium* germplasm resources and the industrialized seedling production of *Dendrobium aurantium*, which has significant practical implications.

[0004] Furthermore, while there are reports on tissue culture of *Dendrobium purpureum*, current techniques are mostly focused on common varieties or germplasm from other producing areas. *Dendrobium purpureum* from the Guizhou Plateau region, due to its long-term adaptation to its native habitat of high altitude, low latitude, low sunlight, and large diurnal temperature variations, has developed genetic and morphological characteristics distinct from those of *Dendrobium purpureum* from other regions. Its plants grow rapidly, have vibrant flower colors, numerous flower buds, and strong differentiation ability, demonstrating significant development potential. However, when using existing common culture media and methods to treat Guizhou germplasm, technical bottlenecks are common, including severe explant browning and death, a high proportion of vitrified seedlings during propagation, and poor root development, resulting in extremely low effective seedling rates and hindering the rapid and pure propagation of its superior germplasm resources. Therefore, developing a tissue culture method for rapid propagation that precisely adapts to the physiological characteristics of *Dendrobium purpureum* from Guizhou germplasm is of significant industrial importance for the protection and utilization of *Dendrobium purpureum* germplasm resources. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing a rapid tissue culture propagation method for Dendrobium nobile germplasm from Guizhou.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A rapid propagation method for Dendrobium purpureus germplasm from Guizhou through tissue culture includes the following steps: S1. Pretreatment of explant materials of Guizhou germplasm Dendrobium purpureum: Select mature 3-4 year old Dendrobium purpureum plants, transplant them into a clean greenhouse, spray the plants with carbendazim solution every week, and harvest the seed pods as explant materials after they mature. S2. Aseptic protocorm induction culture of Guizhou germplasm Dendrobium purpureum: The seed pods containing seeds from step S1 were disinfected with alcohol 3-4 times, then disinfected with mercuric chloride solution for 4-8 minutes, and rinsed with sterile water 3-4 times, 1 minute each time. After treatment, the seed pods were cut open, and the seeds inside were inoculated into the protocorm induction culture medium under aseptic conditions. After culturing for 25-40 days, the seeds of Dendrobium purpureum grew protocorms. S3. Subculture and propagation of clustered shoots of Dendrobium purpureum from Guizhou: The original tubers of Dendrobium purpureum obtained in step S2 were inoculated into the clustered shoot induction medium and cultured for 30-40 days to grow clustered shoots of Dendrobium purpureum. S4. Induction of rooting of vigorous seedlings of Dendrobium purpureum from Guizhou: The clustered buds of Dendrobium purpureum obtained in step S3 were cut and inoculated into rooting induction medium. After 20-25 days of culture, the adventitious buds began to root. After 40-45 days of culture, test-tube seedlings suitable for transplanting were obtained. S5. Cultivation of Guizhou Dendrobium officinale seedlings: Place the rooted Dendrobium officinale seedlings obtained in step S4 in a hardening greenhouse to adapt to the temperature and humidity. After 1-3 days, open the bottle cap and spray water mist. After 5-8 days, take out the tissue culture seedlings and transplant them into seedling trays containing sphagnum moss substrate. Spray water mist on the substrate for the first 5 days after transplanting to keep it moist. After 5 days, place them in an outdoor shade shed to continue growing for about 30 days to obtain Dendrobium officinale tissue culture seedlings.

[0007] Preferably, in step S2, the formulation of the protocorm induction medium is as follows: WPM medium is used as the base medium, with the addition of 0.1-0.3 mg / L 6-BA, 25-40 g / L sucrose, and 4.0-6.0 g / L agar, and the pH is 5.8-6.2. The medium needs to be sterilized by high-temperature moist heat at 116℃ for 20 minutes and then cooled before use.

[0008] More preferably, in step S2, the optimal formulation of the protocorm induction medium is: WPM medium as the base medium, with the addition of 0.1 mg / L 6-BA, 30 g / L sucrose, 4.3 g / L agar, and a pH of 5.9.

[0009] Preferably, in step S3, the formulation of the shoot proliferation induction medium is as follows: WPM medium is used as the basal medium, with the addition of 0.1–0.5 mg / L 6-BA, 0.05–0.2 mg / L NAA, 12.5–25 g / L mashed potato or banana, 25–40 g / L sucrose, and 4.3–6.0 g / L agar, with a pH of 5.8–6.2. The medium needs to be sterilized by high-temperature moist heat at 116°C for 20 minutes and then cooled before use.

[0010] More preferably, in step S3, the optimal formulation of the shoot proliferation induction medium is as follows: WPM medium as the base medium, with the addition of 0.2 mg / L 6-BA, 0.2 mg / L NAA, 12.5 g / L mashed potatoes, 30 g / L sucrose, and 4.3 g / L agar, and the pH is 5.9.

[0011] Preferably, in step S4, the rooting induction medium is formulated as follows: 1 / 2 MS medium is used as the basal medium, with the addition of 0.1–0.5 mg / L IBA, 30 g / L banana puree, 15 g / L activated carbon, 25–40 g / L sucrose, 4.3–6.0 g / L agar, and a pH of 5.8–6.2. The medium needs to be sterilized by high-temperature moist heat at 116°C for 20 minutes and then cooled before use.

[0012] More preferably, in step S4, the optimal formulation of the rooting induction medium is: based on 1 / 2 MS or N6 medium, with the addition of 0.2 mg / L IBA, 30 g / L banana puree, 15 g / L activated carbon, 30 g / L sucrose, 4.3 g / L agar, and pH 5.9.

[0013] Preferably, in step S1, the mass-volume concentration of the carbendazim solution is 0.08%-0.1%.

[0014] Preferably, in step S2, the volume concentration of the alcohol is 75%.

[0015] Preferably, in step S2, the mass-volume concentration of the mercuric chloride solution is 0.1% (g / mL).

[0016] Preferably, in step S5, the sphagnum moss substrate is soaked in purified water with 0.1% Huabao No. 1 added for 24 hours, the water is squeezed out, and it is then disinfected with 0.5% carbendazim before use.

[0017] Preferably, in steps S2, S3 and S4, the culture temperature is 25-26℃, the humidity is 50%, the light intensity is 1200-2000 lx, and the light duration is 10-12 hours / day.

[0018] More preferably, in steps S2, S3 and S4, the culture temperature is 25°C, the humidity is 50%, the light intensity is 1200 lx, and the light exposure time is 12 hours / day.

[0019] This invention primarily utilizes the direct organogenesis pathway. Protocorms are induced from seeds of *Dendrobium nobile* from Guizhou germplasm. These protocorms then proliferate to produce clustered buds. Following a series of processes including seedling vigorous rooting, hardening off, and transplanting, in vitro regenerated plants of *Dendrobium nobile* from Guizhou germplasm are obtained, establishing a rapid tissue culture propagation system for *Dendrobium nobile* from Guizhou germplasm. The direct organogenesis pathway bypasses the callus induction stage, resulting in a significantly shorter regeneration cycle and extremely strong genetic stability, which is crucial for maintaining the physiological characteristics of *Dendrobium nobile*.

[0020] The core of this invention lies in the optimization of each step to suit the unique characteristics of Guizhou Dendrobium officinale. In the design of the proliferation culture medium, a special formula and optimized culture process suitable for Guizhou Dendrobium officinale are adopted, which significantly promotes the formation of protocorms and the proliferation of clustered buds, maintaining vigorous differentiation capacity and significantly increasing the proliferation coefficient compared to conventional methods. During the rooting and hardening-off stages, appropriate addition of nutrients and hormones induces robust root development, improving the plant's adaptability to transplanting.

[0021] This invention selects seeds of *Dendrobium purpureum* from Guizhou as explants, achieving a high success rate in inducing cluster buds. By screening and optimizing the culture medium formula and conditions for the processes of subculturing, rooting, hardening, and transplanting of cluster buds from protocorms, rapid seedling production of *Dendrobium purpureum* from Guizhou can be achieved within 160 days through induction, subculturing, rooting, and transplanting. This invention features high success rate, high induction rate, high proliferation coefficient, high genetic stability, good seedling quality, low cost, uniform seedling growth, and a short seedling cycle. This invention establishes a factory-scale rapid propagation system for *Dendrobium purpureum* from Guizhou using plant tissue culture technology, possessing broad applicability and providing technical support for the large-scale propagation, rapid reproduction, and high-quality variety protection of *Dendrobium purpureum* from Guizhou. Attached Figure Description

[0022] Figure 1 The seed pods of Dendrobium purpureum from Guizhou are shown.

[0023] Figure 2 The image shows seeds of *Dendrobium purpureum* from Guizhou germplasm inoculated on WPM blank medium.

[0024] Figure 3 The induced growth of protocorms of Dendrobium purpureum from Guizhou is shown.

[0025] Figure 4 The study shows the induced growth of clustered shoots from the protocorms of *Dendrobium purpureum* from Guizhou.

[0026] Figure 5 The growth of clustered shoots of Dendrobium purpureum from Guizhou on different culture media is shown.

[0027] Figure 6 The rooting induction of the Guizhou germplasm of *Lithops purpurea* is shown.

[0028] Figure 7 The rooting of *Dendrobium purpureum* from Guizhou was shown on different culture media.

[0029] Figure 8 The hardening-off process of Guizhou germplasm Dendrobium officinale seedlings is shown. Detailed Implementation

[0030] The application method of the present invention is further illustrated below with reference to specific embodiments. The following embodiments and accompanying drawings are for illustrative purposes only and should not be construed as limiting the present invention. Unless otherwise specified, the reagents used in the following embodiments are conventionally commercially available biochemical reagents, and the methods and equipment used in the following embodiments are methods and equipment conventionally used in the art.

[0031] The term "Guizhou germplasm Dendrobium purpureum" as used in this article refers to Dendrobium purpureum distributed or cultivated in Guizhou Province. Dendrobium parishii The germplasm resource of *Dendrobium* (Rchb. f.) belongs to the genus *Dendrobium* of the Orchidaceae family. It has adapted to the unique climate, soil and other ecological environment of Guizhou, forming genetic characteristics and morphological features that are different from similar *Dendrobium* species in other regions. Its plants grow rapidly, have bright flower colors, a large number of flower buds, and strong differentiation ability, and have development potential.

[0032] In this invention, the concentration fractions of carbendazim and mercuric chloride solutions are both mass-volume percentages (%, g / mL).

[0033] The rapid propagation method of Guizhou germplasm Dendrobium purpureum by tissue culture of the present invention comprises the following steps: 1. Pretreatment and explant acquisition of Guizhou Dendrobium purpureum explant material Select mature 3-4 year old Guizhou Dendrobium officinale plants and transplant them into a clean greenhouse. Spray the plants with a 0.08-0.1% (mass volume concentration, 0.08-0.1g carbendazim per 100mL of water) carbendazim solution once a week. Harvest the seeds when the seed pods are nearly mature but have not yet burst open as explant material.

[0034] Figure 1 The seed pods of Dendrobium purpureum from Guizhou are shown.

[0035] 2. Aseptic protocorm induction culture of Guizhou germplasm Dendrobium purpureus After harvesting the *Dendrobium nobile* seed pods (containing seeds) treated in step 1, place them in a clean bench. Wipe the surface 3-4 times with 75% (volume concentration) alcohol wipes for initial disinfection, then disinfect with 0.1% (mass concentration) mercuric chloride solution for 4-8 minutes (ideally 6.5 minutes), followed by rinsing with sterile water 3-4 times, 1 minute each time. After treatment, cut open the *Dendrobium nobile* seed pods and inoculate the yellow seeds inside onto a protocorm induction medium under sterile conditions. After inoculation, provide 12 hours of light daily at a light intensity of 1200 lx, an air temperature of 25℃, and a humidity of 50%. After 25-40 days of culture, the *Dendrobium nobile* seeds will germinate into protocorms.

[0036] The formulation of the protocorm induction medium is as follows: Based on WPM medium (Woody Plant Medium, Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., catalog number HBZ0609), 0.1–0.3 mg / L of 6-BA (6-benzylaminopurine), 25–40 g / L of sucrose, and 4.0–6.0 g / L of agar are added, with a pH of 5.8–6.2. The medium needs to be sterilized by high-temperature moist heat sterilization at 116℃ for 20 minutes and then cooled before use.

[0037] This embodiment uses the optimal formulation of the protocorm induction medium: WPM medium as the base medium, with the addition of 0.1 mg / L 6-BA (6-benzylaminopurine), 30 g / L sucrose, 4.3 g / L agar, and pH 5.9.

[0038] Figure 2 The image shows seeds of *Dendrobium purpureum* from Guizhou germplasm inoculated onto protocorm induction medium.

[0039] Figure 3 The induced growth of protocorms of Dendrobium purpureum from Guizhou is shown.

[0040] 3. Subculture and propagation of clustered shoots of Dendrobium nobile from Guizhou germplasm The *Dendrobium purpureum* protocorms obtained in step 2 above were divided and inoculated into a bud proliferation induction medium. After inoculation, the plants were exposed to 12 hours of light per day at a light intensity of 1200 lx, with an air temperature of 25°C and a humidity of 50%. After 30-40 days of culture, axillary buds emerged from the *Dendrobium purpureum* stem segments.

[0041] The formulation of the bud proliferation induction medium is as follows: WPM medium is used as the basal medium, supplemented with 0.1–0.5 mg / L 6-BA, 0.05–0.2 mg / L NAA (naphthaleneacetic acid), 12.5–25 g / L mashed potato or banana, 25–40 g / L sucrose, and 4.3–6.0 g / L agar, with a pH of 5.8–6.2. The medium should be sterilized by high-temperature moist heat sterilization at 116℃ for 20 minutes and then cooled before use.

[0042] This embodiment uses the optimal formulation of the bud proliferation induction medium: WPM medium as the base medium, with the addition of 0.2 mg / L 6-BA, 0.2 mg / L NAA, 12.5 g / L mashed potatoes, 30 g / L sucrose, 4.3 g / L agar, and pH 5.9.

[0043] Figure 4 The study shows the induced growth of clustered shoots from the protocorms of *Dendrobium purpureum* from Guizhou.

[0044] Figure 5 The proliferation of clustered shoots of Dendrobium purpureum from Guizhou on different culture media is shown.

[0045] Culture medium A: WPM + 0.2 mg / L 6-BA + 0.2 mg / L NAA + 12.5 g / L mashed potatoes + 30 g / L sucrose + 4.3 g / L agar.

[0046] Culture medium B: WPM + 0.2 mg / L 6-BA + 0.2 mg / L NAA + 25 g / L mashed potatoes + 30 g / L sucrose + 4.3 g / L agar.

[0047] Culture medium C: WPM + 0.2 mg / L 6-BA + 0.2 mg / L NAA + 15 g / L banana puree + 30 g / L sucrose + 4.3 g / L agar.

[0048] Culture medium D: WPM + 0.2 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L banana puree + 30 g / L sucrose + 4.3 g / L agar.

[0049] Table 1 shows the effect of different culture media on the proliferation coefficient of clustered shoots of Dendrobium nobile from Guizhou germplasm.

[0050] Table 1. Effects of different culture media on the proliferation coefficient of clustered shoots of Dendrobium nobile from Guizhou germplasm.

[0051] The proliferation coefficient is calculated as follows:

[0052] Figure 5 The results in Table 1 show that, among the four culture media, group A had the highest proliferation coefficient of clustered shoots (1.57±0.50), significantly better than groups B, C, and D. Therefore, group A is the optimal culture medium formulation for the subculture proliferation of clustered shoots of *Dendrobium nobile* from Guizhou germplasm in this invention.

[0053] 4. Induction of rooting in vigorous seedlings of Dendrobium nobile from Guizhou germplasm The *Dendrobium nobile* buds obtained in step 3 above (using the optimal culture medium formula) were cut into appropriate sizes and inoculated into rooting induction medium. After inoculation, the plants were exposed to 12 hours of light per day at a light intensity of 1200 lx, an air temperature of 25°C, and a humidity of 50%. After 20-25 days of culture, the adventitious buds began to root, and after 40-45 days of culture, plantlets suitable for transplanting were obtained.

[0054] The rooting induction medium is formulated as follows: Based on 1 / 2 MS medium (Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., catalog number HB8469-12) or N6 medium (N6 medium (excluding agar and sucrose), Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., catalog number HBZ0601-2), add 0.1–0.5 mg / L IBA (indolebutyric acid), 30 g / L banana puree, 15 g / L activated carbon, 25–40 g / L sucrose, 4.3–6.0 g / L agar, and maintain a pH of 5.8–6.2. The medium should be sterilized by high-temperature moist heat sterilization at 116℃ for 20 minutes and then cooled before use.

[0055] This embodiment uses the optimal formulation of the rooting induction medium: 1 / 2MS medium as the base medium, with the addition of 0.2 mg / L IBA, 30 g / L banana puree, 15 g / L activated carbon, 30 g / L sucrose, 4.3 g / L agar, and pH 5.9.

[0056] Figure 6 The image shows the rooting induction process of *Dendrobium purpureum* from Guizhou. The left image is a top view, and the right image is a side view.

[0057] Figure 7 The rooting of *Dendrobium purpureum* from Guizhou was shown on different culture media.

[0058] Culture medium 1: 1 / 2 MS + 0.2 mg / L IBA + 30 g / L banana puree + 15 g / L activated carbon + 30 g / L sucrose + 4.3 g / L agar.

[0059] Culture medium 2: 1 / 2 MS + 0.5 mg / L IBA + 30 g / L banana puree + 15 g / L activated carbon + 30 g / L sucrose + 4.3 g / L agar.

[0060] Culture medium 3: N6 + 0.2 mg / L IBA + 30 g / L banana puree + 15 g / L activated carbon + 30 g / L sucrose + 4.3 g / L agar.

[0061] Culture medium 4: N6 + 0.5 mg / L IBA + 30 g / L banana puree + 15 g / L activated carbon + 30 g / L sucrose + 4.3 g / L agar.

[0062] Table 2 shows the rooting of Guizhou Dendrobium nobile on different culture media.

[0063] Table 2. Rooting of *Dendrobium nobile* from Guizhou germplasm on different culture media

[0064] The method for calculating the root number growth factor is as follows:

[0065] Figure 7 The results in Table 2 show that among the four rooting media, Group 1 had the highest root growth rate (2.08±0.55), significantly better than Groups 2, 3, and 4. Therefore, Group 1 is the optimal culture medium formula for the vigorous rooting of *Dendrobium nobile* seedlings from Guizhou germplasm in this invention.

[0066] 5. Hardening-off cultivation of Guizhou germplasm Dendrobium purpureus seedlings The *Dendrobium nobile* rooted seedlings obtained in step 4 above were placed in a hardening-off greenhouse to adapt to the temperature and humidity. After 1-3 days, the bottle caps were opened and water mist was sprayed. After 5-8 days, the tissue culture seedlings were removed and transplanted into seedling trays containing sphagnum moss. The sphagnum moss was soaked in purified water with 0.1% (mass concentration) Hyponex No. 1 added for 24 hours, and then the water was squeezed out before use. For the first 5 days after transplanting, water mist should be sprayed to keep the substrate moist. After 5 days, the seedlings were placed in an outdoor shade structure to continue growing for 30 days to obtain *Dendrobium nobile* tissue culture seedlings.

[0067] Figure 8 The hardening-off process of Guizhou germplasm Dendrobium officinale seedlings is shown.

[0068] Experimental data show that this method exhibits significant specificity and high efficiency for *Dendrobium purpureum* germplasm from Guizhou. Using the culture medium and optimized culture process provided by this invention, the survival rate of explants of *Dendrobium purpureum* germplasm from Guizhou is consistently increased to over 90%, the proliferation coefficient of clustered shoots reaches over 1.30, and the root number can increase by up to approximately 2 times. More importantly, the tissue culture seedlings propagated by this method, after being transplanted to a cultivation base simulating the Guizhou habitat, have a survival rate exceeding 90%, and chemical composition analysis shows that the content of its characteristic medicinal components is not significantly different from that of the wild population, fully maintaining the excellent traits and genetic stability of the Guizhou germplasm. This invention not only solves the technical problem of difficult propagation of this specific germplasm, but also provides a reliable technical guarantee for its large-scale, standardized production and subsequent sustainable development and utilization.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A rapid propagation method for Dendrobium purpureus germplasm from Guizhou through tissue culture, characterized in that, Includes the following steps: S1. Pretreatment of explant materials of Guizhou germplasm Dendrobium purpureum: Select mature 3-4 year old Dendrobium purpureum plants, transplant them into a clean greenhouse, spray the plants with carbendazim solution every week, and harvest the seed pods as explant materials after they mature. S2. Aseptic protocorm induction culture of Guizhou germplasm Dendrobium purpureum: The seed pods containing seeds from step S1 were disinfected with alcohol 3-4 times, then disinfected with mercuric chloride solution for 4-8 minutes, and rinsed with sterile water 3-4 times, 1 minute each time. After treatment, the seed pods were cut open, and the seeds inside were inoculated into the protocorm induction culture medium under aseptic conditions. After culturing for 25-40 days, the seeds of Dendrobium purpureum grew protocorms. S3. Subculture and propagation of clustered shoots of Dendrobium purpureum from Guizhou: The original tubers of Dendrobium purpureum obtained in step S2 were inoculated into the clustered shoot induction medium and cultured for 30-40 days to grow clustered shoots of Dendrobium purpureum. S4. Induction of rooting of vigorous seedlings of Dendrobium purpureum from Guizhou: The clustered buds of Dendrobium purpureum obtained in step S3 were cut and inoculated into rooting induction medium. After 20-25 days of culture, the adventitious buds began to root. After 40-45 days of culture, test-tube seedlings suitable for transplanting were obtained. S5. Cultivation of Guizhou Dendrobium officinale seedlings: Place the rooted Dendrobium officinale seedlings obtained in step S4 in a hardening greenhouse to adapt to the temperature and humidity. After 1-3 days, open the bottle cap and spray water mist. After 5-8 days, take out the tissue culture seedlings and transplant them into seedling trays containing sphagnum moss substrate. Spray water mist on the substrate for the first 5 days after transplanting to keep it moist. After 5 days, place them in an outdoor shade shed to continue growing for about 30 days to obtain Dendrobium officinale tissue culture seedlings.

2. The method according to claim 1, characterized in that, In step S2, the formulation of the protocorm induction medium is as follows: WPM medium is used as the base medium, with the addition of 0.1~0.3 mg / L of 6-BA, 25~40 g / L of sucrose, and 4.0~6.0 g / L of agar, and the pH is 5.8~6.

2.

3. The method according to claim 1, characterized in that, In step S2, the optimal formulation of the protocorm induction medium is as follows: WPM medium as the base medium, with the addition of 0.1 mg / L 6-BA, 30 g / L sucrose, 4.3 g / L agar, and pH 5.

9.

4. The method according to claim 1, characterized in that, In step S3, the formulation of the bud proliferation induction medium is as follows: WPM medium is used as the base medium, with the addition of 0.1~0.5 mg / L of 6-BA, 0.05~0.2 mg / L of NAA, 12.5~25 g / L of mashed potatoes or mashed bananas, 25~40 g / L of sucrose, 4.3~6.0 g / L of agar, and the pH is 5.8~6.

2.

5. The method according to claim 1, characterized in that, In step S3, the optimal formulation of the bud proliferation induction medium is as follows: WPM medium is used as the base medium, with the addition of 0.2 mg / L 6-BA, 0.2 mg / L NAA, 12.5 g / L mashed potatoes, 30 g / L sucrose, and 4.3 g / L agar, with a pH of 5.

9.

6. The method according to claim 1, characterized in that, In step S4, the rooting induction medium is formulated as follows: 1 / 2 MS medium is used as the base medium, with the addition of 0.1-0.5 mg / L IBA, 30 g / L banana puree, 15 g / L activated carbon, 25-40 g / L sucrose, 4.3-6.0 g / L agar, and pH 5.8-6.

2.

7. The method according to claim 1, characterized in that, In step S4, the optimal formulation of the rooting induction medium is as follows: 1 / 2 MS or N6 medium as the base medium, with the addition of 0.2 mg / L IBA, 30 g / L banana puree, 15 g / L activated carbon, 30 g / L sucrose, 4.3 g / L agar, and pH 5.

9.

8. The method according to claim 1, characterized in that, In step S1, the mass-volume concentration of the carbendazim solution is 0.08%-0.1%.

9. The method according to claim 1, characterized in that, In step S5, the sphagnum moss substrate is soaked in purified water with 0.1% Huabao No. 1 added for 24 hours, the water is squeezed out, and it is then disinfected with 0.5% carbendazim before use.

10. The method according to claim 1, characterized in that, In steps S2, S3 and S4, the culture temperature is 25-26℃, the humidity is 50%, the light intensity is 1200-2000 lx, and the light duration is 10-12 hours / day.

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