Tissue culture medium and seedling rapid propagation method of dendrobium nobile and application thereof

Abstract

The application belongs to the technical field of plant tissue culture and specifically relates to a tissue culture medium and seedling rapid propagation method of Dendrobium nobile and application. The tissue culture medium of the Dendrobium nobile comprises a seed germination medium, a cluster bud induction medium and a rooting and seedling strengthening medium; under the joint action of 1 / 2MS medium, 6-BA, NAA, potato juice, banana mud and activated carbon, the seed germination of the Dendrobium nobile is induced, the protocorm differentiation is induced into cluster buds, the cluster buds are induced to root and obtain seedlings, especially, the banana mud is added in the seed germination medium, the seed germination time is shortened, and the potato juice is added in the cluster bud induction medium and the rooting and seedling strengthening medium, the protocorm development and growth of the Dendrobium nobile are promoted, a large number of luxuriant cluster buds and strong seedlings are obtained, and the water moss is used as a transplanting substrate, and the transplanting survival rate of the Dendrobium nobile seedlings is further improved.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a tissue culture medium for Dendrobium nobile and a method for rapid propagation of seedlings, as well as their applications. Background Technology

[0002] Nobile-type Dendrobium is a perennial epiphytic herb belonging to the genus Dendrobium in the Orchidaceae family, named for its spring flowering. Nobile-type Dendrobium is favored by growers for its compact plant shape, abundant and vibrant flowers, fragrance, and ease of management. Currently, propagation of Nobile-type Dendrobium relies on conventional methods such as division and cuttings, which have relatively low efficiency, resulting in low yields that fall far short of production needs. Therefore, developing rapid propagation and cultivation of Nobile-type Dendrobium seedlings is essential. However, current rapid seed propagation methods still suffer from low seedling transplant survival rates, limiting the scale of Nobile-type Dendrobium production. Summary of the Invention

[0003] The purpose of this invention is to provide a tissue culture medium and a method for rapid propagation of Dendrobium officinale seedlings, as well as their applications, which can improve the transplant survival rate of Dendrobium officinale tissue culture seedlings and obtain robust Dendrobium officinale seedlings.

[0004] To address the above problems, the present invention provides the following technical solution:

[0005] This invention provides a tissue culture medium for Dendrobium nobile, the tissue culture medium comprising a seed germination medium, a shoot cluster induction medium, and a rooting and seedling strengthening medium;

[0006] The seed germination medium uses 1 / 2 MS medium as the basic medium and also includes: 0.1-0.2 mg / L NAA, 15-25 g / L sucrose, 65-75 g / L banana puree and 6-8 g / L agar;

[0007] The bud induction medium uses 1 / 2 MS medium as the basic medium and also includes: 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 70-90 g / L potato juice, 20-40 g / L sucrose and 6-8 g / L agar;

[0008] The rooting and seedling strengthening culture medium uses 1 / 2 MS medium as the basic medium and also includes: 0.2-0.5 mg / L NAA, 70-90 g / L potato juice, 0.1-0.3 g / L activated carbon, 20-40 g / L sucrose and 6-8 g / L agar.

[0009] Preferably, the pH value of the seed germination medium is 5.8-6.2; the pH value of the shoot induction medium is 5.8-6.2; and the pH value of the rooting and seedling strengthening medium is 5.8-6.2.

[0010] This invention provides a method for rapid propagation of Dendrobium officinale seedlings, comprising the following steps:

[0011] After sterilizing the capsules of Dendrobium nobile, extract the seeds of Dendrobium nobile;

[0012] The Dendrobium nobile seeds were inoculated onto the seed germination culture medium described in the above technical solution to obtain protocorms;

[0013] The original bulbs are then inoculated onto the shoot induction medium described in the above technical solution to induce shoot growth and obtain shoot clusters.

[0014] The clustered buds were inoculated onto the rooting and seedling strengthening culture medium described in the above technical solution to obtain Dendrobium officinale tissue culture seedlings;

[0015] The Dendrobium officinale tissue culture seedlings were hardened off and transplanted to obtain Dendrobium officinale seedlings.

[0016] Preferably, the method for disinfecting the Dendrobium nobile capsules includes: after washing, first soaking in 70%-75% alcohol for 10-30 seconds, then rinsing with sterile water 1-2 times; then surface disinfecting with 0.1% mercuric chloride solution for 5-10 minutes; finally rinsing with sterile water 5-8 times; each second rinse with sterile water lasts 3 minutes; the washing includes rinsing with running water for 5-10 minutes.

[0017] Preferably, before inoculating the protocorms into the bud induction medium, the process further includes dividing the protocorm clusters into protocorm clusters with a size of (3-5) mm × (3-5) mm before inoculation; and dividing the bud clusters into single buds or double buds before inoculating them into the rooting and seedling strengthening medium.

[0018] Preferably, the germination culture temperature is 25–27°C;

[0019] The germination culture was carried out under alternating light and dark conditions, with the light culture time in the light-dark alternation being 12-14 h / d and the dark culture time in the light-dark alternation being 10-12 h / d; the light intensity for the light culture was 1500-2000 lx; ​​and each light-dark alternation culture cycle was 24 h.

[0020] The germination culture time is 18–22 days.

[0021] Preferably, the induction culture temperature is 25–27°C;

[0022] The induction culture was carried out under alternating light and dark conditions, with the light culture time in the light-dark alternation being 12-14 h / d and the dark culture time in the light-dark alternation being 10-12 h / d; the light intensity of the light culture was 1500-2000 lx; ​​and each light-dark alternation culture cycle was 24 h.

[0023] The induction culture time is 90–368 days.

[0024] Preferably, the temperature for the rooting culture of the robust seedlings is 25–27°C;

[0025] The seedling rooting culture was carried out under alternating light and dark conditions. The light culture time during the light-dark alternation was 12-14 h / d, and the dark culture time during the light-dark alternation was 10-12 h / d. The light intensity during the light culture was 1500-2000 lx. Each light-dark alternation culture cycle was 24 h.

[0026] The rooting culture time for the robust seedlings is 25–30 days.

[0027] Preferably, the hardening-off and transplanting process includes hardening off the seedlings in bottles for 9-11 days in a hardening-off room before transplanting them into a greenhouse; the transplanting substrate includes sphagnum moss.

[0028] This invention provides the application of the tissue culture medium or the seedling rapid propagation method described in the above-mentioned technical solutions in improving the survival rate of Dendrobium officinale seedlings after transplanting.

[0029] The beneficial effects of the present invention are as follows: The present invention provides a tissue culture medium for Dendrobium nobile, the tissue culture medium comprising a seed germination medium, a shoot cluster induction medium, and a rooting and seedling strengthening medium;

[0030] The seed germination medium uses 1 / 2 MS medium as the basic medium and also includes: 0.1-0.2 mg / L NAA, 15-25 g / L sucrose, 65-75 g / L banana puree and 6-8 g / L agar;

[0031] The bud induction medium uses 1 / 2 MS medium as the basic medium and also includes: 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 70-90 g / L potato juice, 20-40 g / L sucrose and 6-8 g / L agar;

[0032] The rooting and seedling strengthening culture medium uses 1 / 2 MS medium as the basic medium and also includes: 0.2-0.5 mg / L NAA, 70-90 g / L potato juice, 0.1-0.3 g / L activated carbon, 20-40 g / L sucrose and 6-8 g / L agar.

[0033] In the tissue culture medium provided by this invention, NAA (1-naphthaleneacetic acid) promotes the growth of adventitious buds and roots of *Dendrobium nobile*; banana puree promotes seed germination; 6-BA (6-benzylaminopurine) stimulates cell division and promotes the growth and development of clustered buds; potato juice promotes the growth and development of protocorms and rooting of robust seedlings; and activated carbon is beneficial for rooting. The combined effects of 1 / 2 MS medium, 6-BA, NAA, banana puree, potato juice, and activated carbon facilitate the induction of *Dendrobium nobile* seed germination and protocorm differentiation into clustered buds, resulting in a large number of tissue culture seedlings. After hardening and transplanting, robust seedlings are obtained. Using sphagnum moss as the transplanting substrate further improves the transplant survival rate of the tissue culture seedlings. The results of the examples show that the tissue culture medium of this invention can improve the transplant survival rate of *Dendrobium nobile* tissue culture seedlings, with a survival rate of 95%. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0035] Figure 1 This is the result of aseptic germination culture of Dendrobium officinale seeds on day 22 of Example 1;

[0036] Figure 2 This is the situation on day 60 of the induction culture of Dendrobium nobile shoots in Example 1;

[0037] Figure 3 This is the situation on day 28 of the rooting and seedling cultivation of Dendrobium nobile in Example 1;

[0038] Figure 4 This is the situation on day 20 after transplanting the Dendrobium officinale tissue culture seedlings in Example 1. Detailed Implementation

[0039] This invention provides a tissue culture medium for Dendrobium nobile, the tissue culture medium comprising a seed germination medium, a shoot cluster induction medium, and a rooting and seedling strengthening medium;

[0040] The seed germination medium uses 1 / 2 MS medium as the basic medium and also includes: 0.1-0.2 mg / L NAA, 15-25 g / L sucrose, 65-75 g / L banana puree and 6-8 g / L agar;

[0041] The bud induction medium uses 1 / 2 MS medium as the basic medium and also includes: 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 70-90 g / L potato juice, 20-40 g / L sucrose and 6-8 g / L agar;

[0042] The rooting and seedling strengthening culture medium uses 1 / 2 MS medium as the basic medium and also includes: 0.2-0.5 mg / L NAA, 70-90 g / L potato juice, 0.1-0.3 g / L activated carbon, 20-40 g / L sucrose and 6-8 g / L agar.

[0043] In this invention, the seed germination medium uses 1 / 2 MS medium as the basic medium and further includes: 0.1-0.2 mg / L NAA, 15-25 g / L sucrose, 65-75 g / L banana puree and 6-8 g / L agar; more preferably, using 1 / 2 MS medium as the basic medium, it also contains only 0.1-0.2 mg / L NAA, 15-25 g / L sucrose, 65-75 g / L banana puree and 6-8 g / L agar. The NAA concentration in the seed germination medium of this invention is 0.1–0.2 mg / L, more preferably 0.13–0.16 mg / L, and even more preferably 0.15 mg / L; the concentration of banana puree in the seed germination medium is 65–75 g / L, more preferably 67–71 g / L, and even more preferably 70 g / L; the concentration of sucrose in the seed germination medium is 15–25 g / L, more preferably 17–22 g / L, and even more preferably 20 g / L; the concentration of agar in the seed germination medium is 6–8 g / L, more preferably 6.5–7.5 g / L, and even more preferably 7 g / L. In this invention, the pH value of the sterilized seed germination medium is preferably 5.8–6.2, and even more preferably 5.8.

[0044] In this invention, the shoot induction medium uses 1 / 2 MS medium as the basic medium and further includes: 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 70-90 g / L potato juice, 20-40 g / L sucrose and 6-8 g / L agar; more preferably, using 1 / 2 MS medium as the basic medium, it also contains only 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 70-90 g / L potato juice, 20-40 g / L sucrose and 6-8 g / L agar. The 6-BA concentration in the shoot induction medium of the present invention is 0.1–0.3 mg / L, more preferably 0.2 mg / L; the NAA concentration in the shoot induction medium of the present invention is 0.1–0.2 mg / L, more preferably 0.15 mg / L; the potato juice concentration in the shoot induction medium of the present invention is 70–90 g / L, more preferably 80 g / L; the sucrose concentration in the shoot induction medium of the present invention is 20–40 g / L, more preferably 30 g / L; and the agar concentration in the shoot induction medium of the present invention is 6–8 g / L, more preferably 7 g / L. In the present invention, the pH value of the sterilized culture medium is preferably 5.8–6.2, more preferably 5.8. The preferred method for preparing the potato juice of the present invention includes: peeling and removing the sprouts from potatoes, cutting them into potato strips of approximately 5 cm × 1 cm × 1 cm, boiling them in water for 30 minutes, filtering them through gauze, and obtaining the filtrate for later use. Add 70-90g of potato juice prepared from potatoes to every 1L of shoot induction medium.

[0045] In this invention, the rooting and seedling strengthening culture medium uses 1 / 2 MS medium as the basic culture medium and further includes: 0.2-0.5 mg / L NAA, 70-90 g / L potato juice, 0.1-0.3 g / L activated carbon, 20-40 g / L sucrose and 6-8 g / L agar; more preferably, using 1 / 2 MS medium as the basic culture medium, it also contains only 0.2-0.5 mg / L NAA, 70-90 g / L potato juice, 0.1-0.3 g / L activated carbon, 20-40 g / L sucrose and 6-8 g / L agar. The NAA concentration in the rooting and seedling strengthening culture medium of the present invention is 0.2-0.5 mg / L, more preferably 0.25-0.4 mg / L, and more preferably 0.3 mg / L; the potato juice concentration in the rooting and seedling strengthening culture medium of the present invention is 70-90 g / L, more preferably 75-85 g / L, and more preferably 80 g / L; the activated carbon concentration in the rooting and seedling strengthening culture medium of the present invention is 1-3 g / L, more preferably 1-1.5 g / L, and more preferably 1 g / L; the sucrose concentration in the rooting and seedling strengthening culture medium of the present invention is 20-40 g / L, more preferably 25-35 g / L, and more preferably 30 g / L; the agar concentration in the rooting and seedling strengthening culture medium of the present invention is 6-8 g / L, more preferably 6.5-7.5 g / L, and more preferably 7 g / L. In the present invention, the pH value of the sterilized rooting and seedling strengthening culture medium is preferably 5.8-6.2, and more preferably 5.8.

[0046] In the seed germination medium of this invention, NAA (1-naphthaleneacetic acid) promotes the growth of adventitious buds of *Dendrobium nobile*, while banana pulp promotes seed germination. An appropriate concentration ratio of banana pulp and NAA shortens the germination culture time. In the shoot induction medium of this invention, 6-BA (6-benzylaminopurine) stimulates cell division and promotes shoot growth and development, NAA promotes the growth of adventitious buds of *Dendrobium nobile*, and potato juice promotes protocorm growth and development. A large number of plants were obtained at an appropriate concentration ratio of 6-BA, NAA, and potato juice. In the rooting and seedling strengthening medium of this invention, activated carbon, potato juice, and NAA all promote rooting. An appropriate concentration ratio increases the rooting rate of seedlings and shortens the rooting time. This invention utilizes 1 / 2 MS medium, 6-BA, NAA, potato juice, banana puree, and activated carbon to induce the germination of Dendrobium nobile seeds and the differentiation of protocorms into clustered buds, resulting in a large number of seedlings. The use of sphagnum moss as a transplanting substrate further improves the transplant survival rate of the seedlings.

[0047] This invention does not have any special limitations on the source of each component in the seed germination medium, the shoot cluster induction medium, and the rooting and seedling strengthening medium; conventional commercially available products can be used.

[0048] This invention provides a method for rapid propagation of Dendrobium officinale seedlings, comprising the following steps:

[0049] After sterilizing the capsules of Dendrobium nobile, extract the seeds of Dendrobium nobile;

[0050] After the Dendrobium nobile seeds were inoculated onto the seed germination culture medium described in the above technical solution, protocorms were obtained;

[0051] The protocorms were inoculated onto the shoot induction medium described in the above technical solution to induce culturing, thereby obtaining shoot clusters;

[0052] The clustered buds were inoculated onto the rooting and seedling strengthening culture medium described in the above technical solution to obtain Dendrobium officinale tissue culture seedlings;

[0053] The Dendrobium officinale tissue culture seedlings were hardened off and transplanted to obtain Dendrobium officinale seedlings.

[0054] This invention involves sterilizing the capsules of *Dendrobium nobile* and then extracting the seeds. The maturity of the *Dendrobium nobile* capsules used in this invention is preferably 80%–90%. The *Dendrobium nobile* capsules are used as explants in this invention because they are abundant in material and relatively easy to sterilize.

[0055] After harvesting, the Dendrobium nobile capsules of this invention are preferably cleaned and disinfected before the seeds are extracted. The cleaning process preferably includes rinsing with running water for 5-10 minutes, more preferably 8 minutes. After cleaning, the Dendrobium nobile capsules are preferably disinfected. The disinfection method preferably includes: first soaking in 70%-75% alcohol for 10-30 seconds, then rinsing 1-2 times with sterile water; then surface disinfecting with 0.1% mercuric chloride solution for 5-10 minutes; and finally rinsing 5-8 times with sterile water for 3 minutes each time. The volume concentration of alcohol described in this invention is preferably 70%–75%, more preferably 75%; the number of times the first rinse with sterile water is preferably 1–2 times, more preferably 2 times; the surface disinfection time with the 0.1% mercuric chloride solution is preferably 5–10 minutes, more preferably 7 minutes; the number of times the second rinse with sterile water is preferably 5–8 times, more preferably 6 times. The time for each second rinse with sterile water is preferably 3 minutes. Mercuric chloride has the best disinfection effect, but its residue is difficult to remove. Therefore, this invention controls the disinfection time of mercuric chloride and increases the number of rinses and the rinsing time with sterile water to remove mercuric chloride residue as much as possible and reduce the harm to Dendrobium officinale seeds.

[0056] After extracting the Dendrobium nobile seeds, the present invention inoculates the Dendrobium nobile seeds on a seed germination medium to germinate and culture them to obtain protocorms.

[0057] In this invention, the germination culture temperature is preferably 25–27°C, more preferably 26°C; the germination culture is preferably carried out under alternating light and dark conditions, with the light culture time preferably 12–14 h / d, more preferably 12 h / d; the dark culture time preferably 10–12 h / d, more preferably 12 h / d; each light-dark alternation cycle is 24 h; the light culture intensity is preferably 1500–2000 lx, more preferably 1800 lx; ​​the germination culture time is preferably 18–22 days, more preferably 20 days; the germination culture humidity is preferably 60%–80%, more preferably 65%–75%, more preferably 70%.

[0058] After obtaining the protocorms, the present invention inoculates the protocorms into a bud induction medium for induction culture to obtain bud clusters.

[0059] In this invention, the original bulbs are preferably inoculated into a shoot induction medium. Before inoculating the original bulbs into the shoot induction medium, they are preferably cut into pieces with a size of (3-5) mm × (3-5) mm.

[0060] The preferred temperature for induction culture in this invention is 25–27°C, more preferably 26°C. The induction culture is preferably carried out under alternating light and dark conditions. The light culture time during the light-dark alternation is preferably 12–14 h / d, more preferably 12–13 h / d, and more preferably 12 h / d. The dark culture time during the light-dark alternation is preferably 10–12 h / d, more preferably 12 h / d. Each light-dark alternation culture cycle is preferably 24 h. The light intensity during the light culture is preferably 1500–2000 lx, more preferably 1800 lx–2000 lx. The humidity during the induction culture is preferably 60%–80%, more preferably 65%–75%, and more preferably 70%. The preferred induction culture time is 90–368 days, more preferably 92 days, 187 days, 270 days, or 360 days. The induction culture process preferably also includes 1–3 transfer cultures, more preferably 2. The transfer culture described in this invention preferably begins after 85-95 days of induction culture, more preferably after 90-92 days; the culture medium for the transfer culture described in this invention is preferably a shoot induction medium. The transfer culture described in this invention is for obtaining more Dendrobium nobile shoots; if the demand for Dendrobium nobile shoots is small, transfer culture may not be performed.

[0061] After obtaining the clustered buds, the present invention preferably divides the clustered buds into single or double buds and inoculates them onto a rooting and seedling strengthening culture medium for seedling rooting culture to obtain Dendrobium officinale tissue culture seedlings. The preferred temperature for seedling rooting culture in the present invention is 25–27℃, more preferably 26℃; the seedling rooting culture is preferably carried out under alternating light and dark conditions, with the light culture time preferably 12–14 h / d, more preferably 12 h / d; the dark culture time preferably 10–12 h / d, more preferably 12 h / d; each light-dark alternation cycle is 24 h; the preferred light intensity is 1500–2000 lx, more preferably 1700–1900 lx; ​​the preferred humidity is 60%–80%, more preferably 65%–70%; the preferred seedling rooting culture time is 25–30 days, more preferably 26–29 days, more preferably 28 days.

[0062] The strong seedlings of the present invention are rooted and cultured to obtain Dendrobium officinale tissue culture seedlings. The tissue culture seedlings are preferably 4cm in height, have 4-5 leaves and 2-3 roots, and are then hardened off and transplanted.

[0063] The present invention involves hardening off and transplanting the tissue culture seedlings of Dendrobium nobile to obtain Dendrobium nobile seedlings.

[0064] The preferred method of hardening off and transplanting in this invention includes hardening off the Dendrobium officinale tissue culture seedlings in bottles for 9-11 days in a hardening-off room before transplanting them into a greenhouse; the transplanting substrate includes sphagnum moss. Preferably, the Dendrobium officinale tissue culture seedlings are hardened off in bottles for 9-11 days in a hardening-off room, more preferably for 10 days. The greenhouse preferred in this invention includes a greenhouse structure. After transplanting, the tissue culture seedlings are preferably managed using conventional methods to obtain Dendrobium officinale seedlings.

[0065] This invention provides the application of the tissue culture medium or the seedling rapid propagation method described in the above-mentioned technical solutions in improving the survival rate of Dendrobium officinale seedlings after transplanting.

[0066] This invention provides a method for rapid propagation of Dendrobium officinale seedlings, including explant selection, disinfection, seed germination and protocorm induction, protocorm proliferation and cluster bud induction, seedling strengthening and rooting culture, and acclimatization and transplanting steps. The survival rate of Dendrobium officinale seedlings transplanted using this invention is 93-96%.

[0067] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1

[0069] This invention provides a method for rapid propagation of Dendrobium officinale seedlings, comprising the following steps: explant selection, cleaning and disinfection, seed germination and protocorm induction, proliferation and cluster bud induction, seedling strengthening and rooting culture, and acclimatization and transplanting. The details are as follows:

[0070] (1) Explant selection: On August 1, 2019, seeds from the full, mature capsules of the Dendrobium nobile × (Tsinghua No. 2 * White and Blue Musk) hybrid were collected as explants. Dendrobium nobile was the female parent, and (Tsinghua No. 2 * White and Blue Musk) was also a hybrid Dendrobium and was the male parent.

[0071] (2) Cleaning and disinfection of explants: Cut Dendrobium capsules from the Dendrobium officinale plant in step (1), wash them with running tap water for 10 minutes, immerse them in 75% alcohol for 30 seconds on a clean bench or inoculation box, rinse them twice with sterile water, immerse them in 0.1% mercuric chloride solution for surface disinfection for 10 minutes, and rinse them five times with sterile water for 3 minutes each time. After disinfection, place the Dendrobium officinale capsules on sterilized filter paper to absorb excess water for later use.

[0072] (3) Seed germination culture: On August 1, 2019, the sterilized capsules from step (2) were cut open on a sterilized steel plate, and the seeds were removed. The seeds were inoculated into seed germination medium for seed germination culture, and protocorms were obtained after 22 days of culture. The seed germination medium consisted of 1 / 2 MS medium, 0.2 mg / L NAA, 20 g / L sucrose, 70 g / L banana puree, and 7 g / L agar, with a pH of 6.0. The seed germination culture temperature was 27℃, the humidity was 70%, the light intensity was 1600 lx, and the photoculture time in the photoperiod was 12 h / d. The status of aseptic germination of seeds on day 22 is shown in the figure. Figure 1 The seed germination culture time was 22 days.

[0073] (4) Induction of shoot clusters: On August 23, 2019, the protocorm clusters obtained in step (3) were cut into (3-5) mm × (3-5) mm pieces and inoculated into shoot cluster induction medium for 187 days to obtain abundant shoot clusters. The shoot cluster induction medium consisted of 1 / 2 MS medium, 0.1 mg / L 6-BA, 0.2 mg / L NAA, 80 g / L potato juice, 30 g / L sucrose, and 7 g / L agar, with a pH of 5.8. The induction culture temperature was 27℃, the humidity was 70%, the light intensity was 1800 lx, and the photoculture time was 12 h / d. The results of shoot cluster induction culture on day 60 are shown in [link to relevant documentation]. Figure 2 On day 92 of the bud induction culture, the seedlings were transferred to the bud induction medium. After another 95 days of culture, they were inoculated into the rooting and seedling strengthening medium.

[0074] (5) Rooting and Seedling Strengthening Culture: On February 27, 2020, the clustered buds obtained in step (4) were cut into single or double buds and then transferred to a rooting and seedling strengthening culture medium for 30 days to obtain complete Dendrobium officinale tissue culture seedlings after rooting. The rooting and seedling strengthening culture medium consisted of 1 / 2 MS, 0.4 mg / L NAA, 70 g / L potato juice, 30 g / L sucrose, 7 g / L agar, and 0.2 g / L activated carbon, with a pH of 5.8. The temperature for rooting and seedling strengthening culture was 27℃, humidity 80%, light intensity 2000 lx, and photoperiod 12 h / d. After the Dendrobium officinale tissue culture seedlings grew to a height of 4 cm, with 4-5 leaves and 2-3 roots, they were hardened off and transplanted. The condition of the rooting and seedling strengthening culture on day 28 is shown in the figure. Figure 3 .

[0075] (6) Hardening off and transplanting: On March 26, 2020, the rooted Dendrobium officinale tissue culture seedlings obtained in step (5) were placed in a hardening-off room with bottles for 10 days before being transplanted into a greenhouse. Transplanting was carried out at 10:00 am at a temperature of 25°C. The temperature in the greenhouse was 25°C and the humidity was 80%.

[0076] The transplanting substrate used in the greenhouse is sphagnum moss, with a pH value of 5.8–6.2. The sphagnum moss is disinfected with a 1.0 g / L carbendazim solution before transplanting.

[0077] Routine management after transplanting: Within 7-10 days after transplanting, strengthen heat preservation and humidity control, spray the leaves daily, and spray with nutrient solution every 10 days. The nutrient solution should mainly consist of potassium dihydrogen phosphate at a concentration of 0.1%.

[0078] The condition of Dendrobium officinale tissue culture seedlings on day 20 after transplanting is shown in the figure. Figure 4 Sixty days after transplanting, *Dendrobium nobile* seedlings were obtained, and their survival rate was investigated. The survival rate of the *Dendrobium nobile* seedlings in Example 1 was 95%.

[0079] Comparative Example 1

[0080] Similar to Example 1, except that the shoot induction medium formula in step (4) does not contain 6-BA. Specifically, the shoot induction medium uses 1 / 2 MS as the basic medium and contains only 0.2 mg / L NAA, 80 g / L potato juice, 30 g / L sucrose and 7 g / L agar, with a pH of 5.8. Comparative Example 1 only proceeded to shoot induction culture.

[0081] The induction of shoot clusters in Example 1 and Comparative Example 1 was recorded 60 days after inoculation, as shown in Table 1. Table 1 shows that, compared to Comparative Example 1, Example 1 produced more shoot clusters, had a higher budding rate, and exhibited better growth. The budding rate was calculated using the formula: Budding rate = Number of buds / Number of inoculated protocorms × 100%. See Table 1 for details.

[0082] Table 1. Shoot induction in Example 1 and Comparative Example 1 60 days after inoculation.

[0083] Group Number of protocorms inoculated (units) Number of buds Germination rate (%) Bud height (cm) growth Example 1 100 96 96 2.5 good Comparative Example 1 100 68 68 1 Weak

[0084] Comparative Example 2

[0085] The steps (1) to (4) and (6) of Example 1 are the same, except that potato juice is not added to the rooting and seedling strengthening culture medium formula described in step (5). Specifically, the rooting and seedling strengthening culture medium uses 1 / 2 MS as the basic culture medium and contains only 0.4 mg / L NAA, 30 g / L sucrose, 7 g / L agar, and 0.2 g / L activated carbon. The pH of the culture medium is 5.8.

[0086] Comparative Example 3

[0087] The steps (1) to (4) and (6) of Example 1 are the same, except that activated carbon is not added to the rooting and seedling strengthening culture medium formula described in step (5). The specific composition is as follows: the rooting and seedling strengthening culture medium uses 1 / 2 MS as the basic culture medium and contains only 0.4 mg / L NAA, 30 g / L sucrose, 7 g / L agar, and 70 g / L potato juice. The pH of the culture medium is 5.8.

[0088] The number of roots, root length, and growth vigor of plants in Example 1, Comparative Examples 2 and 3 were recorded on day 28 of rooting culture, as shown in Table 2. The rooting rate was calculated using the formula: Rooting rate (%) = Number of rooted plants / Number of inoculated plants × 100%. According to Table 2, Example 1, by adding 70 g / L potato juice and 0.2 g / L activated carbon to the rooting medium formulation, achieved higher rooting quality, as shown in Table 2.

[0089] Table 2 shows the rooting status of Example 1, Comparative Examples 2 and 3 on day 28 of rooting culture.

[0090]

[0091] Comparative Example 4

[0092] The same steps (1) to (5) are used in Example 1, except that the transplanting substrate in step (6) is peat moss.

[0093] Comparative Example 5

[0094] The same steps (1) to (5) are used in Example 1, except that the transplanting substrate in step (6) is vermiculite.

[0095] The survival rates of tissue culture seedlings in Example 1 and Comparative Examples 2-5 were statistically analyzed on the 20th and 60th day after transplanting, as shown in Table 3.

[0096] Table 3. Transplant survival rate of tissue culture seedlings obtained in Example 1 and Comparative Examples 2-5.

[0097]

[0098] According to Tables 1, 2, and 3,

[0099] (1) Comparing Example 1 and Comparative Example 1, it can be seen that the budding induction medium is different. Example 1 has more buds, higher budding rate, and better growth.

[0100] (2) Comparison of Example 1 with Comparative Example 2 and Comparative Example 3 shows that the rooting and seedling strengthening culture media are different. The plants in Example 1 are more robust, have better rooting effect, and have a higher transplant survival rate.

[0101] (3) Comparing Example 1 with Comparative Example 4 and Comparative Example 5, it can be seen that the transplanting substrate is different. In Example 1, the transplanting survival rate is higher.

[0102] In summary, the culture medium of this invention produces a greater number of clustered shoots from the seeds of hybrid Dendrobium, with a higher germination rate, better growth, higher rooting rate, stronger roots, and a higher survival rate after transplanting. The plants are also more robust.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture medium for Dendrobium nobile, characterized in that, The tissue culture medium consists of seed germination medium, shoot cluster induction medium, and rooting and seedling strengthening medium. The seed germination medium consisted of 1 / 2 MS medium + 0.2 mg / L NAA + 20 g / L sucrose + 70 g / L banana puree + 7 g / L agar; The shoot induction medium consisted of 1 / 2 MS medium + 0.1 mg / L 6-BA + 0.2 mg / L NAA + 80 g / L potato juice + 30 g / L sucrose + 7 g / L agar; The rooting and seedling strengthening culture medium consists of 1 / 2 MS medium + 0.4 mg / L NAA + 70 g / L potato juice + 0.2 g / L activated carbon + 30 g / L sucrose + 7 g / L agar; The pH value of the seed germination medium is 6; the pH value of the shoot induction medium is 5.8; the pH value of the rooting and seedling strengthening medium is 5.8; The explants used in the tissue culture medium were Dendrobium nobile seeds.

2. A method for rapid propagation of Dendrobium officinale seedlings, characterized in that, Includes the following steps: After sterilizing the capsules of Dendrobium nobile, extract the seeds of Dendrobium nobile; The Dendrobium nobile seeds were inoculated onto the seed germination medium described in claim 1 and germinated to obtain protocorms; The original bulbs are then inoculated onto the shoot induction medium of claim 1 to induce shoot growth and obtain shoot clusters. The clustered buds were inoculated onto the rooting and seedling strengthening culture medium of claim 1 to obtain Dendrobium officinale tissue culture seedlings; The Dendrobium officinale tissue culture seedlings were hardened off and transplanted to obtain Dendrobium officinale seedlings; The substrate used for transplanting is sphagnum moss.

3. The rapid propagation method for seedlings according to claim 2, characterized in that, The method for disinfecting the Dendrobium nobile capsules includes: after cleaning, first soaking in 70%~75% alcohol for 10~30 seconds, then rinsing with sterile water 1~2 times; then surface disinfecting with 0.1% mercuric chloride solution for 5~10 minutes; finally rinsing with sterile water 5~8 times; each second rinse with sterile water lasts 3 minutes; the cleaning includes rinsing with running water for 5~10 minutes.

4. The method for rapid propagation of seedlings according to claim 2, characterized in that, Before inoculating the protocorms into the bud induction medium, the process further includes dividing the protocorm clusters into protocorm clusters of size (3~5)mm×(3~5)mm before inoculation; and dividing the bud clusters into single buds or double buds before inoculating them into the rooting and seedling strengthening medium.

5. The method for rapid propagation of seedlings according to claim 2, characterized in that, The germination culture temperature is 25~27℃; The germination culture was carried out under alternating light and dark conditions, with the light culture time being 12-14 h / d and the dark culture time being 10-12 h / d; the light intensity for the light culture was 1500-2000 lx. Each light-dark cycle of incubation lasts 24 hours; The germination culture time is 18-22 days.

6. The method for rapid propagation of seedlings according to claim 2, characterized in that, The induction culture temperature is 25~27℃; The induction culture was carried out under alternating light and dark conditions, with the light culture time in the light-dark alternation being 12-14 h / d and the dark culture time in the light-dark alternation being 10-12 h / d; the light intensity of the light culture was 1500-2000 lx; ​​and each light-dark alternation culture cycle was 24 h. The induction culture time is 90~368 days.

7. The method for rapid propagation of seedlings according to claim 2, characterized in that, The temperature for the rooting culture of the robust seedlings is 25~27℃; The seedling rooting culture was carried out under alternating light and dark conditions, with the light culture time being 12-14 h / d and the dark culture time being 10-12 h / d; the light intensity for the light culture was 1500-2000 lx. Each light-dark cycle of incubation lasts 24 hours; The rooting culture time for the robust seedlings is 25-30 days.

8. The method for rapid propagation of seedlings according to claim 2, characterized in that, The seedling hardening and transplanting process includes hardening the seedlings in bottles for 9-11 days in a hardening room before transplanting them into a greenhouse.

9. The application of the tissue culture medium according to claim 1 or the rapid propagation method of seedlings according to any one of claims 2 to 8 in improving the survival rate of Dendrobium officinale seedlings after transplanting.

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