A method for culturing adventitious roots of Dendrobium officinale

Abstract

The present invention relates to the technical field of plant tissue culture, and particularly relates to a method for culturing adventitious roots of Dendrobium officinale. The present invention provides a method for culturing adventitious roots of Dendrobium officinale, which comprises the following steps: (1) inoculating Dendrobium officinale seeds onto a starting medium, a proliferation medium, and a strong seedling rooting medium in sequence, and performing germination culture, proliferation culture, and rooting culture respectively to obtain sterile seedlings of Dendrobium officinale; (2) performing callus induction culture or adventitious root induction culture on the sterile seedlings of Dendrobium officinale obtained in step (1) to obtain adventitious roots of Dendrobium officinale; The present invention uses multiple explants of sterile seedlings of Dendrobium officinale to induce adventitious roots and then perform propagation culture. The induction rate of adventitious roots is between 72.30% and 88.74%, which lays a foundation for the future industrial production of important active ingredients of Dendrobium officinale using adventitious roots of Dendrobium officinale, and at the same time can solve the problem of the supply-demand contradiction of raw materials of Dendrobium officinale.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for culturing adventitious roots of Dendrobium officinale. Background Art

[0002] Adventitious roots are roots that grow on the stems or leaves of plants. In most cases, adventitious roots grow due to injury to plant organs or stimulation by external factors such as hormones, pathogenic microorganisms, etc., and thus manifest as a regeneration response of the plant. The occurrence and formation of adventitious roots is an important way of asexual reproduction of plants. Adventitious root culture has the advantages of short growth cycle, controllable conditions with small errors, single source of materials, consistent genetic background, economic convenience, strong repeatability, high efficiency, and year-round testing or growth. The adventitious root culture of medicinal plants can overcome the shortcomings of low content of active ingredients and instability in cell suspension culture. Therefore, the adventitious root culture of medicinal plants is an effective way to develop Chinese medicine resources.

[0003] Dendrobium officinale Kimura et Migo is a perennial epiphytic herb of the genus Dendrobium in the orchid family. It is called Dendrobium officinale because of its iron green stem color. The Taoist Canon lists Dendrobium officinale as the first of the nine immortal herbs, and scientists in the field of plant research today call it the "giant panda" of the plant world. Modern scientific research has found that Dendrobium officinale has a series of effects such as anti-tumor, accelerating saliva secretion, improving human immunity, resisting liver damage, lowering blood sugar, and relieving fatigue, and has been approved as one of the medicinal materials that can be used to produce health foods.

[0004] In its natural state, Dendrobium candidum is mainly distributed on cliffs of 1000-3400m or on broad-leaved trees with thick diameter, vigorous moss growth and wild vines. It likes a relatively cool and humid environment. Since the seeds of Dendrobium candidum do not have fully developed embryos and endosperm tissues, the germination rate of seeds is not high and the natural reproduction capacity is also weak. Due to excessive excavation and the destruction of the natural ecological environment, the wild resources of Dendrobium candidum are extremely endangered and are listed as the second-level endangered medicinal plants for key protection. In the mid-1970s, the tissue culture technology of Dendrobium candidum was studied, and complete regenerated plants were successfully obtained. In the 1980s, Zhejiang Academy of Medical Sciences took the lead in using Dendrobium candidum tissue culture technology for production. In the following decades, domestic scholars have set off a craze for the tissue culture of Dendrobium candidum, and great breakthroughs have been made in the research of Dendrobium candidum tissue culture.

[0005] At present, the tissue culture and rapid propagation technology problems of Dendrobium officinale have been basically solved, but the production of medicinal components of Dendrobium officinale by means of biotechnology has not been reported yet. The adventitious root culture technology, as a newly developed technology, has become the main way to produce secondary metabolites in rhizome medicinal plants due to the stable and rapid production of secondary metabolites. The purpose of the present invention is to solve the problems of unstable production of secondary metabolites by cell culture of Dendrobium officinale and the lack of an adventitious root culture system. Based on this, a method for directly inducing and proliferating adventitious roots of Dendrobium officinale is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for culturing adventitious roots of Dendrobium officinale.

[0007] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides a method for culturing adventitious roots of Dendrobium officinale, which comprises the following steps:

[0009] (1) Inoculate Dendrobium officinale seeds into a starting medium, a proliferation medium and a strong seedling rooting medium in sequence, and carry out germination culture, proliferation culture and rooting culture respectively to obtain sterile seedlings of Dendrobium officinale;

[0010] (2) Carry out callus induction culture or adventitious root induction culture on the sterile seedlings of Dendrobium officinale obtained in step (1) to obtain adventitious roots of Dendrobium officinale;

[0011] The method for callus induction culture is as follows:

[0012] a. Inoculate the leaves, petioles or stem segments of the sterile seedlings of Dendrobium officinale described in step (1) into a callus induction medium and a callus proliferation medium in sequence, and carry out light culture and dark culture respectively to obtain callus of Dendrobium officinale;

[0013] b. Cut the callus of Dendrobium officinale described in step a and inoculate it into an adventitious root induction medium for dark culture to obtain adventitious roots of Dendrobium officinale;

[0014] The method for adventitious root induction culture is as follows:

[0015] Inoculate the leaves, petioles or stem segments of the sterile seedlings of Dendrobium officinale described in step (1) into an adventitious root medium for adventitious root culture to obtain adventitious roots of Dendrobium officinale.

[0016] Preferably, the starting medium described in step (1) is: MS + agar 7.0 - 7.5 g / L + sucrose 25 - 30 g / L, and the pH is 5.8 - 6.0.

[0017] The proliferation medium is MS + 7.0 - 7.5 g / L of agar + 1.5 - 2.0 mg / L of 6-BA + 0.1 - 0.2 mg / L of NAA + 25 - 30 g / L of sucrose, with a pH of 5.8 - 6.0:

[0018] The strong seedling rooting medium is: 1 / 2MS + 7.0 - 7.5 g / L of agar + 0.2 - 0.5 mg / L of IBA + 0.2 - 0.5 g / L of activated carbon + 25 - 30 g / L of sucrose, with a pH value of 5.8 - 6.0.

[0019] Preferably, the time for germination culture in step (1) is 10 - 16 d; the time for proliferation culture is 20 - 25 d; the time for rooting culture is 15 - 25 d.

[0020] Preferably, the callus induction medium in step a is MS + 2.0 - 3.0 mg / L of 2,4-D + 1.0 - 2.0 mg / L of NAA + 30 - 40 g / L of sucrose + 7.2 - 7.6 g / L of agar, pH = 5.8 - 6.0.

[0021] The callus proliferation medium is MS + 1.0 - 1.5 mg / L of 6-BA + 0.1 - 0.2 mg / L of 2,4-D + 30 - 40 g / L of sucrose + 7.2 - 7.6 g / L of agar, pH = 5.8 - 6.0.

[0022] Preferably, the time for light culture in step a is 10 - 15 d, and the light intensity is 2500 - 3000 lx; the time for dark culture is 20 - 30 d.

[0023] Preferably, the adventitious root induction medium in step b is: MS + 7.2 - 7.6 g / L of agar + 1.5 - 2.5 mg / L of NAA + 25 - 30 g / L of sucrose, pH = 5.8 - 6.0.

[0024] Preferably, the time for adventitious root induction culture in step b is 10 - 18 d.

[0025] Preferably, the adventitious root medium is MS + 7.2 - 7.6 g / L of agar + 0.5 - 0.8 mg / L of NAA + 15% of banana juice + 30 - 40 g / L of sucrose, pH = 5.8 - 6.0; the adventitious root culture is in the dark for 30 - 40 d.

[0026] Preferably, after obtaining the adventitious roots of Dendrobium officinale, it further includes inoculating the adventitious roots of Dendrobium officinale into an adventitious root proliferation medium for adventitious root proliferation culture to obtain a large number of adventitious roots of Dendrobium officinale.

[0027] Preferably, the adventitious root proliferation culture method is to first culture in the dark for 30 - 40 days, and then culture under light of 2500 - 3000 lx for 10 - 20 days; the adventitious root proliferation culture medium is MS + 7.2 - 7.6 g / L of agar + 0.5 - 0.8 mg / L of NAA + 15% of banana juice + 30 - 40 g / L of sucrose, pH = 5.8 - 6.0.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention uses multiple explants (callus, leaves, protocorms, stem segments) of Dendrobium officinale sterile seedlings to induce adventitious roots and then carry out propagation culture. The induction rate of adventitious roots is between 72.30% and 88.74%, which lays a foundation for the future industrial production of important active ingredients of Dendrobium officinale using adventitious roots of Dendrobium officinale, and at the same time can solve the contradiction between supply and demand of raw materials of Dendrobium officinale. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to the provided drawings.

[0031] Figure 1 is the Dendrobium officinale sterile seedling.

[0032] Figure 2 is the adventitious root of Dendrobium officinale. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The following will combine the embodiments to detail the technical solutions provided by the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0034] Example 1

[0035] The present invention provides a method for culturing adventitious roots of Dendrobium officinale, and the steps are as follows:

[0036] (1) Disinfect the Dendrobium officinale seeds with 70% (v / v) alcohol for 30 s, wash them with water twice, then disinfect them with 0.08% (w / v) mercuric chloride for 6 min, and wash them with water twice.

[0037] (2) Inoculate the Dendrobium officinale seeds onto the initiation medium, proliferation medium, and strong seedling rooting medium in sequence, and carry out germination culture for 10 days, proliferation culture for 20 days, and rooting culture for 15 days respectively to obtain Dendrobium officinale sterile seedlings;

[0038] The starting medium is: MS + 7.0 g / L agar + 25 g / L sucrose, with a pH of 5.8.

[0039] The proliferation medium is MS + 7.0 g / L agar + 1.5 mg / L 6-BA + 0.1 mg / L NAA + 25 g / L sucrose, with a pH of 5.8:

[0040] The strong-seedling rooting medium is: 1 / 2 MS + 7.0 g / L agar + 0.2 mg / L IBA + 0.2 g / L activated carbon + 25 g / L sucrose, with a pH value of 5.8.

[0041] (3) Inoculate the leaves (0.4 cm × 0.4 cm in size) of the aseptic Dendrobium officinale seedlings into the callus induction medium and the callus proliferation medium in sequence, and perform light cultivation at 2500 lx for 1 d and dark cultivation for 20 d respectively to obtain Dendrobium officinale callus;

[0042] The callus induction medium is MS + 2.0 mg / L 2,4-D + 1.0 mg / L NAA + 30 g / L sucrose + 7.2 g / L agar, pH = 5.8.

[0043] The callus proliferation medium is MS + 1.0 mg / L 6-BA + 0.1 mg / L 2,4-D + 30 g / L sucrose + 7.2 g / L agar, pH = 5.8.

[0044] (4) Cut the Dendrobium officinale callus and inoculate it into the adventitious root induction medium for dark cultivation for 10 d to obtain Dendrobium officinale adventitious roots;

[0045] The adventitious root induction medium is: MS + 7.2 g / L agar + 1.5 mg / L NAA + 25 g / L sucrose, pH = 5.8;

[0046] (5) Cut the Dendrobium officinale adventitious roots into 1 cm lengths and inoculate them into the adventitious root proliferation medium for adventitious root proliferation culture. First, perform dark cultivation for 30 d, and then perform light cultivation at 2500 lx for 10 d to obtain a large number of Dendrobium officinale adventitious roots.

[0047] The adventitious root proliferation medium is MS + 7.2 g / L agar + 0.5 mg / L NAA + 15% banana juice + 30 g / L sucrose, pH = 5.8.

[0048] Example 2

[0049] The present invention provides a method for culturing adventitious roots of Dendrobium officinale, and the steps are as follows:

[0050] (1) Disinfect Dendrobium officinale seeds with 80% (v / v) alcohol for 40 s, rinse them with water three times, then disinfect them with 0.12% (w / v) mercuric chloride for 8 min, and rinse with water three times.

[0051] (2) Inoculate the Dendrobium officinale seeds onto the initiation medium, proliferation medium, and strong seedling rooting medium in sequence, and conduct germination culture for 16 d, proliferation culture for 25 d, and rooting culture for 25 d respectively to obtain axenic seedlings of Dendrobium officinale;

[0052] The initiation medium is: MS + 7.5 g / L agar + 30 g / L sucrose, with a pH of 6.0.

[0053] The proliferation medium is MS + 7.5 g / L agar + 2.0 mg / L 6 - BA + 0.15 mg / L NAA + 30 g / L sucrose, with a pH of 6.0:

[0054] The strong seedling rooting medium is: 1 / 2MS + 7.5 g / L agar + 0.5 mg / L IBA + 0.5 g / L activated carbon + 30 g / L sucrose, with a pH value of 6.0.

[0055] (3) Inoculate the petioles (0.5 - cm segments) of the axenic seedlings of Dendrobium officinale onto the callus induction medium and callus proliferation medium in sequence, and conduct light culture at 3000 lx for 15 d and dark culture for 25 d respectively to obtain callus of Dendrobium officinale;

[0056] The callus induction medium is MS + 3.0 mg / L 2,4 - D + 1.5 mg / L NAA + 40 g / L sucrose + 7.6 g / L agar, pH = 6.0.

[0057] The callus proliferation medium is MS + 1.5 mg / L 6 - BA + 0.2 mg / L 2,4 - D + 40 g / L sucrose + 7.6 g / L agar, pH = 6.0.

[0058] (4) Cut the callus of Dendrobium officinale and inoculate it onto the adventitious root induction medium for dark culture for 18 d to obtain adventitious roots of Dendrobium officinale;

[0059] The adventitious root induction medium is: MS + 7.6 g / L agar + 2.5 mg / L NAA + 30 g / L sucrose, pH = 6.0;

[0060] (5) Cut the adventitious roots of Dendrobium officinale into 1.5 - cm lengths and inoculate them into the adventitious root proliferation medium for adventitious root proliferation culture. First, conduct dark culture for 40 d, and then light culture at 3000 lx for 20 d to obtain a large number of adventitious roots of Dendrobium officinale.

[0061] The adventitious root proliferation medium is MS + 7.6 g / L agar + 0.8 mg / L NAA + 15% banana juice + 40 g / L sucrose, pH = 6.0.

[0062] Example 3

[0063] The present invention provides a method for culturing adventitious roots of Dendrobium officinale, and the steps are as follows:

[0064] (1) Disinfect Dendrobium officinale seeds with 75% (v / v) alcohol for 35 s, rinse them 3 times with water, then disinfect them with 0.1% (w / v) mercuric chloride for 7 min, and rinse them 3 times with water.

[0065] (2) Inoculate the Dendrobium officinale seeds onto the initiation medium, proliferation medium, and strong seedling rooting medium in sequence, and carry out germination culture for 13 d, proliferation culture for 23 d, and rooting culture for 20 d respectively to obtain axenic seedlings of Dendrobium officinale;

[0066] The initiation medium is: MS + 7.3 g / L agar + 28 g / L sucrose, pH 5.9.

[0067] The proliferation medium is MS + 7.3 g / L agar + 1.8 mg / L 6 - BA + 0.15 mg / L NAA + 28 g / L sucrose, pH 5.9:

[0068] The strong seedling rooting medium is: 1 / 2MS + 7.3 g / L agar + 0.3 mg / L IBA + 0.4 g / L activated carbon + 28 g / L sucrose, pH value 5.9.

[0069] (3) Inoculate the stem segments (0.7 - cm small segments) of the axenic seedlings of Dendrobium officinale onto the callus induction medium and callus proliferation medium in sequence, and carry out light culture at 2800 lx for 13 d and dark culture for 25 d respectively to obtain callus of Dendrobium officinale;

[0070] The callus induction medium is MS + 2.5 mg / L 2,4 - D + 1.5 mg / L NAA + 35 g / L sucrose + 7.4 g / L agar, pH = 5.9.

[0071] The callus proliferation medium is MS + 1.3 mg / L 6 - BA + 0.15 mg / L 2,4 - D + 35 g / L sucrose + 7.4 g / L agar, pH = 5.9.

[0072] (4) Cut the callus of Dendrobium officinale and inoculate it onto the adventitious root induction medium for dark culture for 14 d to obtain adventitious roots of Dendrobium officinale;

[0073] The adventitious root induction medium is: MS + 7.4 g / L agar + 2 mg / L NAA + 28 g / L sucrose, pH = 5.9;

[0074] (5) Cut the adventitious roots of Dendrobium officinale into lengths of 1.3 cm and inoculate them into the adventitious root proliferation medium for adventitious root proliferation culture. First, culture them in the dark for 35 days, and then culture them under 2800 lx light for 15 days to obtain a large number of adventitious roots of Dendrobium officinale.

[0075] The adventitious root proliferation medium is MS + 7.4 g / L agar + 0.7 mg / L NAA + 15% banana juice + 35 g / L sucrose, pH = 5.9.

[0076] Example 4

[0077] The present invention provides a method for culturing adventitious roots of Dendrobium officinale, and the steps are as follows:

[0078] (1) Disinfect the seeds of Dendrobium officinale with 70% (v / v) alcohol for 30 s, rinse them twice with water, then disinfect them with 0.08% (w / v) mercuric chloride for 6 min, and rinse them twice with water.

[0079] (2) Inoculate the seeds of Dendrobium officinale into the initiation medium, proliferation medium and strong seedling rooting medium in sequence, and carry out germination culture for 10 days, proliferation culture for 20 days and rooting culture for 15 days respectively to obtain axenic seedlings of Dendrobium officinale;

[0080] The initiation medium is: MS + 7.0 g / L agar + 25 g / L sucrose, pH is 5.8.

[0081] The proliferation medium is MS + 7.0 g / L agar + 1.5 mg / L 6 - BA + 0.1 mg / L NAA + 25 g / L sucrose, pH is 5.8:

[0082] The strong seedling rooting medium is: 1 / 2MS + 7.0 g / L agar + 0.2 mg / L IBA + 0.2 g / L activated carbon + 25 g / L sucrose, pH value is 5.8.

[0083] (3) Inoculate the leaves (0.4 cm × 0.4 cm in size) of the axenic seedlings of Dendrobium officinale into the adventitious root medium for adventitious root culture to obtain adventitious roots of Dendrobium officinale;

[0084] The adventitious root medium is MS + 7.2 g / L agar + 0.5 mg / L NAA + 15% banana juice + 30 g / L sucrose, pH = 5.8; The adventitious root culture is carried out in the dark for 30 days.

[0085] (4) Cut the adventitious roots of Dendrobium officinale into lengths of 1 cm and inoculate them into the adventitious root proliferation medium for adventitious root proliferation culture. First, culture them in the dark for 30 days, and then culture them under a light intensity of 2500 lx for 10 days to obtain a large number of adventitious roots of Dendrobium officinale.

[0086] The adventitious root proliferation medium is MS + 7.2 g / L agar + 0.5 mg / L NAA + 15% banana juice + 30 g / L sucrose, with a pH of 5.8.

[0087] Example 5

[0088] The present invention provides a method for culturing adventitious roots of Dendrobium officinale, and the steps are as follows:

[0089] (1) Disinfect the seeds of Dendrobium officinale with 80% (v / v) alcohol for 40 s, rinse them with water 3 times, then disinfect them with 0.12% (w / v) mercuric chloride for 8 min, and rinse them with water 3 times.

[0090] (2) Inoculate the seeds of Dendrobium officinale into the initiation medium, proliferation medium, and strong seedling rooting medium in sequence, and carry out germination culture for 16 days, proliferation culture for 25 days, and rooting culture for 25 days respectively to obtain aseptic seedlings of Dendrobium officinale;

[0091] The initiation medium is: MS + 7.5 g / L agar + 30 g / L sucrose, with a pH of 6.0.

[0092] The proliferation medium is MS + 7.5 g / L agar + 2.0 mg / L 6 - BA + 0.15 mg / L NAA + 30 g / L sucrose, with a pH of 6.0:

[0093] The strong seedling rooting medium is: 1 / 2MS + 7.5 g / L agar + 0.5 mg / L IBA + 0.5 g / L activated carbon + 30 g / L sucrose, with a pH value of 6.0.

[0094] (3) Inoculate the petioles (0.5 cm small segments) of the aseptic seedlings of Dendrobium officinale into the adventitious root medium for adventitious root culture to obtain adventitious roots of Dendrobium officinale;

[0095] The adventitious root medium is MS + 7.6 g / L agar + 0.8 mg / L NAA + 15% banana juice + 40 g / L sucrose, with a pH = 6.0; The adventitious root culture is carried out in the dark for 40 days.

[0096] (4) Cut the adventitious roots of Dendrobium officinale into lengths of 1.5 cm and inoculate them into the adventitious root proliferation medium for adventitious root proliferation culture. First, culture them in the dark for 40 days, and then culture them under a light intensity of 3000 lx for 20 days to obtain a large number of adventitious roots of Dendrobium officinale.

[0097] The adventitious root proliferation medium is MS + 7.6 g / L agar + 0.8 mg / L NAA + 15% banana juice + 40 g / L sucrose, pH = 6.0.

[0098] Example 6

[0099] The present invention provides a method for culturing adventitious roots of Dendrobium officinale, and the steps are as follows:

[0100] (1) Disinfect Dendrobium officinale seeds with 75% (v / v) alcohol for 35 s, rinse with water 3 times, then disinfect with 0.1% (w / v) mercuric chloride for 7 min, and rinse with water 3 times.

[0101] (2) Inoculate the Dendrobium officinale seeds onto the initiation medium, proliferation medium and strong seedling rooting medium in sequence, and carry out germination culture for 13 d, proliferation culture for 23 d and rooting culture for 20 d respectively to obtain sterile seedlings of Dendrobium officinale;

[0102] The initiation medium is: MS + 7.3 g / L agar + 28 g / L sucrose, pH 5.9.

[0103] The proliferation medium is MS + 7.3 g / L agar + 1.8 mg / L 6 - BA + 0.15 mg / L NAA + 28 g / L sucrose, pH 5.9:

[0104] The strong seedling rooting medium is: 1 / 2MS + 7.3 g / L agar + 0.3 mg / L IBA + 0.4 g / L activated carbon + 28 g / L sucrose, pH value 5.9.

[0105] (3) Inoculate the stem segments (0.7 cm small segments) of the sterile seedlings of Dendrobium officinale into the adventitious root medium for adventitious root culture to obtain adventitious roots of Dendrobium officinale;

[0106] The adventitious root medium is MS + 7.4 g / L agar + 0.7 mg / L NAA + 15% banana juice + 35 g / L sucrose, pH = 5.9; the adventitious root culture is carried out in the dark for 35 d.

[0107] (4) Cut the adventitious roots of Dendrobium officinale into lengths of 1.3 cm and inoculate them into the adventitious root proliferation medium for adventitious root proliferation culture. First, culture in the dark for 35 d, and then culture under 2800 lx light for 15 d to obtain a large number of adventitious roots of Dendrobium officinale.

[0108] The adventitious root proliferation medium is MS + 7.4 g / L agar + 0.7 mg / L NAA + 15% banana juice + 35 g / L sucrose, pH = 5.9.

[0109] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A cultivation method for adventitious roots of Dendrobium officinale, characterized in that, It includes the following steps: (1) Sequentially inoculate Dendrobium officinale seeds onto a starting medium, a proliferation medium, and a strong-seedling rooting medium, and conduct germination culture, proliferation culture, and rooting culture respectively to obtain axenic seedlings of Dendrobium officinale; (2) Conduct callus induction culture on the axenic seedlings of Dendrobium officinale obtained in step (1) to obtain adventitious roots of Dendrobium officinale; The method for the callus induction culture is as follows: a. Inoculate the leaves of the axenic seedlings of Dendrobium officinale described in step (1) onto a callus induction medium and a callus proliferation medium, and conduct light culture and dark culture respectively to obtain Dendrobium officinale callus; b. Cut the Dendrobium officinale callus described in step a and inoculate it onto an adventitious root induction medium for dark culture to obtain adventitious roots of Dendrobium officinale; The starting medium described in step (1) is: MS + agar 7.0 - 7.5 g / L + sucrose 25 - 30 g / L, with a pH of 5.8 - 6.0; The proliferation medium is MS + agar 7.0 - 7.5 g / L + 6 - BA 1.5 - 2.0 mg / L + NAA 0.1 - 0.2 mg / L + sucrose 25 - 30 g / L, with a pH of 5.8 - 6.0: The strong-seedling rooting medium is: 1 / 2MS + agar 7.0 - 7.5 g / L + IBA 0.2 - 0.5 mg / L + activated carbon 0.2 - 0.5 g / L + sucrose 25 - 30 g / L, with a pH value of 5.8 - 6.0; The callus induction medium described in step a is MS + 2,4 - D 2.0 - 3.0 mg / L + 1.0 - 2.0 mg / L NAA + sucrose 30 - 40 g / L + agar 7.2 - 7.6 g / L, pH = 5.8 - 6.0; The callus proliferation medium is MS + 6 - BA 1.0 - 1.5 mg / L + 2,4 - D 0.1 - 0.2 mg / L + sucrose 30 - 40 g / L + agar 7.2 - 7.6 g / L, pH = 5.8 - 6.0; The adventitious root induction medium described in step b is: MS + agar 7.2 - 7.6 g / L + 1.5 - 2.5 mg / L NAA + sucrose 25 - 30 g / L, pH = 5.8 - 6.

0.

2. The culture method of adventitious roots of Dendrobium officinale according to claim 1, characterized in that, The time for the germination culture described in step (1) is 10 - 16 d; the time for the proliferation culture is 20 - 25 d; the time for the rooting culture is 15 - 25 d.

3. The culture method of adventitious roots of Dendrobium officinale according to claim 1, characterized in that, The time for the light culture described in step a is 10 - 15 d, and the light intensity is 2500 - 3000 lx; the time for the dark culture is 20 - 30 d.

4. The culture method of the adventitious roots of Dendrobium officinale according to claim 1, characterized in that, The time for the dark culture described in step b is 10 - 18 d.

5. The culture method of adventitious roots of Dendrobium officinale according to claim 1, characterized in that After obtaining the adventitious roots of Dendrobium officinale, it further includes inoculating the adventitious roots of Dendrobium officinale onto an adventitious root proliferation medium for adventitious root proliferation culture to obtain a large number of adventitious roots of Dendrobium officinale.

6. The culture method of adventitious roots of Dendrobium officinale according to claim 5, characterized in that, The adventitious root proliferation culture method is as follows: first, culture in the dark for 30 - 40 d, and then culture under light of 2500 - 3000 lx for 10 - 20 d; the adventitious root proliferation medium is MS + 7.2 - 7.6 g / L of agar + 0.5 - 0.8 mg / L of NAA + 15% of banana juice + 30 - 40 g / L of sucrose, with pH = 5.8 - 6.0.

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