Tissue culture method of rhodiola crenulata

Abstract

The invention provides a rhodiola crenulata tissue culture method, and belongs to the technical field of plant tissue culture. According to the method, multiple explants (calluses, leaves and stems) of rhodiola crenulata tissue culture seedlings are utilized to induce the adventitious roots, then propagation culture is performed, the composition of a culture medium is limited, the induction rate of the adventitious roots is 58.3%-72.6%, the browning rate in the culture process of the rhodiola crenulata tissue culture adventitious roots is low, and the tissue culture yield is high. A foundation is laid for industrial production of important active ingredients of rhodiola crenulata by using adventitious roots of rhodiola crenulata in future, and meanwhile, the problem of contradiction between supply and demand of rhodiola crenulata raw materials can be solved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a Rhodiola rosea tissue culture method. Background Art

[0002] Rhodiola crenulata is a rare perennial medicinal plant of the genus Rhodiola in the Crassulaceae family. Its roots and rhizomes are rich in active ingredients such as salidroside and tyrosol, and have significant pharmacological effects such as anti-oxidation, anti-fatigue, and immune enhancement. It is widely used in the fields of medicine and health products. However, the current wild Rhodiola crenulata resources are on the verge of depletion. In order to meet market demand and protect wild resources, plant tissue culture technology has become an important way to mass-produce the active ingredients of Rhodiola crenulata.

[0003] The main bottlenecks of the current tissue culture technology of Rhodiola rosea are the high browning rate of explants, low efficiency of adventitious root induction, strong hormone dependence, and unstable proliferation system. In view of these problems, it is urgent to develop an efficient and low-cost tissue culture method to achieve large-scale and standardized production of adventitious roots of Rhodiola rosea by optimizing the composition of the culture medium, regulating the hormone ratio and culture conditions, and providing a reliable source of raw materials for subsequent active ingredient extraction and commercial application. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a tissue culture method for Rhodiola rosea.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for tissue culture of Rhodiola rosea, comprising the following steps:

[0007] 1) Cultivating sterilized Rhodiola rosea seeds to obtain sterile seedlings;

[0008] 2) Inoculate the sterile seedling explants into adventitious root induction medium and culture them in the dark for 10 to 30 days to obtain adventitious roots;

[0009] 3) inoculating the adventitious roots into a proliferation medium, culturing in the dark for 20 to 30 days, then switching to a light condition of 12 to 20 hours of light / 4 to 12 hours of darkness for 12 to 18 days to obtain proliferated adventitious roots;

[0010] Step 2) the explants include leaves, stem segments and / or callus tissues of sterile seedlings;

[0011] The callus tissue is cultured from leaves or stem segments of sterile seedlings;

[0012] Step 2) The adventitious root induction medium is B5+IBA 1.0-2.0 mg / L+potato juice 80-120 g / L+sucrose 25-30 g / L+agar 7.2-7.6 g / L, pH 5.8-6.0;

[0013] Step 3) The proliferation medium is B5+IBA 1.0-2.0 mg / L+NAA 0.5-1.0 mg / L+coenzyme Q10 20-30 μM+sucrose 30-45 g / L, pH 5.8-6.0.

[0014] Preferably, the callus culture method is:

[0015] Inoculating the leaves or stem segments of the sterile seedlings in step 1) into a callus induction medium, and culturing under light for 15 to 18 days to obtain callus;

[0016] The callus induction medium is B5+2,4-D 1.5-3.0 mg / L+0.2-0.5 mg / L NAA+sucrose 30-40 g / L+agar 7.2-7.6 g / L.

[0017] Preferably, the culturing in step 1) includes primary culture, subculture and rooting culture;

[0018] The culture medium for the primary culture is MS+agar 7.0-7.5 g / L+sucrose 25-30 g / L, pH 5.8-6.0;

[0019] The subculture medium is MS+KT 0.5-1.0 mg / L+indoleacetonitrile 0.2-0.5 mg / L+agar 7.0-7.5 g / L+sucrose 25-30 g / L, pH 5.8-6.0;

[0020] The culture medium for rooting culture is 1 / 2MS+NAA 0.5-0.8 mg / L+potato juice 100-150 g / L+agar 7.0-7.5 g / L+sucrose 25-30 g / L, pH 5.8-6.0.

[0021] Preferably, the primary culture time is 10 to 15 days;

[0022] The subculture time is 20 to 25 days;

[0023] The rooting culture time is 18 to 22 days;

[0024] The culture temperature of the primary culture, subculture culture and rooting culture is independently 23-27° C., the light conditions of the culture are independently 12-20 hours of light / 4-12 hours of darkness, and the light intensity is independently 2000-3000 lx.

[0025] Preferably, the disinfection method in step 1) is to soak in alcohol for 30 to 40 seconds and then soak in hydrogen peroxide solution for 8 to 10 minutes.

[0026] Preferably, the concentration of alcohol is 70% to 80%.

[0027] Preferably, the mass concentration of hydrogen peroxide is 3% to 8%.

[0028] Preferably, after alcohol soaking, washing with water 2 to 4 times;

[0029] After soaking in hydrogen peroxide, wash with water 2 to 4 times.

[0030] Preferably, the length of the adventitious roots in step 3) is 0.8 to 1.2 cm.

[0031] The invention provides application of the method in rapid propagation of Rhodiola rosea.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The invention utilizes a variety of explants (callus tissue, leaf, stem segment) of Rhodiola rosea tissue culture seedlings to induce adventitious roots and then performs propagation culture, limits the composition of the culture medium, ensures that the induction rate of the adventitious roots is between 58.3% and 72.6%, and the browning rate is low during the culture process of the Rhodiola rosea tissue culture adventitious roots, lays a foundation for utilizing the Rhodiola rosea adventitious roots to carry out industrialized production of important active ingredients of Rhodiola rosea in the future, and can solve the contradiction between supply and demand of Rhodiola rosea raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 These are sterile seedlings of Rhodiola rosea;

[0035] Figure 2 The adventitious roots were induced from the stem segments of Rhodiola rosea tissue culture seedlings as explants.

[0036] Figure 3 These are adventitious roots induced from leaves of Rhodiola rosea tissue culture seedlings as explants;

[0037] Figure 4 The adventitious roots were induced from the callus of Rhodiola rosea tissue culture seedlings as explants.

[0038] Figure 5 This is the effect of solid culture medium proliferation of adventitious roots of Rhodiola rosea. DETAILED DESCRIPTION

[0039] The present invention provides a method for tissue culture of Rhodiola rosea, comprising the following steps:

[0040] 1) Cultivating sterilized Rhodiola rosea seeds to obtain sterile seedlings;

[0041] 2) Inoculate the sterile seedling explants into adventitious root induction medium and culture them in the dark for 10 to 30 days to obtain adventitious roots;

[0042] 3) inoculating the adventitious roots into a proliferation medium, culturing in the dark for 20 to 30 days, then switching to 12 to 20 hours of light / 4 to 12 hours of darkness for 12 to 18 days to obtain proliferated adventitious roots;

[0043] In the present invention, firstly, the seeds of Rhodiola rosea are disinfected, and the disinfection comprises alcohol soaking and hydrogen peroxide soaking performed sequentially; the alcohol soaking time is 30 to 40 seconds, more preferably 33 to 37 seconds, and more preferably 35 seconds, and the volume concentration of the alcohol is preferably 70% to 80%, and more preferably 75%; the hydrogen peroxide solution soaking time is preferably 8 to 10 minutes, more preferably 8.5 to 9.5 minutes, and more preferably 9 minutes, and the mass concentration of the hydrogen peroxide solution is preferably 3% to 8%, more preferably 4% to 7%, and more preferably 5%; after the alcohol soaking, preferably washing with water, the number of washings is preferably 2 to 4 times, and more preferably 3 times; after the hydrogen peroxide soaking, preferably washing with water, the number of washings is preferably 2 to 4 times, and more preferably 3 times.

[0044] In the present invention, the sterilized Rhodiola rosea seeds are cultured; the culture is preferably primary culture, subculture and rooting culture;

[0045] The culture medium for the primary culture is preferably MS + agar 7.0-7.5 g / L + sucrose 25-30 g / L, more preferably MS + agar 7.1-7.4 g / L + sucrose 26-29 g / L, and further preferably MS + agar 7.3 g / L + sucrose 27 g / L, pH 5.8-6.0; the time for the primary culture is preferably 10-15 days, more preferably 11-14 days, and further preferably 13 days.

[0046] The culture medium for the subculture is preferably MS+KT 0.5-1.0 mg / L+indoleacetonitrile 0.2-0.5 mg / L+agar 7.0-7.5 g / L+sucrose 25-30 g / L, further preferably MS+KT 0.6-0.8 mg / L+indoleacetonitrile 0.25-0.4 mg / L+agar 7.1-7.4 g / L+sucrose 26-29 g / L, further preferably MS+KT 0.7 mg / L+indoleacetonitrile 0.3 mg / L+agar 7.3 g / L+sucrose 27 g / L, pH 5.8-6.0; the time for the subculture is preferably 20-25 days, further preferably 21-24 days, and further preferably 23 days.

[0047] The culture medium for rooting culture is preferably 1 / 2MS + NAA 0.5-0.8 mg / L + potato juice 100-150 g / L + agar 7.0-7.5 g / L + sucrose 25-30 g / L, further preferably 1 / 2MS + NAA 0.6-0.7 mg / L + potato juice 110-140 g / L + agar 7.1-7.4 g / L + sucrose 26-29 g / L, further preferably 1 / 2MS + NAA 0.65 mg / L + potato juice 130 g / L + agar 7.3 g / L + sucrose 27 g / L, pH 5.8-6.0; the time for rooting culture is preferably 18-22 days, further preferably 19-21 days, and further preferably 20 days.

[0048] In the present invention, the culture temperature of the primary culture, subculture and rooting culture is independently preferably 23-27°C, further preferably 24-26°C, and further preferably 25°C; the lighting conditions of the culture are independently: culture is carried out using alternating light and dark, the light culture time is preferably 12-20 hours, further preferably 14-18h, and further preferably 16h, the light intensity is independently preferably 2000-3000lx, further preferably 2200-2800lx, and further preferably 2500lx; the dark culture time is preferably 4-12 hours, further preferably 6-10 hours, and further preferably 8 hours.

[0049] In the present invention, explants are obtained from sterile seedlings obtained by culture, and the explants preferably include leaves, stem segments and / or callus tissue of sterile seedlings, and more preferably stem segments of sterile seedlings; the callus tissue is obtained by culturing leaves or stem segments of sterile seedlings.

[0050] In the present invention, the method for culturing callus is preferably:

[0051] Inoculating the leaves or stem segments of the sterile seedlings in step 1) into a callus induction medium, preferably culturing under light for 15 to 18 days, more preferably 16 days, to obtain callus;

[0052] In the present invention, the lighting conditions are independently: preferably, light and dark alternation is used for cultivation, the light cultivation time is preferably 12 to 20 hours, more preferably 14 to 18 hours, and more preferably 16 hours, the light intensity is independently preferably 2000 to 3000 lx, more preferably 2200 to 2800 lx, and more preferably 2500 lx; ​​the dark cultivation time is preferably 4 to 12 hours, more preferably 6 to 10 hours, and more preferably 8 hours.

[0053] The callus induction medium is preferably B5+2,4-D 1.5-3.0 mg / L+0.2-0.5 mg / L NAA+sucrose 30-40 g / L+agar 7.2-7.6 g / L, further preferably B5+2,4-D 1.7-2.5 mg / L+0.3-0.45 mg / L NAA+sucrose 33-37 g / L+agar 7.3-7.5 g / L, and further preferably B5+2,4-D 2.0 mg / L+0.4 mg / L NAA+sucrose 35 g / L+agar 7.4 g / L.

[0054] The present invention inoculates the explants of sterile seedlings into an adventitious root induction culture medium and cultures them in the dark for 10 to 30 days to obtain adventitious roots;

[0055] In the present invention, the adventitious root induction medium is preferably B5+IBA 1.0-2.0 mg / L+potato juice 80-120 g / L+sucrose 25-30 g / L+agar 7.2-7.6 g / L, more preferably B5+IBA 1.3-1.8 mg / L+potato juice 90-110 g / L+sucrose 26-29 g / L, and further preferably B5+IBA 1.5 mg / L+potato juice 100 g / L+sucrose 27 g / L, and the pH is preferably 5.8-6.0, and further preferably 5.9;

[0056] In the present invention, the dark culture time is 10 to 30 days, preferably 15 to 25 days, more preferably 20 days, and the culture temperature is preferably 23 to 27°C, more preferably 24 to 26°C, and further preferably 25°C.

[0057] In the present invention, the length of the adventitious roots is preferably 0.8 to 1.2 cm, more preferably 0.9 to 1.1 cm, and even more preferably 1.0 cm.

[0058] In the present invention, the adventitious roots are inoculated into a proliferation medium, cultured in the dark for 20 to 30 days, and then cultured in a light condition of 12 to 20 hours of light / 4 to 12 hours of darkness for 12 to 18 days to obtain proliferated adventitious roots; the proliferation medium is preferably B5+IBA 1.0 to 2.0 mg / L+NAA 0.5 to 1.0 mg / L+coenzyme Q10 20 to 30 μM+sucrose 30 to 45 g / L, more preferably B5+IBA 1.2 to 1.8 mg / L+NAA 0.6 to 0.9 mg / L+coenzyme Q10 22 to 28 μM+sucrose 33 to 40 g / L, and further preferably B5+IBA 1.5 mg / L+NAA 0.7mg / L+coenzyme Q1025μM+sucrose 35g / L, pH is preferably 5.8-6.0, more preferably 5.9; the dark culture time is 20-30 days, preferably 22-28 days, and more preferably 25 days; after dark culture, it is preferably cultured using alternating light and dark, the light culture time is preferably 12-20 hours, more preferably 14-18h, and more preferably 16h, and the light intensity is independently preferably 2000-3000lx, more preferably 2200-2800lx, and more preferably 2500lx; the dark culture time is preferably 4-12 hours, more preferably 6-10 hours, and more preferably 8 hours; the number of days of alternating light and dark culture is preferably 12-18 days, more preferably 14-16 days, and more preferably 15 days.

[0059] The invention provides application of the method in rapid propagation of Rhodiola rosea.

[0060] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Example 1

[0062] (1) Cultivation of sterile seedlings: Soak the seeds of Rhodiola rosea in 75% alcohol for 35 seconds, rinse with water three times, soak in 5% hydrogen peroxide for 10 minutes, rinse with water five times, and then culture in a primary culture medium, proliferation culture medium, and seedling rooting culture medium at 25±2°C. The culture time is 13 days, 23 days, and 20 days, respectively. The light intensity of plant tissue culture is 2500 lx. The primary culture medium is: MS + agar 7.3g / L + sucrose 27g / L, pH value is 5.8-6.0; the proliferation culture medium is: MS + KT 0.7mg / L + indoleacetonitrile 0.3mg / L + agar 7.3g / L + sucrose 27g / L, pH value is 5.8-6.0; the seedling rooting culture medium is: 1 / 2MS + NAA 0.65mg / L + potato juice 130g / L + agar 7.3g / L + sucrose 27g / L, pH value is 5.8-6.0. Figure 1 .

[0063] (2) Differentiation of adventitious roots from stem segments of Rhodiola rosea: The stem segments obtained from sterile seedlings in step (1) were inoculated on an adventitious root induction medium and cultured in the dark at 25±2°C for 14 days to obtain adventitious roots; the adventitious root induction medium was B5+IBA 1.5 mg / L+potato juice 100 g / L+sucrose 27 g / L+agar 7.4 g / L, pH = 5.8-6.0. Figure 3 .

[0064] (3) Proliferation culture of adventitious roots in tissue culture: The adventitious roots obtained in step (2) were cut into 1 cm lengths on a clean bench and inoculated into a solid culture medium for adventitious root proliferation: B5 + IBA 1.5 mg / L + NAA 0.7 mg / L + Coenzyme Q10 25 μM + Sucrose 35 g / L + Agar 7.4 g / L, pH = 5.8-6.0; the culture method was dark culture for 25 days and then transferred to a 16-hour light / 8-hour dark environment for 15 days. The light intensity for plant tissue culture was 2500 lx.

[0065] Example 2

[0066] (1) Cultivation of sterile seedlings: Soak the seeds of Rhodiola rosea in 75% alcohol for 35 seconds, rinse with water three times, soak in 5% hydrogen peroxide for 10 minutes, rinse with water five times, and then culture in a primary culture medium, proliferation culture medium, and seedling rooting culture medium at 25±2°C. The culture time is 13 days, 23 days, and 20 days, respectively. The light intensity of plant tissue culture is 2500 lx. The primary culture medium is: MS+agar 7.3g / L+sucrose 27g / L, with a pH value of 5.8-6.0; the proliferation culture medium is: MS+KT 0.7mg / L+indoleacetonitrile 0.3mg / L+agar 7.3g / L+sucrose 27g / L, with a pH value of 5.8-6.0; the seedling rooting culture medium is: 1 / 2MS+NAA 0.65mg / L+potato juice 130g / L+agar 7.3g / L+sucrose 27g / L, with a pH value of 5.8-6.0.

[0067] (2) Differentiation of adventitious roots from leaves of Rhodiola rosea: Take the leaves of the sterile seedlings obtained in step (1) and inoculate them on an adventitious root induction medium, and culture them in the dark at 25±2°C for 14 days to obtain adventitious roots; the adventitious root induction medium is B5+IBA1.5mg / L+potato juice 100g / L+sucrose 27g / L+agar 7.4g / L, pH=5.8-6.0. For details on adventitious roots induced from leaves, see Figure 4 .

[0068] (3) Proliferation culture of adventitious roots in tissue culture: The adventitious roots obtained in step (2) were cut into 1 cm lengths on a clean bench and inoculated into a solid culture medium for adventitious root proliferation: B5 + IBA 1.5 mg / L + NAA 0.7 mg / L + Coenzyme Q10 25 μM + Sucrose 35 g / L + Agar 7.4 g / L, pH = 5.8-6.0; the culture method was dark culture for 25 days and then transferred to a 16-hour light / 8-hour dark environment for 15 days. The light intensity for plant tissue culture was 2500 lx. The light intensity for plant tissue culture was 2500 lx.

[0069] Example 3

[0070] (1) Cultivation of sterile seedlings: Soak the seeds of Rhodiola rosea in 75% alcohol for 35 seconds, rinse with water three times, soak in 5% hydrogen peroxide for 10 minutes, rinse with water five times, and then culture in a primary culture medium, proliferation culture medium, and seedling rooting culture medium at 25±2°C. The culture time is 13 days, 23 days, and 20 days, respectively. The light intensity of plant tissue culture is 2500 lx. The primary culture medium is: MS+agar 7.3g / L+sucrose 27g / L, with a pH value of 5.8-6.0; the proliferation culture medium is: MS+KT 0.7mg / L+indoleacetonitrile 0.3mg / L+agar 7.3g / L+sucrose 27g / L, with a pH value of 5.8-6.0; the seedling rooting culture medium is: 1 / 2MS+NAA 0.65mg / L+potato juice 130g / L+agar 7.3g / L+sucrose 27g / L, with a pH value of 5.8-6.0.

[0071] (2) Induction of callus tissue: Leaves and stem segments cut from sterile seedlings of Rhodiola rosea after the culture is completed are inoculated into callus tissue induction medium in a clean bench and cultured under light for 15 to 18 days to obtain a large amount of Rhodiola rosea callus tissue. The callus tissue induction medium is B5+2,4-D 2.0 mg / L+0.4 mg / L NAA+sucrose 35 g / L+agar 7.4 g / L.

[0072] (3) Callus differentiation into adventitious roots: The Rhodiola rosea callus obtained in step (2) was cut into small pieces, inoculated on an adventitious root induction medium, and cultured in the dark at 25±2°C for 10 to 18 days to obtain adventitious roots; the adventitious root induction medium was B5+IBA 1.5 mg / L+potato juice 100 g / L+sucrose 27 g / L+agar 7.4 g / L, pH = 5.8 to 6.0. For details on adventitious roots induced by callus, see Figure 5 .

[0073] (4) Proliferation culture of adventitious roots in tissue culture: The adventitious roots obtained in step (3) were cut into 1 cm lengths on a clean bench and inoculated into a solid culture medium for adventitious root proliferation: B5 + IBA 1.5 mg / L + NAA 0.7 mg / L + Coenzyme Q10 25 μM + Sucrose 35 g / L + Agar 7.4 g / L, pH = 5.8-6.0; the culture method was dark culture for 25 days and then transferred to a 16-hour light / 8-hour dark environment for 15 days. The light intensity for plant tissue culture was 2500 lx. The light intensity for plant tissue culture was 2500 lx.

[0074] Example 4

[0075] (1) Cultivation of sterile seedlings: Soak the seeds of Rhodiola rosea in 70% alcohol for 40 seconds, rinse twice with water, soak in 3% hydrogen peroxide for 10 minutes, rinse five times with water, and then culture in a primary culture medium, proliferation culture medium, and seedling rooting culture medium at 25±2°C. The culture time is 15 days, 20 days, and 22 days, respectively. The light intensity of the plant tissue culture is 2000 lx. The primary culture medium is: MS+agar 7.0g / L+sucrose 25g / L, with a pH value of 5.8-6.0; the proliferation culture medium is: MS+KT 1.0mg / L+indoleacetonitrile (IAN) 0.5mg / L+agar 7.5g / L+sucrose 30g / L, with a pH value of 5.8-6.0; the seedling rooting culture medium is: 1 / 2MS+NAA 0.8 mg / L+potato juice 150g / L+agar 7.5g / L+sucrose 30g / L, with a pH value of 5.8-6.0.

[0076] (2) Adventitious roots of leaf stem segments of Rhodiola rosea: Take the leaves of the sterile seedlings obtained in step (1) and inoculate them on adventitious root induction medium: B5 + IBA 1.5 mg / L + potato juice 120 g / L + sucrose 30 g / L + agar 7.6 g / L, pH = 5.8-6.0, and culture in the dark at 25±2°C for 30 days to obtain adventitious roots.

[0077] (3) Proliferation culture of tissue culture adventitious roots: The adventitious roots obtained in step (3) were cut into 0.8 cm lengths on a clean bench and inoculated into a liquid proliferation medium for adventitious root proliferation: B5 + IBA 1.0 mg / L + NAA 1.0 mg / L + 30 μM coenzyme Q10 + sucrose 45 g / L, pH = 5.8-6.0; the culture method was dark culture for 20 days and then transferred to a 16-hour light / 8-hour dark environment for 20 days. The light intensity of plant tissue culture was 2500 lx.

[0078] Example 5

[0079] (1) Cultivation of sterile seedlings: Soak the seeds of Rhodiola rosea in 80% alcohol for 30 seconds, rinse with water three times, soak in 8% hydrogen peroxide for 8 minutes, rinse with water five times, and then culture in a primary culture medium, proliferation culture medium, and seedling rooting culture medium at 25±2°C. The culture time is 10 days, 25 days, and 18 days, respectively. The light intensity of the plant tissue culture is 3000 lx. The primary culture medium is: MS+agar 7.5g / L+sucrose 30g / L, with a pH value of 5.8-6.0; the proliferation culture medium is: MS+KT 0.5mg / L+indoleacetonitrile (IAN) 0.2mg / L+agar 7.0g / L+sucrose 25g / L, with a pH value of 5.8-6.0; the seedling rooting culture medium is: 1 / 2MS+NAA 0.5 mg / L+potato juice 100g / L+agar 7.0g / L+sucrose 40g / L, with a pH value of 5.8-6.0.

[0080] (2) Differentiation of adventitious roots from stem segments of Rhodiola rosea: The stem segments of the sterile seedlings obtained in step (1) were inoculated on an adventitious root induction medium: B5 + IBA 1.0 mg / L + potato juice 80 g / L + sucrose 25 g / L + agar 7.2 g / L, pH = 5.8-6.0, and cultured in the dark at 25±2°C for 30 days to obtain adventitious roots.

[0081] (3) Proliferation culture of tissue culture adventitious roots: The adventitious roots obtained in step (3) were cut into 1.2 cm lengths on a clean bench and inoculated into a liquid proliferation medium for adventitious root proliferation: B5 + IBA 1.0 mg / L + NAA 0.5 mg / L + 20 μM coenzyme Q10 + sucrose 35 g / L, pH = 5.8-6.0; the culture method was dark culture for 30 days and then transferred to a 16-hour light / 8-hour dark environment for 18 days. The light intensity for plant tissue culture was 2500 lx.

[0082] Experimental Example 1

[0083] The adventitious root induction rates of the explants in Examples 1 to 3 were statistically analyzed, and the results are shown in Table 1.

[0084] Table 1 Statistics of adventitious root induction rate of different Rhodiola rosea explants

[0085]

[0086] From the results in Table 1, it can be seen that the induction efficiency of leaves is the fastest, and the induction rate can reach 16.67% in two weeks. However, as time goes by, the induction effect of stem segments is the best.

[0087] Experimental Example 2

[0088] The stem segments of Rhodiola rosea were used as explants to explore the effects of different hormone combinations (adventitious root induction medium) on the adventitious root induction of Rhodiola rosea. The results are shown in Table 2.

[0089] Table 2 Effects of different hormone ratios on adventitious root induction in tissue culture of Rhodiola rosea

[0090]

[0091]

[0092] From the results in Table 2, it can be seen that the best adventitious root induction medium is B5+IBA 1.5 mg / L+potato juice 100 g / L+sucrose 27 g / L.

[0093] Experimental Example 3

[0094] B5+IBA1.3mg / L+NAA0.7mg / L+10-30μM coenzyme Q10+sucrose 35g / L+agar 7.4g / L, pH=5.8-6.0, was used as the solid proliferation medium for adventitious roots of Rhodiola rosea tissue culture, and the effects of different concentrations of coenzyme Q10 on the growth status and browning of adventitious roots of Rhodiola rosea tissue culture were investigated.

[0095] Table 3 Effects of different concentrations of coenzyme Q10 on the growth of adventitious roots of Rhodiola rosea in tissue culture

[0096] Coenzyme Q10 concentration / μM Browning rate / % Growth 10 36.2 The root system is slender and grows slowly 15 28.3 The root system is slender and grows slowly 20 21.6 Slender roots 25 15.3 The root system is slender and has many branches 30 20.8 The root system is slender and has few branches

[0097] The results showed that when the concentration of coenzyme Q10 was 25 μM, the browning rate was the lowest and the growth state was the best.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for tissue culture of Rhodiola rosea, characterized in that: The following steps are involved: 1) Cultivating sterilized Rhodiola rosea seeds to obtain sterile seedlings; 2) Inoculate the sterile seedling explants into adventitious root induction medium and culture them in the dark for 10 to 30 days to obtain adventitious roots; 3) inoculating the adventitious roots into a proliferation medium, culturing in the dark for 20 to 30 days, then switching to a light condition of 12 to 20 hours of light / 4 to 12 hours of darkness for 12 to 18 days to obtain proliferated adventitious roots; Step 2) the explants include leaves, stem segments and / or callus tissues of sterile seedlings; The callus tissue is cultured from leaves or stem segments of sterile seedlings; Step 2) The adventitious root induction medium is B5+IBA 1.0-2.0 mg / L+potato juice 80-120 g / L+sucrose 25-30 g / L+agar 7.2-7.6 g / L, pH 5.8-6.0; Step 3) The proliferation medium is B5+IBA 1.0-2.0 mg / L+NAA 0.5-1.0 mg / L+coenzyme Q10 20-30 μM+sucrose 30-45 g / L, pH 5.8-6.

0.

2. The method according to claim 1, characterized in that The callus culture method is: Inoculating the leaves or stem segments of the sterile seedlings in step 1) into a callus induction medium, and culturing under light for 15 to 18 days to obtain callus; The callus induction medium is B5+2,4-D1.5-3.0 mg / L+0.2-0.5 mg / L NAA+sucrose 30-40 g / L+agar 7.2-7.6 g / L.

3. The method according to claim 2, characterized in that The culture in step 1) includes primary culture, subculture and rooting culture; The culture medium for the primary culture is MS+agar 7.0-7.5 g / L+sucrose 25-30 g / L, pH 5.8-6.0; The subculture medium is MS+KT 0.5-1.0 mg / L+indoleacetonitrile 0.2-0.5 mg / L+agar 7.0-7.5 g / L+sucrose 25-30 g / L, pH 5.8-6.0; The culture medium for rooting culture is 1 / 2MS+NAA 0.5-0.8 mg / L+potato juice 100-150 g / L+agar 7.0-7.5 g / L+sucrose 25-30 g / L, with a pH of 5.8-6.

0.

4. The method according to claim 3, characterized in that The primary culture time is 10 to 15 days; The subculture time is 20 to 25 days; The rooting culture time is 18 to 22 days; The culture temperature of the primary culture, subculture culture and rooting culture is independently 23-27° C., the light conditions of the culture are independently 12-20 hours of light / 4-12 hours of darkness, and the light intensity is independently 2000-3000 lx.

5. The method according to claim 1, characterized in that The disinfection method in step 1) is to soak in alcohol for 30 to 40 seconds and then soak in hydrogen peroxide solution for 8 to 10 minutes.

6. The method according to claim 5, characterized in that The volume concentration of alcohol is 70% to 80%.

7. The method according to claim 6, characterized in that The mass concentration of hydrogen peroxide is 3% to 8%.

8. The method according to claim 7, characterized in that After soaking in alcohol, wash with water 2 to 4 times; After soaking in hydrogen peroxide, wash with water 2 to 4 times.

9. The method according to claim 1, characterized in that: Step 3) The length of the adventitious roots is 0.8 to 1.2 cm.

10. Use of the method according to any one of claims 1 to 9 in the rapid propagation of Rhodiola rosea.

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