A tissue culture method of rhodiola crenulata

Abstract

The application provides a tissue culture method of Rhodiola crenulata, and belongs to the technical field of plant tissue culture. The application uses various explants (callus, leaf, stem section) of the tissue culture seedling of Rhodiola crenulata to induce adventitious roots and then carries out propagation culture, limits the composition of the culture medium, so that the induction rate of the adventitious roots is between 58.3% and 72.6%, and the browning rate is low during the adventitious root culture of the tissue culture of Rhodiola crenulata, which lays a foundation for industrialized production of important active components of Rhodiola crenulata by using the adventitious roots of Rhodiola crenulata in the future, and can solve the contradiction between supply and demand of Rhodiola crenulata raw materials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture method of Rhodiola crenulata. BACKGROUND

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

[0003] The main bottleneck of the current tissue culture technology of Rhodiola crenulata is the high browning rate of explants, low induction efficiency of adventitious roots, 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 realize the large-scale and standardized production of adventitious roots of Rhodiola crenulata by optimizing the medium composition, regulating the hormone ratio and the culture conditions, and to provide a reliable raw material source for the subsequent extraction of active ingredients and commercial application. SUMMARY

[0004] Therefore, the present application aims to provide a tissue culture method of Rhodiola crenulata.

[0005] In order to achieve the above-mentioned application purpose, the present application provides the following technical solutions:

[0006] The present application provides a tissue culture method of Rhodiola crenulata, comprising the following steps:

[0007] 1) Culturing the sterilized seeds of Rhodiola crenulata to obtain sterile seedlings;

[0008] 2) Inoculating the explants of the sterile seedlings into an adventitious root induction medium, and culturing in darkness for 10-30 days to obtain adventitious roots;

[0009] 3) Inoculating the adventitious roots into a proliferation medium, and culturing in darkness for 20-30 days, then culturing under light conditions of 12-20 hours light / 4-12 hours darkness for 12-18 days to obtain proliferated adventitious roots;

[0010] The explants of step 2) include leaf blades, stem segments and / or callus of the sterile seedlings;

[0011] The callus is obtained by culturing the leaf blades or stem segments of the 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 culture method of the callus is as follows:

[0015] the leaf or stem segment of the sterile seedling in step 1) is inoculated into a callus induction medium, and cultured under light for 15-18 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 culture in step 1) comprises primary culture, subculture and rooting culture.

[0018] the 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 medium for the subculture 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 medium for the 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 time for the primary culture is 10-15 days.

[0022] the time for the subculture is 20-25 days;

[0023] the time for the rooting culture is 18-22 days;

[0024] the culture temperature for the primary culture, subculture and rooting culture is independently 23-27°C, the light condition for the culture is 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 of step 1) is soaking in alcohol for 30-40s, and then soaking in hydrogen peroxide solution for 8-10min.

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

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

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

[0029] After hydrogen peroxide soaking, washing with water for 2-4 times.

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

[0031] The application provides the application of the method in rapid propagation of Rhododendron aureum.

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

[0033] The application utilizes various explants (callus, leaves, stem segments) of Rhododendron aureum tissue culture seedlings to induce adventitious roots and then to carry out propagation culture, limits the composition of the culture medium, so that the induction rate of the adventitious roots is between 58.3% and 72.6%, and the browning rate is low during the Rhododendron aureum tissue culture adventitious root culture process, which lays a foundation for future industrial production of important active ingredients of Rhododendron aureum by using Rhododendron aureum adventitious roots, and solves the supply and demand contradiction problem of Rhododendron aureum raw materials. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Rhododendron aureum aseptic seedlings;

[0035] Figure 2 Rhododendron aureum adventitious roots induced by using stem segments of Rhododendron aureum tissue culture seedlings as explants;

[0036] Figure 3 Rhododendron aureum adventitious roots induced by using leaves of Rhododendron aureum tissue culture seedlings as explants;

[0037] Figure 4 Rhododendron aureum adventitious roots induced by using callus of Rhododendron aureum tissue culture seedlings as explants;

[0038] Figure 5 Rhododendron aureum adventitious root solid culture medium propagation culture effect. DETAILED DESCRIPTION

[0039] The application provides a Rhododendron aureum tissue culture method, which comprises the following steps:

[0040] 1) culturing and disinfecting Rhododendron aureum seeds to obtain aseptic seedlings;

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

[0042] 3) inoculate the adventitious roots into proliferation medium, and culture in dark for 20-30 days, then culture under 12-20 hours light / 4-12 hours dark for 12-18 days to obtain proliferated adventitious roots;

[0043] In the present application, the seed of Rhodoleia gigantea is first sterilized, and the sterilization comprises sequentially alcohol immersion and hydrogen peroxide immersion; the time of alcohol immersion is 30-40 s, further preferably 33-37 s, and more further preferably 35 s, and the volume concentration of alcohol is preferably 70%-80%, further preferably 75%; the time of hydrogen peroxide solution immersion is preferably 8-10 min, further preferably 8.5-9.5 min, and more further preferably 9 min, and the mass concentration of hydrogen peroxide solution is preferably 3%-8%, further preferably 4%-7%, and more further preferably 5%; after alcohol immersion, water washing is preferably performed, and the number of washing is preferably 2-4 times, further preferably 3 times; after hydrogen peroxide immersion, water washing is preferably performed, and the number of washing is preferably 2-4 times, further preferably 3 times.

[0044] In the present application, the seed of Rhodoleia gigantea after sterilization is cultured; the culture is preferably primary culture, subculture and rooting culture;

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

[0046] The medium of the subculture is preferably MS+KT 0.5-1.0 mg / L+indole acetonitrile 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+indole acetonitrile 0.25-0.4 mg / L+agar 7.1-7.4 g / L+sucrose 26-29 g / L, and more further preferably MS+KT 0.7 mg / L+indole acetonitrile 0.3 mg / L+agar 7.3 g / L+sucrose 27 g / L, and pH is 5.8-6.0; the time of the subculture is preferably 20-25 days, further preferably 21-24 days, and more further preferably 23 days.

[0047] The rooting culture medium 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, more 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 rooting culture time is preferably 18-22 days, further preferably 19-21 days, more preferably 20 days.

[0048] In the present application, the culture temperature of the primary culture, subculture and rooting culture is independently preferably 23-27℃, further preferably 24-26℃, more preferably 25℃; the light condition of the culture is independently preferably light and dark alternation, the light culture time is preferably 12-20 hours, further preferably 14-18 hours, more preferably 16 hours, the light intensity is independently preferably 2000-3000 lx, further preferably 2200-2800 lx, more preferably 2500 lx; the dark culture time is preferably 4-12 hours, further preferably 6-10 hours, more preferably 8 hours.

[0049] In the present application, the explant is obtained from the sterile seedling obtained by culture, and the explant preferably comprises leaf, stem segment and / or callus of the sterile seedling, further preferably stem segment of the sterile seedling; the callus is obtained by culture of the leaf or stem segment of the sterile seedling.

[0050] In the present application, the culture method of the callus is preferably:

[0051] The leaf or stem segment of the sterile seedling in step 1) is inoculated into the callus induction culture medium, and the light culture time is preferably 15-18 days, further preferably 16 days, to obtain the callus;

[0052] In the present application, the light condition is independently preferably light and dark alternation, the light culture time is preferably 12-20 hours, further preferably 14-18 hours, more preferably 16 hours, the light intensity is independently preferably 2000-3000 lx, further preferably 2200-2800 lx, more preferably 2500 lx; the dark culture time is preferably 4-12 hours, further preferably 6-10 hours, 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 more preferably B5+2,4-D 2.0 mg / L+0.4 mg / L NAA+ sucrose 35 g / L+ agar 7.4 g / L.

[0054] In the present application, the explant of the sterile seedling is inoculated into the adventitious root induction medium, and cultured in the dark for 10-30 days to obtain the adventitious root.

[0055] In the present application, 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, further preferably B5+IBA 1.3-1.8 mg / L+ potato juice 90-110 g / L+ sucrose 26-29 g / L, and more 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 application, the time of the dark culture is 10-30 days, preferably 15-25 days, and further preferably 20 days, and the culture temperature is preferably 23-27℃, further preferably 24-26℃, and more preferably 25℃.

[0057] In the present application, the length of the adventitious root is preferably 0.8-1.2 cm, further preferably 0.9-1.1 cm, and more preferably 1.0 cm.

[0058] In the present application, the adventitious roots are inoculated into a proliferation medium, and after dark culture for 20-30 days, the culture is carried out under light conditions of 12-20 hours light / 4-12 hours dark for 12-18 days to obtain proliferated adventitious roots; the proliferation medium is preferably B5+IBA 1.0-2.0 mg / L+NAA 0.5-1.0 mg / L+coenzyme Q1020-30 μM+sucrose 30-45 g / L, further preferably B5+IBA 1.2-1.8 mg / L+NAA 0.6-0.9 mg / L+coenzyme Q1022-28 μM+sucrose 33-40 g / L, more preferably B5+IBA 1.5 mg / L+NAA 0.7 mg / L+coenzyme Q1025 μM+sucrose 35 g / L, and the pH is preferably 5.8-6.0, further preferably 5.9; the dark culture time is 20-30 days, preferably 22-28 days, further preferably 25 days; after dark culture, the culture is preferably carried out using light-dark alternation, and the light culture time is preferably 12-20 hours, further preferably 14-18 h, more preferably 16 h, and the light intensity is independently preferably 2000-3000 lx, further preferably 2200-2800 lx, more preferably 2500 lx; the dark culture time is preferably 4-12 hours, further preferably 6-10 hours, more preferably 8 hours; and the light-dark alternation culture time is preferably 12-18 days, further preferably 14-16 days, more preferably 15 days.

[0059] The present application provides application of the method in rapid propagation of Rhodiolae Crenulatae.

[0060] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0061] Example 1

[0062] (1) the culture of aseptic seedlings: the seeds of Rhodiola crenulata are soaked with 75% alcohol for 35s, washed with water for 3 times, then soaked with 5% hydrogen peroxide for 10 min, washed with water for 5 times, and then cultured in an environment with temperature of 25±2℃ using primary culture medium, proliferation culture medium and strong seedling rooting culture medium in sequence, and the culture time is 13 days, 23 days and 20 days respectively, and the light intensity of plant tissue culture is 2500lx. The primary culture medium is MS+agar 7.3g / L+sucrose 27g / L, and the pH value is 5.8-6.0; the proliferation culture medium is MS+KT 0.7mg / L+indole acetonitrile 0.3mg / L+agar 7.3g / L+sucrose 27g / L, and the pH value is 5.8-6.0; the strong seedling rooting culture medium is 1 / 2MS+NAA 0.65mg / L+potato juice 130g / L+agar 7.3g / L+sucrose 27g / L, and the pH value is 5.8-6.0. The aseptic seedlings of Rhodiola crenulata are shown in detail in Figure 1 .

[0063] (2) the differentiation of adventitious roots from stem segments of Rhodiola crenulata: the stem segments of the aseptic seedlings obtained in step (1) are inoculated on the adventitious root induction medium, and cultured in darkness at 25±2℃ for 14d to obtain adventitious roots; the adventitious root induction medium is B5+IBA 1.5mg / L+potato juice 100g / L+sucrose 27g / L+agar 7.4g / L, and the pH value is 5.8-6.0. The adventitious roots induced from the stem segments are shown in detail in Figure 3 .

[0064] (3) the proliferation culture of tissue culture adventitious roots: the adventitious roots obtained in step (2) are cut into 1cm in length on the super-clean workbench, inoculated on the adventitious root proliferation solid medium: B5+IBA 1.5mg / L+NAA 0.7mg / L+coenzyme Q1025μM+sucrose 35g / L+agar 7.4g / L, and the pH value is 5.8-6.0; the culture mode is dark culture for 25d, and then transferred into an environment with 16h light / 8h darkness for 15d. The light intensity of plant tissue culture is 2500lx.

[0065] Example 2

[0066] (1) the culture of aseptic seedlings: the seeds of Rhodiola crenulata are soaked with 75% alcohol for 35s, washed with water for 3 times, then soaked with 5% hydrogen peroxide for 10 min, washed with water for 5 times, and then cultured in an environment with a temperature of 25±2℃ using a primary culture medium, a proliferation culture medium, and a strong seedling rooting culture medium in sequence, with the culture time being 13 days, 23 days, and 20 days respectively, and the light intensity for plant tissue culture being 2500lx. 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+indole acetonitrile 0.3mg / L+agar 7.3g / L+sucrose 27g / L, with a pH value of 5.8-6.0; and the strong 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 crenulata: the leaves of the aseptic seedlings obtained in step (1) are inoculated on an adventitious root induction medium, and cultured in darkness at 25±2℃ for 14 days to obtain adventitious roots; the adventitious root induction medium is B5+IBA 1.5mg / L+potato juice 100g / L+sucrose 27g / L+agar 7.4g / L, with a pH value of 5.8-6.0. The adventitious roots induced from leaves are described in detail in Figure 4 .

[0068] (3) proliferation culture of tissue culture adventitious roots: the adventitious roots obtained in step (2) are cut into 1cm in length on a super-clean workbench, and inoculated on an adventitious root proliferation solid culture medium: B5+IBA 1.5mg / L+NAA 0.7mg / L+coenzyme Q1025μM+sucrose 35g / L+agar 7.4g / L, with a pH value of 5.8-6.0; the culture mode is to culture in darkness for 25 days, and then transfer to an environment with 16 hours of light and 8 hours of darkness for 15 days. The light intensity for plant tissue culture is 2500lx.

[0069] Example 3

[0070] (1) The culture of aseptic seedlings: The seeds of Rhodiola crenulata are soaked in 75% alcohol for 35s, washed with water for 3 times, then soaked in 5% hydrogen peroxide for 10 min, washed with water for 5 times, and then cultured in primary culture medium, proliferation culture medium and strong seedling rooting culture medium in turn at 25±2℃, with the culture time being 13 days, 23 days and 20 days respectively, and the light intensity of plant tissue culture being 2500lx. The primary culture medium is MS+agar 7.3g / L+sucrose 27g / L, with pH value being 5.8-6.0; the proliferation culture medium is MS+KT 0.7mg / L+indole acetonitrile 0.3mg / L+agar 7.3g / L+sucrose 27g / L, with pH value being 5.8-6.0; and the strong seedling rooting culture medium is 1 / 2MS+NAA 0.65mg / L+potato juice 130g / L+agar 7.3g / L+sucrose 27g / L, with pH value being 5.8-6.0.

[0071] (2) The induction of callus: the leaves and stems cut from the Rhodiola crenulata aseptic seedling plants after the culture are inoculated in callus induction medium in a clean bench, and a large amount of Rhodiola crenulata callus can be obtained after 15-18d of light culture, wherein the callus induction medium is B5+2.0mg / L 2,4-D+0.4mg / L NAA+sucrose 35g / L+agar 7.4g / L.

[0072] (3) The differentiation of callus into adventitious roots: the Rhodiola crenulata callus obtained in step (2) is cut into small pieces and inoculated in adventitious root induction medium, and the adventitious roots are obtained after 10-18d of 25±2℃ dark culture; the adventitious root induction medium is B5+IBA 1.5mg / L+potato juice 100g / L+sucrose 27g / L+agar 7.4g / L, with pH value being 5.8-6.0. The induction of callus into adventitious roots is shown in detail in Figure 5 .

[0073] (4) The proliferation culture of tissue culture adventitious roots: the adventitious roots obtained in step (3) are cut into 1cm in length in a clean bench, inoculated in adventitious root proliferation solid medium: B5+IBA 1.5mg / L+NAA 0.7mg / L+coenzyme Q1025μM+sucrose 35g / L+agar 7.4g / L, with pH value being 5.8-6.0; and the culture mode is 25d of dark culture, then 15d of culture in an environment of 16h light / 8h dark. The light intensity of plant tissue culture is 2500lx.

[0074] Example 4

[0075] (1) The culture of aseptic seedlings: the seeds of Rhodiola crenulata are soaked with 70% alcohol for 40s, washed with water for 2 times, then soaked with 3% hydrogen peroxide for 10 min, washed with water for 5 times, and then cultured in an environment with temperature of 25±2℃ using primary culture medium, proliferation culture medium and strong seedling rooting culture medium in sequence, and the culture time is 15 days, 20 days and 22 days respectively, and the light intensity of plant tissue culture is 2000lx. The primary culture medium is MS+agar 7.0g / L+sucrose 25g / L, and the pH value is 5.8-6.0; the proliferation culture medium is MS+KT 1.0mg / L+indole acetonitrile (IAN) 0.5mg / L+agar 7.5g / L+sucrose 30g / L, and the pH value is 5.8-6.0; and the strong seedling rooting culture medium is 1 / 2MS+NAA 0.8mg / L+potato juice 150g / L+agar 7.5g / L+sucrose 30g / L, and the pH value is 5.8-6.0.

[0076] (2) The adventitious root of Rhodiola crenulata leaf and stem segment: the leaf of the aseptic seedling obtained in step (1) is inoculated on the adventitious root induction medium: B5+IBA 1.5mg / L+potato juice 120g / L+sucrose 30g / L+agar 7.6g / L, pH=5.8-6.0, and cultured in darkness at 25±2℃ for 30d to obtain the adventitious root.

[0077] (3) The proliferation culture of tissue culture adventitious root: the adventitious root obtained in step (3) is cut into 0.8cm in length on the super-clean workbench, and inoculated on the liquid proliferation culture medium for adventitious root proliferation: B5+IBA 1.0mg / L+NAA 1.0mg / L+30μM coenzyme Q10+sucrose 45g / L, pH=5.8-6.0; and the culture mode is to culture in darkness for 20d, and then transfer to the environment with 16h light / 8h darkness for 20d. The light intensity of plant tissue culture is 2500lx.

[0078] Example 5

[0079] (1) The culture of aseptic seedlings: the seeds of Rhodiola crenulata are soaked in 80% alcohol for 30s, washed with water for 3 times, then soaked in 8% hydrogen peroxide for 8 min, washed with water for 5 times, and then cultured in an environment with a temperature of 25±2°C by using primary culture medium, proliferation culture medium and strong seedling rooting culture medium in sequence, and the culture time is 10 days, 25 days and 18 days respectively, and the light intensity of plant tissue culture is 3000lx. The primary culture medium is MS+agar 7.5g / L+sucrose 30g / L, and the pH value is 5.8-6.0; the proliferation culture medium is MS+KT 0.5mg / L+indole acetonitrile (IAN) 0.2mg / L+agar 7.0g / L+sucrose 25g / L, and the pH value is 5.8-6.0; and the strong seedling rooting culture medium is 1 / 2MS+NAA 0.5mg / L+potato juice 100g / L+agar 7.0g / L+sucrose 40g / L, and the pH value is 5.8-6.0.

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

[0081] (3) The proliferation culture of tissue culture adventitious roots: the adventitious roots obtained in step (3) are cut into 1.2cm in length on a clean bench, inoculated on the liquid proliferation culture medium for adventitious root proliferation: B5+IBA 1.0mg / L+NAA 0.5mg / L+20μM coenzyme Q10+sucrose 35g / L, pH=5.8-6.0, and cultured in darkness for 30d, then transferred into an environment with 16h light / 8h darkness for 18d. The light intensity of plant tissue culture is 2500lx.

[0082] Experimental Example 1

[0083] The adventitious root induction rates of the explants in Experimental Examples 1-3 are counted, and the results are shown in Table 1.

[0084] Table 1 The adventitious root induction rates of different Rhodiola crenulata explants

[0085]

[0086] As can be seen from the results in Table 1, the induction efficiency of leaves is the fastest, and the induction rate can reach 16.67% in two weeks, but the induction effect of stem segments is the best with the increase of time.

[0087] Experimental Example 2

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

[0089] Table 2 Effects of different hormone combinations on the induction of adventitious roots of Rhodiola crenulata

[0090]

[0091]

[0092] As shown in Table 2, the optimal 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+IBA 1.3 mg / L+NAA 0.7 mg / L+10-30 μM coenzyme Q10+sucrose 35 g / L+agar 7.4 g / L, pH=5.8-6.0, is used as a solid proliferation medium for Rhodiola crenulata adventitious roots, and the effects of different concentrations of coenzyme Q10 on the growth state and browning of Rhodiola crenulata adventitious roots are explored.

[0095] Table 3 Effects of different concentrations of coenzyme Q10 on the growth state of Rhodiola crenulata adventitious roots

[0096] Coenzyme Q10 concentration / μM Browning rate / % Growth condition 10 36.2 Root system was slender, and growth speed was slow 15 28.3 Root system was slender, and growth speed was slow 20 21.6 Root system was slender 25 15.3 Root system was slender, and branching was much 30 20.8 Root system was slender, and branching was less

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

[0098] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A tissue culture method of Rhodiola crenulata, characterized by, The method comprises the following steps: 1) culturing the sterilized seeds of Rhodiola crenulata to obtain aseptic seedlings; 2) inoculating the explants of the aseptic seedlings into an adventitious root induction medium and culturing in darkness for 10-30 days to obtain adventitious roots; 3) inoculating the adventitious roots into a proliferation medium, culturing in darkness for 20-30 days, then culturing under light conditions of 12-20 hours light / 4-12 hours darkness for 12-18 days to obtain proliferated adventitious roots; the explants in step 2) are leaf blades or stem segments of the aseptic seedlings; the adventitious root induction medium in step 2) is B5+IBA 1.5 mg / L+potato juice 100 g / L+sucrose 27 g / L+agar 7.2-7.6 g / L, pH 5.8-6.0; the proliferation medium in step 3) is B5+IBA 1.0-2.0 mg / L+NAA 0.5-1.0 mg / L+coenzyme Q10 25 μM+sucrose 30-45 g / L, pH 5.8-6.

0.

2. The method of claim 1, wherein, The sterilization method in step 1) is soaking in alcohol for 30-40 s, and then soaking in hydrogen peroxide solution for 8-10 min.

3. The method of claim 2, wherein, The volume concentration of alcohol is 70%-80%.

4. The method of claim 3, wherein, The mass concentration of hydrogen peroxide is 3%-8%.

5. The method of claim 4, wherein, After alcohol soaking, washing with water for 2-4 times; After hydrogen peroxide soaking, washing with water for 2-4 times.

6. The method of claim 1, wherein, The length of the adventitious roots in step 3) is 0.8-1.2 cm.

7. Application of the method in any one of claims 1-6 in rapid propagation of Rhodiola crenulata.

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