A tissue culture and test-tube seedling acclimatization and transplanting method of rhodiola crenulata

Abstract

The application provides a tissue culture and test-tube seedling acclimatization and transplanting method of Rhodiola crenulata, and belongs to the technical field of plant tissue culture.The tissue culture method of the Rhodiola crenulata comprises the following steps: (1) after the seed of the Rhodiola crenulata is sterilized, the seed is subjected to germination culture to obtain adventitious buds; (2) the adventitious buds obtained in the step (1) are transferred to an adventitious bud proliferation culture medium to perform adventitious bud proliferation culture, and clumpy buds are obtained; (3) the clumpy buds obtained in the step (2) are transferred to a strong seedling rooting culture medium to perform strong seedling rooting culture, and test-tube seedlings are obtained.The tissue culture and test-tube seedling acclimatization and transplanting method of the Rhodiola crenulata provided by the application can provide a new method for large-area and high-efficiency cultivation of the Rhodiola crenulata, and has important application value for resource protection and development and utilization of the Rhodiola crenulata.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for tissue culture and test tube seedling hardening and transplanting of Rhodiola rosea. Background Art

[0002] Rhodiola crenulata (Hook. F. & Thomson) H. Ohba is a perennial herb or subshrub of the genus Rhodiola in the family Crassulaceae. It primarily grows in the Himalayas, northwestern Yunnan, and the western Sichuan Plateau at altitudes of 3,500 to 5,000 meters in the high mountain scree areas of the eastern edge of the Himalayas and the western Sichuan Plateau. Its dried roots and rhizomes are used medicinally, boasting benefits such as invigorating qi and promoting blood circulation, relieving asthma, and promoting blood circulation. Clinically, it is primarily used to treat qi deficiency and blood stasis, chest pain, hemiplegia due to stroke, and fatigue and shortness of breath. Modern pharmacological research has shown that Rhodiola crenulata has anti-hypoxia, anti-fatigue, anti-radiation, anti-viral, anti-tumor effects, cardiovascular system benefits, and anti-aging effects, suggesting broad potential for development.

[0003] In recent years, due to overexploitation and ecological changes, wild Rhodiola rosea resources have become increasingly endangered, making their conservation and rational development and utilization imperative. However, Rhodiola rosea is an alpine plant that cannot tolerate high temperatures and has stringent requirements for its growing environment. Propagation in other locations is difficult, and large-scale cultivation remains unsuccessful. Research on tissue culture techniques for Rhodiola rosea could accelerate its propagation and has important theoretical and practical value for resource conservation and rational development and utilization. Summary of the Invention

[0004] The purpose of the present invention is to provide a tissue culture and test tube seedling hardening and transplanting method for Rhodiola rosea, so as to provide a new method for resource protection and development and utilization of 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 tissue culture method of Rhodiola rosea, comprising the following steps:

[0007] (1) sterilizing Rhodiola rosea seeds and then germinating and culturing them to obtain adventitious buds;

[0008] (2) transferring the adventitious buds obtained in step (1) to an adventitious bud proliferation medium for adventitious bud proliferation culture to obtain clustered buds;

[0009] (3) transferring the clustered buds obtained in step (2) to a seedling rooting medium for seedling rooting culture to obtain test tube plantlets;

[0010] Wherein, the adventitious bud proliferation medium in step (2) is based on MS medium and further contains 0.8-1.2 mg / L LTDZ and 0.15-0.25 mg / L IAA;

[0011] The seedling rooting medium in step (3) uses 1 / 2 MS medium as the basic medium and also contains 0.25-0.35 mg / L IBA and 0.08-0.12 mg / L NAA.

[0012] Preferably, the disinfection method in step (1) is to soak the Rhodiola rosea seeds in a 0.08-0.12% HgCl2 solution or a 1.6-2.4% NaClO solution for 5-11 minutes.

[0013] Preferably, the Rhodiola rosea seeds are subjected to a germination treatment before being disinfected. During the germination treatment, the Rhodiola rosea seeds are immersed in 350-450 mg / L gibberellin for 15-20 minutes.

[0014] Preferably, the culture medium used in the germination culture in step (1) is MS basic culture medium, the culture temperature is 23-27°C, the humidity is 70-80%, the light intensity is 2000-3000Lx, and the lighting conditions are 11-13h light and 11-13h dark alternating culture time for 5-10d.

[0015] Preferably, the adventitious bud proliferation medium in step (2) further contains 25-35 g / L sucrose and 7-8 g / L agar, and the pH of the adventitious bud proliferation medium is 5.8-6.0;

[0016] The temperature during the adventitious bud proliferation culture is 23-27° C., the humidity is 70-80%, the light intensity is 2000-3000 Lx, and the lighting conditions are 11-13 hours of light and 11-13 hours of darkness, and the culture time is 30-40 days.

[0017] Preferably, the seedling rooting medium in step (3) further contains 0.8-1.2 g / L activated carbon, 25-35 g / L sucrose and 7-8 g / L agar, and the pH of the seedling rooting medium is 5.8-6.0;

[0018] The temperature during the seedling rooting culture is 23-27° C., the humidity is 70-80%, the light intensity is 2000-3000 Lx, and the lighting conditions are 11-13 hours of light and 11-13 hours of darkness, and the culture time is 20-25 days.

[0019] The application provides a seedling hardening and transplanting method of Rhodiola crenulata test-tube seedlings.

[0020] The mixed substrate comprises peat, vermiculite and river sand, and the mass ratio of the peat, vermiculite and river sand is 0.8-1.2:0.8-1.2:0.8-1.2.

[0021] Preferably, the temperature during the indoor hardening is 23-27 DEG C, the humidity is 70-80 %, the light intensity is 2000-3000 Lx, the light condition is 11-13 h light and 11-13 h darkness alternately, and the culture time is 3-5 days.

[0022] Preferably, the test-tube seedlings are taken out after the hardening, and the roots of the test-tube seedlings are subjected to sterilization treatment.

[0023] Preferably, the roots of the test-tube seedlings are soaked in a carbendazim solution during the sterilization, the dilution multiple of the carbendazim solution is 500-1000, and the soaking time is 20-35 min.

[0024] The application provides a tissue culture method of Rhodiola crenulata, wherein the seeds of Rhodiola crenulata are used as explants, the explants are sterilized by using a 2 % NaClO solution for 11 min, and the germination rate can reach 96 %; the germinated adventitious buds are cultured in an adventitious bud proliferation culture medium prepared by adding 1.0 mg / L TDZ and 0.2 mg / L IAA to a MS culture medium as a basic culture medium, and the proliferation coefficient can reach 18.0; the proliferated clustered buds are cultured in a strong seedling rooting culture medium prepared by adding 0.1 mg / L IBA and 1.0 mg / L NAA to a 1 / 2MS culture medium as a basic culture medium, and the rooting rate can reach 98.0 %.

[0025] The application further provides a seedling hardening and transplanting method of Rhodiola crenulata test-tube seedlings, wherein the test-tube seedlings obtained through the tissue culture are first subjected to indoor hardening, and then are transplanted into a mixed substrate prepared by mixing peat, vermiculite and river sand at a mass ratio of 1:1:1, so that the survival rate of the transplanted seedlings can reach 90.5 %.

[0026] The tissue culture and seedling hardening and transplanting method of Rhodiola crenulata provided by the application can provide a new method for large-area and high-efficiency cultivation of Rhodiola crenulata, and has important application value for resource protection and development and utilization of Rhodiola crenulata. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0028] Figure 1 The Rhodiola rosea seeds in Example 1;

[0029] Figure 2 The adventitious buds of Rhodiola rosea obtained in treatment group 8 in step (2) of Example 1;

[0030] Figure 3 The Rhodiola rosea clustered buds obtained in step (3) of treatment group B in Example 1;

[0031] Figure 4 The Rhodiola rosea test tube seedlings obtained in step (4) of treatment group c in Example 1;

[0032] Figure 5 This is a diagram showing the state of Rhodiola rosea being transplanted into the seedling medium in Example 2;

[0033] Figure 6 This is a diagram showing the state of the transplanted Rhodiola rosea seedlings being cultured in a light culture room in Example 2. DETAILED DESCRIPTION

[0034] The present invention provides a tissue culture method of Rhodiola rosea, comprising the following steps:

[0035] (1) sterilizing Rhodiola rosea seeds and then germinating and culturing them to obtain adventitious buds;

[0036] (2) transferring the adventitious buds obtained in step (1) to an adventitious bud proliferation medium for adventitious bud proliferation culture to obtain clustered buds;

[0037] (3) transferring the clustered buds obtained in step (2) to a seedling rooting medium for seedling rooting culture to obtain test tube plantlets;

[0038] Wherein, the adventitious bud proliferation medium in step (2) is based on MS medium and further contains 0.8-1.2 mg / L LTDZ and 0.15-0.25 mg / L IAA;

[0039] The seedling rooting medium in step (3) uses 1 / 2 MS medium as the basic medium and also contains 0.25-0.35 mg / L IBA and 0.08-0.12 mg / L NAA.

[0040] In the present application, the method for disinfecting in step (1) is to immerse the Rhodiola crenulata seeds in 0.08-0.12% HgCl2 solution or 1.6-2.4% NaClO solution for 5-11 min, preferably in 0.1% HgCl2 solution or 2.0% NaClO solution for 5-11 min, further preferably in 2.0% NaClO solution for 5-11 min, and more preferably in 2.0% NaClO solution for 11 min.

[0041] In the present application, the Rhodiola crenulata seeds are also subjected to germination treatment before disinfection. During the germination, the Rhodiola crenulata seeds are immersed in 350-450 mg / L gibberellin for 15-20 min, preferably in 380-420 mg / L gibberellin for 16-19 min, and further preferably in 400 mg / L gibberellin for 18 min.

[0042] In the present application, the medium used in the germination culture in step (1) is MS basic medium, the temperature during the culture is 23-27°C, preferably 24-26°C, further preferably 25°C, the humidity is 70-80%, preferably 75%, the light intensity is 2000-3000 Lx, preferably 2200-2800 Lx, further preferably 2400-2600 Lx, and more preferably 2500 Lx, the light condition is 11-13 h light and 11-13 h darkness alternately, preferably 12 h light and 12 h darkness alternately, and the time is 5-10 d, preferably 6-8 d, and further preferably 7 d.

[0043] In the present application, the adventitious bud proliferation medium in step (2) is based on MS medium, and further contains 0.8-1.2 mg / L TDZ and 0.15-0.25 mg / L IAA, preferably 0.9-1.1 mg / L TDZ and 0.18-0.22 mg / L IAA, and further preferably 1.0 mg / L TDZ and 0.2 mg / L IAA.

[0044] In the present application, the adventitious bud proliferation medium in step (2) further contains 25-35 g / L sucrose and 7-8 g / L agar, preferably 30 g / L sucrose and 7.5 g / L agar, and the pH of the adventitious bud proliferation medium is 5.8-6.0, preferably 5.9.

[0045] The temperature during the adventitious bud proliferation culture is 23-27°C, preferably 24-26°C, and more preferably 25°C; the humidity is 70-80%, preferably 75%; the light intensity is 2000-3000Lx, preferably 2200-2800Lx, more preferably 2400-2600Lx, and more preferably 2500Lx; the lighting conditions are 11-13h light and 11-13h dark alternating culture, preferably 12h light and 12h dark alternating culture, and the time is 30-40d, preferably 32-38d, more preferably 34-36d, and more preferably 35d.

[0046] In the present invention, the seedling rooting medium in step (3) uses 1 / 2 MS medium as the basic medium, and further contains 0.25-0.35 mg / L IBA and 0.08-0.12 mg / L NAA, preferably contains 0.28-0.32 mg / L IBA and 0.09-0.11 mg / L NAA, and more preferably contains 0.30 mg / L IBA and 0.10 mg / L NAA.

[0047] In the present invention, the seedling rooting medium in step (3) further contains 0.8-1.2 g / L activated carbon, 25-35 g / L sucrose and 7-8 g / L agar, preferably 1.0 g / L activated carbon, 30 g / L sucrose and 7.5 g / L agar, and the pH of the seedling rooting medium is 5.8-6.0, preferably 5.9;

[0048] The temperature during the rooting culture of the seedlings is 23-27°C, preferably 24-26°C, and more preferably 25°C; the humidity is 70-80%, preferably 75%; the light intensity is 2000-3000Lx, preferably 2200-2800Lx, more preferably 2400-2600Lx, and more preferably 2500Lx; the lighting conditions are 11-13h light and 11-13h dark alternating culture, preferably 12h light and 12h dark alternating culture, and the time is 20-25d, preferably 20-23d, and more preferably 21d.

[0049] The present invention provides a method for hardening and transplanting test tube seedlings of Rhodiola rosea. When the length of the roots of the cultured test tube seedlings reaches 3 to 4 cm, the seedlings are hardened indoors, the test tube seedlings are taken out after hardening, and transplanted into a mixed medium for cultivation.

[0050] The mixed matrix includes peat, vermiculite and river sand, and the mass ratio of the peat, vermiculite and river sand is 0.8-1.2:0.8-1.2:0.8-1.2.

[0051] In the present application, the temperature during indoor seedling raising is 23-27℃, preferably 24-26℃, further preferably 25℃, the humidity is 70-80%, preferably 75%, the light intensity is 2000-3000Lx, preferably 2200-2800Lx, further preferably 2400-2600Lx, more preferably 2500Lx, the light condition is 11-13h light, 11-13h dark alternation culture, preferably 12h light, 12h dark alternation culture, and the time is 3-5d, preferably 4d.

[0052] In the present application, after the test-tube seedlings after seedling raising are taken out, the root of the test-tube seedlings is also subjected to sterilization treatment.

[0053] In the present application, the test-tube seedling root is soaked in the carbendazim solution during sterilization, the dilution ratio of the carbendazim solution is 500-1000 times, preferably 800 times, and the soaking time is 20-35min, preferably 30min.

[0054] 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.

[0055] Example 1

[0056] A tissue culture method of Rhodiola crenulata, the steps are as follows:

[0057] (1) Seed selection and germination treatment

[0058] The mature Rhodiola crenulata seeds (from Tibet, such as shown in the figure) with full seeds and no disease are selected, soaked in 400mg / L gibberellin (GA3) for 18min to improve the seed germination rate, then taken out and washed with running water for 5min, then soaked in washing water for 10min to remove impurities, washed with running water, and then filtered paper is used to absorb water. Figure 1

[0059] (2) Seed sterilization and germination culture

[0060] ​The seeds after step (1) were washed three times with detergent water, and after rinsing away the foam, they were placed in a mesh bag, rinsed under running water for 30 minutes, and disinfected in a clean bench. During disinfection, the seeds were first blotted dry and immersed in 75% alcohol for 30 seconds and washed twice with sterile water. Afterwards, 0.1% HgCl2 or 2% NaClO solution was used for soaking. A total of 8 disinfection treatment groups were set up. Treatment groups 1 to 4 were soaked in 0.1% HgCl2 solution for 5, 7, 9, and 11 minutes, respectively, and treatment groups 5 to 8 were soaked in 2% NaClO solution for 5, 7, 9, and 11 minutes, respectively. After soaking, the seeds were washed 5 times with sterile water, placed on filter paper to blot dry, and inoculated into MS minimal medium. The seeds were cultured for 7 days at a temperature of 25°C, a humidity of 75%, a light intensity of 2500Lx, and 12h light / 12h dark conditions. After the cultivation was completed, the germination rate, contamination rate and mortality rate of the seeds in the different treatment groups were observed and counted. The statistical results of the germination rate, contamination rate and mortality rate of the Rhodiola rosea seeds after treatment with 0.1% HgCl2 solution are shown in Table 1, and the statistical results of the germination rate, contamination rate and mortality rate of the Rhodiola rosea seeds after treatment with 2% NaClO solution are shown in Table 2.

[0061] Table 10. Effects of soaking in 1% HgCl2 solution for different times on Rhodiola rosea seeds

[0062]

[0063]

[0064] Table 2 Effects of soaking in 2% NaClO solution for different times on Rhodiola rosea seeds

[0065] Treatment group Disinfection time (min) Germination rate (%) Contamination rate (%) mortality rate(%) 5 5 89.3 0.7 5.3 6 7 85.0 0 7.0 7 9 95.0 0.3 3.0 8 11 96.0 0.3 1.0

[0066] As shown in Tables 1 and 2, the seeds of R. rosea obtained by soaking in 2% NaClO solution for 11 minutes had the highest germination rate, reaching 96%, the lowest mortality rate, only 1%, and a relatively low contamination rate of 0.3%. Therefore, the adventitious buds of R. rosea obtained in Treatment Group 8 were selected for subsequent adventitious bud proliferation culture.

[0067] (3) Adventitious bud proliferation culture

[0068] The adventitious buds (such as Figure 2 The cells were transferred to a proliferation medium and cultured for 35 days at a temperature of 25°C, a humidity of 75%, a light intensity of 2500 Lx, and a 12-hour light / 12-hour dark cycle. Nine proliferation medium treatment groups (A to I) were set up, each with the following proliferation medium:

[0069] A: MS + TDZ 0.5 mg / L + IAA 0.1 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0070] B: MS + TDZ 1.0 mg / L + IAA 0.2 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0071] C: MS + TDZ 1.5 mg / L + IAA 0.3 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0072] D: 1 / 2MS + TDZ 0.5 mg / L + IAA 0.1 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0073] E: 1 / 2MS + TDZ 1.0 mg / L + IAA 0.3 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0074] F: 1 / 2MS + TDZ 1.5 mg / L + IAA 0.1 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0075] G: 1 / 4MS + TDZ 0.5 mg / L + IAA 0.3 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0076] H: 1 / 4MS + TDZ 1.0 mg / L + IAA 0.1 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0077] I: 1 / 4MS + TDZ 1.5 mg / L + IAA 0.2 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9.

[0078] The proliferation coefficient of the adventitious buds of each treatment group after culture was observed and counted. The results are shown in Table 3. It can be seen that the proliferation coefficient of the adventitious buds of Rhodiola rosea after culture using the medium of treatment group B (MS + TDZ 1.0 mg / L + IAA 0.2 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9) is the highest, reaching 18.0. Therefore, the clustered buds of Rhodiola rosea obtained in treatment group B were selected for subsequent seedling rooting culture.

[0079] Table 3 Effects of different adventitious bud proliferation media on the proliferation of adventitious buds of Rhodiola rosea

[0080] Treatment group Culture medium type TDZ (mg / L) IAA (mg / L) proliferation coefficient A MS 0.5 0.1 11.5 B MS 1.0 0.2 18.0 C MS 1.5 0.3 13.4 D 1 / 2MS 0.5 0.2 8.1 E 1 / 2MS 1.0 0.3 7.4 F 1 / 2MS 1.5 0.1 8.7 G 1 / 4MS 0.5 0.3 5.9 H 1 / 4MS 1.0 0.1 7.5 I 1 / 4MS 1.5 0.2 6.8

[0081] (4) Rooting and cultivation of seedlings

[0082] The buds (about 2.5 cm in length, such as Figure 3 The seedlings were cut into rooting medium and inoculated into the seedling rooting medium. The seedlings were cultured for 21 days at a temperature of 25°C, a humidity of 75%, a light intensity of 2500 Lx, and a 12-hour light / 12-hour dark period. A total of 9 seedling rooting medium treatment groups (a to i) were set up. The seedling rooting medium was as follows:

[0083] a: 1 / 2MS + IBA 0.1 mg / L + NAA 0.1 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0084] b: 1 / 2MS + IBA 0.1 mg / L + NAA 0.5 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0085] c: 1 / 2MS + IBA 0.1 mg / L + NAA 1.0 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0086] d: 1 / 2MS + IBA 0.3 mg / L + NAA 0.1 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0087] e: 1 / 2MS + IBA 0.3 mg / L + NAA 0.5 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0088] f: 1 / 2MS + IBA 0.3 mg / L + NAA 1.0 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0089] g: 1 / 2MS + IBA 0.5 mg / L + NAA 0.1 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0090] h: 1 / 2MS + IBA 0.5 mg / L + NAA 0.5 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9;

[0091] i: 1 / 2MS + IBA 0.5 mg / L + NAA 1.0 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7.5 g / L, pH = 5.9.

[0092] The rooting rate of the test tube seedlings obtained in each treatment group after the seedling rooting culture was observed and counted. The results are shown in Table 4. It can be seen that the test tube seedlings (such as Figure 4 The rooting rate of the plant was the highest, reaching 98.0%.

[0093] Table 4 Effects of different seedling rooting media on the rooting rate of Rhodiola rosea test tube seedlings

[0094]

[0095]

[0096] In summary, the seeds of Rhodiola rosea soaked in 2% NaClO solution for 11 minutes had the highest germination rate and the lowest mortality rate, with the germination rate reaching 96% and the mortality rate being only 1%; the adventitious buds of Rhodiola rosea cultured in MS medium as the basic culture medium and supplemented with 1.0 mg / LTDZ and 0.2 mg / L IAA had the highest proliferation coefficient of 18.0, which was obtained; the rooting rate of Rhodiola rosea test tube plantlets cultured in 1 / 2MS medium as the basic culture medium and supplemented with 0.1 mg / L IBA and 1.0 mg / L NAA was the highest, which was 98.0%.

[0097] Example 2

[0098] A method for hardening and transplanting test tube seedlings of Rhodiola rosea, comprising the following steps:

[0099] When the length of the roots of the Rhodiola rosea test tube seedlings obtained in step (4) of Example 1 in treatment group c reaches about 3.5 cm, the bottle cap is opened and the seedlings are hardened indoors for 4 days. The culture conditions during hardening are: temperature 25°C, humidity 75%, light intensity 2500Lx, 12h light / 12h dark.

[0100] After the hardening is completed, the test tube seedlings are taken out from the rooting medium, the root medium is washed, and the roots of the test tube seedlings are soaked in 800 times diluted carbendazim solution for 30 minutes, and the water is absorbed with absorbent paper, and then transplanted into the seedling medium and watered thoroughly (such as Figure 5 As shown), placed in a light culture room (as Figure 6The survival rate of the transplanted seedlings is counted after the Rhodiola kirilowii tube seedlings are transplanted to different seedling raising substrates, and the type of the seedling raising substrate and the statistical result of the survival rate of the transplanted seedlings are shown in Table 5.

[0101] As shown in Table 5, the survival rate of the transplanted seedlings is the highest after the mixed substrate of peat soil: vermiculite: river sand = 1:1:1 is used as the seedling raising substrate, which is 90.5%.

[0102] Table 5 Influence of different seedling raising substrates on the survival rate of Rhodiola kirilowii transplanted seedlings

[0103]

[0104]

[0105] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A tissue culture method for Rhodiola rosea, characterized in that: The steps include: (1) sterilizing Rhodiola rosea seeds and then germinating and culturing them to obtain adventitious buds; (2) transferring the adventitious buds obtained in step (1) to an adventitious bud proliferation medium for adventitious bud proliferation culture to obtain clustered buds; (3) transferring the clustered buds obtained in step (2) to a seedling rooting medium for seedling rooting culture to obtain test tube plantlets; Wherein, the adventitious bud proliferation medium in step (2) is based on MS medium and further contains 0.8-1.2 mg / L TDZ and 0.15-0.25 mg / L IAA; The seedling rooting medium in step (3) uses 1 / 2 MS medium as the basic medium and also contains 0.25-0.35 mg / L I BA and 0.08-0.12 mg / L NAA.

2. The method according to claim 1, wherein The disinfection method in step (1) is to soak the Rhodiola rosea seeds in a 0.08-0.12% HgCl2 solution or a 1.6-2.4% NaClO solution for 5-11 minutes.

3. The method according to claim 2, wherein The seeds of Rhodiola rosea were also germinated before being disinfected. During the germination process, the seeds of Rhodiola rosea were immersed in 350-450 mg / L gibberellin for 15-20 minutes.

4. The method according to claim 2, wherein The culture medium used in the germination culture in step (1) is MS basic culture medium, the culture temperature is 23-27°C, the humidity is 70-80%, the light intensity is 2000-3000Lx, and the lighting conditions are 11-13h light and 11-13h dark alternating culture for 5-10d.

5. The method according to claim 1, wherein The adventitious bud proliferation medium in step (2) further contains 25-35 g / L sucrose and 7-8 g / L agar, and the pH of the adventitious bud proliferation medium is 5.8-6.0; The temperature during the adventitious bud proliferation culture is 23-27° C., the humidity is 70-80%, the light intensity is 2000-3000 Lx, and the lighting conditions are 11-13 hours of light and 11-13 hours of darkness, and the culture time is 30-40 days.

6. The method according to claim 1, wherein The seedling rooting medium in step (3) further contains 0.8-1.2 g / L activated carbon, 25-35 g / L sucrose and 7-8 g / L agar, and the pH of the seedling rooting medium is 5.8-6.0; The temperature during the seedling rooting culture is 23-27° C., the humidity is 70-80%, the light intensity is 2000-3000 Lx, and the lighting conditions are 11-13 hours of light and 11-13 hours of darkness, and the culture time is 20-25 days.

7. A method for hardening and transplanting test tube seedlings of Rhodiola rosea, characterized in that: When the root length of the test tube seedlings obtained by culturing according to any one of claims 1 to 6 reaches 3 to 4 cm, the test tube seedlings are hardened indoors, the test tube seedlings are taken out after hardening, and transplanted into a mixed medium for cultivation; The mixed matrix includes peat, vermiculite and river sand, and the mass ratio of the peat, vermiculite and river sand is 0.8-1.2:0.8-1.2:0.8-1.

2.

8. The method according to claim 7, wherein The temperature during indoor seedling hardening is 23-27° C., the humidity is 70-80%, the light intensity is 2000-3000 Lx, and the lighting conditions are 11-13 hours of light and 11-13 hours of darkness, and the cultivation time is 3-5 days.

9. The method according to claim 8, wherein After the test tube seedlings are taken out after hardening, the roots of the test tube seedlings are sterilized.

10. The method according to claim 9, wherein During the sterilization, the roots of the test tube seedlings are immersed in a carbendazim solution, the dilution ratio of the carbendazim solution is 500 to 1000 times, and the immersion time is 20 to 35 minutes.

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