Tissue culture and test-tube seedling acclimatization and transplantation method of rhodiola crenulata

Abstract

The invention provides a rhodiola crenulata tissue culture and test-tube seedling acclimatization and transplantation method, and belongs to the technical field of plant tissue culture. The tissue culture method of the rhodiola crenulata comprises the following steps: (1) sterilizing rhodiola crenulata seeds, and performing germination culture to obtain adventitious buds; (2) transferring the adventitious buds obtained in the step (1) into an adventitious bud proliferation culture medium for adventitious bud proliferation culture to obtain cluster buds; and (3) transferring the cluster buds obtained in the step (2) into a seedling strengthening and rooting culture medium for seedling strengthening and rooting culture to obtain test-tube plantlets. The tissue culture and test-tube seedling acclimatization and transplantation method of rhodiola crenulata can provide a new method for large-area efficient cultivation of rhodiola crenulata, and has important application value for resource protection, development and utilization of 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 of Rhodiola rosea and test tube seedling hardening and transplanting. Background Art

[0002] Rhodiola crenulata (Hook.f.&Thomson) H.Ohba is a perennial herb or subshrub plant of the genus Rhodiola in the family Crassulaceae. It mainly grows in the Himalayas, the northwest Yunnan and the western Sichuan Plateau at an altitude of 3500-5000m in the high mountain gravel beach area. The dried roots and rhizomes of Rhodiola crenulata are used as medicine, which has the effects of invigorating qi and activating blood circulation, unblocking meridians and relieving asthma. It is mainly used clinically to treat qi deficiency and blood stasis, chest pain, hemiplegia caused by stroke, fatigue and asthma. Modern pharmacological studies have shown that Rhodiola crenulata has the effects of anti-hypoxia, anti-fatigue, anti-radiation, anti-virus, anti-tumor, improving the cardiovascular system, and delaying the aging of the body, and has broad development prospects.

[0003] In recent years, with the overexploitation and ecological environment changes of Rhodiola rosea, wild resources are gradually endangered, and its resource protection and rational development and utilization are imperative. However, Rhodiola rosea is an alpine plant that is not resistant to high temperatures and has strict requirements for the growth environment. It is difficult to propagate in other places, and large-scale cultivation has not been successful. Research on tissue culture technology of Rhodiola rosea can accelerate its propagation speed, which has important theoretical significance and application value for resource protection and rational development and utilization. Summary of the invention

[0004] The invention aims to provide a tissue culture and test tube seedling hardening and transplanting method of Rhodiola rosea, so as to provide a new method for the 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 seedlings;

[0010] Wherein, the adventitious bud proliferation medium in step (2) is based on MS medium as the basic 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 / 2MS 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 sterilized. During the germination, 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 of 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 2000-3000Lx, the light conditions are 11-13h light and 11-13h dark alternately, and the culture time is 30-40d.

[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 rooting culture of the seedlings is 23-27°C, the humidity is 70-80%, the light intensity is 2000-3000Lx, the light conditions are 11-13h of light and 11-13h of darkness, and the culture time is 20-25d.

[0019] The 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 to a mixed medium for culture.

[0020] 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.

[0021] Preferably, 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 h of light and 11-13 h of darkness for 3-5 days.

[0022] Preferably, after the test-tube seedlings are taken out after the hardening, the roots of the test-tube seedlings are sterilized.

[0023] Preferably, during the sterilization, the roots of the test tube seedlings are immersed in a carbendazim solution, the dilution multiple of the carbendazim solution is 500 to 1000 times, and the immersion time is 20 to 35 minutes.

[0024] The invention provides a tissue culture method of Rhodiola rosea. The method comprises the following steps: using seeds of Rhodiola rosea as explants, sterilizing with a 2% NaClO solution for 11 minutes, and achieving a germination rate of 96%. The germinated adventitious buds are placed in an adventitious bud proliferation medium using an MS medium as a basic medium and supplemented with 1.0 mg / LTDZ and 0.2 mg / LIAA for culture, and achieving a proliferation coefficient of 18.0. The clustered buds obtained by the proliferation culture are placed in a seedling rooting medium using a 1 / 2MS medium as a basic medium and supplemented with 0.1 mg / LIBA and 1.0 mg / LNAA for culture, and achieving a rooting rate of 98.0%.

[0025] The invention also provides a method for hardening and transplanting test tube seedlings of Rhodiola rosea. The test tube seedlings obtained by tissue culture are first hardened indoors and then transplanted to a mixed matrix of peat soil: vermiculite: river sand = 1:1:1 for culture, so that the survival rate of the transplanted seedlings can reach 90.5%.

[0026] The tissue culture and test tube seedling hardening and transplanting method of Rhodiola rosea provided by the invention can provide a new method for large-scale and efficient cultivation of Rhodiola rosea, and has important application value for resource protection and development and utilization of Rhodiola rosea. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying 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 state diagram of the large-flowered Rhodiola rosea transplanted into the seedling medium in Example 2;

[0033] Figure 6 This is a state diagram of the large-flowered Rhodiola rosea transplanted seedlings being cultivated in the 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 seedlings;

[0038] Wherein, the adventitious bud proliferation medium in step (2) is based on MS medium as the basic 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 / 2MS 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 invention, the disinfection method described 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, preferably to soak the Rhodiola rosea seeds in a 0.1% HgCl2 solution or a 2.0% NaClO solution for 5-11 minutes, further preferably to soak the Rhodiola rosea seeds in a 2.0% NaClO solution for 5-11 minutes, and more preferably to soak the Rhodiola rosea seeds in a 2.0% NaClO solution for 11 minutes.

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

[0042] In the present invention, the culture medium used in the germination culture in step (1) is MS basic culture medium, the culture temperature 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, and the lighting conditions are 11-13h light, 11-13h dark alternating culture, preferably 12h light, 12h dark alternating culture, and the time is 5-10d, preferably 6-8d, and more preferably 7d.

[0043] In the present invention, the adventitious bud proliferation medium in step (2) uses MS medium as the basic medium, and also contains 0.8-1.2 mg / L TDZ and 0.15-0.25 mg / L IAA, preferably contains 0.9-1.1 mg / L TDZ and 0.18-0.22 mg / L IAA, and more preferably contains 1.0 mg / L TDZ and 0.2 mg / L IAA.

[0044] In the present invention, 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 of light and 11-13h of dark alternating culture, preferably 12h of light and 12h of 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 / 2MS medium as the basic medium, and also 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, and the lighting conditions are 11-13h of light and 11-13h of dark alternating culture, preferably 12h of light and 12h of dark alternating culture, and the time is 20-25d, preferably 20-23d, and more preferably 21d.

[0049] The 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 to a mixed medium for culture.

[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 invention, the temperature during indoor hardening 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, and the lighting conditions are 11-13h light, 11-13h dark alternating culture, preferably 12h light, 12h dark alternating culture, and the time is 3-5d, preferably 4d.

[0052] In the present invention, after the test-tube seedlings are taken out after the hardening, the roots of the test-tube seedlings are sterilized.

[0053] In the present invention, during the sterilization, the roots of the test tube seedlings are immersed in a carbendazim solution, the dilution multiple of the carbendazim solution is 500 to 1000 times, preferably 800 times, and the immersion time is 20 to 35 minutes, preferably 30 minutes.

[0054] 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.

[0055] Example 1

[0056] A tissue culture method of Rhodiola rosea, comprising the following steps:

[0057] (1) Seed screening and germination treatment

[0058] Select mature Rhodiola rosea seeds with full seeds and no diseases (from Tibet, such as Figure 1 As shown), soak the seeds in 400 mg / L gibberellin (GA3) for 18 minutes to improve the germination rate of the seeds, then take them out and rinse them with running water for 5 minutes, soak them in washing water for 10 minutes to remove impurities, rinse them with running water and then absorb the moisture with filter paper.

[0059] (2) Seed disinfection and germination culture

[0060] The seeds treated in step (1) were washed three times with detergent water, and after the foam was rinsed off, 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 dried and immersed in 75% alcohol for 30 seconds and washed twice with sterile water, and then immersed in 0.1% HgCl2 or 2% NaClO solution. A total of 8 disinfection treatment groups were set up, and treatment groups 1 to 4 were immersed in 0.1% HgCl2 solution for 5, 7, 9 and 11 minutes, respectively, and treatment groups 5 to 8 were immersed in 2% NaClO solution for 5, 7, 9 and 11 minutes, respectively. After the soaking, the seeds were washed 5 times with sterile water, placed on filter paper to dry the water, and inoculated into MS basic culture medium, and 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. The germination rate, contamination rate and mortality rate of seeds in different treatment groups were observed and counted after the cultivation. The statistical results of the germination rate, contamination rate and mortality rate of seeds of Rhodiola rosea treated with 0.1% HgCl2 solution are shown in Table 1, and the statistical results of the germination rate, contamination rate and mortality rate of seeds of Rhodiola rosea treated with 2% NaClO solution are shown in Table 2.

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

[0062]

[0063]

[0064] Table 2 Effects of soaking in 2% NaClO solution for different time 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 can be seen from Tables 1 and 2, the germination rate of Rhodiola rosea seeds obtained by soaking in 2% NaClO solution for 11 minutes was the highest, reaching 96%, the mortality rate was the lowest, only 1%, and the contamination rate was also low, at 0.3%. Therefore, the adventitious buds of Rhodiola 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 12 h light / 12 h dark conditions. A total of 9 proliferation medium treatment groups (A to I) were set up, and the proliferation mediums were:

[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 0.5mg / L + IAA 0.1mg / L + sucrose 30g / L + agar 7.5g / L, pH = 5.9;

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

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

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

[0076] H: 1 / 4MS + TDZ 1.0mg / L + IAA 0.1mg / L + sucrose 30g / L + agar 7.5g / 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 in 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 medium on adventitious bud proliferation 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 clustered buds (about 2.5 cm in length) obtained from the treatment group B in step (3) were Figure 3 The seedlings were cut off and inoculated into the seedling rooting medium, and cultured for 21 days at a temperature of 25°C, a humidity of 75%, a light intensity of 2500Lx, and 12h light / 12h dark conditions. A total of 9 seedling rooting medium treatment groups (a-i) were set up, and the seedling rooting mediums were:

[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 0.1mg / L + NAA 1.0mg / L + activated carbon 1.0g / L + sucrose 30g / L + agar 7.5g / 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 0.3mg / L + NAA 1.0mg / L + activated carbon 1.0g / L + sucrose 30g / L + agar 7.5g / 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 (shown) was the highest, reaching 98.0%.

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

[0094]

[0095]

[0096] In summary, the germination rate of Rhodiola rosea seeds obtained by soaking in 2% NaClO solution for 11 minutes was the highest and the mortality rate was the lowest, the germination rate could reach 96%, and the mortality rate was only 1%; the proliferation coefficient of Rhodiola rosea adventitious buds obtained by culturing with MS medium as the basic medium and adventitious bud proliferation medium supplemented with 1.0 mg / LTDZ and 0.2 mg / L IAA was the highest, which was 18.0; the rooting rate of Rhodiola rosea test tube seedlings obtained by culturing with 1 / 2MS medium as the basic medium and seedling rooting medium supplemented with 0.1 mg / L IBA and 1.0 mg / LNAA 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 root length of the Rhodiola rosea test tube seedlings obtained in step (4) of Example 1 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 of 25° C., humidity of 75%, light intensity of 2500 Lx, 12 h light / 12 h dark.

[0100] After hardening, remove the test-tube seedlings from the rooting medium, wash the root medium, soak the roots of the test-tube seedlings in 800-fold diluted carbendazim solution for 30 minutes, dry them with absorbent paper, transplant them into the seedling medium, and water them thoroughly (such as Figure 5 ), placed in a light culture chamber (as shown Figure 6The culture medium was 25 °C, 75% humidity, 2500 Lx light intensity, and 12 h light / 12 h dark conditions for 30 days. A total of 10 seedling medium treatment groups were set up, and the survival rate of transplanted seedlings after transplanting Rhodiola rosea test tube seedlings to different seedling mediums was statistically analyzed. The statistical results of the types of seedling medium used and the survival rate of transplanted seedlings are shown in Table 5.

[0101] It can be seen from Table 5 that the survival rate of transplanted seedlings after cultivation using a mixed matrix of peat soil: vermiculite: river sand = 1:1:1 as the seedling medium is the highest, at 90.5%.

[0102] Table 5 Effects of different seedling medium on the survival rate of transplanted seedlings of Rhodiola rosea

[0103]

[0104]

[0105] 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 tissue culture method of 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 seedlings; Wherein, the adventitious bud proliferation medium in step (2) is based on MS medium as the basic medium, and further contains 0.8-1.2 mg / LTDZ and 0.15-0.25 mg / LIAA; The seedling rooting medium in step (3) uses 1 / 2MS medium as the basic medium and also contains 0.25-0.35 mg / L IBA and 0.08-0.12 mg / L NAA.

2. The method according to claim 1, characterized in that The disinfection method described 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, characterized in that The seeds of Rhodiola rosea were also germinated before disinfection. During the germination, the seeds of Rhodiola rosea were immersed in 350-450 mg / L gibberellin for 15-20 minutes.

4. The method according to claim 2, characterized in that 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 2000-3000 Lx, and the lighting conditions are 11-13 h of light and 11-13 h of darkness for 5-10 days.

5. The method according to claim 1, characterized in that 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 2000-3000Lx, the light conditions are 11-13h light and 11-13h dark alternately, and the culture time is 30-40d.

6. The method according to claim 1, characterized in that 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 rooting culture of the seedlings is 23-27°C, the humidity is 70-80%, the light intensity is 2000-3000Lx, the light conditions are 11-13h of light and 11-13h of darkness, and the culture time is 20-25d.

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, and the test-tube seedlings after hardening are taken out 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, characterized in that The temperature during indoor seedling hardening is 23-27°C, the humidity is 70-80%, the light intensity is 2000-3000Lx, and the light conditions are 11-13h light and 11-13h dark alternating cultivation for 3-5d.

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

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

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