CN112616674B - Induced proliferation culture medium for tissue culture and rapid propagation of rhodiola rosea and tissue culture and rapid propagation method - Google Patents

Abstract

The invention provides an induced proliferation culture medium for tissue culture and rapid propagation of rhodiola rosea and a tissue culture and rapid propagation method, belonging to the technical field of plant tissue culture seedling cultivation. The invention provides an induced proliferation culture medium for tissue culture and rapid propagation of rhodiola rosea, which takes an MS culture medium as a basic culture medium and also comprises the following components in mass volume concentration on the basis of the MS culture medium: 1.0-2.0 mg/L of 6-BA, 0.1-0.2 mg/L of NAA, 30-35 g/L of sucrose and 6-7 g/L of agar, wherein the pH value of the induced proliferation culture medium is 5.8-6.0. The induced proliferation culture medium provided by the invention can simultaneously realize the induction and proliferation of adventitious buds, simplifies the steps of tissue culture, shortens the seedling period of test-tube seedlings, saves the cost, avoids the pollution to tissue culture seedlings caused by frequent switching, and improves the survival rate of the tissue culture seedlings.

Description

A kind of Rhodiola rosea tissue culture fast propagation induced proliferation medium and tissue culture fast propagation method

technical field

The invention belongs to the technical field of plant tissue culture seedling cultivation, and in particular relates to an induced proliferation medium for Rhodiola rosea tissue culture rapid propagation and a method for tissue culture rapid propagation.

Background technique

Rhodiola rosea (Rhodiola rosea L.) is a perennial herb of the genus Rhodiola, also known as Rhodiola rosea. It is mostly wild in woodlands, hillsides, dry stone crevices, steep rocks, etc. Distributed in Shaanxi, Gansu, Xinjiang, Hebei and other places. In the "Compendium of Materia Medica", Li Shizhen in the Ming Dynasty recorded that Rhodiola rosea is "the top grade in the scriptures, and it is called "Xiancicao". It has a rose fragrance and is more intense when it is fresh. , Commonly used rhizomes, the nature is "sweet, bitter, flat, and returns to the lung and heart meridians". The main active ingredients of Rhodiola rosea include salidroside and its aglycone tyrosol, rosavir and so on. A large number of studies have shown that it has various physiological functions such as anti-fatigue, hypoxia resistance, enhancing learning and memory, stimulating the central nervous system, improving sleep, preventing altitude sickness, and anti-cancer. original" plant.

The growth environment of Rhodiola is extremely harsh and changeable, such as hypoxia, low temperature drying, strong wind, strong ultraviolet radiation, large temperature difference between day and night, etc., which makes it possess special adaptive substances that are rare in other plants, and also leads to its extremely high natural resources. scarcity. The natural germination rate of Rhodiola rosea seeds is low, and when it is introduced from high mountains to low altitude areas for domestication and cultivation, the occurrence of diseases and insect pests is very serious. Therefore, it is extremely urgent to strengthen the tissue culture and rapid propagation of Rhodiola, and to protect its natural resources and genetic diversity.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides an inducing proliferation medium for Rhodiola rosea tissue culture rapid propagation and a method for tissue culture rapid propagation. The induction and proliferation medium for Rhodiola rosea tissue culture and fast propagation provided by the invention can simultaneously realize the induction and proliferation of adventitious buds, the method is simple to operate, and a large number of Rhodiola rosea test-tube seedlings can be obtained in a relatively short time, and the realization of Rhodiola rosea rapid reproduction.

In order to achieve the above object, the present invention provides the following technical solutions:

The present invention provides an induced proliferation medium for Rhodiola rosea tissue culture and fast propagation. The induced proliferation medium is based on MS medium, and on the basis of MS medium, it also includes the following groups of mass and volume concentrations. Divided into: 1.0～2.0mg/L 6-BA, 0.1～0.2mg/L NAA, 30～35g/L sucrose and 6～7g/L agar, the pH value of the proliferation induction medium is 5.8～6.0.

The invention provides a method for tissue culture and rapid propagation of Rhodiola rosea, comprising the following steps:

1) the seeds are placed in the induced proliferation medium described in the above-mentioned technical scheme to carry out induced proliferation culture to obtain Rhodiola rosea cluster buds;

2) Rhodiola rosea cluster buds are inoculated into rooting medium and carry out rooting culture to obtain Rhodiola rosea test-tube seedlings; Described rooting medium is basal medium with 1/2MS medium, in the 1/2MS medium. On the basis, it also includes components with the following mass and volume concentrations: 0.4-0.6 mg/LNAA, 0.5-1.0 g/L activated carbon, 30-35 g/L sucrose and 6-7 g/L agar, and the pH value of the rooting medium is 5.8～6.0;

3) The obtained Rhodiola rosea test-tube seedlings are hardened and transplanted.

Preferably, in step 1), before the seeds are induced to proliferate and culture, the seeds are also subjected to a germination promotion treatment, and the germination promotion treatment includes: soaking in an aqueous solution of gibberellin, and the concentration of gibberellin in the aqueous solution of gibberellin is 350% ～450mg/L, the soaking time is 5～10min.

Preferably, in step 1), before the seeds are induced to proliferate and cultivate, the seeds further include disinfection treatment. The disinfection treatment method includes: immersing the seeds in 75% alcohol for 25-30s, washing them with sterile water, and then transferring them to 0.1% alcohol. Soak in HgCl ₂ for 10-15min to obtain the seeds after disinfection.

Preferably, the conditions for inducing proliferation culture in step 1) include: firstly carrying out dark culture to obtain callus; after obtaining callus, carrying out light culture to obtain Rhodiola rosea cluster buds.

Preferably, the conditions of the dark culture are: temperature 23-25°C, humidity 75%-85%, dark culture time 20-23d; the light culture conditions are: temperature 23-25°C, humidity 75%-85% %, light intensity 1500～2000Lx, light time 12～14h/d, culture time 30～40d.

Preferably, in step 2), the length of the clump buds used for inoculation is 1.5-2.0 cm.

Preferably, the conditions for rooting culture in step 2) are: temperature 23-25°C, humidity 75%-85%, light intensity 1500-2000Lx, light time 12-14h/d, and culture time 20-30d.

Preferably, when the length of the adventitious roots of the obtained Rhodiola rosea test-tube seedlings reaches 2-3 cm, the seedlings are hardened, and the time for the hardening is 2-4 days.

Preferably, the transplanting substrate comprises peat, vermiculite and coconut bran, and in the substrate, the mass ratio of peat, vermiculite and coconut bran is (2-2.5):(1-1.5):1.

beneficial effect

The present invention provides an induced proliferation medium for Rhodiola rosea tissue culture and fast propagation. The induced proliferation medium is based on MS medium, and on the basis of MS medium, it also includes the following groups of mass and volume concentrations. Divided into: 1.0～2.0mg/L 6-BA, 0.1～0.2mg/L NAA, 30～35g/L sucrose and 6～7g/L agar, the pH value of the proliferation induction medium is 5.8～6.0. The induced proliferation medium provided by the invention can realize the induction and proliferation of adventitious buds at the same time, simplifies the steps of tissue culture, shortens the seedling growth cycle of test-tube seedlings, saves costs, and also avoids the frequent transfer of tissue culture seedlings. pollution, and improve the survival rate of tissue culture seedlings.

Further, the present invention also provides a method for tissue culture and rapid propagation of Rhodiola rosea. First, the seeds are placed in an induced proliferation medium for induced proliferation culture to obtain Rhodiola rosea cluster buds. The present invention induces proliferation culture. The substrate can simultaneously conduct adventitious bud induction and proliferation culture, which simplifies the steps of tissue culture, shortens the cycle of test-tube seedlings, saves costs, and avoids the pollution of tissue culture seedlings caused by frequent transfer. In the invention, the clump buds are inoculated into the rooting medium for rooting culture to obtain Rhodiola rosea test-tube seedlings, and the rooting medium of the invention has a good effect of inducing rooting; finally, the obtained Rhodiola rosea test-tube seedlings are hardened, transplant. The tissue culture and rapid propagation method of Rhodiola rosea provided by the invention has simple operation, can obtain a large number of Rhodiola rosea test-tube seedlings in a relatively short time, realizes the rapid propagation of Rhodiola rosea, and has great promotion value.

Description of drawings

Fig. 1 is the situation that embodiment 1 Rhodiola rosea induces proliferation and cultivates 1d;

Fig. 2 is the situation that embodiment 1 Rhodiola rosea induces proliferation and cultivates for 25d;

Fig. 3 is the situation that embodiment 1 Rhodiola rosea induces proliferation and cultivates 40d;

Fig. 4 is the situation that embodiment 1 Rhodiola rosea is rooted and cultivated for 20d;

Figure 5 shows the situation of Rhodiola rosea transplanted for 50 d in Example 1.

Detailed ways

The present invention provides an induced proliferation medium for Rhodiola rosea tissue culture and fast propagation. The induced proliferation medium is based on MS medium, and on the basis of MS medium, it also includes the following groups of mass and volume concentrations. Divided into: 1.0～2.0mg/L 6-BA, 0.1～0.2mg/L NAA, 30～35g/L sucrose and 6～7g/L agar, the pH value of the induced proliferation medium is 5.8～6.0; more preferably The following components were included in mass volume concentrations: 2.0 mg/L 6-BA, 0.2 mg/LNAA, 30 g/L sucrose, and 6.5 g/L agar. In the present invention, the pH of the growth-inducing medium is 5.8 to 6.0, more preferably 5.9. The present invention has no special requirements on the source of each component of the culture medium, and it suffices to use common commercially available products in the field. In the induced proliferation medium of the present invention, 6-BA can promote adventitious bud differentiation and cell division, NAA can promote cell division, and sucrose is a carbon source. Through the combined use of 6-BA and NAA, the induced proliferation medium of the present invention can realize the induction and proliferation of adventitious buds at the same time, simplify the steps of tissue culture, shorten the period of in vitro seedling formation, save costs, and avoid The pollution of tissue culture seedlings caused by frequent transfer improves the survival rate of tissue culture seedlings.

The invention provides a method for tissue culture and rapid propagation of Rhodiola rosea, comprising the following steps:

1) the seeds are placed in the induced proliferation medium described in the above-mentioned technical scheme to carry out induced proliferation culture to obtain Rhodiola rosea cluster buds;

2) Rhodiola rosea cluster buds are inoculated into rooting medium and carry out rooting culture to obtain Rhodiola rosea test-tube seedlings; Described rooting medium is basal medium with 1/2MS medium, in the 1/2MS medium. On the basis, it also includes components with the following mass and volume concentrations: 0.4-0.6 mg/LNAA, 0.5-1.0 g/L activated carbon, 30-35 g/L sucrose and 6-7 g/L agar, and the pH value of the rooting medium is 5.8～6.0;

3) The obtained Rhodiola rosea test-tube seedlings are hardened and transplanted.

In the present invention, the seeds are preferably subjected to a germination-promoting treatment before being subjected to inducing proliferation culture. In the present invention, the seeds are preferably selected from mature Rhodiola rosea seeds with full seeds and no disease to improve the germination rate. In the present invention, the germination promotion treatment preferably includes: soaking with a gibberellin aqueous solution, the concentration of gibberellin in the gibberellin aqueous solution is preferably 350-450 mg/L, more preferably 400 mg/L; the soaking The time is preferably 5 to 10 minutes, more preferably 10 minutes. In the present invention, soaking with the above-mentioned specific concentration of gibberellin can promote the germination of seeds and improve the germination rate of seeds. In the present invention, it is preferable to rinse with running water after the treatment with the aqueous solution of gibberellin, and then soak with detergent after rinsing. In the present invention, the time for washing with running water is preferably 5 to 10 minutes, more preferably 5 minutes; the time for soaking the seeds in the detergent is preferably 5 to 10 minutes, more preferably 10 minutes, during which time is continuously stirred to remove impurities, and the cleaning process is carried out with running water. After rinsing, the filter paper absorbs water.

In the present invention, it is preferable to sterilize the seeds after the germination-promoting treatment. In the present invention, before the disinfection treatment is carried out, the germination-promoting seeds are preferably washed three times with washing powder water, and after rinsing the foam, put them into a mesh bag, soak and rinse under running water for 2 hours to remove impurities and take away to inhibit the germination of the seeds. substance. In the present invention, the method of disinfection treatment includes: immersing the seeds in 75% alcohol for 25-30s, washing them with sterile water, and then transferring them to 0.1% HgCl ₂ for immersing for 10-15min; Immerse in 75% alcohol for 30s, wash with sterile water and then transfer to 0.1% _HgCl2 for 15min. The disinfection treatment can kill germs on the seeds, prevent contamination in the process of tissue culture, and reduce the survival rate of tissue culture.

After the sterilization treatment, the present invention places the seeds in the induced proliferation medium described in the above technical scheme to conduct induced proliferation culture to obtain Rhodiola rosea cluster buds. In the present invention, the conditions for inducing proliferation culture preferably include: firstly carrying out dark culture to obtain callus; after obtaining callus, carrying out light culture to obtain Rhodiola rosea cluster buds. In the present invention, the conditions of the dark culture are preferably temperature 23-25°C, humidity 75%-85%, dark culture time 20-23d; more preferably 24°C, humidity 80%, dark culture time 20d, the present invention The dark culture facilitates callus induction. In the present invention, the conditions of the illumination culture are preferably a temperature of 23 to 25° C., a humidity of 75% to 85%, an illumination intensity of 1500 to 2000 Lx, an illumination time of 12 to 14 h/d, and a cultivation time of 30 to 40 d; more preferably the temperature 24° C., humidity 80%, light intensity 1800Lx, light time 12h/d, culture time 35d, the light culture of the present invention can promote callus to differentiate into adventitious buds. After light cultivation, Rhodiola rosea clumps were obtained.

After obtaining Rhodiola rosea cluster buds, the present invention inoculates Rhodiola rosea cluster buds into a rooting medium for rooting culture to obtain Rhodiola rosea test-tube seedlings. In the present invention, Rhodiola rosea cluster buds are preferably excised for inoculation. In the present invention, the length of the clump buds used for inoculation is preferably 1.5-2.0 cm, more preferably 2 cm. The invention selects clump buds with similar lengths for rooting culture, and the tissue culture seedlings are more uniform and have better commerciality. In the present invention, the rooting medium is based on 1/2MS medium, and on the basis of 1/2MS medium, it also includes the following components with the following mass and volume concentrations: 0.4-0.6 mg/L NAA, 0.5- 1.0g/L activated carbon, 30～35g/L sucrose and 6～7g/L agar, further preferably include the following components in mass volume concentration: 0.5mg/LNAA, 1.0g/L activated carbon, 30g/L sucrose and 6.5g/L L agar. In the present invention, the pH value of the rooting medium is 5.8 to 6.0, more preferably 5.9. The NAA in the rooting medium of the present invention has the effect of promoting cell division and inducing adventitious roots; the activated carbon can adsorb the autotoxic substances released by the tissue culture seedlings, making the tissue culture seedlings more robust; sucrose can provide a suitable carbon source for the tissue culture seedlings , the rooting medium provided by the invention has good effects of strengthening seedlings and promoting rooting. In the present invention, the conditions for the rooting culture are preferably a temperature of 23-25° C., a humidity of 75%-85%, a light intensity of 1500-2000Lx, a light-time of 12-14h/d, and a culture time of 20-30d; more preferably the temperature 24°C, humidity 80%, light intensity 1800Lx, light time 12h/d, culture time 21d, the conditions for rooting culture of the present invention provide a suitable environment for Rhodiola rosea roots to promote rapid rooting.

After rooting and cultivating, the present invention carries out hardening and transplanting of the obtained Rhodiola rosea test-tube seedlings. In the present invention, when the length of the adventitious roots of the obtained Rhodiola rosea test-tube seedlings reaches 2 to 3 cm, the tissue culture bottle cap is opened, and a small amount of sterile water is poured into the seedlings, and the seedlings are hardened for 2 to 4 days. More preferably, it is 3d. In the present invention, the conditions for cultivating seedlings are preferably a temperature of 23 to 25° C., a humidity of 75% to 85%, a light intensity of 1500 to 2000 Lx, a light time of 12 to 14 h/d, and a cultivation time of 2 to 4 days; more preferably The temperature is 24°C, the humidity is 80%, the light intensity is 1800Lx, the light time is 12h/d, and the cultivation time is 3d. The seedling hardening of the invention can improve the transplanting survival rate of Rhodiola rosea. In the present invention, the transplanting substrate preferably comprises peat, vermiculite and coconut bran, and in the substrate, the mass ratio of peat, vermiculite and coconut bran is preferably (2-2.5):(1-1.5):1, The transplanting substrate can improve the survival rate of the transplanted tissue culture seedlings. In the present invention, the transplanted tissue culture seedlings are preferably cultured for 1 to 2 weeks in a greenhouse with a temperature of 21 to 25° C., a light intensity of 1500 to 2000 lx, and a relative air humidity of 70% to 85%. Outdoor field transplanting can further improve the survival rate of the transplanted tissue culture seedlings; the transplanting time is preferably between 5:00 pm and 6:00 pm, and watering is required.

In order to further illustrate the present invention, a kind of Rhodiola rosea tissue culture fast propagation induction medium provided by the invention and the method for tissue culture fast propagation are described in detail below in conjunction with the examples, but they cannot be understood as protection of the present invention Scope limitation.

Example 1

A method for tissue culture and rapid propagation of Rhodiola rosea

(1) Seed selection and germination promotion treatment: Screen mature Rhodiola rosea seeds with plump seeds and no disease and soak them in 400mg/L gibberellin (GA ₃ ) for 20 minutes to increase the germination rate of the seeds, then remove them and rinse with running water for 5 minutes , and then soak the seeds in detergent water for 10 minutes, stirring continuously during the period to remove impurities, rinse with running water, and then filter paper to absorb water.

(2) Seed disinfection and germination culture: The seeds after the above treatment were washed three times with washing powder water, rinsed with clean foam, put into a mesh bag, soaked and rinsed under running water for 2 hours, and disinfected in a clean workbench. Immerse the water-absorbing seeds in 75% alcohol for 30s, wash them with sterile water for 5 times, then transfer them to 0.1% HgCl ₂ for 15 minutes, wash them with sterile water for 4 times, and place them on filter paper to absorb the water. use.

(3) Induction and proliferation of adventitious buds: the above-mentioned sterilized seeds are sown into the induced proliferation medium, wherein the induced proliferation medium is based on MS medium, and the following mass and volume concentrations are added on the basis of MS medium Components: 2.0mg/L 6-BA, 0.2mg/LNAA, 30g/L sucrose and 6.5g/L agar, pH 6.0; culture conditions: dark culture for 20 days, temperature 24±1℃, humidity 80±5 %, post-light culture, the light time is 12h/d, the light intensity is 1500-2000Lx, and the culture is carried out for 35 days to obtain Rhodiola rosea cluster buds. Fig. 1 is the situation of Rhodiola rosea induced proliferation culture for 1d, Fig. 2 is the situation of Rhodiola rosea induced proliferation culture for 25d (dark culture for 20d, light culture for 5d), Fig. 3 is Rhodiola rosea induced proliferation culture for 40d (dark culture) 20 days of culture and 20 days of light culture).

(4) Cultivation of strong seedlings and rooting: the adventitious buds of 1.5-2 cm of Rhodiola rosea are cut off and inoculated into the rooting medium, wherein the rooting medium takes 1/2MS medium as the basic medium, and is cultivated in 1/2MS On the basis of the base, the following components were added with the following mass and volume concentrations: 0.5mg/L NAA, 1.0g/L activated carbon, 30g/L sucrose and 6.5g/L agar, pH value 6.0; culture conditions: temperature 24±1℃, Humidity 80±5%, light 12h/d, light intensity 1500-2000Lx, cultured for 20d, to obtain complete test-tube seedlings of Rhodiola rosea, the results are shown in Figure 4, from the photos in Figure 4, it can be seen that the rooting of test-tube seedlings of Rhodiola rosea In good condition.

5) Seedling hardening and transplanting: when the adventitious root length of Rhodiola rosea tissue culture seedlings reaches 2 to 3 cm, open the bottle cap, pour a small amount of sterile water, and after indoor hardening for 3 days, remove the tissue culture seedlings from the rooting medium. Separation, wash the culture medium with running water, soak the roots of the test tube seedlings with 1000 times diluted carbendazim solution for 20min, absorb the water with absorbent paper, and transplant to the mass ratio of peat, vermiculite and coconut chaff of 2:1:1 In the transplanting medium, pour enough water to fix the roots. The transplanted test-tube seedlings were first cultivated in a greenhouse with a temperature of 24°C, a light intensity of 1500-2000lx, and a humidity of 80±5% for 1 week, and then were transplanted in the outdoor field. The transplanting time was selected from 5:00 to 6 pm. :00, and watered thoroughly. Figure 5 shows the situation 50d after transplanting.

Example 2

A method for tissue culture and rapid propagation of Rhodiola rosea is the same as in Example 1, except that the induced proliferation medium is a component with the following mass and volume concentrations added on the basis of the MS medium: 1.0 mg/L 6 -BA, 0.1 mg/L NAA, 30 g/L sucrose and 6.5 g/L agar, pH 6.0.

Example 3

A method for tissue culture and rapid propagation of Rhodiola rosea is the same as Example 1, except that the proliferation medium is a component with the following mass volume concentrations added on the basis of the MS medium: 1.5 mg/L 6- BA, 0.15 mg/L NAA, 30 g/L sucrose and 6.5 g/L agar, pH 6.0.

Comparative Example 1

A method for tissue culture and rapid propagation of Rhodiola rosea is the same as in Example 1, except that the induced proliferation medium is a component with the following mass and volume concentrations added on the basis of the MS medium: 1.0 mg/L 6 -BA, 0.2 mg/L NAA, 0.1 mg/L KT, 30 g/L sucrose and 6.5 g/L agar, pH 6.0.

Comparative Example 2

A method for tissue culture and rapid propagation of Rhodiola rosea is the same as Example 1, except that the rooting medium is a component with the following mass volume concentration added on the basis of 1/2MS medium: 0.5mg/LIAA , 1.0g/L activated carbon, 30g/L sucrose and 6.5g/L agar, pH 6.0.

Comparative Example 3

A method for tissue culture and rapid propagation of Rhodiola rosea is the same as Example 1, except that the rooting medium is a component with the following mass volume concentrations added on the basis of 1/2MS medium: 0.5mg/LNAA , 30g/L sucrose and 6.5g/L agar, pH 6.0.

Comparative Example 4

A method for tissue culture and rapid propagation of Rhodiola rosea is the same as Example 1, except that the mass ratio of peat and coconut bran of the transplanting substrate is 1:1.

Comparative Example 5

A method for tissue culture and rapid propagation of Rhodiola rosea is the same as Example 1, except that the mass ratio of peat and vermiculite in the transplanting substrate is 1:1.

Evaluation

1. Investigation on the induction effect of adventitious buds

Adventitious buds were induced by the methods of Example 1, Example 2 and Comparative Example 1, and the induction effects of different media on callus and adventitious buds were investigated. The test results are shown in Table 1.

Table 1 Effects of different media on callus and adventitious bud induction of Rhodiola rosea

From the results in Table 1, it can be seen that the induction rate of adventitious buds and the callus of the methods of Examples 1 to 3 of the present invention can reach 84% to 95%, and the induction rate of callus can reach 84% to 96%. The callus induction rate and adventitious bud induction rate were the highest; in Comparative Example 1, kinetin KT was added on the basis of Example 1, which had the effect of promoting cell differentiation and division and inducing callus, but Comparative Example 1 did not further improve callus. The induction rate of wound tissue and adventitious buds inhibited the induction effect of Example 1, indicating that the types and concentrations of hormones suitable for inducing callus and adventitious buds are different for different plant varieties, and not all callus and adventitious buds are promoted. The hormones induced by adventitious buds are all suitable, but the selection of hormones is not appropriate, which can also inhibit the induction of callus and adventitious buds.

2. Investigation on the rooting effect of strong seedlings

The methods of Example 1, Comparative Example 2 and Comparative Example 3 were used to induce Rhodiola rosea cluster shoots and rooting, and the effects of different rooting media on the rooting effect of strong seedlings were investigated. The test results are shown in Table 2.

Table 2 Effects of different rooting media on the growth and rooting of Rhodiola rosea

From the results in Table 2, it can be seen that in the rooting medium for strong seedlings, adding 0.5mg/L NAA has better rooting effect than adding 0.5mg/L IAA, and the plants are more robust, and the rooting effect of adding activated carbon is better than that without adding activated carbon.

3. Investigation on the effect of transplanting tissue culture seedlings

The methods of Example 1, Comparative Example 4 and Comparative Example 5 were used to cultivate Rhodiola rosea, and the effects of different transplanting media on the transplanting of Rhodiola rosea tissue culture seedlings were investigated. The test results are shown in Table 3.

Table 3 Effects of different transplanting media on the transplanting survival rate and growth of Rhodiola rosea

As can be seen from the results in Table 3, in the comparative example 4, the mixed water retention of peat and coconut bran is good, and the air permeability is poor, which causes the transplanted tissue culture seedlings to grow poorly. Example 1 adds vermiculite to increase air permeability, and the plant transplant survival rate is high, The growth is good; the transplanting effect of the mixed substrate in the comparative example 5 is not as good as that of the embodiment 1, indicating that the combined use of peat, vermiculite and coconut bran has the best effect.

The results of the above examples show that the Rhodiola rosea tissue culture rapid propagation method provided by the present invention can simultaneously conduct adventitious bud induction and proliferation culture, simplifies the steps of tissue culture, shortens the test-tube seedling growth cycle, saves costs, and also reduces costs. The pollution of tissue culture seedlings caused by frequent transfer is avoided, a large number of Rhodiola rosea test-tube seedlings can be obtained in a relatively short time, and the rapid propagation of Rhodiola rosea can be realized.

Although the above-mentioned embodiment has made a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments according to the present embodiment without creativity. These embodiments All belong to the protection scope of the present invention.

Claims (8)

1. a method for tissue culture rapid propagation of Rhodiola rosea, is characterized in that, comprises the following steps: 1) The seeds are placed in an induced proliferation medium for induced proliferation culture to obtain Rhodiola rosea cluster buds; 2) Inoculate Rhodiola rosea cluster buds into a rooting medium for rooting culture to obtain Rhodiola rosea test-tube seedlings; the rooting medium uses 1/2 MS medium as a basal medium, and is cultured at 1/2 MS On the basis of the base, it also includes the following components of mass volume concentration: 0.4~0.6mg/L NAA, 0.5~1.0g/L activated carbon, 30~35g/L sucrose and 6~7g/L agar. pH value is 5.8~6.0; 3) Carry out hardening and transplanting of the obtained Rhodiola rosea test-tube seedlings; The inducing proliferation medium is based on MS medium, and on the basis of MS medium, it also includes the following components with the following mass and volume concentrations: 1.0-2.0 mg/L 6-BA, 0.1-0.2 mg/L NAA, 30~35g/L sucrose and 6~7g/L agar, the pH value of the induced proliferation medium is 5.8~6.0; The transplanting substrate comprises peat, vermiculite and coconut bran, and in the substrate, the mass ratio of peat, vermiculite and coconut bran is (2~2.5):(1~1.5):1. 2 . The method according to claim 1 , wherein in step 1) the seeds are further subjected to germination-promoting treatment before inducing proliferation culture, and the germination-promoting treatment comprises: soaking in an aqueous solution of gibberellin; The concentration of gibberellin in the gibberellin aqueous solution is 350~450mg/L, and the soaking time is 5~10min. 3. The method according to claim 1, characterized in that, in step 1), before the seeds are induced to proliferate and cultivate, further comprising a disinfection treatment, and the method for the disinfection treatment comprises: immersing the seeds in 75% alcohol for 25~25~ 30s, washed with sterile water and then transferred to 0.1% HgCl ₂ to soak for 10-15min to obtain the seeds after disinfection treatment. 4 . The method according to claim 1 , wherein the conditions for inducing proliferation culture in step 1) include: firstly carrying out dark culture to obtain callus; after obtaining callus, carrying out light culture to obtain rose bengal Sedum sprouts. 5. method according to claim 4, is characterized in that, the condition of described dark culture is: temperature 23～25 ℃, humidity 75%～85%, dark culture time 20～23d; The condition of described light culture is : Temperature 23~25℃, humidity 75%~85%, light intensity 1500~2000Lx, light time 12~14 h/d, culture time 30~40d. 6. The method according to claim 1, wherein in step 2), the length of the clump buds used for inoculation is 1.5-2.0 cm. 7. The method according to claim 1, wherein the conditions of the rooting culture in step 2) are: temperature 23～25°C, humidity 75%～85%, light intensity 1500～2000Lx, light time 12～14 h/d, culture time 20-30d. 8. method according to claim 1, is characterized in that, when the length of the adventitious root of the Rhodiola rosea test-tube seedling obtained reaches 2～3cm, carry out seedling hardening, and the time of described hardening seedling is 2～4d.

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