CN112616674A - Induced proliferation culture medium for tissue culture and rapid propagation of rhodiola rosea and tissue culture and rapid propagation method - Google Patents
Induced proliferation culture medium for tissue culture and rapid propagation of rhodiola rosea and tissue culture and rapid propagation method Download PDFInfo
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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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
Technical Field
The invention belongs to the technical field of plant tissue culture seedling cultivation, and particularly relates to an induced proliferation culture medium for tissue culture and rapid propagation of rhodiola rosea and a tissue culture and rapid propagation method.
Background
Rhodiola rosea (Rhodiola rosea L.) is a perennial herb of Rhodiola of Crassulaceae, also called Rhodiola rosea, is mostly wild in forest land, hillside, dry stone gap, abrupt rock and the like, and is distributed in Shaanxi, Gansu, Xinjiang, Hebei and the like. The Ming Dynasty Li Shizhen records that the rose rhodiola rosea is the top-grade of the original channel and eliminates evil and bad qi in Ben Cao gang mu, is called as "Xiancao", has rose fragrance, is more intense when fresh, can be used as both medicine and food in the whole herb, and commonly uses rhizome with the nature of "sweet, bitter, flat, entering lung and heart channel". The main active ingredients of the rhodiola rosea comprise salidroside and aglycone tyrosol, Sicelavin and the like. A large number of researches show that the plant has multiple physiological functions of resisting fatigue and oxygen deficit, enhancing learning and memory, exciting the central nervous system, improving sleep, preventing altitude diseases, resisting cancer and the like, and is a popular medicine and food dual-purpose adaptogen plant.
The growth environment of the rhodiola is extremely harsh and variable, such as anoxia, low-temperature drying, high wind, strong ultraviolet irradiation, large day and night temperature difference and the like, so that the rhodiola has special adaptability substances rarely seen by other plants, and the natural resources of the rhodiola are extremely deficient. The rhodiola seeds have low natural germination rate, serious pest and disease damage are caused when the rhodiola seeds are introduced from high mountains to low-altitude areas for domestication cultivation, and a large amount of wild resources are dug by people, so that the rhodiola seeds are in an endangered situation. Therefore, the tissue culture and the rapid propagation of the rhodiola are enhanced, and the natural resources and the genetic diversity of the rhodiola are protected extremely urgently.
Disclosure of Invention
In order to solve the problems, 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. The induction proliferation culture medium for tissue culture and rapid propagation of rhodiola rosea provided by the invention can simultaneously realize the induction and proliferation of adventitious buds, the method is simple to operate, a large number of test-tube plantlets of rhodiola rosea can be obtained in a short time, and the rapid propagation of rhodiola rosea is realized.
In order to achieve the above purpose, the invention provides the following technical scheme:
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 invention provides a tissue culture and rapid propagation method of rhodiola rosea, which comprises the following steps:
1) placing the seeds in the induced proliferation culture medium in the technical scheme for induced proliferation culture to obtain roselle buds;
2) inoculating the roselle buds of the rhodiola rosea into a rooting culture medium for rooting culture to obtain test-tube plantlets of the rhodiola rosea; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises the following components in mass volume concentration on the basis of 1/2MS culture medium: 0.4-0.6 mg/LNAA, 0.5-1.0 g/L active carbon, 30-35 g/L sucrose and 6-7 g/L agar, wherein the pH value of the rooting medium is 5.8-6.0;
3) and hardening and transplanting the obtained rhodiola rosea test-tube plantlet.
Preferably, the seeds of step 1) are further subjected to germination-promoting treatment before being subjected to proliferation-inducing culture, wherein the germination-promoting treatment comprises: soaking the bamboo shoots in gibberellin water solution, wherein the concentration of gibberellin in the gibberellin water solution is 350-450 mg/L, and the soaking time is 5-10 min.
Preferably, the seeds in step 1) further comprise a disinfection treatment before the proliferation-inducing culture, and the disinfection treatment method comprises the following steps: soaking the seeds in 75% alcohol for 25-30 s, washing with sterile water, and transferring to 0.1% HgCl2Soaking for 10-15 min to obtain the disinfected seeds.
Preferably, the conditions for inducing proliferation culture in step 1) include: dark culture is carried out firstly to obtain callus; and after obtaining the callus, carrying out illumination culture to obtain the roselle rhodiola rosea cluster buds.
Preferably, the dark culture conditions are: the temperature is 23-25 ℃, the humidity is 75-85%, and the dark culture time is 20-23 d; the illumination culture conditions are as follows: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination intensity is 1500-2000 Lx, the illumination time is 12-14 h/d, and the culture time is 30-40 d.
Preferably, in the step 2), the length of the cluster buds for inoculation is 1.5-2.0 cm.
Preferably, the rooting culture conditions in step 2) are as follows: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination intensity is 1500-2000 Lx, the illumination time is 12-14 h/d, and the culture time is 20-30 d.
Preferably, when the length of the adventitious root of the obtained rhodiola rosea test-tube plantlet reaches 2-3 cm, hardening off the plantlet, wherein the hardening-off time is 2-4 days.
Preferably, the matrix for transplanting comprises peat, vermiculite and coconut coir, wherein the mass ratio of the peat to the vermiculite to the coconut coir in the matrix is (2-2.5): (1-1.5): 1.
advantageous effects
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.
Furthermore, the invention also provides a tissue culture and rapid propagation method of rhodiola rosea, firstly, seeds are placed in an induced proliferation culture medium for induced proliferation culture to obtain rhodiola rosea clustered buds, the induced proliferation culture medium can simultaneously carry out adventitious bud induction and proliferation culture, the step of tissue culture is simplified, the seedling period of test-tube seedlings is shortened, the cost is saved, and meanwhile, the pollution to the tissue culture seedlings caused by frequent transfer is avoided; after the cluster buds are obtained, the cluster buds are inoculated to a rooting culture medium for rooting culture to obtain the rhodiola rosea test-tube plantlet, and the rooting culture medium has a good effect of inducing rooting; and finally, hardening and transplanting the obtained rhodiola rosea test-tube plantlet. The tissue culture and rapid propagation method of rhodiola rosea provided by the invention is simple to operate, can obtain a large number of test-tube plantlets of rhodiola rosea in a short time, realizes rapid propagation of rhodiola rosea, and has great popularization value.
Drawings
FIG. 1 shows the condition of rhodiola rosea induced proliferation culture 1d in example 1;
FIG. 2 shows the case of culturing rhodiola rosea in the proliferation-inducing culture of rhodiola rosea for 25d in example 1;
FIG. 3 shows the case of culturing rhodiola rosea in the proliferation-inducing culture of 40d according to example 1;
FIG. 4 shows the case of rooting culture of strong seedlings of rhodiola rosea for 20 days in example 1;
FIG. 5 shows the case of transplanting rhodiola rosea 50d according to example 1.
Detailed Description
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; further preferred are compositions comprising the following mass volume concentrations: 2.0 mg/L6-BA, 0.2mg/LNAA, 30g/L sucrose and 6.5g/L agar. In the present invention, the pH of the proliferation induction medium is 5.8 to 6.0, and more preferably 5.9. The invention has no special requirements on the sources of all components of the culture medium, and can adopt common commercial products in the field. In the induced proliferation culture medium, 6-BA can promote adventitious bud differentiation and cell division, NAA can promote cell division, and cane sugar is a carbon source. The induced proliferation culture medium can simultaneously realize the induction and proliferation of adventitious buds by using the 6-BA and the NAA in a combined way, simplifies the steps of tissue culture, shortens the seedling period of test-tube seedlings, saves the cost, avoids the pollution to the tissue culture seedlings caused by frequent switching, and improves the survival rate of the tissue culture seedlings.
The invention provides a tissue culture and rapid propagation method of rhodiola rosea, which comprises the following steps:
1) placing the seeds in the induced proliferation culture medium in the technical scheme for induced proliferation culture to obtain roselle buds;
2) inoculating the roselle buds of the rhodiola rosea into a rooting culture medium for rooting culture to obtain test-tube plantlets of the rhodiola rosea; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises the following components in mass volume concentration on the basis of 1/2MS culture medium: 0.4-0.6 mg/LNAA, 0.5-1.0 g/L active carbon, 30-35 g/L sucrose and 6-7 g/L agar, wherein the pH value of the rooting medium is 5.8-6.0;
3) and hardening and transplanting the obtained rhodiola rosea test-tube plantlet.
In the present invention, it is preferable to subject the seeds to germination acceleration treatment before the seeds are subjected to induced growth culture. In the invention, the seeds are preferably selected from full-grain and disease-free mature rhodiola rosea seeds so as to improve the germination rate. In the present invention, the germination acceleration treatment preferably includes: soaking the bamboo shoot in a gibberellin water solution, wherein the concentration of gibberellin in the gibberellin water solution is preferably 350-450 mg/L, and more preferably 400 mg/L; the soaking time is preferably 5-10 min, and more preferably 10 min. The gibberellin with the specific concentration is selected for soaking, so that the germination of seeds can be promoted, and the germination rate of the seeds is improved. The invention preferably uses running water to wash after the gibberellin aqueous solution is treated, and then uses the detergent to soak after the washing. In the invention, the time for flushing with flowing water is preferably 5-10 min, and more preferably 5 min; the time for soaking the seeds by the detergent is preferably 5-10 min, more preferably 10min, continuously stirring the seeds during the time, removing impurities, washing the seeds by running water, and then sucking water by using filter paper.
The present invention preferably sterilizes the seeds after the germination-promoting treatment. Before the disinfection treatment, the seeds subjected to germination promotion treatment are preferably washed with laundry powder water for three times, washed clean with foam, put into a mesh bag, soaked and washed under running water for 2 hours to remove impurities and take away substances inhibiting seed germination. In the present invention, the method of sterilization treatment includes: soaking the seeds in 75% alcohol for 25-30 s, washing with sterile water, and transferring to 0.1% HgCl2Soaking for 10-15 min; more preferably, the seeds are immersed in 75% ethanol for 30s, washed with sterile water, and then transferred to 0.1% HgCl2Soaking for 15 min. The sterilization treatment can kill germs on seeds, prevent pollution in the tissue culture process and reduce the tissue culture survival rate.
After the disinfection treatment, the seeds are placed in the induced proliferation culture medium in the technical scheme for induced proliferation culture, and the roselle buds of the rhodiola rosea are obtained. In the present invention, the conditions for inducing proliferation culture preferably include: dark culture is carried out firstly to obtain callus; and after obtaining the callus, carrying out illumination culture to obtain the roselle rhodiola rosea cluster buds. In the invention, the conditions of dark culture preferably comprise the temperature of 23-25 ℃, the humidity of 75-85% and the dark culture time of 20-23 d; further preferably 24 ℃, the humidity is 80 percent, and the dark culture time is 20d, wherein the dark culture is favorable for the induction of the callus. In the invention, the illumination culture conditions are preferably 23-25 ℃, 75-85% of humidity, 1500-2000 Lx of illumination intensity, 12-14 h/d of illumination time and 30-40 d of culture time; further preferably, the temperature is 24 ℃, the humidity is 80%, the illumination intensity is 1800Lx, the illumination time is 12h/d, and the culture time is 35 d. And (5) obtaining the rosette buds after illumination culture.
After obtaining the rose rhodiola rosea cluster buds, inoculating the rose rhodiola rosea cluster buds into a rooting culture medium for rooting culture to obtain the rose rhodiola rosea test-tube plantlet. The invention preferably cuts off the rosette buds of rhodiola rosea for inoculation. In the invention, the preferred length of the cluster buds for inoculation is 1.5-2.0 cm, the further preferred length is 2cm, the cluster buds with similar length are selected for rooting culture, the tissue culture seedlings are more uniform, and the commodity is better. In the invention, the rooting medium takes 1/2MS culture medium as a basic culture medium, and also comprises the following components in mass volume concentration on the basis of 1/2MS culture medium: 0.4-0.6 mg/L NAA, 0.5-1.0 g/L active carbon, 30-35 g/L sucrose and 6-7 g/L agar, and further preferably comprises the following components in mass volume concentration: 0.5mg/LNAA, 1.0g/L activated carbon, 30g/L sucrose and 6.5g/L agar. In the invention, the pH value of the rooting medium is 5.8-6.0, and the preferable pH value is 5.9. NAA in the rooting culture medium has the effects of promoting cell division and inducing the generation of adventitious roots; the activated carbon can adsorb the self-toxic substances released by the tissue culture seedlings, so that the tissue culture seedlings are more robust; the sucrose can provide a proper carbon source for the tissue culture seedling, and the rooting medium provided by the invention has good effects of strengthening the seedling and promoting rooting. In the invention, the conditions for rooting culture are preferably 23-25 ℃, 75-85% of humidity, 1500-2000 Lx of illumination intensity, 12-14 h/d of illumination time and 20-30 d of culture time; further preferably, the temperature is 24 ℃, the humidity is 80%, the illumination intensity is 1800Lx, the illumination time is 12h/d, and the culture time is 21 d.
After rooting culture, the invention carries out seedling hardening and transplanting on the obtained rhodiola rosea test-tube plantlet. According to the method, preferably, when the length of the adventitious root of the obtained rhodiola rosea test-tube plantlet reaches 2-3 cm, a tissue culture bottle cap is opened, a small amount of sterile water is poured in, and the seedling hardening is carried out, wherein the seedling hardening time is preferably 2-4 d, and is further preferably 3 d. In the invention, the seedling exercising culture conditions are preferably 23-25 ℃ of temperature, 75-85% of humidity, 1500-2000 Lx of illumination intensity, 12-14 h/d of illumination time and 2-4 d of culture time; further preferably, the temperature is 24 ℃, the humidity is 80%, the illumination intensity is 1800Lx, the illumination time is 12h/d, and the culture time is 3 d. In the invention, the transplanting substrate preferably comprises peat, vermiculite and coconut coir, and the mass ratio of the peat to the vermiculite to the coconut coir in the substrate is preferably (2-2.5): (1-1.5): 1, the matrix for transplanting can improve the survival rate of the transplanted tissue culture seedlings. In the invention, preferably, the transplanted tissue culture seedlings are firstly cultured in a greenhouse with the temperature of 21-25 ℃, the illumination intensity of 1500-2000 lx and the relative air humidity of 70-85% for 1-2 weeks, and then are transplanted outdoor in a field, so that the survival rate of the transplanted tissue culture seedlings can be further improved; the transplanting time is preferably between 5:00 and 6:00 in the afternoon, and the water is thoroughly poured.
For further illustration of the present invention, the tissue culture and rapid propagation inducing medium and the tissue culture and rapid propagation method of rhodiola rosea provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Tissue culture and rapid propagation method for rhodiola rosea
(1) Selecting seeds and promoting germination: 400mg/L Gibberellin (GA) for screening full-seed and disease-free mature rhodiola rosea seeds3) Soaking for 20min to increase seed germination rate, taking out, washing with running water for 5min, soaking the seeds with detergent water for 10min while stirring, removing impurities, washing with running water, and draining.
(2) Seed disinfection and germination culture: and cleaning the treated seeds with the laundry powder water for three times, flushing the seeds with foam, putting the seeds into a mesh bag, soaking and flushing the seeds for 2 hours under running water, and disinfecting the seeds in a super-clean workbench. Soaking the seeds with water in 75% ethanol for 30s, washing with sterile water for 5 times, and transferring to 0.1% HgCl2Soaking for 15min, washing with sterile water for 4 times, and placing on filter paper to suck water for use.
(3) Adventitious bud induction and proliferation: sowing the sterilized seeds into an induced proliferation culture medium, wherein the induced proliferation culture medium takes an MS culture medium as a basic culture medium, and the following components with mass volume concentration are added on the basis of the MS culture medium: 2.0mg/L of 6-BA, 0.2mg/L of LNAA, 30g/L of sucrose and 6.5g/L of agar, and the pH value is 6.0; the culture conditions are as follows: culturing in the dark for 20 days at 24 +/-1 ℃ and with the humidity of 80 +/-5%, and culturing in the post-illumination for 12h/d and the illumination intensity of 1500-2000 Lx for 35 days to obtain the rhodiola rosea tufted buds. FIG. 1 shows the case of rhodiola rosea induced proliferation culture for 1d, FIG. 2 shows the case of rhodiola rosea induced proliferation culture for 25d (dark culture for 20d, light culture for 5d), and FIG. 3 shows the case of rhodiola rosea induced proliferation culture for 40d (dark culture for 20d, light culture for 20 d).
(4) Strong seedling and rooting culture: cutting 1.5-2 cm adventitious buds of rhodiola rosea, inoculating the cut adventitious buds into a rooting culture medium, wherein the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and adding the following components in mass volume concentration on the basis of 1/2MS culture medium: 0.5mg/L NAA, 1.0g/L active carbon, 30g/L sucrose and 6.5g/L agar, and the pH value is 6.0; the culture conditions are as follows: the temperature is 24 +/-1 ℃, the humidity is 80 +/-5%, the illumination is 12h/d, the illumination intensity is 1500-2000 Lx, and the whole rhodiola rosea test-tube plantlet is obtained after culturing for 20d, the result is shown in figure 4, and the picture in figure 4 shows that the rhodiola rosea test-tube plantlet has good rooting condition.
5) Hardening and transplanting seedlings: when the length of the adventitious root of the rhodiola rosea tissue culture seedling reaches 2-3 cm, opening a bottle cap, pouring a small amount of sterile water, hardening the seedling for 3d indoors, separating the tissue culture seedling from a rooting culture medium, washing the culture medium with running water, soaking the root of the test-tube seedling for 20min with carbendazim solution diluted by 1000 times, sucking water with absorbent paper, transplanting the test-tube seedling into a transplanting medium with the mass ratio of peat, vermiculite and coconut chaff of 2:1:1, and watering enough rooting water. Culturing the transplanted test-tube seedlings in a greenhouse at the temperature of 24 ℃, the illumination intensity of 1500-2000 lx and the humidity of 80 +/-5% for 1 week, then transplanting the test-tube seedlings in an outdoor field, wherein the transplanting time is selected to be between 5:00 and 6:00 in the afternoon, and watering the test-tube seedlings thoroughly. FIG. 5 shows the condition 50d after transplantation.
Example 2
The tissue culture and rapid propagation method of rhodiola rosea is the same as that in example 1, and is different from the following steps: the induced proliferation culture medium is prepared by adding the following components in mass volume concentration on the basis of an MS culture medium: 1.0mg/L of 6-BA, 0.1mg/L of NAA, 30g/L of cane sugar and 6.5g/L of agar, and the pH value is 6.0.
Example 3
The tissue culture and rapid propagation method of rhodiola rosea is the same as that in example 1, and is different from the following steps: the proliferation culture medium is prepared by adding the following components in mass volume concentration on the basis of MS culture medium: 1.5 mg/L6-BA, 0.15mg/L NAA, 30g/L sucrose and 6.5g/L agar, and the pH value is 6.0.
Comparative example 1
The tissue culture and rapid propagation method of rhodiola rosea is the same as that in example 1, and is different from the following steps: the induced proliferation culture medium is prepared by adding the following components in mass volume concentration on the basis of an MS culture medium: 1.0 mg/L6-BA, 0.2mg/L NAA, 0.1mg/L KT, 30g/L sucrose and 6.5g/L agar, and the pH value is 6.0.
Comparative example 2
The tissue culture and rapid propagation method of rhodiola rosea is the same as that in example 1, and is different from the following steps: the rooting medium is prepared by adding the following components in mass-volume concentration on the basis of 1/2MS culture medium: 0.5mg/LIAA, 1.0g/L active carbon, 30g/L sucrose and 6.5g/L agar, and the pH value is 6.0.
Comparative example 3
The tissue culture and rapid propagation method of rhodiola rosea is the same as that in example 1, and is different from the following steps: the rooting medium is prepared by adding the following components in mass-volume concentration on the basis of 1/2MS culture medium: 0.5mg/LNAA, 30g/L sucrose and 6.5g/L agar, pH 6.0.
Comparative example 4
The tissue culture and rapid propagation method of rhodiola rosea is the same as that in example 1, and is different from the following steps: the mass ratio of peat to coconut chaff of the transplanting matrix is 1: 1.
Comparative example 5
The tissue culture and rapid propagation method of rhodiola rosea is the same as that in example 1, and is different from the following steps: the mass ratio of peat to vermiculite of the transplanting medium is 1: 1.
Evaluation of Effect
1 examination of adventitious bud Induction Effect
Adventitious buds were induced by the methods of example 1, example 2 and comparative example 1, and the callus and adventitious bud induction effects of different media were examined, and the results were shown in Table 1.
TABLE 1 Effect of different media on callus and adventitious bud induction of rhodiola rosea
The results in table 1 show that the induction rate of adventitious buds can reach 84% -95% and the induction rate of callus can reach 84% -96% in the methods of examples 1-3 of the present invention, wherein the induction rate of callus and the induction rate of adventitious buds are the highest in the method of example 1; comparative example 1 adding kinetin KT on the basis of example 1 has the effects of promoting cell differentiation, division and inducing callus, but comparative example 1 does not further improve the induction rate of callus and adventitious buds, but inhibits the induction effect of example 1, and shows that the types and concentrations of hormones suitable for inducing callus and adventitious buds of different plant varieties are different, not all hormones for promoting the induction of callus and adventitious buds are suitable, the types of hormones are not properly selected, and the hormones also have the effect of inhibiting the induction of callus and adventitious buds.
2 investigation of rooting effect of strong seedlings
The method of example 1, comparative example 2 and comparative example 3 is used for inducing the rhodiola rosea clustered shoots to root, the influence of different rooting culture media on the rooting effect of strong seedlings is examined, and the detection results are shown in table 2.
TABLE 2 Effect of different rooting media on rhodiola rosea growth and rooting
As can be seen from the results in Table 2, the rooting medium for strong seedlings has better rooting effect than the rooting medium with 0.5mg/L IAA, and the plants are stronger, and the rooting effect is better than the rooting medium without the activated carbon.
3 investigation of transplanting effect of tissue culture seedling
The rhodiola rosea is cultured by using the methods of example 1, comparative example 4 and comparative example 5, the transplanting effect of different transplanting culture media on the rhodiola rosea tissue culture seedlings is examined, and the detection results are shown in table 3.
TABLE 3 influence of different transplanting media on the survival rate and growth vigor of transplanting rhodiola rosea
From the results in table 3, it can be seen that the peat coconut coir mixture in the comparative example 4 has good water retention and poor air permeability, which results in poor growth of the transplanted tissue culture seedlings, and the vermiculite added in the example 1 increases air permeability, so that the survival rate of the transplanted plants is high and the growth is good; the effect of transplanting the mixed substrate in comparative example 5 was not as good as that in example 1, indicating that the effect of using peat, vermiculite and coir together was the best.
The results of the above embodiments show that the tissue culture and rapid propagation method of rhodiola rosea provided by the invention can simultaneously perform adventitious bud induction and proliferation culture, simplify the steps of tissue culture, shorten the seedling period of test-tube plantlets, save the cost, avoid the pollution to the tissue culture plantlets caused by frequent switching, obtain a large amount of test-tube plantlets of rhodiola rosea in a shorter time, and realize rapid propagation of rhodiola rosea.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. The tissue culture and rapid propagation induced proliferation culture medium for rhodiola rosea is characterized by taking an MS culture medium as a basic culture medium and further comprising 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.
2. A tissue culture and rapid propagation method of rhodiola rosea is characterized by comprising the following steps:
1) placing the seeds in the induced proliferation culture medium of claim 1 for induced proliferation culture to obtain rosette buds of rhodiola rosea;
2) inoculating the roselle buds of the rhodiola rosea into a rooting culture medium for rooting culture to obtain test-tube plantlets of the rhodiola rosea; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises the following components in mass volume concentration on the basis of 1/2MS culture medium: 0.4-0.6 mg/L NAA, 0.5-1.0 g/L active carbon, 30-35 g/L sucrose and 6-7 g/L agar, wherein the pH value of the rooting medium is 5.8-6.0;
3) and hardening and transplanting the obtained rhodiola rosea test-tube plantlet.
3. The method according to claim 2, wherein the seeds of step 1) are further subjected to germination-promoting treatment before being subjected to induced proliferation culture, the germination-promoting treatment comprising: soaking the bamboo shoots in gibberellin water solution, wherein the concentration of gibberellin in the gibberellin water solution is 350-450 mg/L, and the soaking time is 5-10 min.
4. The method of claim 2, wherein the seeds of step 1) are further subjected to a sterilization treatment prior to the induced proliferation culture, and the sterilization treatment comprises: soaking the seeds in 75% alcohol for 25-30 s, washing with sterile water, and transferring to 0.1% HgCl2Soaking for 10-15 min to obtain the disinfected seeds.
5. The method of claim 2, wherein the conditions of step 1) for inducing proliferation culture comprise: dark culture is carried out firstly to obtain callus; and after obtaining the callus, carrying out illumination culture to obtain the roselle rhodiola rosea cluster buds.
6. The method according to claim 5, wherein the dark culture conditions are: the temperature is 23-25 ℃, the humidity is 75-85%, and the dark culture time is 20-23 d; the illumination culture conditions are as follows: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination intensity is 1500-2000 Lx, the illumination time is 12-14 h/d, and the culture time is 30-40 d.
7. The method according to claim 2, wherein the clumped buds to be inoculated in step 2) have a length of 1.5-2.0 cm.
8. The method according to claim 2, wherein the rooting culture conditions of step 2) are: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination intensity is 1500-2000 Lx, the illumination time is 12-14 h/d, and the culture time is 20-30 d.
9. The method according to claim 2, wherein the seedling exercising is carried out when the length of the adventitious root of the obtained rhodiola rosea test-tube plantlet reaches 2-3 cm, and the seedling exercising time is 2-4 days.
10. The method according to claim 2, wherein the transplanting substrate comprises peat, vermiculite and coconut coir, and the mass ratio of the peat, the vermiculite and the coconut coir in the substrate is (2-2.5): (1-1.5): 1.
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