CN114176004A - Regeneration culture method of saussurea involucrate - Google Patents
Regeneration culture method of saussurea involucrate Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a regeneration culture method of saussurea involucrate. The method for regenerating and culturing the saussurea involucrate comprises a new culture mode of the saussurea involucrate, namely development of a water culture system and optimization of a tissue culture and rapid propagation system of the saussurea involucrate, and the two are organically combined, so that the problem of wild resource shortage of the saussurea involucrate is effectively solved, the limitation of flat ground growth of the saussurea involucrate is broken through, the tissue culture and rapid propagation time is maximally shortened, and the manpower, material resources and financial resources are saved. Provides an important foundation for the resource and protection research of the saussurea involucrate.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a regeneration culture method of saussurea involucrate, in particular to a dynamic water culture method of saussurea involucrate and a tissue culture and rapid propagation improvement method of saussurea involucrate in low altitude and flat environment.
Background
Saussurea involucrate (Saus-surea involucrate (Kar. et Kir.) Sch. -Bip.) is perennial high mountain herbaceous plant of saussurea of Compositae. Since ancient times, the saussurea involucrate is always a traditional famous and precious Chinese medicinal material, and is recorded in Chinese pharmacopoeia, has warm and slightly bitter properties, has the effects of warming kidney and tonifying yang, strengthening bones and muscles, nourishing nerves, stimulating the menstrual flow and activating blood and the like, and is mainly used for treating wind-cold-dampness arthralgia, lower abdominal pain, irregular menstruation and the like clinically. The main chemical components of the snow lotus are alkaloid, flavonoid and phenylpropanoid compounds, and the snow lotus has extremely high medicinal value.
The saussurea involucrate grows in the mountain range of the Tianshan, is mainly distributed among the mountain stream rock beach, the mountain grass slope, the mountain marsh grassland and the mountain top broken stones near the 2400-4000m mountain snow line, has specific habitat, low natural reproduction rate and slow growth, can bloom and fruit in 3-5 years, and is difficult to artificially cultivate. Since the 20 th century, the wild resources are endangered due to the long-term predatory mining of people.
The wild resource of the saussurea involucrate is seriously insufficient due to the severe growth condition of the saussurea involucrate, extremely low population self-renewal capacity and excessive mining of people. Although there have been reports of artificial propagation using seeds, artificial propagation has been little effective due to low germination rate and high seedling death rate. In addition, under the background of global warming, the snow accumulation area of the Tianshan mountain area is gradually reduced, the snow line is raised, and the environment suitable for the survival of the wild saussurea involucrate is less and less. In order to protect wild resources and biodiversity of saussurea involucrate, establishing a high-efficiency regeneration culture system of the saussurea involucrate in a low-altitude environment by utilizing modern biotechnology means is particularly important.
Disclosure of Invention
The invention aims to provide a regeneration culture method of saussurea involucrate.
The regeneration culture method of the saussurea involucrate provided by the invention comprises the following steps:
1) soaking the saussurea involucrate seeds in gibberellin solution, and culturing to obtain germinated saussurea involucrate seeds;
2) culturing the germinated saussurea involucrate seeds in a water culture mode to obtain water culture seedlings;
3) taking the leaves or petioles of the water culture seedlings as explants to perform callus induction and differentiation culture in a callus induction culture medium to obtain regenerated seedlings;
4) and culturing the regenerated seedling in a rooting culture medium to obtain a saussurea involucrate regenerated plant.
In the method, in the step 1), the concentration of the gibberellin solution may be 50-200 mg/L; further 50-100mg/L or 100-200 mg/L; still further, it may be 50mg/L or 100mg/L or 200 mg/L.
The soaking conditions were as follows: soaking in dark at low temperature for 12-24 hr; in particular to soaking for 12 hours in the dark at the temperature of 4 ℃.
The culture conditions were as follows: culturing for 3-5 days under the conditions of illumination intensity of 2000-3000Lx, temperature of 24-25 ℃ and humidity of 50-60%.
The culture method is to spread the soaked seeds in filter paper or a culture dish for culture.
In the method, in the step 2), the culture solution for water culture is an MS culture solution; the MS culture solution is obtained by uniformly mixing MS powder and water (tertiary water).
The concentration of the MS culture solution may be 1.10 to 4.43g/L, further 1.10 to 2.43g/L or 2.43 to 4.43g/L, and further 1.10g/L or 2.43g/L or 4.43 g/L.
The pH value of the MS culture solution is 5.6-6.0.
The hydroponic conditions were as follows: the illumination intensity is 2000-3000 Lx; the illumination time is 16 h/d; the temperature is 24-25 ℃; humidity is 50-60%; the oxygen content is more than 5 mg/L; the flow rate of the water pump is 50-350L/h. Further, when the culture is just started, the flow rate of the water pump is 50L/h, and the culture solution is replaced once in two weeks; after culturing for 1 month, the flow rate of the water pump can be adjusted to 150L/h, and the culture solution is replaced once a week; after 2 months of culture, the flow of the water pump can be adjusted to 350L/h, and the culture solution is replaced twice a week.
The method for culturing comprises the step of culturing the germinated saussurea involucrate seeds in a water culture device filled with ceramsite. The hydroponic device can be a hydroponic device or a soilless culture device or a hydroponic culture box, etc. which are common in the field, and in one example of the invention, the hydroponic device and the ceramsite are products of Taobao [ Dry hydroponic culture ], and can also be obtained from Hebei Dry hydroponic electronic technology, Inc.
In the above method, in step 3), the explant further comprises a step of sterilizing before callus induction and differentiation culture: the disinfection method sequentially comprises the following steps: tap water washing, sterile water washing, NaClO solution soaking, alcohol disinfection and sterile water washing;
further, the time for flushing with tap water may be 1 h.
The number of times of the sterile water washing can be 3-5 times.
The concentration of the NaClO solution may be 10%.
The soaking time can be 5-10 min.
The alcohol may be 75% alcohol.
The time for the sterilization may be 30 s.
The size of the explant is 1cm2。
In the above method, in the step 3), the callus induction and differentiation culture method comprises the following steps:
3-1) carrying out induction culture on the explant in a callus induction culture medium to obtain callus;
3-2) carrying out subculture on the callus in a subculture medium to obtain cluster buds;
3-3) carrying out differentiation culture on the cluster buds in a differentiation culture medium to obtain regenerated seedlings;
the subculture medium and the differentiation medium are both the callus induction medium;
the callus induction culture medium comprises naphthylacetic acid (NAA) and 6-benzylamino adenine (6-BA).
Further, the concentration of the NAA in the callus induction culture medium can be 1-2 mg/L; further 1-1.5mg/L or 1.5-2 mg/L; further, the concentration of the compound may be 1mg/L, 1.5mg/L or 2 mg/L.
The concentration of the 6-BA in the callus induction culture medium can be 0.2-2 mg/L; further 0.2-1 mg/L or 1-2 mg/L; further, it may be 0.2mg/L, 1mg/L or 2 mg/L.
The culture conditions of 3-1) are as follows: culturing in the dark at 24-25 deg.C and 50-60% humidity for 3 weeks;
the culture conditions of 3-2) are as follows: culturing in dark at 24-25 deg.C and 50-60% humidity for 2-3 weeks;
the culture conditions of 3-3) are as follows: culturing in the dark at 24-25 deg.C and 50-60% humidity for 1 week, culturing at 24-25 deg.C and 50-60% illumination intensity of 1000Lx for 1 week, and culturing at 24-25 deg.C and 50-60% humidity for 2-3 weeks at 2500-3000 Lx.
Further, the callus induction medium consists of MS powder, sucrose, NAA, 6-BA, plant gel and water, wherein the concentration of the MS powder in the callus induction medium is 3.32g/L, the concentration of the sucrose in the callus induction medium is 30g/L, the concentration of the NAA in the callus induction medium is 1.5mg/L, the concentration of the 6-BA in the callus induction medium is 1mg/L, and the concentration of the plant gel in the callus induction medium is 4 g/L. The pH value of the callus induction culture medium is 5.9-6.0.
In the method, the step of performing bud re-induction culture on the tissue culture seedling in a bud induction culture medium is further included between the step 3) and the step 4);
the shoot induction medium comprises 6-BA;
the concentration of the 6-BA in the bud induction medium can be 0.5-2 mg/L; further, the concentration of the compound is 0.5 to 1mg/L or 1 to 2mg/L, and further 0.5mg/L or 1mg/L or 2 mg/L.
The conditions for the shoot re-induction culture were as follows: culturing for 4-5 weeks under the conditions of illumination intensity of 2500-3000Lx, temperature of 24-25 deg.C and humidity of 50-60%.
Further, the bud induction medium consists of MS powder, sucrose, 6-BA, plant gel and water, wherein the concentration of the MS powder in the callus induction medium is 2.21g/L, the concentration of the sucrose in the callus induction medium is 15g/L, the concentration of the 6-BA in the callus induction medium is 0.5mg/L, and the concentration of the plant gel in the callus induction medium is 4 g/L.
Further, the pH of the shoot induction medium is 5.9 to 6.0.
In the above method, in the step 4), the rooting medium comprises NAA;
the concentration of the NAA in the rooting culture medium is 0.1-0.3 mg/L; further 0.1-0.2 mg/L0.2-0.3 mg/L; further, it may be 0.1mg/L, 0.2mg/L or 0.3 mg/L.
The rooting culture conditions are as follows: culturing at the illumination intensity of 2000-3000Lx, temperature of 24-25 deg.C and humidity of 50-60% for 3-5 weeks.
Further, the rooting medium consists of MS powder, sucrose, NAA and water, wherein the concentration of the MS powder in the callus induction medium is 2.21g/L, the concentration of the sucrose in the callus induction medium is 15g/L, and the concentration of the NAA in the callus induction medium is 0.3 mg/L.
Further, the rooting medium has a pH of 5.9 to 6.0.
The invention also provides a water culture method of the saussurea involucrate.
The water culture method of the saussurea involucrate provided by the invention comprises the steps 1) and 2) in the regeneration culture method of the saussurea involucrate.
Further, the site of the water culture method is located in a low-altitude flat ground environment.
Furthermore, the water culture method is performed in a laboratory of molecular breeding team of energy crops of Qingdao biological energy and process research institute of Qingdao institute of Chinese academy of sciences, Qingdao, Shandong province.
The invention also provides a tissue culture method of the saussurea involucrate.
The tissue culture method of the saussurea involucrate provided by the invention comprises the steps 3) and 4) in the regeneration culture method of the saussurea involucrate.
The invention also provides any one of the following products A1) -A3):
A1) the reagent for water culture of saussurea involucrate comprises gibberellin solution and/or MS culture solution in the regeneration culture method of saussurea involucrate;
A2) the reagent for tissue culture of saussurea involucrate comprises a callus induction culture medium and/or a bud induction culture medium and/or a rooting culture medium in the regeneration culture method of saussurea involucrate;
A3) the complete set of reagents for the regeneration culture of the saussurea involucrate comprises gibberellin solution and/or MS culture solution and/or callus induction culture medium and/or bud induction culture medium and/or rooting culture medium in the regeneration culture method of the saussurea involucrate.
The invention finally provides the following applications of B1) -B4):
B1) the method or the application of the product in culturing the saussurea involucrate;
B2) the method or the product is applied to breeding or expanding propagation of the saussurea involucrate;
B3) the method or the product is applied to shortening the propagation or expanding propagation time of the saussurea involucrate;
B4) application of gibberellin or gibberellin solution in promoting germination of saussurea involucrate seed.
The invention has the following beneficial effects:
1. the invention adopts the water culture mode to culture the seeds of the saussurea involucrate in the regeneration culture technical system of the saussurea involucrate, develops the water culture mode of the saussurea involucrate for the first time, compared with the prior art which can only obtain the aseptic seed seedling of the saussurea involucrate by the tissue culture mode, the invention breaks through the limitation of the growth of the saussurea involucrate on the flat ground, solves the problem that the saussurea involucrate is difficult to survive on the flat ground, successfully obtains the water culture seedling of the saussurea involucrate by the water culture mode, and realizes the normal growth of the saussurea involucrate under the flat ground environment with low altitude.
2. According to the invention, gibberellin is adopted to treat the saussurea involucrate seeds in the saussurea involucrate regeneration culture technology system, so that the germination of the saussurea involucrate seeds is effectively promoted, the germplasm resource utilization rate is improved, and wild saussurea involucrate germplasm resources are saved.
3. The tissue culture and rapid propagation system of the saussurea involucrate is optimized in the technical system of the regeneration culture of the saussurea involucrate, the callus induction and the bud induction are integrated, the explant generates cluster buds after being cultured in the callus induction culture medium for 5 to 6 weeks, and compared with the prior art that the process of 'seed-aseptic seedling-induced callus-induced bud-rooting' requires 6 to 8 months, the regeneration culture of the saussurea involucrate can be completed only by 5 months in the invention, thereby effectively shortening the breeding time, and saving the manpower, the material resources and the financial resources.
4. In the tissue culture and rapid propagation method in the saussurea involucrate regeneration culture technical system, a bud re-induction step is added before rooting culture, and 2-3 new buds can grow at the base of each single seedling, compared with the method that in the prior art, after cluster buds are induced, the cluster buds are cultured for a period of time, and then are separated, and single seedlings are selected to be directly subjected to rooting culture, the propagation coefficient after the bud re-induction step is added can be improved by 2-3 times.
The technical system for regeneration culture of the saussurea involucrate provided by the invention comprises a new cultivation mode of the saussurea involucrate, namely development of a water culture system and optimization of a tissue culture and rapid propagation system of the saussurea involucrate, and the two are organically combined, so that the problem of wild resource shortage of the saussurea involucrate is effectively solved, the limitation of flat land growth of the saussurea involucrate is broken through, the tissue culture and rapid propagation time is maximally shortened, and the manpower, material resources and financial resources are saved. Provides an important foundation for the resource and protection research of the saussurea involucrate.
Drawings
FIG. 1: and (4) germinating the snow lotus seeds.
FIG. 2: the water culture of saussurea involucrate seeds.
FIG. 3: water cultured seedlings (1 month old).
FIG. 4: water cultured seedlings (3 months old).
FIG. 5: and (4) callus induction of the explant.
FIG. 6: and (4) bud induction.
FIG. 7: and (4) bud differentiation.
FIG. 8: culturing in tissue culture bottle.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical stores. In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Example 1: dynamic water culture method for saussurea involucrate
1.1 germination of saussurea involucrate seeds
Putting saussurea involucrate seeds into a 50mL centrifuge tube, adding 200mL/L gibberellin (GA3), placing in a refrigerator at 4 ℃, processing in the dark at low temperature for 12h, spreading the processed seeds into filter paper or a culture dish, and culturing under the light, wherein the culture conditions are as follows: the illumination intensity is 2000-3000Lx, the temperature is 25 ℃, the humidity is 50-60%, and the seeds can germinate after being cultured for 3-5 days, so as to obtain the seeds (figure 1) germinated by the experimental group.
Taking saussurea involucrate seeds, putting the saussurea involucrate seeds into a 50mL centrifuge tube, placing the centrifuge tube in a refrigerator at 4 ℃, processing the seeds in the dark at low temperature for 12 hours, spreading the processed seeds into filter paper or a culture dish, and culturing the seeds under the light, wherein the culture conditions are as follows: the illumination intensity is 2000-3000Lx, the temperature is 25 ℃, the humidity is 50-60%, and the seeds can germinate after being cultured for 3-5 days, so as to obtain the germinated seeds of the control group.
And (5) counting the seed germination rates of the experimental group and the control group. The results show that: the germination rate of the control group seeds is 20 percent, and the germination rate of the experimental group seeds is 65 percent.
1.2 hydroponic liquid preparation
1.1g of MS powder (from Biochemical Co., Ltd., product No. F3150901) was weighed out and dissolved in 1L of tertiary water at pH 5.6-6.0.
1.3 hydroponic culture of seeds
Placing the germinated seeds obtained in the step 1.1 in a water culture device (the water culture device and the ceramsite are products of Taobao [ dry hydroponics ]) filled with ceramsite, placing 3-5 germinated saussurea involucrata seeds in each water culture hole (shown in figure 2), then placing the water culture device with the seeds on an illumination culture rack for culture under the conditions of illumination intensity of 2000 and 3000Lx, illumination time of 16h/d, oxygen content of more than 5mg/L, temperature of 25 ℃, humidity of 50-60% and water pump flow of 50L/h, and replacing the culture solution once for two weeks (the water culture solution in the step 1.2).
After 1 month of water culture seedling culture (water culture seedling is shown in figure 3), the flow of the water pump is adjusted to 150L/h, the culture solution is changed once a week, and the culture is continued under the same other conditions.
After 2 months of culture of the hydroponic seedlings, adjusting the flow of a water pump to 350L/h, replacing culture solution twice a week, keeping other conditions unchanged, and continuously culturing until the saussurea involucrate of 3 months old is obtained. The water culture seedling of saussurea involucrate of 3 months old is shown in figure 4.
Example 2: regeneration culture method of saussurea involucrate
2.1 explant Disinfection
Explants (leaves, petioles, roots) of the 2-month-old saussurea involucrata obtained in example 1 were taken as materials and sterilized. The disinfection process comprises the following steps: washing with tap water for 1h, washing with sterile water for 3-5 times in a clean bench, soaking in 10% NaClO for 5-10min, sterilizing with 75% alcohol for 30s, and cleaning with sterile water.
2.2 callus induction
Shearing the explant to 1cm with scissors2Size (FIG. 5), then placed on callus induction medium for callus induction culture.
Callus induction culture conditions were as follows: culturing in dark at 25 deg.C and humidity of 50-60%.
Each liter of callus induction culture medium comprises 3.32g of MS powder, 30g of sucrose, 1.5mg of NAA, 1mg of 6-BA, 4g of plant gel and 1L of water. The pH value of the callus induction culture medium is 5.9-6.0.
Each liter of callus induction culture medium' is composed of 3.32g of MS powder, 30g of sucrose, 2mg of NAA, 0.2mg of 6-BA, 4g of plant gel and 1L of water. The pH value of the callus induction culture medium' is 5.9-6.0.
After 3 weeks of culture in the callus induction medium or callus induction medium', the callus induction rate was counted. In the callus induction medium', the callus induction rates of roots, leaves and petioles were 90%, 90% and 90%, respectively. In the callus induction medium, the callus induction rates of roots, leaves and petioles were 90%, 90% and 90%, respectively.
2.3 subculture of callus
After 3 weeks in callus induction medium, the medium was changed to a new subculture medium for subculture.
The subculture medium was the same as the callus induction medium or callus induction medium' of 2.2.
Subculture conditions were the same as those in callus induction culture in 2.2.
After callus induction on subculture medium for 2-3 weeks, leaf and petioles showed clustered shoots (FIG. 6), while roots showed no clustered shoots.
The bud induction rate was counted after 3 weeks of culture in subculture medium. In the callus induction medium', the shoot induction rates of the leaf and petiole were 89% and 90%, respectively. In the callus induction medium, the shoot induction rates of the leaf and the petiole were 90% and 95%, respectively.
2.4 Cluster bud Induction and differentiation
And (4) picking out cluster buds which appear after 2-3 weeks of induction on a subculture medium, replacing the cluster buds with a new differentiation medium, and continuously culturing in the dark. The differentiation medium was the same as callus induction medium or callus induction medium' in 2.2.
Culturing in dark for 1 week, culturing in low light for 1 week under the following conditions: the illumination intensity is 500-1000Lx, the temperature is 25 ℃, and the humidity is 50-60%.
Culturing for 1 week under weak light, placing on a tissue culture rack, and culturing under normal conditions: the illumination intensity is 2500-3000Lx, the temperature is 25 ℃, and the humidity is 50-60%. After further culturing for 2-3 weeks, the cluster buds differentiated into shoots as shown in FIG. 7.
2.5 shoot Re-Induction
The tissue culture seedling of 1 month old is replaced on a bud induction culture medium, the culture is carried out for 1 to 2 weeks, the base part of the tissue culture seedling can induce new buds, and the culture is continued for 1 to 2 weeks, so that the tissue culture seedling can develop into a seedling. The culture conditions were as follows: the illumination intensity is 2500-3000Lx, the temperature is 25 ℃, and the humidity is 50-60%.
Each liter of bud induction culture medium comprises 2.21g of MS powder, 15g of sucrose, 0.5mg of 6-BA, 4g of plant gel and 1L of water. The pH value of the bud induction culture medium is 5.9-6.0.
2.6 rooting of tissue culture seedlings
And (3) replacing the tissue culture seedling which grows on the differentiation culture medium for 4-5 weeks in the step 2.4 or grows on the bud induction culture medium for 4-5 weeks in the step 2.5 on a rooting culture medium, wherein the culture conditions are as follows: the illumination intensity is 2000-3000Lx, the temperature is 25 ℃, the humidity is 50-60%, and the regeneration plant is obtained after 3 weeks of culture (figure 8).
Each liter of rooting medium consists of 2.21g of MS powder, 15g of cane sugar, 0.3mg of NAA and 1L of water. The pH value of the rooting culture medium is 5.9-6.0.
And (3) replacing the tissue culture seedling which grows on the differentiation culture medium for 4-5 weeks in the step 2.4 or grows on the bud induction culture medium for 4-5 weeks in the step 2.5 to a rooting culture medium for culturing for 3 weeks, and then counting the rooting rate. The results show that: the rooting rate of both is 60%. But compared with the method that the cluster buds obtained in the step 2.4 are directly subjected to rooting culture without bud re-induction, after the bud re-induction step in the step 2.5, 2-3 new buds can be generated at the base part of each single seedling, and the propagation coefficient is improved by 2-3 times.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A regeneration culture method of saussurea involucrate comprises the following steps:
1) soaking the saussurea involucrate seeds in gibberellin solution, and culturing to obtain germinated saussurea involucrate seeds;
2) culturing the germinated saussurea involucrate seeds in a water culture mode to obtain water culture seedlings;
3) taking the leaves or petioles of the water culture seedlings as explants to perform callus induction and differentiation culture in a callus induction culture medium to obtain regenerated seedlings;
4) and culturing the regenerated seedling in a rooting culture medium to obtain a saussurea involucrate regenerated plant.
2. The method of claim 1, wherein: in the step 1), the concentration of the gibberellin solution is 50-200 mg/L;
and/or, the soaking conditions are as follows: soaking in dark at low temperature for 12-24 hr;
and/or, the culture conditions are as follows: culturing for 3-5 days under the conditions of illumination intensity of 2000-3000Lx, temperature of 24-25 ℃ and humidity of 50-60%.
3. The method according to claim 1 or 2, characterized in that: in the step 2), the culture solution for water culture is an MS culture solution;
and/or the concentration of the MS culture solution is 1.10-4.43 g/L;
and/or the pH value of the MS culture solution is 5.6-6.0;
and/or, the water culture conditions are as follows: the illumination intensity is 2000-3000 Lx; the illumination time is 16 h/d; the temperature is 24-25 ℃; humidity is 50-60%; the oxygen content is more than 5 mg/L; the flow rate of the water pump is 50-350L/h.
4. A method according to any one of claims 1 to 3, wherein: in the step 3), the explant further comprises a disinfection step before callus induction and differentiation culture:
and/or the disinfection method sequentially comprises the following steps: tap water washing, sterile water washing, NaClO solution soaking, alcohol disinfection and sterile water washing;
and/or the time of the tap water washing is 1 h;
and/or the frequency of the sterile water washing is 3-5 times;
and/or the concentration of the NaClO solution is 10 percent;
and/or, the soaking time is 5-10 min;
and/or, the alcohol is 75% alcohol;
and/or the time for sterilization is 30 s.
5. The method according to any one of claims 1 to 4, wherein: in the step 3), the callus induction and differentiation culture method comprises the following steps:
3-1) carrying out induction culture on the explant in a callus induction culture medium to obtain callus;
3-2) carrying out subculture on the callus in a subculture medium to obtain cluster buds;
3-3) carrying out differentiation culture on the cluster buds in a differentiation culture medium to obtain regenerated seedlings;
the subculture medium and the differentiation medium are both the callus induction medium;
the callus induction culture medium comprises NAA and 6-BA.
6. The method of claim 5, wherein: the concentration of the NAA in the callus induction culture medium is 1-2 mg/L;
and/or the concentration of the 6-BA in the callus induction culture medium is 0.2-2 mg/L;
and/or, the culture conditions of 3-1) are as follows: culturing in dark at 24-25 deg.C and 50-60% humidity for 3 weeks;
and/or, the culture conditions of 3-2) are as follows: culturing in dark at 24-25 deg.C and 50-60% humidity for 2-3 weeks;
and/or, the culture conditions of 3-3) are as follows: culturing in the dark at 24-25 deg.C and 50-60% humidity for 1 week, culturing at 24-25 deg.C and 50-60% illumination intensity of 1000Lx for 1 week, and culturing at 24-25 deg.C and 50-60% humidity for 2-3 weeks at 2500-3000 Lx.
7. The method according to any one of claims 1 to 6, wherein: the step 3) or the step 4) further comprises the step of carrying out bud re-induction culture on the tissue culture seedling in a bud induction culture medium;
and/or, the shoot induction medium comprises 6-BA;
and/or the concentration of the 6-BA in the bud induction culture medium is 0.5-2 mg/L;
and/or, the conditions for the shoot re-induction culture are as follows: culturing for 4-5 weeks under the conditions of illumination intensity of 2500-;
and/or, in the step 4), the rooting medium comprises NAA;
and/or the concentration of the NAA in the rooting medium is 0.1-0.3 mg/L;
and/or, the rooting culture conditions are as follows: culturing at the illumination intensity of 2000-3000Lx, temperature of 24-25 deg.C and humidity of 50-60% for 3-5 weeks.
8. A water culture method of saussurea involucrate comprises the steps 1) and 2) of claims 1-7.
Or, a tissue culture method of saussurea involucrate, comprising the steps 3) and 4) of claims 1-7.
9. The following A1) -A3):
A1) a reagent for aqueous culture of saussurea involucrate comprising gibberellin solution and/or MS culture solution of any one of claims 1 to 7;
A2) a reagent for tissue culture of saussurea involucrate comprising the callus induction medium and/or shoot induction medium and/or rooting medium of any one of claims 1 to 7;
A3) a kit for regeneration culture of saussurea involucrate, comprising gibberellin solution and/or MS culture solution and/or callus induction medium and/or shoot induction medium and/or rooting medium according to any one of claims 1 to 7.
10. The following B1) -B4):
B1) use of the method of any one of claims 1-8 or the product of claim 9 in culturing saussurea involucrate;
B2) use of the method of any one of claims 1-8 or the product of claim 9 for propagating or expanding saussurea involucrate;
B3) the method of any one of claims 1 to 8 or the product of claim 9 for shortening the propagation time of saussurea involucrate;
B4) application of gibberellin or gibberellin solution in promoting germination of saussurea involucrate seed.
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