CN117770140B - Tissue culture and breeding method for gastrodia elata - Google Patents
Tissue culture and breeding method for gastrodia elata Download PDFInfo
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- 241000305491 Gastrodia elata Species 0.000 title claims abstract description 54
- 238000009395 breeding Methods 0.000 title claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 64
- 244000221226 Armillaria mellea Species 0.000 claims abstract description 40
- 235000011569 Armillaria mellea Nutrition 0.000 claims abstract description 40
- 230000001954 sterilising effect Effects 0.000 claims abstract description 10
- 239000000284 extract Substances 0.000 claims abstract description 9
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- 239000008103 glucose Substances 0.000 claims description 11
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- 239000001888 Peptone Substances 0.000 claims description 8
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 4
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a tissue culture and breeding method of gastrodia elata, and relates to the technical field of tissue culture of gastrodia elata. The tissue culture and breeding method of gastrodia elata mainly comprises the steps of obtaining gastrodia elata explant, circularly sterilizing the explant, adding culture medium armillaria mellea extract, inoculating activated armillaria mellea during culture and the like. The invention overcomes the defects of the prior art, effectively improves the growth efficiency of tissue breeding of the gastrodia elata, and reduces the subsequent experimental time for experimental study of the gastrodia elata, thereby ensuring the continuity of the whole experiment, accelerating the experimental progress and reducing the investment of the experiment.
Description
Technical Field
The invention relates to the technical field of tissue culture of gastrodia elata, in particular to a tissue culture and breeding method of gastrodia elata.
Background
Gastrodia elata is perennial parasitic herbaceous plant of Gastrodia genus of Orchidaceae family, and is used as a medicine with dry tuber, is a common rare Chinese medicinal material in China, has effects of calming endogenous wind, relieving spasm, suppressing liver yang, dispelling pathogenic wind, and dredging collaterals, and can be used for treating infantile convulsion, epilepsia, tetanus, headache, dizziness, hand and foot paralysis, limb numbness, rheumatalgia, etc. The Chinese medicinal materials are firstly carried in Shennong Ben Cao Jing and are parallel to the Chinese medicinal materials, have medicinal history of over 2000 in China, and are also health-care food materials which are favored by modern people.
Modern research on planting of gastrodia elata has begun to go deep into genomics, and different varieties of gastrodia elata in different areas finally cause differences of nutritional ingredients and beneficial effects in the gastrodia elata, and the key genes of biosynthesis paths of traditional Chinese medicine ingredients in the gastrodia elata are researched and verified by sequencing of gastrodia elata transcriptome through going deep into genomics.
In the research direction, homologous hemp with consistent genetic characteristics and close quality is often required to be selected for detection, the detection is usually required to be planted in a test way in 3 months each year, liquid nitrogen is frozen for preservation after picking in 11-12 months in the following year, the whole planting period is long, and different gastrodia elata are greatly influenced by environment and weather in the planting process, so that the operation of a laboratory is complicated, and the experimental effect is also easily influenced.
Tissue culture is a vegetative propagation technology of plants, a complete individual can be bred through induction of one tissue of the plants, for a transcriptome sequencing experiment of gastrodia elata, the supply stability of raw materials can be effectively improved, the influence of external environment is reduced, and meanwhile, the whole experimental period is shortened, but the current culture period of gastrodia elata tissue culture is mostly about 60 days, the whole time consumption is long, the gastrodia elata tissue culture is not required to be completed in a laboratory for sequencing the gastrodia elata, and experiments can be put into only by breeding larger tubers, so that the experimental efficiency can be effectively improved by designing a rapid gastrodia elata tissue culture method.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the tissue culture breeding method for the gastrodia elata, which effectively improves the growth efficiency of tissue breeding of the gastrodia elata and reduces the experimental time for carrying out subsequent experimental study on the gastrodia elata, thereby ensuring the continuity of the whole experiment, accelerating the experimental progress and reducing the investment of the experiment.
In order to achieve the above object, the technical scheme of the present invention is realized by the following technical scheme:
a tissue culture and breeding method of gastrodia elata, which comprises the following steps:
(1) Obtaining a gastrodia elata explant: brushing rhizoma Gastrodiae with clear water to remove surface sediment, repeatedly washing with ice water, and cutting into small pieces of 0.5-1.0cm to obtain rhizoma Gastrodiae explant;
(2) Sterilization of explants: soaking the gastrodia elata explant in 75% alcohol for 10s, soaking in 1% mercuric chloride for 3min, repeating the alcohol soaking and mercuric chloride soaking operations for 2-3 times, washing with sterile water at 45-50 ℃ in a water bath for 2-3 times, continuing washing with normal-temperature sterile water for 1-2 times, and sucking water to obtain a sterilized explant for later use;
(3) Preparation of the culture medium: selecting a culture medium of MS+zein 0.5 mg/L+naphthylacetic acid 0.1 mg/L+armillaria mellea extract 80g/L to obtain a preparation culture medium;
(4) Tissue culture: inoculating activated Armillariella mellea on the surface of the sterilized explant, transferring to the prepared culture medium, and culturing at constant temperature in darkroom to obtain rhizoma Gastrodiae tissue culture product.
Preferably, the number of repeated rinsing with ice water in the step (1) is 3-5.
Preferably, the tall gastrodia tuber explant in the step (1) is selected as a tall gastrodia tuber stem tip or a stem body cutting block.
Preferably, in the step (2), the explant is subjected to moisture adsorption drying treatment by using sterile paper.
Preferably, the preparation method of the armillaria mellea extract comprises the following steps:
s1-1, preparing a solid culture medium: adding 5g of yeast extract, 10 g g of glucose, 3g of peptone and 10 g agar into a beaker, and cooling the plate after autoclaving to obtain a solid culture medium;
S1-2, preparing a liquid culture medium: 10g/L of yeast extract, 20g/L of glucose, 6g/L of peptone and the balance of water, and performing high-pressure sterilization for later use;
S1-3, inoculating armillaria mellea spores in a solid culture medium, performing activation culture, inoculating the armillaria mellea spores into a liquid culture medium for culture propagation after the armillaria mellea spores grow vigorously, and taking a liquid matrix of the liquid culture medium for autoclaving after the armillaria mellea spores grow vigorously to obtain an armillaria mellea extract.
Preferably, the solid culture medium and the liquid culture medium in the step S1-3 are cultivated in dark room at constant temperature of 25 ℃ and cut into 0.5cm square blocks and placed in 100ml of liquid culture medium after hypha grows fully in the solid culture medium, and then the culture is continued for 2-3d.
Preferably, the obtaining method of the activated armillaria mellea in the step (4) includes the following steps:
S2-1, preparing a solid culture medium: adding 5g of yeast extract, 10 g g of glucose, 3g of peptone and 10 g agar into a beaker, and cooling the plate after autoclaving to obtain a solid culture medium;
S2-2, preparing a liquid shake flask culture medium: 200g/L of potato, 20g/L of glucose, 3g/L of monopotassium phosphate, 1.5g/L, VB 1 mg/L of magnesium sulfate heptahydrate and the balance of water;
S2-3, inoculating armillaria mellea spores in a solid culture medium, performing activation culture, and inoculating the armillaria mellea spores into a liquid shake flask culture medium for culture propagation after the armillaria mellea spores grow vigorously to obtain activated armillaria mellea.
Preferably, after the hyphae in the solid medium grow fully in the step S2-3, the solid medium is cut into 0.5cm squares and placed in 100ml of liquid shake flask medium.
Preferably, the culture in the step S2-3 is carried out in a solid culture medium, an activation culture medium and a liquid shake flask culture medium at a constant temperature of 25+/-2 ℃ in the dark, and the shake flask culture medium also needs to be subjected to shake flask treatment at a speed of 120 r/min.
Preferably, the temperature of the culture in the step (4) is 20.+ -. 2 ℃.
The invention provides a tissue culture breeding method of gastrodia elata, which has the advantages compared with the prior art that:
(1) The invention adopts ice water flushing when taking the gastrodia elata explant, simultaneously stimulates the gastrodia elata tissue through subsequent warm water flushing, promotes the subsequent higher differentiation activity of the tissue, promotes the tissue culture efficiency, and can effectively ensure the sterilization effect and promote the survival rate of the subsequent tissue culture of the gastrodia elata explant and prevent the browning death of the gastrodia elata tissue culture through the cyclic treatment of alcohol and mercuric chloride.
(2) According to the invention, the armillaria mellea extracting solution is added into the tissue culture medium, and the activated armillaria mellea is inoculated to the explant, so that the differentiated growth of the gastrodia elata tissue can be effectively ensured, meanwhile, death of the gastrodia elata explant caused by overgrowth of the armillaria mellea is prevented, the tissue culture efficiency of the whole gastrodia elata is improved, the gastrodia elata explant can be rapidly cultivated and maintained to grow into large and small stem blocks for laboratory detection, the experimental period is shortened, and the laboratory detection convenience is improved.
Drawings
FIG. 1 is a schematic diagram of the experimental group 1 explant culture 0d in the example of the present invention;
FIG. 2 is a schematic diagram of the experimental group 1 explant culture 5d in the example of the present invention;
FIG. 3 is a schematic representation of the culture 15d of the explant of experimental group 1 in the example of the present invention;
FIG. 4 is a schematic representation of the culture 30d of the explant of experimental group 1 in the example of the present invention;
FIG. 5 is a schematic representation of experimental group 2 explant culture 0d in an example of the present invention;
FIG. 6 is a schematic diagram of experimental group 2 explant culture 5d in an example of the present invention;
FIG. 7 is a schematic representation of experimental group 2 explant culture 15d in an example of the present invention;
FIG. 8 is a schematic representation of experimental group 2 explant culture 30d in an example of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples:
1. material preparation:
1-1, preparation of explants: digging fresh Gastrodia elata Blume planted in Zhaotong Yiliang small dam country (road and land producing area) for 6 months, brushing the surface to remove sediment, washing with clear water, cutting the stem into small pieces of 0.5-1.0cm, serving as an explant, sterilizing the explant, washing with sterile water, and finally absorbing surface moisture with sterile paper to obtain the sterilized explant.
And specific preparation methods of sterilized explants are shown in table 1 below:
table 1: preparation method of sterilized explant
1-2, Preparation of a culture medium:
① Preparation of medium a: MS+zeatin 0.5 mg/L+naphthylacetic acid 0.1 mg/L+Armillaria mellea extract 80g/L;
② Preparation of medium B: MS+zeatin 0.5 mg/L+naphthylacetic acid 0.1 mg/L;
③ Solid medium: 5g of yeast extract, 10 g g of glucose, 3g of peptone and 10 g agar are added into a beaker, and after being autoclaved at 121 ℃ for 30min, a plate is cooled down to obtain a solid culture medium;
④ Liquid medium: 10g/L yeast extract, 20g/L glucose, 6g/L peptone and the balance water, and performing high-pressure sterilization at 121 ℃ for 30min and cooling to obtain a liquid culture medium;
⑤ Liquid shake flask medium: 200g/L of potato, 20g/L of glucose, 3g/L of monopotassium phosphate, 1.5g/L, VB 1 mg/L of magnesium sulfate heptahydrate and the balance of water, and sterilizing at 121 ℃ for 30min, and cooling to obtain the liquid shake flask culture medium.
Wherein the content of each element in MS is: ammonium nitrate (NH 4NO3) 1650mg/L, potassium nitrate (KNO 3) 1900mg/L, magnesium sulfate heptahydrate (MgSO 4·7H2 O) 370mg/L, potassium dihydrogen phosphate (KH 2PO4) 170mg/L, calcium chloride (CaCl 2·2H2 O) 440mg/L, manganese sulfate (MnSO 4·4H2 O) 22.3mg/L, zinc sulfate (ZnSO 4·7H2 O) 8.6mg/L, boric acid (H 3BO3) 6.2mg/L, potassium iodide (KI) 0.83mg/L, sodium molybdate (Na 2MoO4·2H2 O) 0.25mg/L, copper sulfate (CuSO 4·5H2 O) 0.025mg/L, cobalt chloride (CoCl 2·6H2 O) 0.025mg/L, iron sulfate (FeSO 4·7H2 O) 27.8mg/L,37.3Mg/L glycine 0.025mg/L thiamine hydrochloride 0.4mg/L pyridoxine hydrochloride 0.5mg/L nicotinic acid 0.5mg/L inositol 100mg/L.
2. Culturing Armillariella mellea:
Inoculating Armillariella mellea spores into the solid culture medium, culturing at a constant temperature at a dark place and 25 ℃ until mycelia in the solid culture medium are full, cutting into 0.5cm square blocks, placing into 100ml liquid shake flask culture medium, and continuously culturing at a dark place and 25 ℃ for 3d at a speed of 120r/min to obtain activated Armillariella mellea liquid;
3. preparation of armillaria mellea extract:
Inoculating Armillaria mellea spores into a solid culture medium, culturing at constant temperature in the absence of light at 25deg.C until mycelia grow fully in the solid culture medium, cutting into 0.5cm square blocks, placing into 100ml liquid culture medium, culturing at constant temperature in the absence of light at 25deg.C for 3d, taking out liquid in the liquid culture medium, sterilizing at 121deg.C under high pressure, and cooling to obtain Armillariella mellea extractive solution.
4. Tissue culture:
dipping the gastrodia elata explant in an activated armillaria mellea bacterial solution, inoculating the gastrodia elata explant onto a preparation culture medium, and carrying out tissue culture at a constant temperature of 20 ℃ in dark room.
5. And (3) experimental verification:
(1) Detecting survival effects of explants with different treatment modes, and respectively carrying out tissue culture on the sterilized explant A, the sterilized explant B, the sterilized explant C and the sterilized explant D according to the method under the same condition by adopting a preparation culture medium A as a unified culture medium, wherein each group of sterilized explants is cultured for 50 groups, and the explants with browning deactivation after 15D culture are observed and recorded, wherein the specific results are shown in the following table 2:
table 2: number of deaths after culture of different sterilized explants 15d
As can be seen from table 2 above, the use of ice water rinse and 48 ℃ sterile water rinse resulted in a somewhat increased number of deaths (comparison of sterilized explant a and sterilized explant D), but the cyclic treatment of alcohol soak and mercuric chloride soak effectively prevented the death of the explants treated with ice water rinse and 48 ℃ sterile water rinse, whereas the cyclic treatment of alcohol soak and mercuric chloride soak had little effect on the survival of the explants rinsed with normal temperature clear water rinse and normal temperature sterilized water rinse.
(2) The growth of explants under different culture conditions of the preparation medium was examined, namely sterilized explant A was selected as explant, and the following culture medium and culture conditions were used for treatment culture:
table 3: different treatment culture conditions of sterilized explant A
Mass change detection (calculated as living body only) was performed on explants cultured in the above experimental groups 1 to 4 (30 per group) in cultures 5d, 15d, 30d, and the specific results are shown in table 4 below:
Table 4: weight change of explants cultured in Experimental groups 1-4
From the table, the tissue culture is performed by adopting the mode of the experimental group 1, so that the growth of the large-amplitude weight increment can be realized within 30 days, and the detection requirement of a laboratory can be met.
(3) After different sterilized explants were examined and light dip activated armillaria mellea bacteria liquid, the culture was performed at 20 ℃ in a prepared medium a under the condition of avoiding light, namely, tissue culture was performed by using sterilized explant a, sterilized explant B, sterilized explant C and sterilized explant D in the above manner, each sterilized explant was subjected to tissue culture for 30 groups, each sterilized explant was subjected to culture for 30D, the weight change of each group of explants was calculated (calculated as living body only), and the following table 5 was recorded:
table 5: the different explants were cultured in the same way for 30d weight change
In summary, with the results shown in tables 2 and 5, when the difference in survival rate was small, the whole culture efficiency of sterilized explant A was the highest.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; while the invention has been described in detail with reference to the foregoing embodiments, it will be appreciated by those skilled in the art that variations may be made in the techniques described in the foregoing embodiments, or equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The tissue culture and breeding method of gastrodia elata is characterized by comprising the following steps of:
(1) Obtaining a gastrodia elata explant: brushing rhizoma Gastrodiae with clear water to remove surface sediment, repeatedly washing with ice water, and cutting into small pieces of 0.5-1.0cm to obtain rhizoma Gastrodiae explant;
(2) Sterilization of explants: soaking the gastrodia elata explant in 75% alcohol for 10s, soaking in 1% mercuric chloride for 3min, repeating the alcohol soaking and mercuric chloride soaking operations for 2-3 times, washing with sterile water at 45-50 ℃ in a water bath for 2-3 times, continuing washing with normal-temperature sterile water for 1-2 times, and sucking water to obtain a sterilized explant for later use;
(3) Preparation of the culture medium: selecting a culture medium of MS+zein 0.5 mg/L+naphthylacetic acid 0.1 mg/L+armillaria mellea extract 80g/L to obtain a preparation culture medium;
(4) Tissue culture: inoculating activated Armillariella mellea on the surface of the sterilized explant, transferring to the prepared culture medium, and culturing at constant temperature in darkroom to obtain rhizoma Gastrodiae tissue culture product.
2. The tissue culture and propagation method of gastrodia elata according to claim 1, which is characterized in that: the number of times of repeated flushing of the ice water in the step (1) is 3-5 times.
3. The tissue culture and propagation method of gastrodia elata according to claim 1, which is characterized in that: in the step (1), the gastrodia elata explant is selected as a gastrodia elata stem tip or a stem body cutting block.
4. The tissue culture and propagation method of gastrodia elata according to claim 1, which is characterized in that: and (3) performing moisture adsorption drying treatment on the explant by adopting sterile paper in the step (2).
5. The tissue culture and propagation method of gastrodia elata according to claim 1, which is characterized in that: the preparation method of the armillaria mellea extract comprises the following steps:
s1-1, preparing a solid culture medium: adding 5g of yeast extract, 10 g g of glucose, 3g of peptone and 10 g agar into a beaker, and cooling the plate after autoclaving to obtain a solid culture medium;
S1-2, preparing a liquid culture medium: 10g/L of yeast extract, 20g/L of glucose, 6g/L of peptone and the balance of water, and performing high-pressure sterilization for later use;
S1-3, inoculating armillaria mellea spores in a solid culture medium, performing activation culture, inoculating the armillaria mellea spores into a liquid culture medium for culture propagation after the armillaria mellea spores grow vigorously, and taking a liquid matrix of the liquid culture medium for autoclaving after the armillaria mellea spores grow vigorously to obtain an armillaria mellea extract.
6. The tissue culture and propagation method of gastrodia elata according to claim 5, which is characterized in that: the solid culture medium and the liquid culture medium in the step S1-3 are cultivated in dark room at constant temperature of 25 ℃ and cut into 0.5cm square blocks and placed in 100ml of liquid culture medium after hypha grows fully in the solid culture medium, and then the culture is continued for 2-3d.
7. The tissue culture and propagation method of gastrodia elata according to claim 1, wherein the obtaining method of the activated armillaria mellea in the step (4) comprises the following steps:
S2-1, preparing a solid culture medium: adding 5g of yeast extract, 10 g g of glucose, 3g of peptone and 10 g agar into a beaker, and cooling the plate after autoclaving to obtain a solid culture medium;
S2-2, preparing a liquid shake flask culture medium: 200g/L of potato, 20g/L of glucose, 3g/L of monopotassium phosphate, 1.5g/L, VB 1 mg/L of magnesium sulfate heptahydrate and the balance of water;
S2-3, inoculating armillaria mellea spores in a solid culture medium, performing activation culture, and inoculating the armillaria mellea spores into a liquid shake flask culture medium for culture propagation after the armillaria mellea spores grow vigorously to obtain activated armillaria mellea.
8. The tissue culture and propagation method of gastrodia elata according to claim 7, wherein the tissue culture and propagation method is characterized in that: after hyphae in the solid culture medium grow fully in the step S2-3, cutting into 0.5cm square blocks, and placing the square blocks in 100ml of liquid shake flask culture medium.
9. The tissue culture and propagation method of gastrodia elata according to claim 7, wherein the tissue culture and propagation method is characterized in that: the mode of culturing in the solid culture medium, the activation culture medium and the liquid shake flask culture medium in the step S2-3 is light-proof constant temperature culture at 25+/-2 ℃, and the shake flask culture medium is also required to be subjected to shake flask treatment at the speed of 120 r/min.
10. The tissue culture and propagation method of gastrodia elata according to claim 1, which is characterized in that: the temperature of the culture in the step (4) is 20+/-2 ℃.
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