CN112575053A - Identification method for early bolting seeds of angelica sinensis - Google Patents
Identification method for early bolting seeds of angelica sinensis Download PDFInfo
- Publication number
- CN112575053A CN112575053A CN202011601174.5A CN202011601174A CN112575053A CN 112575053 A CN112575053 A CN 112575053A CN 202011601174 A CN202011601174 A CN 202011601174A CN 112575053 A CN112575053 A CN 112575053A
- Authority
- CN
- China
- Prior art keywords
- seeds
- angelica
- colony
- angelica sinensis
- seed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 17
- 241000382455 Angelica sinensis Species 0.000 title claims description 67
- 241000125175 Angelica Species 0.000 claims abstract description 29
- 235000001287 Guettarda speciosa Nutrition 0.000 claims abstract description 29
- 230000001580 bacterial effect Effects 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 15
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 238000005070 sampling Methods 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 238000005406 washing Methods 0.000 claims description 15
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 238000011049 filling Methods 0.000 claims description 5
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000008223 sterile water Substances 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 3
- 241000238631 Hexapoda Species 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 239000006004 Quartz sand Substances 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 229910052802 copper Inorganic materials 0.000 claims description 3
- 239000010949 copper Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 235000013399 edible fruits Nutrition 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 239000004570 mortar (masonry) Substances 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 claims description 2
- 230000002745 absorbent Effects 0.000 claims description 2
- 239000002250 absorbent Substances 0.000 claims description 2
- 238000011156 evaluation Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 12
- 239000003814 drug Substances 0.000 description 8
- 238000009395 breeding Methods 0.000 description 7
- 230000001488 breeding effect Effects 0.000 description 7
- 229940079593 drug Drugs 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 240000001810 Angelica gigas Species 0.000 description 2
- 235000018865 Angelica gigas Nutrition 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 244000275105 Angelica sylvestris Species 0.000 description 1
- 235000014429 Angelica sylvestris Nutrition 0.000 description 1
- 241000208173 Apiaceae Species 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 235000021060 food property Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 239000012499 inoculation medium Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to an identification method of early bolting seeds of angelica, which comprises the steps of a, preparing seeds for sampling; b. preparing a culture medium; c. extracting and separating and culturing angelica seed endophyte; d. and (3) identifying the bacterial colony in the angelica seeds. The method can be used for rapidly and accurately identifying early bolting seeds of angelica, identifying the truth of the seeds, protecting the application of high-quality angelica seeds in production and promoting the sustainable production of the Chinese medicinal material angelica in the genuine producing area.
Description
Technical Field
The invention relates to identification of a genuine medicinal plant angelica sinensis seed, in particular to an identification method of an early bolting seed of angelica sinensis.
Background
Chinese angelica root (Angelica sinensis (oliv.) DielsAngelica sinensis) The medicinal plant is a perennial herb medicinal plant belonging to the family Umbelliferae, has various effects of enriching blood, promoting blood circulation and the like (Chinese pharmacopoeia, 2020), is a high-quality region of angelica on the way in Gansu Mincounty, is also a country of Chinese angelica, and has annual output accounting for more than 90% of the total output in China. Chinese angelica is used as a medicinal and edible traditional Chinese medicinal material. Wild angelica in China is unevenly distributed and rare, and the medicine source is mainly a cultivated product. However, as the planting age is prolonged and global climate warming is added, the early bolting rate of angelica is increased year by yearThe land ecological area is reduced. The angelica sinensis cultivation period is 3 years, the 1 st year is a seedling cultivation period, the 2 nd year is a patent medicine cultivation period, and the 3 rd year is a seed reproduction period. And (4) normally breeding Chinese angelica mature drug roots without picking, reserving plants for overwintering, and normally bolting and seeding the regenerated plants in the 3 rd year. Early bolting, also known as immature bolting, is a phenological phenomenon that Chinese angelica does not become a drug in the drug forming period of the 2 nd year, plants bolt early, and roots lose medicinal food property due to lignification. Early bolting is not only a great obstacle to angelica production, but also increases the labor intensity of pulling out early bolting plants in the field and greatly increases the production cost. Generally, the bolting rate in the field is 10% -20% and belongs to a normal phenomenon, but at present, the bolting rate reaches 40%, even 60% -80%, the yield is almost no longer acceptable, and the cultivation effect of the angelica is seriously influenced. We investigate and find that there is a certain proportion of plant pesticides under the same cultivation conditions and complete the normal life history, which shows that there is a difference in the individual phenological conditions of Chinese angelica. Therefore, breeding of the angelica varieties with strong winterness and early bolting resistance is an effective way for relieving the early bolting problem, but at present, identification indexes are lacked, and the seed selection blindness is large. The seed, as a biological system, contains its performance characteristics. Seed contamination leads to accelerated sexual degradation. We find that the breeding capacity of early bolting species is stronger, the winter property is weak, and all late generations bolt early. The problem of early bolting is more severe by mixing commercially available angelica seeds, and the identification of early bolting characteristics from seed sources is of great importance. Endophyte (A)Endophyte) Is a microbial population in a plant body, and can generate population specificity with the same functions as a host in the long-term mutual benefit symbiosis process with the host. In the period of Chinese angelica mature drug cultivation, early bolting plants do not have any morphological signs in the early green turning stage of the field and gradually appear along with the development of individuals, which shows that the early bolting plants have close relation with the synergistic effect of the seed properties and endophytes thereof, but the research on Chinese angelica seed endophytes is not reported yet. Therefore, the identification method of the early bolting seeds of the angelica sinensis is searched from the comparative research of endophytic bacterial colonies of the early bolting seeds and the normal mature seeds of the angelica sinensis, and a scientific and effective identification method can be provided for breeding new bolting-resistant varieties of the angelica sinensis and identifying the authenticity of the angelica seeds.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for identifying early bolting seeds of angelica so as to rapidly detect early bolting seeds. The method adopts seed endophytic bacteria extraction and colony isolation culture identification, has good effect, and can quickly identify early bolting seeds of angelica.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an identification method for early bolting seeds of angelica comprises the following steps:
1. seed sampling preparation
Seed sampling: randomly sampling commercially available seeds, and removing impurities and insect seeds;
storing seeds: filling the seed samples of the step a and the step b into a paper bag, covering the paper bag with a self-sealing bag, adding absorbent paper into the paper bag, and placing the paper bag in a refrigerator at 4 ℃ for later use;
2. preparation of the Medium
Preparing a nutrient medium: accurately weighing 3g of beef extract, 10g of peptone, 5g of sodium chloride and 15g of agar, and fixing the volume to 1000ml by using distilled water;
preparing a culture dish; selecting culture dishes with consistent specifications, cleaning and drying for later use;
subpackaging and sterilizing the culture medium; subpackaging the prepared culture medium of step b, step one into culture dishes, filling 30 ml of culture medium into each dish, and carrying out autoclaving for later use;
3. extraction and separation culture of angelica sinensis seed endophyte
Seed surface sterilization: firstly, removing peel and fruit stalk of angelica sinensis seeds, washing the angelica sinensis seeds with running water, drying the angelica sinensis seeds under aseptic condition, weighing 1 g of angelica sinensis seeds, washing the angelica sinensis seeds with the running water for 10 min, placing the angelica sinensis seeds on a superclean workbench, washing the angelica sinensis seeds with the running water for 3-4 times, sterilizing the surfaces of the angelica sinensis seeds with 75% ethanol for 1 min, washing the angelica sinensis seeds with the running water for 3-4 times, fully shaking the angelica sinensis seeds with 2.5% NaClO for 10 min, then washing the angelica sinensis seeds with the running water for 3-4 times again to ensure that the surfaces of the angelica sinensis seeds are free of bacteria, and collecting the finally washed sterile water in a sterile culture dish for detecting the bacteria-carrying condition;
② extracting endophytes from angelica seeds: and (3) placing the c, the phi sterile angelica seeds into a sterile mortar under the sterile condition, adding 9.9 mL of sterile water, adding 2 g of sterile quartz sand, and grinding for 10 min. Stock solution after grindingDensity fraction of 1X 10-1And then graded and diluted to 1 × 10-2And 1X 10-3. Storing in a refrigerator at 4 ℃ for separating and culturing microorganisms;
③ separating and culturing the endophyte of the angelica seeds: the culture place should be selected from sterile environment, and inoculated by coating method. Taking an inoculation culture medium as a control, and culturing the coated culture medium in an incubator at 28 ℃;
4. identification of endophyte colony of angelica sinensis seed
Determining a colony identification period: counting the number of colonies after culturing for 7 days, counting by dishes during counting, and multiplying by the dilution times;
secondly, colony identification: the colony types are classified according to colony morphology, color, size and structure, and the richness index (richness) is calculated to reveal the richness of colony types in the biological community, the richness index (richness index,R）＝(S－1)/lnNin the formula (I), wherein,Sin order to determine the number of colonies,Nthe total number of colonies;
thirdly, comprehensive evaluation: the early bolting seeds can culture the endophytic bacteria with a large number of colonies, the arrangement is dense, and the average number of the colonies is more than 50.0 multiplied by 105 CFU g-1The same bacterial colony is various in morphology, the dominance of the copper green bacterial colony is outstanding, the bacterial colony is single in type, 10-15 bacterial colonies can be seen on a flat plate, the number of bacterial colonies growing in the normal mature Chinese angelica seed is small, the bacterial colonies are arranged and dispersed, golden yellow bacterial colonies are clear and visible, and the total number of the average bacterial colonies is less than 1.0 multiplied by 105 CFU g-1The same bacterial colony has single shape and strong specificity, but the bacterial colony has multiple types, and 20-30 bacterial colonies can be seen on the plate.
The technology has the following advantages and benefits:
by adopting the angelica seed endophyte bacterial colony separation culture identification method, the identification time can be shortened, the vernalization characteristic identification of the field seeds needs 2 years, the technology only needs about 10 d, the identification accuracy is high, the high-quality and high-efficiency production of the genuine Chinese medicinal material angelica is realized, the identification technical guarantee is provided for the breeding of new varieties of the early bolting resistant angelica, and the breeding efficiency is improved.
The specific implementation mode is as follows:
the invention relates toAnd the species of Angelica gigas nakai refers to Angelica gigas nakai of UmbelliferaeAngelica sinensis(Oliv.) Diels. The identification technology related to the application of the invention refers to the key technology for identifying the bacterial colony of the angelica sinensis.
Example 1
In the Min county angelica production area in Gansu province in 8 months in 2018, early bolting seeds in the two-year development period are collected in the field, meanwhile, normal mature seeds are collected in the three-year-old seed breeding field, and in the Chinese herbal medicine cultivation and identification laboratory in Gansu agriculture university in 10 months in 2018, microorganism separation cultivation and identification are carried out. Meanwhile, early bolting seeds and normal mature seeds are sown and raised in the Min county field in 2019, and the seeds are transplanted to the field in 2020 for patent drug cultivation. The invention is implemented according to the following steps:
1. an identification method for early bolting seeds of angelica comprises the following steps:
a. seed sampling preparation
Seed sampling: harvesting seeds of bolting plants in early biennial period and normal seed reproduction plants in third year, removing impurities and insect seeds, hanging for cooling and air drying;
storing seeds: putting the collected seed sample into a paper bag, covering the paper bag with a self-sealing bag, and placing the paper bag in a refrigerator at 4 ℃ for later use;
b. preparation of the Medium
Preparing a nutrient medium: accurately weighing 3g of beef extract, 10g of peptone, 5g of sodium chloride and 15g of agar, and fixing the volume to 1000ml by using distilled water;
preparing a culture dish; selecting culture dishes with consistent specifications, cleaning and drying for later use;
subpackaging and sterilizing the culture medium; subpackaging the prepared culture medium into ② culture dishes, filling 30 ml of culture medium into each dish, and performing autoclaving for later use;
c. extraction and separation culture of angelica sinensis seed endophyte
Seed surface sterilization: b, removing fruit stalks of angelica sinensis seeds, washing the angelica sinensis seeds clean by running water, weighing 1 g of angelica sinensis seeds after drying under aseptic conditions, washing the angelica sinensis seeds for 10 min by the running water, placing the angelica sinensis seeds on a superclean workbench, washing the angelica sinensis seeds by the running water for 3-4 times, then sterilizing the surfaces of the angelica sinensis seeds by 75% of ethanol for 1 min, washing the angelica sinensis seeds by the running water for 3-4 times, finally fully shaking the angelica sinensis seeds by 2.5% of NaClO for 10 min to achieve the effect of thorough surface sterilization, then washing the angelica sinensis seeds by the running water for 3-4 times again to ensure that the surfaces of the angelica sinensis seeds are free of bacteria, and finally collecting the washed sterile water in a sterile culture dish for detecting bacteria;
② extracting endophytes from angelica seeds: and c, placing the angelica sinensis seeds subjected to surface sterilization in the c in a sterile mortar under a sterile condition, and extracting endophytes in the angelica sinensis seeds, namely adding 9.9 mL of sterile water at first, and then adding 2 g of sterile quartz sand for grinding for 10 min. The density fraction of the stock solution after grinding was 1X 10-1And then graded and diluted to 1 × 10-2And 1X 10-3. Preparing for microbial isolation culture;
③ separating and culturing the endophyte of the angelica seeds: the coating is carried out in sequence by the coating plate method. Counting was performed plate by plate on the culture plate, and finally the average number of the 2 similar plates was multiplied by the dilution factor. Inoculation medium was used as control;
d. culture conditions
Selecting a culture place: the culture place should be selected to be in a sterile environment;
② culture conditions: culturing the coated culture medium in an incubator at 28 ℃;
e. identification of endophyte colony of angelica sinensis seed
Determining a colony identification period: counting the number of colonies after culturing for 7 d;
secondly, colony identification: the colony types are classified according to colony morphology, color, size and structure, and the richness index (richness) is calculated to reveal the richness of colony types in the biological community, the richness index (richness index,R）＝(S－1)/lnNin the formula (I), wherein,Sin order to determine the number of colonies,Nthe total number of colonies;
thirdly, comprehensive judgment: the early bolting seeds can culture the endophytic bacteria with a large number of colonies, the arrangement is dense, and the average number of the colonies is more than 50.0 multiplied by 105 CFU g-1The same bacterial colony has various morphologies, the dominance of the copper green bacterial colony is outstanding, but the bacterial colony has single type, 13 bacterial colonies can be seen on a flat plate, the bacterial colonies generated in the culture of the normal mature Chinese angelica seeds are few, the arrangement is dispersed, and the total number of the average bacterial colonies is 0.9 multiplied by 105 CFU g-1The same colony has single shape and strong specificity, but the colony types are various, and 23 colonies can be seen on the plate. The traditional identification result in the field shows that the Chinese angelica seeds undergo a seedling raising period of 1a and a medicine transplanting and cultivating period of 1a, and early bolting begins to occur in 6 months in 2020. Compared with the traditional method, the method for identifying the early bolting seeds of the angelica sinensis only takes 10 days, greatly shortens the identification time, and saves land and manpower resources.
Claims (1)
1. An identification method of early bolting seeds of angelica sinensis comprises the following steps:
a. seed sampling preparation
Seed sampling: randomly sampling commercially available seeds, and removing impurities and insect seeds;
storing seeds: filling the seed samples of the step a and the step b into a paper bag, covering the paper bag with a self-sealing bag, adding absorbent paper into the paper bag, and placing the paper bag in a refrigerator at 4 ℃ for later use;
b. preparation of the Medium
Preparing a nutrient medium: accurately weighing 3g of beef extract, 10g of peptone, 5g of sodium chloride and 15g of agar, and fixing the volume to 1000ml by using distilled water;
preparing a culture dish; selecting culture dishes with consistent specifications, cleaning and drying for later use;
subpackaging and sterilizing the culture medium; subpackaging the prepared culture medium of step b, step one into culture dishes, filling 30 ml of culture medium into each dish, and carrying out autoclaving for later use;
c. extraction and separation culture of angelica sinensis seed endophyte
Seed surface sterilization: firstly, removing peel and fruit stalk of angelica sinensis seeds, washing the angelica sinensis seeds with running water, drying the angelica sinensis seeds under aseptic condition, weighing 1 g of angelica sinensis seeds, washing the angelica sinensis seeds with the running water for 10 min, placing the angelica sinensis seeds on a superclean workbench, washing the angelica sinensis seeds with the running water for 3-4 times, sterilizing the surfaces of the angelica sinensis seeds with 75% ethanol for 1 min, washing the angelica sinensis seeds with the running water for 3-4 times, fully shaking the angelica sinensis seeds with 2.5% NaClO for 10 min, then washing the angelica sinensis seeds with the running water for 3-4 times again to ensure that the surfaces of the angelica sinensis seeds are free of bacteria, and collecting the finally washed sterile water in a sterile culture dish for detecting the bacteria-carrying condition;
② extracting endophytes from angelica seeds: under the aseptic condition, putting the aseptic angelica seeds of the step c, the step c into an aseptic mortar, adding 9.9 mL of aseptic water, adding 2 g of aseptic quartz sand, and grinding for 10 min; the density fraction of the ground stock solution is 1 x 10-1, and the ground stock solution is graded and diluted to 1 x 10-2 and 1 x 10-3; storing in a refrigerator at 4 ℃ for separating and culturing microorganisms;
③ separating and culturing the endophyte of the angelica seeds: selecting a sterile environment at a culture place, inoculating by using a coating method, taking an inoculation culture medium as a control, and culturing the coated culture medium in an incubator at 28 ℃;
d. identification of endophyte colony of angelica sinensis seed
Determining a colony identification period: counting the number of colonies after culturing for 7 days, counting by dishes during counting, and multiplying by the dilution times;
secondly, colony identification: classifying colony types according to colony morphology, color, size and structure, and calculating a richness index (richness index) to reveal the richness degree of the colony types in the biological community, wherein S is a certain colony number, and N is the total number of the colonies;
thirdly, comprehensive evaluation: the early bolting seed can cultivate endogenetic bacterial colony quantity many, arrange densely, the average colony number is greater than 50.0X 105 CFU g-1, the colony morphology of the same kind is various, the copper green colony dominance is outstanding, but the colony kind is single, the dull and stereotyped visible 10 ~ 15 colonies, and the normal ripe chinese angelica seed can cultivate endogenetic bacterial colony quantity few, arrange dispersedly, golden yellow colony is clear visible, the average bacterial colony total number is less than 1.0X 105 CFU g-1, the colony morphology of the same kind is single, the specificity is strong, but the colony kind is many, the dull and stereotyped visible 20 ~ 30 colonies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011601174.5A CN112575053A (en) | 2020-12-30 | 2020-12-30 | Identification method for early bolting seeds of angelica sinensis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011601174.5A CN112575053A (en) | 2020-12-30 | 2020-12-30 | Identification method for early bolting seeds of angelica sinensis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112575053A true CN112575053A (en) | 2021-03-30 |
Family
ID=75144189
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011601174.5A Pending CN112575053A (en) | 2020-12-30 | 2020-12-30 | Identification method for early bolting seeds of angelica sinensis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112575053A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114586497A (en) * | 2022-03-15 | 2022-06-07 | 岷县大河追溯农产品基地有限公司 | Angelica sinensis seed storage method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150230478A1 (en) * | 2013-02-05 | 2015-08-20 | University Of Saskatchewan | Endophytic Microbial Symbionts in Plant Prenatal Care |
| US20150335029A1 (en) * | 2012-06-22 | 2015-11-26 | Ait Austrian Institute Of Technology Gmbh | Method for Producing Plant Seed Containing Endophytic Micro-Organisms |
-
2020
- 2020-12-30 CN CN202011601174.5A patent/CN112575053A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150335029A1 (en) * | 2012-06-22 | 2015-11-26 | Ait Austrian Institute Of Technology Gmbh | Method for Producing Plant Seed Containing Endophytic Micro-Organisms |
| US20150230478A1 (en) * | 2013-02-05 | 2015-08-20 | University Of Saskatchewan | Endophytic Microbial Symbionts in Plant Prenatal Care |
Non-Patent Citations (2)
| Title |
|---|
| TAO LU等: "Rhizosphere microorganisms can influence the timing of plant flowering", MICROBIOME, vol. 6, pages 1 - 12, XP021265986, DOI: 10.1186/s40168-018-0615-0 * |
| 江曙;段金廒;严辉;于光;: "当归根际微生物种群结构与生态分布的研究", 中国中药杂志, no. 12, pages 1483 - 1488 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114586497A (en) * | 2022-03-15 | 2022-06-07 | 岷县大河追溯农产品基地有限公司 | Angelica sinensis seed storage method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101061781A (en) | Chinese truffle and India truffle mycorrhiza fungi seeding cultivating method | |
| CN111763629B (en) | Bacillus belgii and application thereof | |
| CN110468057B (en) | Endophytic pestalotiopsis fungus M7SB41 and application thereof | |
| CN104164472B (en) | A kind of indoor appraising method of eggplant Resistance to brown streak disease | |
| CN115537346B (en) | Mucillus mucilaginosus for promoting growth and differentiation of sansevieria trifasciata and application thereof | |
| CN105039181A (en) | Metarhizium anisopliae MAYX130921 and application thereof | |
| CN113604363B (en) | Alternaria solani and application thereof | |
| CN109628550B (en) | Screening method of anti-fusarium wilt varieties of ground-cover chrysanthemum | |
| CN112575053A (en) | Identification method for early bolting seeds of angelica sinensis | |
| US20210120749A1 (en) | Artificial inoculation method for bacterial panicle blight of rice | |
| CN111733083B (en) | New endophytic fungus strain PG2 and application thereof | |
| CN109804913B (en) | Method for constructing aegilops tauschii-endophytic fungi symbiont | |
| CN111748603A (en) | Artificial inoculation identification method for stem basal rot resistance of corn variety | |
| CN108076962B (en) | Artificial cultivation method of Thelephora ganbajun zang | |
| CN103205501A (en) | Method for identifying rice blast-resistant gene of wild rice | |
| CN108148768B (en) | Tortomium globosum strain and application thereof | |
| CN110804556B (en) | Endophytic fungus LZ3 and application thereof | |
| CN111304097A (en) | Wild pleurotus ferulae strain separation and purification method | |
| CN109699495A (en) | A method of improving scythian lamb rhizome spore germination rate | |
| Fares et al. | Isolation and diagnosis of the pathological cause of dieback disease in orange Citrus sinensis tree In Baghdad | |
| Yan et al. | Simulation of the spreading trend of Camellia oleifera Anthracnose in Guangdong Province | |
| CN117187083B (en) | Photosensitive hericium erinaceus strain suitable for industrialization and cultivation method thereof | |
| CN112940994B (en) | Bacillus subtilis, culture method and application | |
| CN111567179B (en) | Method for promoting direct seeding germination of bletilla striata seeds and germination agent | |
| CN111440729B (en) | Application and method of dendrobium officinale endophytic fungi in seed seedling culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210330 |