CN107699531A - A kind of hippophae plant Frankia isolated culture method - Google Patents
A kind of hippophae plant Frankia isolated culture method Download PDFInfo
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- CN107699531A CN107699531A CN201711258256.2A CN201711258256A CN107699531A CN 107699531 A CN107699531 A CN 107699531A CN 201711258256 A CN201711258256 A CN 201711258256A CN 107699531 A CN107699531 A CN 107699531A
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- endophyte
- hippophae
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- root
- nodule
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
Abstract
The invention discloses a kind of hippophae plant Frankia isolated culture method, it is after carrying out surface sterilization to Hippophae nodule sample, using root nodule microtomy and flat board partition method, seabuckthorn root leachate is separately added on different culture mediums, carries out being separately cultured for variety classes Hippophae nodule endophyte.Research shows, the culture medium nutrition added when Hippophae nodule endogenetic bacteria separates, in culture medium after seabuckthorn root leachate is more abundant, is more suitable for the growth of endophyte.During endophyte is separated, the faster bacterium lawn of growth is paved with around root nodule section quickly, hinders the growth for growing slower bacterium and actinomyces, especially frankia；And in the expansion culture of later stage bacterial strain, the growth of bacterial strain can also be remarkably promoted by adding the culture medium of seabuckthorn root leachate, the type and quantity of Frankia bacterial strain can be cultivated by significantly increasing, and obtain more endophyte resources, and can shorten the time that endophyte is separately cultured.
Description
Technical field
The present invention relates to a kind of isolated culture method of plant root nodule endophyte, more particularly to a kind of hippophae plant root nodule
The isolated culture method of endophyte, belong to microorganism and be separately cultured technical field.
Background technology
Endophyte of plant（Endophyte）It is the tissue and organ that health plant is moved in certain phase or whole stages
An internal big quasi-microorganism, they can form the relation such as parasitism, symbiosis, saprophytic with plant.Research is found, not only from each plantation
Can be with isolated endophyte in thing, and have endophyte in the root in same plant, stem, leaf, flower, fruit and seed etc.
It is found.Endophyte substantial amounts, huge number, including endogenetic fungus, endogenetic bacteria and endogeny rayungus etc..Endophyte of plant
With abundant bio-diversity, for a kind of plant, the endogenetic fungus or bacterium therefrom separated is usually several
To tens kinds, some is even up to hundreds of kinds.Endophyte of plant is also a kind of new microorganism money with potential using value
Source, new bioactive substance is found from endophyte of plant turns into the focus of research.
Sea-buckthorn is a kind of shrub or arbor, is the actinomyces dross plant of Elaeangnaceae Hippophne.There is scholar's use to cultivate
The root of the wild sea-buckthorn health of technique study, stem, the endophyte in leaf texture, it is found that it is abundant various Endophytes from Hippophae has
Property.In order to verify in Hippophae nodule with the presence or absence of various endophyte, it is necessary to deploy further resource investigation, collection is as far as possible
The bacterial strain in more different hosts sources, the form of accumulating and enriching, Physiological and biochemical index, foundation and abundant hippophae plant root nodule
Endophyte strain resource library database.
For the multifarious research of endophyte, traditional method is mainly microbe culture technique, from specific sample
The pure culture of microorganism is obtained by separating, cultivating etc. in product, then carries out the taxonomic identification of bacterial strain.But due to culture medium and certainly
Nutrition between right environment differs greatly, so original condition of Hippophae nodule endophyte habitat can not be simulated, a large amount of micro- lifes
Thing can not be cultivated.PDA culture medium, beef extract-peptone is respectively adopted when being separately cultured Hippophae nodule endophyte in conventional method
Culture medium and Gao Shi I culture mediums etc., the endophyte type and quantity being separated on flat board are less, it is impossible to which fully reflection is whole
The value volume and range of product of plant endophyte.
The content of the invention
The purpose of the present invention is a kind of isolated culture method of hippophae plant Frankia, is desirably to obtain substantial amounts of bacterium
Kind resource, material is provided for further development endophyte correlative study later.
Hippophae nodule size, color and shape etc. all can because of the difference of sea-buckthorn species, and sea-buckthorn growth habitat and when
Between difference and be varied from.Therefore, fresh tender Hippophae nodule is chosen, after carrying out surface sterilization to Hippophae nodule sample,
Using root nodule microtomy and flat board partition method, the separation training of variety classes Hippophae nodule endophyte is carried out on different culture mediums
Support.Its concrete technology is as follows：
（1）Surface sterilization is carried out to Hippophae nodule sample：Standby Hippophae nodule sample is taken, is rushed successively with the running water of flowing
Wash, and after scrubbing off the impurity such as silt with writing brush, with aseptic water washing 3 ~ 5 times, with 75% 8 ~ 10min of ethanol surface sterilization, with 1%
NaClO solution surfaces sterilize 5 ~ 7min, then with aseptic water washing 3 ~ 5 times（Hippophae nodule sample table is kept away during whole operation
Face is damaged）.
（2）Endophyte culture of isolated：Sterile-processed Hippophae nodule is cut into slices, is respectively placed in culture medium on different flat boards
On, using different culture mediums, and seabuckthorn root leachate is added, carry out sterile culture, cultivation temperature is 25 ~ 30 DEG C, during culture
Between be 3 ~ 7d；Endogenetic fungus uses PDA culture medium, and endogenetic bacteria uses beef-protein medium, and endogeny rayungus uses
Gao Shi I culture mediums.The volume ratio of culture medium and seabuckthorn root leachate is 1:15~1:20.
The preparation method of above-mentioned seabuckthorn root leachate：Seabuckthorn root is cleaned and shredded, 1 is pressed with distilled water:1~1:1.5 volume
Than mixing, 0.5 ~ 1h is cooked by slow fire after boiling, filters, takes clear liquid, 121 DEG C of sterilizings store for future use.
（3）The purifying of endophyte：Fresh flat board is forwarded to after tissue block edge grows bacterium colony, is purified by ruling,
Produce endophyte single bacterium colony.
The present invention has advantages below compared with the prior art：Added when Hippophae nodule endophyte separates, in culture medium husky
Culture medium after spine root leachate is more nearly the prototroph condition of endophyte growth, is more suitable for the growth of endophyte.Dividing
During endophyte, the faster endophyte lawn of growth is paved with around root nodule section quickly；And in the expansion of later stage bacterial strain
In big culture, the growth of bacterial strain can also be remarkably promoted by adding the culture medium of seabuckthorn root leachate, and root nodule can be cultivated by significantly increasing
The type and quantity of endophyte bacterial strain, more endophyte resources are obtained, and the time that endophyte is separately cultured can be shortened.
Brief description of the drawings
Fig. 1 is the Hippophae nodule endophyte culture figure for not adding seabuckthorn root leachate（It is left）And the sand of addition seabuckthorn root leachate
Spine Frankia culture figure（It is right）.
Embodiment
The method and effect of Hippophae nodule endophyte culture of isolated of the present invention are done further below by specific embodiment
Explanation.
Embodiment 1
（1）Surface sterilization is carried out to Hippophae nodule sample
Fresh tender Hippophae nodule is chosen, surface sterilization is carried out to Hippophae nodule sample：Standby Hippophae nodule sample is taken, according to
The secondary running water with flowing rinses, and after scrubbing off the impurity such as silt with writing brush, with aseptic water washing 3 ~ 5 times, with 75% ethanol table
Face sterilizes 8 ~ 10min, 5 ~ 7min is sterilized with 1%NaClO solution surfaces, then with aseptic water washing 3 ~ 5 times（In whole operation process
In keep away Hippophae nodule sample surfaces breakage）.
（2）Endophyte is separately cultured
The preparation of seabuckthorn root leachate：Seabuckthorn root is cleaned and shredded, 1 is pressed with distilled water:1 volume ratio is mixed, and slow fire is used after boiling
About 1h is boiled, filters, takes clear liquid, 121 DEG C of sterilizings store for future use.
Endophyte is separately cultured：Root nodule is cut into 1~2mm thin slices with sterile scalpel, and is inserted into three kinds of differences
Agar medium on, 25 DEG C of cultures, after knurl piece grows thalline, it chosen to be connected on flat board with transfer needle be further purified.
Endogenetic fungus uses PDA culture medium, and seabuckthorn root leachate is added in PDA culture medium（Culture medium soaks with seabuckthorn root
The volume ratio for going out liquid is 1:20）, flat board is placed at 25 DEG C and cultivates 7d.
Endogenetic bacteria uses beef-protein medium, and seabuckthorn root leachate is added in beef-protein medium
（The volume ratio of culture medium and seabuckthorn root leachate is 1:20）, flat board is placed at 25 DEG C and cultivates 7d.
Endogeny rayungus uses Gao Shi I culture mediums, adds seabuckthorn root leachate in the medium（Culture medium and seabuckthorn root
The volume ratio of leachate is 1:20）, flat board is placed at 25 DEG C and cultivates 7d.
Control group：Seabuckthorn root leachate is not added with all plating mediums, carries out the separation of endophyte under the same conditions
Culture.
（3）The purifying of endophyte：Fresh flat board is forwarded to after tissue block edge grows bacterium colony, is purified by ruling,
Then observe under the microscope and detect its purity, until obtaining endophyte single bacterium colony, inclined-plane saves backup.
The bacterium colony kind for judging to isolate according to features such as colonial morphology, color, rim condition, transparency, surface humidity
Class simultaneously counts.It the results are shown in Table 1.
Embodiment 2
（1）Surface sterilization is carried out to Hippophae nodule sample：With embodiment 1.
（2）Endophyte is separately cultured：Endophyte is separately cultured：It is thin that root nodule is cut into 1~2mm with sterile scalpel
Piece, and be inserted on three kinds of different agar mediums, 28 DEG C of cultures, after knurl piece grows thalline, it is chosen with transfer needle
It is connected on flat board and is further purified.
Endogenetic fungus uses PDA culture medium, and seabuckthorn root leachate is added in PDA culture medium（Culture medium soaks with seabuckthorn root
The volume ratio for going out liquid is 1:15）, flat board is placed at 28 DEG C and cultivates 5d.
Endogenetic bacteria uses beef-protein medium, and seabuckthorn root leachate is added in beef-protein medium
（The volume ratio of culture medium and seabuckthorn root leachate is 1:15）, flat board is placed at 28 DEG C and cultivates 5d.
Endogeny rayungus uses Gao Shi I culture mediums, adds seabuckthorn root leachate in the medium（Culture medium and seabuckthorn root
The volume ratio of leachate is 1:15）, flat board is placed at 28 DEG C and cultivates 5d.
Control group：Seabuckthorn root leachate is not added with all plating mediums, carries out the separation of endophyte under the same conditions
Culture.
（3）The purifying of endophyte：Fresh flat board is forwarded to after tissue block edge grows bacterium colony, is purified by ruling,
Then observe under the microscope and detect its purity, until obtaining endophyte single bacterium colony, inclined-plane saves backup.
The bacterium colony kind for judging to isolate according to features such as colonial morphology, color, rim condition, transparency, surface humidity
Class simultaneously counts.It the results are shown in Table 1.
Embodiment 3
（1）Surface sterilization is carried out to Hippophae nodule sample：With embodiment 1.
（2）Endophyte is separately cultured：Endophyte is separately cultured：It is thin that root nodule is cut into 1~2mm with sterile scalpel
Piece, and be inserted on three kinds of different agar mediums, 30 DEG C of cultures, after knurl piece grows thalline, it is chosen with transfer needle
It is connected on flat board and is further purified.
Endogenetic fungus uses PDA culture medium, and seabuckthorn root leachate is added in PDA culture medium（Culture medium soaks with seabuckthorn root
The volume ratio for going out liquid is 1:18）, flat board is placed at 30 DEG C and cultivates 3d.
Endogenetic bacteria uses beef-protein medium, and seabuckthorn root leachate is added in beef-protein medium
（The volume ratio of culture medium and seabuckthorn root leachate is 1:15）, flat board is placed at 30 DEG C and cultivates 3d.
Endogeny rayungus uses Gao Shi I culture mediums, adds seabuckthorn root leachate in the medium（Culture medium and seabuckthorn root
The volume ratio of leachate is 1:15）, flat board is placed at 30 DEG C and cultivates 3d.
Control group：Seabuckthorn root leachate is not added with all plating mediums, carries out the separation of endophyte under the same conditions
Culture.
（3）The purifying of endophyte：Fresh flat board is forwarded to after tissue block edge grows bacterium colony, is purified by ruling,
Then observe under the microscope and detect its purity, until obtaining endophyte single bacterium colony, inclined-plane saves backup.
The bacterium colony kind for judging to isolate according to features such as colonial morphology, color, rim condition, transparency, surface humidity
Class simultaneously counts.It the results are shown in Table 1.
In the various embodiments described above, the preparation method of seabuckthorn root leachate is：Seabuckthorn root is cleaned and shredded, 1 is pressed with distilled water:
1 volume ratio mixes, and simmer in water about 1h after boiling, and filtering, takes clear liquid, 121 DEG C of sterilizings store for future use.
The separating effect of the Hippophae nodule endophyte of table 1
Result in table 1 shows that the culture medium nutrition added using the present invention after seabuckthorn root leachate is more nearly endophyte life
Long prototroph condition, is more suitable for the growth of endophyte.Obtain more endophyte bacterial strains.With being not added with seabuckthorn root leachate
Culture medium compare, the type and quantity that can cultivate Frankia bacterial strain can be dramatically increased, obtain more endophytes money
Source, and the time that endophyte is separately cultured can be shortened.
Claims (3)
1. a kind of hippophae plant Frankia isolated culture method, is comprised the following steps that：
（1）Surface sterilization is carried out to Hippophae nodule sample：Standby Hippophae nodule sample is taken, is rushed successively with the running water of flowing
Wash, and after scrubbing off the impurity such as silt with writing brush, with aseptic water washing 3 ~ 5 times, with 75% 8 ~ 10min of ethanol surface sterilization, with 1%
NaClO solution surfaces sterilize 5 ~ 7min, then with aseptic water washing 3 ~ 5 times；
（2）Endophyte culture：Sterile-processed Hippophae nodule is cut into slices, is respectively placed on different plating mediums, is used
Different culture medium is simultaneously separately added into seabuckthorn root leachate, carries out sterile culture；Condition of culture is：At 25 ~ 30 DEG C culture 3 ~
7d；
Endogenetic fungus uses PDA culture medium, and endogenetic bacteria uses beef-protein medium, and endogeny rayungus uses Gao Shi I
Number culture medium；
（3）Endophyte isolates and purifies：Fresh flat board is forwarded to after tissue block edge grows bacterium colony, is purified by ruling,
Then observe under the microscope and detect its purity, until obtaining endophyte single bacterium colony, inclined-plane saves backup.
A kind of 2. hippophae plant Frankia isolated culture method as claimed in claim 1, it is characterised in that：Culture medium with
The volume ratio of seabuckthorn root leachate is 1:15~1:20.
A kind of 3. hippophae plant Frankia isolated culture method as claimed in claim 1 or 2, it is characterised in that：Sea-buckthorn
The preparation method of root leachate is：Seabuckthorn root is cleaned and shredded, 1 is pressed with distilled water:1~1:1.5 volume ratio mixing, after boiling
0.5 ~ 1h is cooked by slow fire, filters, takes clear liquid, 121 DEG C of sterilizings store for future use.
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| CN201711258256.2A CN107699531A (en) | 2017-12-04 | 2017-12-04 | A kind of hippophae plant Frankia isolated culture method |
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| CN201711258256.2A CN107699531A (en) | 2017-12-04 | 2017-12-04 | A kind of hippophae plant Frankia isolated culture method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819568A (en) * | 2019-11-21 | 2020-02-21 | 山西金科海生物制品有限公司 | Sea-buckthorn nodule endophyte culture medium and preparation and isolated culture method thereof |
| CN112877263A (en) * | 2021-04-09 | 2021-06-01 | 黑龙江省科学院高技术研究院 | Separation method of hemp endophytes |
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| CN104250616A (en) * | 2014-09-03 | 2014-12-31 | 中国热带农业科学院热带生物技术研究所 | Separation method for plant endophyte |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819568A (en) * | 2019-11-21 | 2020-02-21 | 山西金科海生物制品有限公司 | Sea-buckthorn nodule endophyte culture medium and preparation and isolated culture method thereof |
| CN110819568B (en) * | 2019-11-21 | 2023-11-03 | 山西金科海生物制品有限公司 | Sea buckthorn rhizobium endophyte culture medium and preparation and separation culture methods thereof |
| CN112877263A (en) * | 2021-04-09 | 2021-06-01 | 黑龙江省科学院高技术研究院 | Separation method of hemp endophytes |
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Application publication date: 20180216 |