CN106350452A - Separation method of grass endophytic fungi - Google Patents
Separation method of grass endophytic fungi Download PDFInfo
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- CN106350452A CN106350452A CN201610722008.8A CN201610722008A CN106350452A CN 106350452 A CN106350452 A CN 106350452A CN 201610722008 A CN201610722008 A CN 201610722008A CN 106350452 A CN106350452 A CN 106350452A
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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
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- 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
- A01C1/08—Immunising seed
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- 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
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- 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
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention provides a separation method of grass endophytic fungi. The method comprises the following steps: (1) sterilizing surfaces of plant seeds; (2) culturing plant aseptic seedlings: placing the sterilized plant seeds in a culture medium, carrying out sprouting culture until the seeds grow into seedlings; (3) managing the grass seed sprouted seedlings: observing in the process when the seeds sprout and grow to be the seedlings, removing the polluted seeds in time, thus obtaining the plant aseptic seedlings; and (4) after the plant aseptic seedlings are formed, cutting off hypocotyledonary axes of the aseptic seedlings under an aseptic condition, performing culture, thus obtaining the endophytic fungi. The effect of the method for separating the plant endophytic fungi is better than that of a conventional separation method, and the method is particularly suitable for the plant materials difficult for separating the endophytic fungi. Therefore, the separation method is an excellent method for researching the plant endophytic fungi. In addition, the separation method of the grass endophytic fungi is wide in application range and can be used for efficiently separating epichlo-endophytic fungi carried by early-mature agrostidoideae among the cold-season grasses.
Description
Technical field
The invention belongs to microbial technology field is and in particular to a kind of detached method of dogstail endogenetic fungus.
Background technology
Plant endogenesis epiphyte homobium is a kind of common symbiosises in nature.Plant provides photosynthetic for endogenetic fungus
Product and mineral simultaneously help its propagation by seed, and endogenetic fungus (epichloë) then can check the herbivores pair such as insecticide
Searching for food of host plant, also can promote plant growing by improving stress resistance of plant, increase tiller and Biomass simultaneously, improve
Its competitiveness in population and group.
In endogenetic fungus-host plant-herbivore ecosystem, dogstail endogenetic fungus play strange heavy
Want ecological effects, on the one hand can improve the resistance of host plant, on the other hand because it produces alkaloid with dogstail symbiosis
And lead to Livestock poisoned.Dogstail endogenetic fungus are concentrated mainly on ascomycetous Clavicipitaceae epichloeepichloëIn, wherein
The phorozoon of epichloeepichloëReceive extensive concern because of its substantial connection with tame forage grass, asexualepichloë
Belong to endogenetic fungus to be settled in host plant body with mycelia form all the year round, do not produce Stroma and spore, only entered by mycelial growth
Enter the organ of multiplication of host plant, through seed dispersal.
Plant endogenesis epiphyte is the microbial resources that a class has potentiality to be exploited very much, separates the technology of endogenetic fungus at present still not
Perfect, the rate that is generally separated is low, in addition easily by external source saprophytic fungus and other microorganism pollutions in separation process.At present, interior
Raw funguses separation method is mainly: (1) piece of tissue partition method, plant surface is sterilized, is cut into small pieces, carry out culture of isolated；(2)
Using plant exudates method, plant tissue is crushed by the method for chemistry or physics after directly sterilizing, and then extracts transudate,
Then carry out culture of isolated.As insufficient in surface sterilization in both the above method, surface bacterium easily pollutes and suppression endogenetic fungus
Growth；In addition, disinfecting time is long, endogenetic fungus may be killed.(3) plant tissue surface sterilization pretreatment, is then carried out
Plant tissue culture, aseptically pulverizes after plant tissue restoration ecosystem and then extracts transudate, then cultivated
Separate.Although the method has carried out organized renewing, the unavoidable destruction to plant internal body portion endophyte.
Content of the invention
As insufficient in surface sterilization in additive method in order to solve, surface bacterium easily pollutes and suppression endogenetic fungus growth；
In addition, disinfecting time is long, the problems such as may kill endogenetic fungus, the present invention provides a kind of plant endogenesis epiphyte detached
Method.
The present invention provides a kind of detached method of dogstail endogenetic fungus, and step is as follows:
(1) plant seed surface is carried out disinfection；
(2) aseptic seedlings of plants culture: the plant seed after sterilization is placed in culture medium, carries out culture of germinateing, until seed is long
Become seedling；
(3) Grass seed germination seedling management: observed to during grow up to seedling in germination, reject contaminated in time
Seed, obtain aseptic seedlings of plants；
(4) after aseptic seedlings of plants is formed, aseptically, cut aseptic seedling hypocotyls, cultivated, obtain endogenetic fungus
Bacterium colony.
Preferably, in step (1), described method that plant seed surface carries out disinfection is: by plant seed first
Being placed in volumetric concentration is soaking disinfection 3-5min in 75% ethanol, after sterile water wash, then is placed in volumetric concentration for 3% sodium hypochlorite
Middle soaking disinfection 8-10min, sterile water wash.
Preferably, in step (2), the method for described aseptic seedlings of plants culture is: the plant seed after sterilization is placed in
In ms culture medium, carry out culture of germinateing, until seed grows up to seedling；Condition of culture is temperature 15-25 DEG C, humidity 30-70%, light
It is 1000-2500lux according to condition, light application time 8-14h/d.
Preferably, in step (4), the culture medium using during described culture is pda culture medium.
Preferably, in step (4), the condition of culture of described culture is: temperature 15-20 DEG C, humidity 50-80%, dark bar
Part is cultivated.
Preferably, step is as follows:
(1) plant seed is processed
It is soaking disinfection 4min in 75% ethanol that plant seed is placed in volumetric concentration, sterile water wash, then is 3% with volumetric concentration
Sodium hypochlorite soaking disinfection 9min, then uses sterile water wash, blots standby after surface moisture；
(2) aseptic seedlings of plants culture
Plant seed after sterilization is placed in ms culture medium, carries out culture of germinateing, until seed grows up to seedling；Condition of culture is
20 DEG C of temperature, humidity 50%, illumination condition is 1800lux, light application time 10h/d；
(3) Grass seed germination seedling management
Regularly observe to during grow up to seedling in germination, reject contaminated seed in time, obtain aseptic seedlings of plants；
(4) separation of endogenetic fungus
After aseptic seedlings of plants is formed, aseptically, cut aseptic seedling hypocotyls, be cut into 1-2cm segment, be placed in pda training
Cultivated on foster base, condition of culture is 18 DEG C of temperature, humidity 60%, and dark condition is cultivated, and obtains final product.
The separation method of the dogstail endogenetic fungus that the present invention provides, first passes through and plant seed is sterilized, then cultivate aseptic seedling,
Method using the separating plant endogenous funguses of aseptic seedling hypocotyls.In separating endogenetic fungus operation, need not carry out disinfection place again
Reason, thus decreasing the injury to plant endogenesis epiphyte for the disinfectant, and reduction environment and plant material surface are saprophytic true in a large number
Microbial pollution problem.In addition, the method is simple to operate, separation costs are relatively low and additive method separation efficiency relatively is higher.
More than conventional method separation method effect is significant using the separating plant endogenous antifungal effect of the method for the present invention, special
It is not the vegetable material being suitable for being difficult to isolate endogenetic fungus.Therefore, the present invention be research plant endogenesis epiphyte one kind excellent
Method.
Epichlo Nei Shengzhen additionally, the scope of application of the present invention is wider, entrained by cold-season-type dogstail mid-early maturity Agrostidoideae
Bacterium all can be obtained by efficient separation.
Brief description
Accompanying drawing is used for providing a further understanding of the present invention, and constitutes a part for description, the reality with the present invention
Apply example and be used for explaining the present invention together, be not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the flow chart of general law separation endogenetic fungus；
Fig. 2 is the flow chart of the inventive method separation endogenetic fungus.
Specific embodiment
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is city
Sell.
The dogstail endogenetic fungus of the present invention are detached method particularly includes:
(1) plant seed is processed
It is soaking disinfection 3-5min in 75% ethanol that plant seed is placed in volumetric concentration, sterile water wash 3-5 time, then uses volume
Concentration is 3% sodium hypochlorite soaking disinfection 8-10min, then uses sterile water wash 5-8 time, is placed in aseptic filter paper and blots surface water
Standby after point；
(2) aseptic seedlings of plants culture
Plant seed after sterilization is placed in ms culture medium, carries out culture of germinateing, until seed grows up to seedling；Condition of culture is
Temperature 15-25 DEG C, humidity 30-70%, illumination condition is 1000-2500lux, light application time 8-14h/d；
(3) Grass seed germination seedling management
Regularly observe to during grow up to seedling in germination, reject contaminated seed in time, obtain aseptic seedlings of plants；
(4) separation of endogenetic fungus
Formed after (seedling of growth four week old) after aseptic seedlings of plants, aseptically, cut aseptic seedling hypocotyls, be cut into 1-
2cm segment, is placed in and is cultivated in pda culture medium, and condition of culture is temperature 15-20 DEG C, humidity 50-80%, and dark condition is trained
Support, obtain final product endogenetic fungus bacterium colony.
Embodiment 1
The dogstail endogenetic fungus of the present invention are detached method particularly includes:
(1) plant seed is processed
It is soaking disinfection 4min in 75% ethanol that plant seed is placed in volumetric concentration, sterile water wash 4 times, then uses volumetric concentration
For 3% sodium hypochlorite soaking disinfection 9min, then use sterile water wash 6 times, be placed in aseptic filter paper blot standby after surface moisture；
(2) aseptic seedlings of plants culture
Plant seed after sterilization is placed in ms culture medium, carries out culture of germinateing, until seed grows up to seedling；Condition of culture is
20 DEG C of temperature, humidity 50%, illumination condition is 1800lux, light application time 10h/d；
(3) Grass seed germination seedling management
Regularly observe to during grow up to seedling in germination, reject contaminated seed in time, obtain aseptic seedlings of plants；
(4) separation of endogenetic fungus
Formed after (seedling of growth four week old) after aseptic seedlings of plants, aseptically, cut aseptic seedling hypocotyls, be cut into 1-
2cm segment, is placed in and is cultivated in pda culture medium, and condition of culture is 18 DEG C of temperature, humidity 60%, and dark condition is cultivated, and obtains final product
Endogenetic fungus bacterium colony.
Embodiment 2
The dogstail endogenetic fungus of the present invention are detached method particularly includes:
(1) plant seed is processed
It is soaking disinfection 3min in 75% ethanol that plant seed is placed in volumetric concentration, sterile water wash 5 times, then uses volumetric concentration
For 3% sodium hypochlorite soaking disinfection 10min, then use sterile water wash 5 times, be placed in aseptic filter paper blot standby after surface moisture；
(2) aseptic seedlings of plants culture
Plant seed after sterilization is placed in ms culture medium, carries out culture of germinateing, until seed grows up to seedling；Condition of culture is
25 DEG C of temperature, humidity 30%, illumination condition is 2500lux, light application time 8h/d；
(3) Grass seed germination seedling management
Regularly observe to during grow up to seedling in germination, reject contaminated seed in time, obtain aseptic seedlings of plants；
(4) separation of endogenetic fungus
Formed after (seedling of growth four week old) after aseptic seedlings of plants, aseptically, cut aseptic seedling hypocotyls, be cut into 1-
2cm segment, is placed in and is cultivated in pda culture medium, and condition of culture is 20 DEG C of temperature, humidity 50%, and dark condition is cultivated, and obtains final product
Endogenetic fungus bacterium colony.
Embodiment 3
The dogstail endogenetic fungus of the present invention are detached method particularly includes:
(1) plant seed is processed
It is soaking disinfection 5min in 75% ethanol that plant seed is placed in volumetric concentration, sterile water wash 3 times, then uses volumetric concentration
For 3% sodium hypochlorite soaking disinfection 8min, then use sterile water wash 8 times, be placed in aseptic filter paper blot standby after surface moisture；
(2) aseptic seedlings of plants culture
Plant seed after sterilization is placed in ms culture medium, carries out culture of germinateing, until seed grows up to seedling；Condition of culture is
15 DEG C of temperature, humidity 70%, illumination condition is 1000lux, light application time 14h/d；
(3) Grass seed germination seedling management
Regularly observe to during grow up to seedling in germination, reject contaminated seed in time, obtain aseptic seedlings of plants；
(4) separation of endogenetic fungus
Formed after (seedling of growth four week old) after aseptic seedlings of plants, aseptically, cut aseptic seedling hypocotyls, be cut into 1-
2cm segment, is placed in and is cultivated in pda culture medium, and condition of culture is 15 DEG C of temperature, humidity 80%, and dark condition is cultivated, and obtains final product
Endogenetic fungus bacterium colony.
Using two methods respectively to 11 kinds of plant endogenesis such as wild barley, Herba oxytropis glabrae, Festuca sinensis, lyme grass, rye grass
Funguses carry out separating；Flow process such as Fig. 1 using general law separation endogenetic fungus；Using the inventive method separation endogenetic fungus flow process
As Fig. 2.By the flat board obtaining respectively through above two method through 25 DEG C of constant temperature culture, statistical result is as shown in table 1.
The separation rate that table 1 should differently separate dogstail endogenetic fungus compares
As can be seen here, using the inventive method to the separation rate of various plants endogenetic fungus apparently higher than being provided without side of the present invention
Method, its average separation rate exceeds nearly more than 20%, and pollution rate is relatively low.Sterilization in separation process is decreased using the inventive method
The damage to plant endogenesis epiphyte vigor for the agent, thus indirectly improve the separation rate of endogenetic fungus.
Finally it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention,
Although being described in detail to the present invention with reference to the foregoing embodiments, for a person skilled in the art, it still may be used
To modify to the technical scheme described in foregoing embodiments, or equivalent is carried out to wherein some technical characteristics.
All any modification, equivalent substitution and improvement within the spirit and principles in the present invention, made etc., should be included in the present invention's
Within protection domain.
Claims (6)
1. a kind of detached method of dogstail endogenetic fungus it is characterised in that: step is as follows:
(1) plant seed surface is carried out disinfection；
(2) aseptic seedlings of plants culture: the plant seed after sterilization is placed in culture medium, carries out culture of germinateing, until seed is long
Become seedling；
(3) Grass seed germination seedling management: observed to during grow up to seedling in germination, reject contaminated in time
Seed, obtain aseptic seedlings of plants；
(4) after aseptic seedlings of plants is formed, aseptically, cut aseptic seedling hypocotyls, cultivated, obtain endogenetic fungus
Bacterium colony.
2. method according to claim 1 it is characterised in that: in step (1), described plant seed surface is carried out disinfection
Method be: by plant seed be initially positioned at volumetric concentration be 75% ethanol in soaking disinfection 3-5min, after sterile water wash, then
Being placed in volumetric concentration is soaking disinfection 8-10min in 3% sodium hypochlorite, sterile water wash.
3. method according to claim 1 it is characterised in that: in step (2), the method for described aseptic seedlings of plants culture
For: the plant seed after sterilization is placed in ms culture medium, carries out culture of germinateing, until seed grows up to seedling；Condition of culture is
Temperature 15-25 DEG C, humidity 30-70%, illumination condition is 1000-2500lux, light application time 8-14h/d.
4. method according to claim 1 it is characterised in that: in step (4), during described culture, the culture medium that uses is
Pda culture medium.
5. method according to claim 4 it is characterised in that: in step (4), the condition of culture of described culture is: temperature
15-20 DEG C, humidity 50-80%, dark condition is cultivated.
6. method according to claim 1 it is characterised in that: step is as follows:
(1) plant seed is processed
It is soaking disinfection 4min in 75% ethanol that plant seed is placed in volumetric concentration, sterile water wash, then is 3% with volumetric concentration
Sodium hypochlorite soaking disinfection 9min, then uses sterile water wash, blots standby after surface moisture；
(2) aseptic seedlings of plants culture
Plant seed after sterilization is placed in ms culture medium, carries out culture of germinateing, until seed grows up to seedling；Condition of culture is
20 DEG C of temperature, humidity 50%, illumination condition is 1800lux, light application time 10h/d；
(3) Grass seed germination seedling management
Regularly observe to during grow up to seedling in germination, reject contaminated seed in time, obtain aseptic seedlings of plants；
(4) separation of endogenetic fungus
After aseptic seedlings of plants is formed, aseptically, cut aseptic seedling hypocotyls, be cut into 1-2cm segment, be placed in pda training
Cultivated on foster base, condition of culture is 18 DEG C of temperature, humidity 60%, and dark condition is cultivated, and obtains final product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109121778A (en) * | 2018-08-07 | 2019-01-04 | 广西田东县东辰农业开发有限公司 | It is a kind of slowly take root tumor fungus extract extracting method and its mango grafting on application |
| CN110713938A (en) * | 2019-09-20 | 2020-01-21 | 兰州大学 | Method for improving content of ergot alkaloid serving as secondary metabolite of buddleia-endophytic fungus strain JZ |
| CN110713937A (en) * | 2019-09-20 | 2020-01-21 | 兰州大学 | Method for improving content of boswellin (a secondary metabolite of PA) of Eremorhedi sinensis-endophytic fungi strain |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119658A (en) * | 2010-12-07 | 2011-07-13 | 东南大学 | Method for culturing aseptic seedlings of plants |
| CN102870671A (en) * | 2011-07-11 | 2013-01-16 | 中国科学院植物研究所 | High-efficiency aseptic seedling acquisition method from grass seed |
| 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 |
-
2016
- 2016-08-25 CN CN201610722008.8A patent/CN106350452A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119658A (en) * | 2010-12-07 | 2011-07-13 | 东南大学 | Method for culturing aseptic seedlings of plants |
| CN102870671A (en) * | 2011-07-11 | 2013-01-16 | 中国科学院植物研究所 | High-efficiency aseptic seedling acquisition method from grass seed |
| 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 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109121778A (en) * | 2018-08-07 | 2019-01-04 | 广西田东县东辰农业开发有限公司 | It is a kind of slowly take root tumor fungus extract extracting method and its mango grafting on application |
| CN110713938A (en) * | 2019-09-20 | 2020-01-21 | 兰州大学 | Method for improving content of ergot alkaloid serving as secondary metabolite of buddleia-endophytic fungus strain JZ |
| CN110713937A (en) * | 2019-09-20 | 2020-01-21 | 兰州大学 | Method for improving content of boswellin (a secondary metabolite of PA) of Eremorhedi sinensis-endophytic fungi strain |
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