CN106047757A - Separation method of endophytic bacteria of ammodendron argenteum root and seed germination promoting method - Google Patents
Separation method of endophytic bacteria of ammodendron argenteum root and seed germination promoting method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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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
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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
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Abstract
The invention discloses a separation method of endophytic bacteria of ammodendron argenteum root. The separation method comprises the following steps of: primary shearing, surface disinfection, secondary shearing and separation and culture of endophytic bacteria. The invention provides a seed germination promoting method using the separated endophytic bacteria, wherein the method comprises the following steps of: seed pretreatment, bacterial suspension preparation, seed soaking using the bacterial suspension and seeding and germination culture to obtain AG18 endophytic bacteria which have a seed germination promoting function. In the separation method disclosed by the invention, the risk of introducing exogenous microorganism is reduced, and the number and varieties of endophytic bacteria are increased; in the seed germination promoting method, seed germination promotion using a biological method is realized, the endophytic bacteria AG18 promoting seed germination are obtained, and sustainable seed germination promotion is realized.
Description
Technical field
The present invention relates to microbiological art, is specifically related to the separation method of a kind of rermilion Radix sophorae portion endogenetic bacteria and promotees seed
Germination method.
Background technology
Endophytic bacterium and host form harmonious symbiosis in long-term common evolutionary.Existing for of symbiotic microorganism
Plant adapts to environment, strengthens the aspects such as resistance and plays a significant role.Rermilion Chinese scholartree is that the one in desert drought environment is excellent fixes the sand
Plant, owing to it is not enough at nature seedling magnitude of recruitment, population recruitment ability, it is listed in Chinese Second Class Key Protected Plant, its
The research of endophyte and plant interaction is just becoming the focus in the fields such as biology, engineering, environmental science.
Existing endophyte of plant separation method is divided into four steps, and the first step is cut into 1-2cm segment after being cleaned by plant, the
Two steps utilize mercuric chloride that tissue fragment carries out surface sterilization, and tissue fragment is put in normal saline and carries out underhand polish by the 3rd step
Or tissue mashing crusher machine, the 4th step take homogenate after supernatant or diluent coat culture medium and carry out 37 DEG C of cultivations.Existing
Rermilion Chinese scholartree is promoted the method for seed germination and is mainly soaked seed by sulphuric acid: 98% soak with sulphuric acid 120min, and redistilled water cleans seed soaking
12h.Germinating bed uses filter paper method to carry out sprouting experiment.
Above-mentioned separation method has the disadvantage in that antiseptic solution is easily accessible inside and causes substantial amounts of endogenetic bacteria dead,
Decreasing the value volume and range of product of endogenetic bacteria, interior tissue microorganism is easily damaged by mercuric chloride, and environment is caused by its leakage
Pollute.Additionally, tissue mashing crusher machine exists the risk introducing inoculating microbe, make the quantity of the endogenetic bacteria that final separation goes out
Kind antibacterial on the low side with kind and each is impure, and then the endogenetic bacteria utilizing said method to prepare is promoting seed germination method
In, seed germination rate is the highest.Additionally, break seed dormancy at present to promote that the method for seed germination is broadly divided into physical method and change
Method, physical method such as mechanical treatment, Stratificated treatment, chemical method such as HORMONE TREATMENT, strong acid treatment etc., different seeds needs
Using different methods, the seed for combinational dormancy then must use complex method to process.Kind due to rermilion Chinese scholartree seed
Skin is waterproof and there is physics dormancy, but still can not sprout after seed coat cut, illustrates to there is also other kinds of dormancy, needs to adopt
Improve with complex method Dormancy breaking and sprout, but utilize current complex method to process, easily to environment,
And easily hurt seed, not there is persistence.
Summary of the invention
It is an object of the invention to, it is provided that a kind of separation method obtaining rermilion Radix sophorae portion endogenetic bacteria, it is possible to obtain more
Many quantity and a greater variety of endogenetic bacteria, and utilize this endogenetic bacteria to realize bioanalysis rush seed germination, and then obtain rush
The endogenetic bacteria of seed germination.
To achieve these goals, the invention provides the separation method of a kind of rermilion Radix sophorae portion endogenetic bacteria, including such as
Lower step:
Step 1, once shearing: after rermilion Radix sophorae portion tissue wash, be cut into 4-6cm fragment；
Step 2, surface sterilization: with ethanol and sodium hypochlorite, described 4-6cm fragment is carried out surface sterilization process successively；
Step 3, secondary shearing: the described 4-6cm fragment disinfected aseptically is cut into 0.5-1cm sheet
Section；
Step 4, the separation of endogenetic bacteria and cultivation: described 0.5-1cm fragment is inserted directly in culture medium flat plate and carries out
Cultivate, picking different shape bacterium colony, carrying out streak culture until obtaining the pure culture of each bacterium colony, preserving.
Wherein, step 2 is particularly as follows: first with 75% alcohol disinfecting 3min, aseptic water washing at least 1 time, then uses 2% hypochlorous acid
Sodium sterilization 3min, aseptic water washing 6 times, take 100 flushing water detection Disinfection Effects last for μ l.
Wherein, in step 4, described culture medium is beef-protein medium, and its pH value is 7.0.
Wherein, in step 4, after described 0.5-1cm fragment is cultivated 7 days with 28～30 DEG C in described culture medium, then carry out
Streak culture, it is saved in the glycerol of 20%, is stored in-80 DEG C.
Secondly, it is provided that a kind of rush seed germination method, endogenetic bacteria prepared by above-mentioned method is used for this plant and promotees to plant
In sub-germination method, comprise the steps:
Step a, Seeds preprocess: use liquor natrii hypochloritis to carry out disinfection described seed, carry out afterwards cutting into slices brokenly and plant；
Prepared by step b, bacteria suspension: be inoculated in beef extract-peptone fluid medium by described endogenetic bacteria, be placed in
Incubated overnight in shaking table, is centrifuged afterwards, and the precipitation taking described endogenetic bacteria is resuspended in PBS, obtains described bacterium
Suspension；
Step c, seed soaking: immerse in described bacteria suspension by the described seed of sterilization, soak seed 4h；
Step d, sowing: carry out sprouting by the described seed after seed soaking and cultivate, and periodic statistical germination percentage and radicle length.
Wherein, step a particularly as follows: first with 75% alcohol disinfecting 12min, aseptic water washing at least 1 time, then with 5%
Liquor natrii hypochloritis sterilizes 15min, aseptic water washing 6 times.
Wherein, the concussion frequency of shaking table described in step b is 150rpm.
Wherein, in step b, OD600 being adjusted to 1.0, centrifugation rate is 8000rmp, and centrifugation time is 10min.
Wherein, the described seed germination in step d is cultivated and is used culture dish filter paper method.
Again, it is provided that a kind of AG18 endogenetic bacteria obtained according to above-mentioned rush seed germination method, raw thin in described AG18
Bacterium has the effect promoting seed germination.
Compared with prior art, the beneficial effects of the present invention is: in the separation method in the present invention, it is not necessary to plant
Tissue carries out underhand polish or Mechanical Crushing, greatly reduces the risk introducing inoculating microbe, cultivates bar by strict control
Part, such as the growth of inferior position flora in pH value and the setting of cultivation temperature, beneficially endogenetic bacteria, by increasing capacitance it is possible to increase endogenetic bacteria
Quantity and kind.And utilize isolated endogenetic bacteria to carry out seed promoting to sprout, it is achieved that bioanalysis promotees seed germination,
And obtain the endogenetic bacteria AG18 promoting seed germination, it is to avoid use complex method to promote seed germination and the environment that causes is dirty
Dye, and it is substantially reduced the injury to seed, and owing to AG18 endogenetic bacteria and host form mutualism, it is achieved that sustainable
The rush seed germination of property.
Accompanying drawing explanation
Fig. 1 is the seed germination rate bar diagram that in the present invention, isolated endogenetic bacteria promotees seed germination；
Fig. 2 is the bar diagram of seed root length in isolated endogenetic bacteria rush seed germination in the present invention.
Detailed description of the invention
Below by specific embodiment and combine accompanying drawing the present invention is described in further detail.
Rermilion Chinese scholartree (Ammodendron bifolium (Pall.) Yakovl.) is the pulse family rermilion raw deciduous plant of Sophora sand.
This platymiscium whole world has 8 kinds, only a kind of China, i.e. rermilion Chinese scholartree, is only distributed in Xinjiang, China Huocheng County Plutarch that not that
Desert, is excellent plant of checking winds and fixing drifting sand, and the rermilion Chinese scholartree in the present invention also derives from this.
The separation method of the rermilion Radix sophorae portion endogenetic bacteria in the present invention comprises the steps:
Step 1, once shearing: after rermilion Radix sophorae portion tissue wash, be cut into 4-6cm fragment.This fragment is bigger, it is possible to keep away
Exempt from the follow-up surface sterilization destruction to its endogenetic bacteria, retain complete Microflora and kind in tissue.
Step 2, surface sterilization: with ethanol and sodium hypochlorite, described 4-6cm fragment is carried out surface sterilization process successively.Tool
Body for being first 75% alcohol disinfecting 3min by volume fraction, aseptic water washing at least 1 time, then be 2% hypochlorous acid by volume fraction
Sodium sterilization 3min, aseptic water washing 6 times, in flushing water the last time, take its 100 μ l and detect Disinfection Effect.
Step 3, secondary shearing: the described 4-6cm fragment disinfected aseptically is cut into 0.5-1cm sheet
Section.Aseptically, it is carried out secondary shearing, be not required to use underhand polish or machine to crush, it is to avoid introducing external source is micro-
Biology and pollution, simplify step, improve survival rate and the purity of bacterial strain.
Step 4, the separation of endogenetic bacteria and cultivation: described 0.5-1cm fragment is inserted directly in culture medium flat plate and carries out
Cultivate, picking different shape bacterium colony, carrying out streak culture until obtaining the pure culture of each bacterium colony, preserving.
Culture medium in step 4 is beef-protein medium, regulates and controls its pH value 7.0, in this culture medium 28
～30 DEG C cultivate 7 days, carry out streak culture the most again, control the pH value of culture medium to control 30 in 7.0 and temperature herein
DEG C inferior position flora growth can being of value in endogenetic bacteria, improves kind and the quantity of endogenetic bacteria further.Obtain pure training
After supporting thing, being placed on volume fraction is in 20% glycerol, is stored in-80 DEG C standby.
Utilize above-mentioned separation method, separate out 69 strain endophytes from rermilion Radix sophorae part, identify through 16S rRNA, belong to 11
Individual genus.
Separation method in the present invention is generally separated the bacterium colony situation of method acquisition as shown in Table 1 with of the prior art:
Table one
From table one it is apparent that use the separation method phase that the separation method the present invention is general with prior art
Ratio is seen, its colony counts is greatly increased, and the kind of endophyte also increases a lot, remains most endogenetic bacteria.
In separation method in the present invention, it is not necessary to plant tissue is carried out underhand polish or Mechanical Crushing, greatly reduces
Introduce the risk of inoculating microbe, by strictly controlling condition of culture, such as pH value and the setting of cultivation temperature, in being conducive to
The growth of inferior position flora in endophytic bacteria, by increasing capacitance it is possible to increase the quantity of endogenetic bacteria and kind.
Owing to endogenetic bacteria and host plant interact, the invention provides and a kind of utilize above-mentioned separation method to obtain
Endogenetic bacteria promotees the method for seed germination, comprises the steps:
Step a, Seeds preprocess: use liquor natrii hypochloritis to carry out disinfection described seed, carry out afterwards cutting into slices brokenly and plant.
The most first with the alcohol disinfecting 12min that volume fraction is 75%, aseptic water washing at least 1 time, then use volume integral
Number be 5% liquor natrii hypochloritis sterilize 15min, aseptic water washing 6 times.
Prepared by step b, bacteria suspension: be inoculated in beef extract-peptone fluid medium by described endogenetic bacteria, be placed in
Incubated overnight in shaking table, is centrifuged afterwards, and the precipitation taking described endogenetic bacteria is resuspended in PBS, obtains described bacterium
Suspension.
In above-mentioned steps, the concussion frequency setting of shaking table is 150rmp, and 0D600 is adjusted to 1.0, at 8000rmp
Centrifugal 10min under rotating speed, the concentration of PBS (phosphate buffered saline(PBS)) is 10mM, and pH value is 7.0.
Wherein, OD600, is to utilize the light absorption value at spectrophotometric determination 600nm wavelength.When bacterium solution OD value is 1.0,
Bacterial growth is preferable, and cell density is relatively reasonable, and its OD value is adjusted to 1.0, and another advantage is unified standard, it is simple to different bacterium
Comparison between strain process.
Step c, seed soaking: immerse in described bacteria suspension by the described seed of sterilization, soak seed 4h；
Step d, sowing: carry out sprouting by the described seed after seed soaking and cultivate, and periodically (as every 5 days) statistics germination percentage
And radicle length.Sprouting cultivation and use culture dish filter paper method, the method is that international Seed Inspection association (ISTA) regulation is for planting
Son sprouts the standardization program of experiment, i.e. spreads in culture dish into filter paper, and after water-soaked, the seed after processing is placed on filter paper
Cultivate.
Utilizing the separation method in the present invention can isolate 69 strain endogenetic bacterias, (GeneBank sequence is stepped in 29 strains below
Record number: KR045813-KR045835, KR045852-KR045857) it is representative strain, wherein 5 strains belong to bacillus
(Bacillus), 8 strains belong to general Pseudomonas (Pantoea), and 4 strains belong to Erwinia (Erwinia), and 2 strains belong to acinetobacter
(Acinetobacter), 2 strains belong to Rhodopseudomonas (Pseudomonas), and 2 strains belong to Sporosarcina
(Sporosarcina), 2 strains belong to Klebsiella (Klebsiella), and 1 strain belongs to Arthrobacter (Arthrobacter), 1 strain
Belonging to cock Bordetella (Kocuria), 1 strain belongs to Cellulomonas (Cellulomonas), and 1 strain belongs to series bacillus and belongs to
(Paenibacillus).The numbering of its correspondence and accession number such as table two.
Table two
Utilizing above-mentioned 29 strain antibacterials to carry out the experiment of above-mentioned rush seed germination, at the end of experiment, (after sprouting 15 days) is surveyed and is sprouted
Send out rate and radicle length such as table three.Arranging PBS buffer solution in table three is blank, i.e. in step c, and this blank group
In the seed of sterilization be immersed in PBS.Numerical value is meansigma methods ± SE (SE is standard error), and " * " represents at bacterial strain
Manage significant difference (P < 0.05) compared with the control.
Table three
AG18 endogenetic bacteria can be obtained by table three and there is the effect of rush rermilion Chinese scholartree seed germination.Reflect according to 16SrDNA
Fixed result and the sequence similarity comparison in GeneBank, this AG18 endogenetic bacteria and bacillus megaterium bacterial strain
(Bacillus megaterium strain G1-7) has the similarity of 100%, and it belongs to the one of bacillus.
As depicted in figs. 1 and 2, during it is respectively the present invention, isolated endogenetic bacteria promotees the seed germination rate of seed germination
Bar diagram and isolated endogenetic bacteria promote the block diagram of seed root length in seed germination, it can be seen that AG18
Promote seed germination effect the most obvious." * " in figure represents that bacterial strain processes significant difference, P < 0.05 compared with matched group.
P value is probability, and P < 0.05 is notable, and P < 0.01 is extremely notable, i.e. refers to that the difference between sample is by caused by sampling error
Probability less than 0.05 or 0.01.
Owing to endophyte cell suspension is in PBS, carry out seed promoting sprouting effect, so the seed processed by PBS is made
For matched group.
It can be seen that the AG18 endogenetic bacteria obtained by the separation method of the present invention to promote seed germination with
The growth of root has obvious advantage.
Rermilion self germination rate of Chinese scholartree seed is extremely low, the rich and varied interior life obtained by the separation method in the present invention
Bacterium, reduces the risk that microbial resources are lost, and uses mechanical treatment (cut) seed, and by the side of plant with microbial interaction
Formula provide endophyte promote plant seed germination method, and obtain endophyte AG18 rermilion Chinese scholartree seed is had rush sprout
Effect.Not having the growth-promoting functions of this endophyte strains on plant of reported in literature at present, this is also to obtain from rermilion Chinese scholartree first
Growth-promoting bacterial strain.
These are only the preferred embodiments of the present invention, be not limited to the present invention, for those skilled in the art
For Yuan, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, any amendment of being made,
Equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. the separation method of a rermilion Radix sophorae portion endogenetic bacteria, it is characterised in that comprise the steps:
Step 1, once shearing: after rermilion Radix sophorae portion tissue wash, be cut into 4-6cm fragment；
Step 2, surface sterilization: with ethanol and sodium hypochlorite, described 4-6cm fragment is carried out surface sterilization process successively；
Step 3, secondary shearing: the described 4-6cm fragment disinfected aseptically is cut into 0.5-1cm fragment；
Step 4, the separation of endogenetic bacteria and cultivation: described 0.5-1cm fragment is inserted directly in culture medium flat plate and cultivates,
Picking different shape bacterium colony, carrying out streak culture until obtaining the pure culture of each bacterium colony, preserving.
The separation method of rermilion Radix sophorae portion the most according to claim 1 endogenetic bacteria, it is characterised in that
Step 2 is particularly as follows: first with 75% alcohol disinfecting 3min, aseptic water washing at least 1 time, then uses 2% hypochlorite disinfectant
3min, aseptic water washing 6 times, take 100 flushing water detection Disinfection Effects last for μ l.
The separation method of rermilion Radix sophorae portion the most according to claim 1 endogenetic bacteria, it is characterised in that in step 4, described
Culture medium is beef-protein medium, and its pH value is 7.0.
The separation method of rermilion Radix sophorae portion the most according to claim 3 endogenetic bacteria, it is characterised in that in step 4, described
After 0.5-1cm fragment is cultivated 7 days with 28～30 DEG C in described culture medium, then carry out streak culture, be saved in the glycerol of 20%
In, it is stored in-80 DEG C.
5. one kind promotees seed germination method, it is characterised in that the interior life described method arbitrary in Claims 1-4 prepared
Antibacterial promotees, in seed germination method, to comprise the steps: for this plant
Step a, Seeds preprocess: use liquor natrii hypochloritis to carry out disinfection described seed, carry out afterwards cutting into slices brokenly and plant；
Prepared by step b, bacteria suspension: be inoculated in beef extract-peptone fluid medium by described endogenetic bacteria, be placed in shaking table
Middle incubated overnight, is centrifuged afterwards, and the precipitation taking described endogenetic bacteria is resuspended in PBS, obtains described bacteria suspension；
Step c, seed soaking: immerse in described bacteria suspension by the described seed of sterilization, soak seed 4h；
Step d, sowing: carry out sprouting by the described seed after seed soaking and cultivate, and periodic statistical germination percentage and radicle length.
Rush seed germination method the most according to claim 5, it is characterised in that step a particularly as follows: first with 75% ethanol
Sterilization 12min, aseptic water washing at least 1 time, then sterilize 15min with the liquor natrii hypochloritis of 5%, aseptic water washing 6 times.
Rush seed germination method the most according to claim 5, it is characterised in that the concussion frequency of shaking table described in step b
For 150rpm.
Rush seed germination method the most according to claim 5, it is characterised in that in step b, OD600 is adjusted to 1.0,
Centrifugation rate is 8000rmp, and centrifugation time is 10min.
Rush seed germination method the most according to claim 5, it is characterised in that the described seed germination in step d is cultivated
Use culture dish filter paper method.
10. according to the arbitrary described AG18 endogenetic bacteria promoting the acquisition of seed germination method of claim 5 to 9, its feature
Being, described AG18 endogenetic bacteria has the effect promoting seed germination.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110915356A (en) * | 2019-12-06 | 2020-03-27 | 怀化学院 | Germination promoting method for plant seeds |
| CN111019856A (en) * | 2019-12-11 | 2020-04-17 | 榆林市中泰农业科技有限公司 | Cockerella rosea SDB9 and preparation method and application thereof |
| CN111826319A (en) * | 2020-07-31 | 2020-10-27 | 西南林业大学 | Microbial growth promoter and application thereof |
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| CN101565676A (en) * | 2008-04-24 | 2009-10-28 | 中国医学科学院药用植物研究所 | Three fungus strains for promoting germination of arethusa seeds |
| CN101622922A (en) * | 2009-08-07 | 2010-01-13 | 昆明市林业科学研究所 | Symbiotic germination method of Chinese cymbidium hybrid seeds and bacteria |
| CN102676398A (en) * | 2012-03-14 | 2012-09-19 | 安徽农业大学 | Separation and purification method of endophytic fungi from ginkgo biloba |
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| CN101225430A (en) * | 2008-02-14 | 2008-07-23 | 江苏科技大学 | Separation screening method for antibiotic antituberculotic plant endophyte |
| CN101565676A (en) * | 2008-04-24 | 2009-10-28 | 中国医学科学院药用植物研究所 | Three fungus strains for promoting germination of arethusa seeds |
| CN101622922A (en) * | 2009-08-07 | 2010-01-13 | 昆明市林业科学研究所 | Symbiotic germination method of Chinese cymbidium hybrid seeds and bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110915356A (en) * | 2019-12-06 | 2020-03-27 | 怀化学院 | Germination promoting method for plant seeds |
| CN111019856A (en) * | 2019-12-11 | 2020-04-17 | 榆林市中泰农业科技有限公司 | Cockerella rosea SDB9 and preparation method and application thereof |
| CN111826319A (en) * | 2020-07-31 | 2020-10-27 | 西南林业大学 | Microbial growth promoter and application thereof |
| CN111826319B (en) * | 2020-07-31 | 2023-02-24 | 西南林业大学 | Microbial growth promoter and application thereof |
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Application publication date: 20161026 |