CN101899430A - High-efficiency nitrogen-fixing microorganism mutation breeding method - Google Patents
High-efficiency nitrogen-fixing microorganism mutation breeding method Download PDFInfo
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- CN101899430A CN101899430A CN2010102238675A CN201010223867A CN101899430A CN 101899430 A CN101899430 A CN 101899430A CN 2010102238675 A CN2010102238675 A CN 2010102238675A CN 201010223867 A CN201010223867 A CN 201010223867A CN 101899430 A CN101899430 A CN 101899430A
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Abstract
The invention discloses a high-efficiency nitrogen-fixing microorganism mutation breeding method. In the method, nitrogen-fixing microorganism culture solution is irradiated by 2,400-2,500 MHz microwaves with the centre wavelength of 122mm. The microwaves with the specific wavelength can perform mutation on some specific DNA fragments of the nitrogen-fixing microorganisms so as to initiate hereditary variation of the nitrogen-fixing capacity of strains, and the radiation mutation treatment effect of 2,450 MHz microwaves is the best. By performing multi-generation directional selection on high-efficiency nitrogen-fixing mutant strains through nitrogen-free medium isolation and the testing of nitrogenase activity of the strains, stable and high-efficiency mutant strains with stronger nitrogen-fixing capacity than that of original strains are bred. Compared with ultraviolet mutation, high-energy particle beam mutation, chemical mutation, and other mutation modes, the high-efficiency nitrogen-fixing microorganism mutation breeding method has the characteristics of safety, cleanliness, simple equipment, low cost, high efficiency, and capacities of improving the breeding efficiency of high-efficiency nitrogen-fixing microorganisms and reducing the breeding cost.
Description
Technical field
The present invention relates to the microorganism mutation breeding field, particularly, relate to a kind of nitrogen-fixing microorganism mutation breeding method.
Background technology
Vinelandii are meant can free living and airborne nitrogen is fixed into can be by the microorganism of the available nitrogen of plant utilization.The short fertilizer of giving birth to of microorganism so that nitrogen-fixing microorganism is made has been widely used in the agriculture production at present, and is nontoxic, free from environmental pollution, production-increasing function is obvious, and amount of increase in production generally reaches 5%～17.9%, is applied to the interior effect of increasing production of the lower anthropogenic soil of available nitrogen and reaches 22.1%～34%.Both at home and abroad all in active research and Application and Development.The nitrogen-fixing microorganism fertiliser production of China is used existing nearly 50 years history, has obtained good economic benefit.As the basis that vinelandii fertilizer is produced and used, the seed selection of high-efficiency nitrogen-fixing bacterial strain is development of vinelandii fertilizer and exploitation most crucial steps.But the nitrogen fixing capacity of wild azotobacter strain often a little less than, do not reach effectively short purpose of giving birth to.
At present, the high-efficiency nitrogen-fixing microorganism mutagenic and breeding often adopts methods such as ultraviolet irradiation mutagenesis, chemomorphosis, and its defective is to exist certain danger, may cause serious injury to operator's health, and the efficient bacterial strain that selects easily produces reverse mutation, and inherited character is often unstable.The method of energetic particle beam and laser beam irradiation mutagenesis then cost is higher, and its concrete operations also are subjected to appointed condition and Personnel Skill Levels's influence, and the seed selection efficient of strain excellent is restricted and weakens.Therefore how to improve the seed selection efficient of high-efficiency nitrogen-fixing microorganism, always be the emphasis of scientific research institution and microbial inoculum research and development department research.
Summary of the invention
The objective of the invention is at above-mentioned defective of the prior art, propose that a kind of safely cleaning, equipment are simple, the nitrogen-fixing microorganism mutation breeding method of Cheap highly effective, can improve the breeding efficiency of high-efficiency nitrogen-fixing microorganism and reduce the breeding cost.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of high-efficiency nitrogen-fixing microorganism mutation breeding method, comprise mutagenic treatment, the screening and many of nitrogen-fixing microorganism for orthoselection, described mutagenic treatment is to adopt the microwave exposure of 2400～2500MHz, centre wavelength 122mm to handle the nitrogen-fixing microorganism nutrient solution, irradiation power is 100W～1050W, and irradiation time is 5s～240s.
The present invention adopts the microwave of 2400～2500MHz, centre wavelength 122mm to nitrogen-fixing microorganism nutrient solution and pure yeast culture thing radiation treatment certain hour.The micro-wave energy of this specific wavelength causes the heritable variation of bacterial strain nitrogen fixing capacity to some specific DNA fragments generation mutagenesis of nitrogen-fixing microorganism, and is best with the microwave radiation mutagenic treatment effect of 2450MHz.
Further, the OD value of described nitrogen-fixing microorganism nutrient solution when 600nm be 0.5 and more than.
Further, described nitrogen-fixing microorganism is rhizobium melioti (Rhizobium meliloti), and microwave exposure power is 800W, and irradiation time is 12s.
Further, described nitrogen-fixing microorganism is azotobacter chroococcum (Azotobocter chroococcum), and microwave exposure power is 150W, and irradiation time is 178s, or irradiation power is 100W, and irradiation time is 240s.
Further, described nitrogen-fixing microorganism is klebsiella (Klebsiella sp.), and microwave exposure power is 250W, and irradiation time is 36s.
Further, described nitrogen-fixing microorganism is sainfoin root nodule bacterium (Rhizobium), and microwave exposure power is 1050W, and irradiation time is 5s.
Above-mentioned different nitrogen-fixing microorganism, under above specific irradiation power, irradiation time parameter, it is the highest that positive mutation rate all reaches.
Further, in the present invention, the step of described screening is: will coat Winogradsky through the bacteria suspension of the nitrogen-fixing microorganism after the microwave exposure mutagenic treatment does not have on the nitrogen solid medium and cultivates, select the colony growth diameter obviously greater than the mutant strain of original strain, the back adopts the acetylene reduction method to finish the nitrogenase activity quantitative assay of pure growth, tentatively obtains good mutant strain.
It is as follows that wherein Winogradsky does not have the collocation method of nitrogen solid medium:
Substratum mother liquor preparation: KH
2PO
4(50.0g/L), MgSO
47H
2O (25.0g/L), NaCl (25.0g/L), FeSO
47H
2O (1.0g/L), Na
2MoO
42H
2O (1.0g/L), MnSO
44H
2O (1.0g/L) adds that to regulate pH with 1M NaOH behind the 1000ml dissolved in distilled water be 7.2, and 5ml substratum mother liquor is settled to 1000ml with the mannitol solution of 10g/L, adding CaCO
3Powder (0.1g/L) is with 2M H
2SO
4Adjust pH is 6.2.Solid medium adds agar 15g/L behind the filtering with microporous membrane of 0.45 μ m, regulating the pH value is 6.8 back autoclave sterilizations.
Further, in the present invention, described many steps for orthoselection are: will apply short the come into force fruit of cultivated plant root with the checking mutant strain through the mutant strain nutrient solution that screening obtains, the mutant strain that nitrogenase activity and plant growth-promoting effect all significantly is better than starting strain goes down to posterity test to measure its genetic stability, finally obtains the short stable bacterial strain of inheritance of ability of giving birth to of fixed nitrogen.
The present invention compares mutant strain nitrogen fixing capacity synergy height, low, the simple to operate safety of mutagenic and breeding cost, breeding efficiency height with chemomorphosis, uv irradiation mutagenesis, energetic particle beam irradiation mutagenesis, space flight etc.The mutagenic and breeding of at present short living microorganism excellent species mainly adopts the method for uv irradiation mutagenesis, chemomorphosis, energetic particle beam and high-energy electron beam irradiation, its mutagen mode is different, but ultimata all is by destroying original dna structure of thalline and base sequence, and make it produce reorganization and reparation, in this process, produce heritable variation.Ultraviolet mutagenesis since penetrance a little less than, need manually bacterium liquid to be stirred, make a large amount of thalline touch uviolizing, and that the employed medicine of chemomorphosis has is very strong carcinogenic pathogenic to human body, so ultraviolet irradiation mutagenesis and chemomorphosis are bigger to the injury that human body may cause.The energetic particle beam irradiation mutagenesis is then comparatively safe, but after surpassing doses, secondary radioactivity can appear in bacterium liquid.In addition, the apparatus expensive of energetic particle beam irradiation only can be finished in minority research and development unit with good conditionsi.There is not secondary radioactivity problem in microwave exposure mutagenesis nitrogen-fixing microorganism, does not have the residual of chemical agent and pollution yet, and is safe.Because the equipment that microwave exposure uses is cheap and easy to get, easy and simple to handle, also improve the efficient of strain selection and reduced the seed selection cost, have good practicability and reliability.
The major advantage of high-efficiency nitrogen-fixing microorganism microwave irradiation of the present invention breeding is:
1, equipment cheapness, radiation parameter controllability and security are good:
Because it is irradiation source that microwave exposure is handled with civilian microwave oven, equipment is cheap and easy to get.The significant parameter of radiation treatment is irradiation power and irradiation time, all can control by adjusting irradiation firepower gear and irradiation time, has reduced the operation easier of bacterial strain irradiation mutagenesis.Common microwave stove security at present is all than higher, microwave leakage can not occur, the principle of microwave exposure is different with particle beam irradiation simultaneously, no matter the dosage size, there is not secondary radioactive possibility in bacterium liquid after the processing, and this safety to operator provides guarantee.
2, efficient mutant strain seed selection efficient height:
After the mutagenesis of nitrogen-fixing microorganism bacterial strain process microwave exposure, under optimum irradiation parameters, klebsiella (250W, 36s), rhizobium melioti (800W, 12s), azotobacter chroococcum (150W, 178s or 100W, 240s), the sainfoin root nodule bacterium (1050W 5s) waits nitrogen-fixing microorganism all can reach positive mutation rate more than 14%.Come the qualitative relatively nitrogen fixing capacity of mutant strain in conjunction with the single colony diameter size on the nitrogen-free agar, finish the nitrogenase activity quantitative assay of pure growth with the acetylene reduction method, can in 1～2 week, finish the mutagenesis and the screening process of bacterial strain, tentatively obtain good mutant strain.
3, mutant strain nitrogen fixing capacity synergy height:
When microwave exposure, have electromagnetism polar water molecules generation HF oscillation, the dna double chain structure of nitrogen-fixing microorganism is subjected to the violent friction of water molecules, and the synergy variation of generation is bigger.The good mutant strain thalline pure growth that obtains or the nitrogen fixing capacity synergy of its symbiotic nitrogen fixation system (root nodule) reach 47.03%～3884.01% (vinelandii pure growth) and 26.02%～159.28% (root nodule of symbiotic nitrogen-fixing bacteria) respectively, all reach utmost point conspicuous level (P＜0.01) with original strain difference;
4, easy and simple to handle, be easy to grasp:
The simple easy to understand of this know-why, the nitrogen-fixing microorganism elite plant screening method that relates to is comparative maturity, is convenient to operation, can adapt to different production and development environment.
Embodiment
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for description and interpretation the present invention, and be not used in qualification the present invention.
With fixed nitrogen bacterial classifications such as rhizobium melioti, azotobacter chroococcum, klebsiella, sainfoin root nodule bacterium is that example illustrates microwave exposure method for mutation breeding of the present invention:
Adopt the cultivation bacterium liquid (OD of the microwave of 2400～2500MHz, centre wavelength 122mm to nitrogen-fixing microorganism
600nm=0.5) carrying out microwave exposure and handle (according to the difference of fixed nitrogen bacterial classification, irradiation power is 100W to 1050W, and irradiation time 5s to 240s does not wait), is the best irradiation parameters of azotobacter strain with the highest irradiation power of positive mutation rate.Will be under best irradiation parameters postradiation root nodule bacterium (800W, 12s), azotobacter chroococcum (150W, 178s or 100W, 240s), klebsiella (250W, 36s), sainfoin root nodule bacterium (1050W, 5s) bacteria suspension (bacterial content about 1 * 10 of nitrogen-fixing microorganism such as grade
4Cfu.ml
-1) the 0.2ml separate application do not have on the nitrogen solid medium (substratum mother liquor preparation: KH in Winogradsky
2PO
4(50.0g/L), MgSO
47H
2O (25.0g/L), NaCl (25.0g/L), FeSO
47H
2O (1.0g/L), Na
2MoO
42H
2O (1.0g/L), MnSO
44H
2O (1.0g/L) adds that to regulate pH with 1M NaOH behind the 1000ml dissolved in distilled water be 7.2.5ml substratum mother liquor is settled to 1000ml with the mannitol solution of 10g/L, adds CaCO
3Powder (0.1g/L) is with 2M H
2SO
4Regulating the pH value is 6.2.Solid medium adds agar 15g/L behind the filtering with microporous membrane of 0.45 μ m, regulating the pH value is 6.8 back autoclave sterilizations), cultivate 7d (rhizobium melioti respectively, the sainfoin root nodule bacterium) or 5d (azotobacter chroococcum, amphimicrobian vinelandii such as klebsiella) back selects single colony growth diameter obviously greater than the mutant strain of original strain, carry out the mensuration of pure growth or symbiosis root nodule (by root nodule bacterium mutant strain inducing plant root system and the symbiotic nitrogen fixation nodules that forms) nitrogenase activity by mutant strain, filter out the high-efficiency nitrogen-fixing mutant strain of nitrogenase activity apparently higher than original strain to primary election.The mutant strain nutrient solution is applied the cultivation plant, and (non-nodulation and nitrogen fixation bacterium inoculation plant is an oat; The inoculation plant of rhizobium melioti and sainfoin root nodule bacterium is respectively alfalfa and sainfoin) root with the checking mutant strain short come into force the fruit, the mutant strain that nitrogenase activity and plant growth-promoting effect all significantly is better than starting strain goes down to posterity test to measure its genetic stability, finally obtains the short stable bacterial strain of inheritance of ability of giving birth to of fixed nitrogen.The bacterial strain nitrogenase activity synergy that obtains with this irradiation mutagenesis method reaches 47.03%～3884.01%.The high-efficiency nitrogen-fixing bacterium mutant strain that induces reaches 14.44%～36.43% to the increasing amount of oat dry weight, improve 33.58%～137.10% than original strain, increasing amount to the clover dry weight reaches 11.02～58.54%, improves 14.98%～90.50% than original strain.Increasing amount to the sainfoin dry weight reaches 14.13～35.96%, improves 21.22%～111.76% than original strain.Statistical results show, the nitrogenase activity of efficient mutant strain and the increasing amount and the difference between original strain of plant dry weight reached significantly (P＜0.05) and utmost point conspicuous level (P＜0.01) respectively.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment put down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. high-efficiency nitrogen-fixing microorganism mutation breeding method, comprise mutagenic treatment, the screening and many of nitrogen-fixing microorganism for orthoselection, it is characterized in that: described mutagenic treatment is to adopt the microwave exposure of 2400～2500MHz, centre wavelength 122mm to handle the nitrogen-fixing microorganism nutrient solution, irradiation power is 100W～1050W, and irradiation time is 5s～240s.
2. high-efficiency nitrogen-fixing microorganism mutation breeding method according to claim 1 is characterized in that, the frequency of described microwave exposure is 2450MHz.
3. high-efficiency nitrogen-fixing microorganism mutation breeding method according to claim 1 is characterized in that, the OD value of described nitrogen-fixing microorganism nutrient solution when 600nm be 0.5 and more than.
4. high-efficiency nitrogen-fixing microorganism mutation breeding method according to claim 1 is characterized in that, described nitrogen-fixing microorganism is a rhizobium melioti, and microwave exposure power is 800W, and irradiation time is 12s.
5. high-efficiency nitrogen-fixing microorganism mutation breeding method according to claim 1 is characterized in that, described nitrogen-fixing microorganism is an azotobacter chroococcum, and microwave exposure power is 150W, and irradiation time is 178s, or irradiation power is 100W, and irradiation time is 240s.
6. high-efficiency nitrogen-fixing microorganism mutation breeding method according to claim 1 is characterized in that, described nitrogen-fixing microorganism is a klebsiella, and microwave exposure power is 250W, and irradiation time is 36s.
7. high-efficiency nitrogen-fixing microorganism mutation breeding method according to claim 1 is characterized in that, described nitrogen-fixing microorganism is the sainfoin root nodule bacterium, and microwave exposure power is 1050W, and irradiation time is 5s.
8. according to the described high-efficiency nitrogen-fixing microorganism mutation breeding method of claim 1～7, it is characterized in that the step of described screening is:
To coat Winogradsky through the bacteria suspension of the nitrogen-fixing microorganism after the microwave exposure mutagenic treatment does not have on the nitrogen solid medium and cultivates, select the colony growth diameter obviously greater than the mutant strain of original strain, the back adopts the acetylene reduction method to finish the nitrogenase activity quantitative assay of pure growth, tentatively obtains good mutant strain.
9. high-efficiency nitrogen-fixing microorganism mutation breeding method according to claim 8 is characterized in that, described many steps for orthoselection are:
To apply short the come into force fruit of cultivated plant root through the mutant strain nutrient solution that screening obtains with the checking mutant strain, the mutant strain that nitrogenase activity and plant growth-promoting effect all significantly is better than starting strain goes down to posterity test to measure its genetic stability, finally obtains the short stable bacterial strain of inheritance of ability of giving birth to of fixed nitrogen.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586102A (en) * | 2012-03-09 | 2012-07-18 | 张星昊 | Strain mutagenic instrument and method for carrying out composite mutagenesis on radiation rhizobium by utilizing strain mutagenic instrument |
| CN104450671A (en) * | 2014-11-25 | 2015-03-25 | 张吉科 | Breeding method of fast-growing type Frankia bacterial strain |
| CN109825496A (en) * | 2019-03-20 | 2019-05-31 | 南京康之春生物科技有限公司 | The breeding method of water body high-efficiency nitrogen-fixing bacterium |
| CN112739202A (en) * | 2018-07-11 | 2021-04-30 | 皮沃特生物股份有限公司 | Dynamic nitrogen delivery by remodeling microorganisms for temporal and spatial targeting |
| CN110438036B (en) * | 2019-07-10 | 2022-05-17 | 西北农林科技大学 | Nitrogen-fixing bacterium N24 with nitrogen-fixing effect and application thereof |
| US11565979B2 (en) | 2017-01-12 | 2023-01-31 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| EP3921293A4 (en) * | 2019-02-05 | 2023-04-05 | Pivot Bio, Inc. | Improved consistency of crop yield through biological nitrogen fixation |
| US11739032B2 (en) | 2015-07-13 | 2023-08-29 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
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2010
- 2010-07-12 CN CN2010102238675A patent/CN101899430A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586102A (en) * | 2012-03-09 | 2012-07-18 | 张星昊 | Strain mutagenic instrument and method for carrying out composite mutagenesis on radiation rhizobium by utilizing strain mutagenic instrument |
| CN104450671A (en) * | 2014-11-25 | 2015-03-25 | 张吉科 | Breeding method of fast-growing type Frankia bacterial strain |
| US11739032B2 (en) | 2015-07-13 | 2023-08-29 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US11565979B2 (en) | 2017-01-12 | 2023-01-31 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| CN112739202A (en) * | 2018-07-11 | 2021-04-30 | 皮沃特生物股份有限公司 | Dynamic nitrogen delivery by remodeling microorganisms for temporal and spatial targeting |
| JP2021530984A (en) * | 2018-07-11 | 2021-11-18 | ピボット バイオ， インコーポレイテッド | Dynamic nitrogen delivery targeting time and space by remodeled microorganisms |
| EP3820279A4 (en) * | 2018-07-11 | 2022-04-13 | Pivot Bio, Inc. | Temporally and spatially targeted dynamic nitrogen delivery by remodeled microbes |
| EP3921293A4 (en) * | 2019-02-05 | 2023-04-05 | Pivot Bio, Inc. | Improved consistency of crop yield through biological nitrogen fixation |
| CN109825496A (en) * | 2019-03-20 | 2019-05-31 | 南京康之春生物科技有限公司 | The breeding method of water body high-efficiency nitrogen-fixing bacterium |
| CN110438036B (en) * | 2019-07-10 | 2022-05-17 | 西北农林科技大学 | Nitrogen-fixing bacterium N24 with nitrogen-fixing effect and application thereof |
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Application publication date: 20101201 |