CN105453923A - Method for promoting target rhizobium to migrate and colonize in alfalfa plant - Google Patents
Method for promoting target rhizobium to migrate and colonize in alfalfa plant Download PDFInfo
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Abstract
The invention discloses a method for promoting a target rhizobium to migrate and colonize in an alfalfa plant. An endogenous fluorescence labeling rhizobium R. GN5f liquid with cyan fluorescent protein (CFP) characteristics is obtained through a tri-parent conjugation method, natural plant source bacteriostatic agent matrine is added, and the endogenous fluorescence labeling rhizobium R. GN5f liquid and the matrine are mixed and inoculated to the root of gannong No.5 alfalfa in the reproductive growth stage. The method for promoting the target rhizobium to migrate and colonize in the alfalfa plant is advantaged in that (1), the target rhizobium R. GN5f is obtained through the tri-parent conjugation method, is good in genetic stability and convenient to use, and can be obtained easily; (2) the matrine restrains air source and soil source microorganisms, and does not restrain rhizobium, and promotes the migration and colonization of the rhizobium in the alfalfa, and is low in price, and is innocuous and unpoisonous to the environment; (3) the fluorescence labeling rhizobium liquid containing the matrine can be obtained easily, the inoculation operation is simple, short in consuming time, and inoculation can be carried out at any moment in the growth period; and (4) by adding the matrine, the migration and colonization of the target rhizobium are promoted, and the foundation is laid to obtain seeds with a lot of target rhizobia.
Description
Technical field
The present invention relates to a kind of promotion method that target rhizobium are migrated and surely grow in alfalfa plants body.
Background technology
Rhizobium and fabaceous symbiotic nitrogen fixation not only have huge economic and social benefit, and simultaneously in agricultural production, artificial infection rhizobium promote plant growth and improve seed production also to become a kind of common agricultural measures.But due to floristics and rhizobium kind and external special natural environment, inoculation method, with the competition of Indigenous Rhizobia and multiple biology and abioticly coerce the marker technique in situ that all can affect Rhizobium Inoculation, make part rhizobium be difficult to carry out nodulation and nitrogen fixation efficiently, be difficult to reach due effect of increasing production.Studies have found that endogenous rhizobium that alfalfa seed carries is compared with Indigenous Rhizobia, has obvious advantage in Competitive Nodulation.Rhizobium in seed and plant body is in endophyte category, and the vaccination ways of endophyte and the health status of plant corpus are surely grown the success of endophyte in plant corpus and played decisive role with Function.But at present, on rhizobium migrate in alfalfa plants body the approach that enters seed and seed decided at the higher level but not officially announced grow mechanism and affect rhizobium migrate in plant and seed surely to grow etc. study less.When research finds that vegetative growth phase, clover marked bacterium by injection inoculation in the middle part of stem, the migration of IAA to endogenous bacterium has promotion.Find to adopt root system tieback simultaneously and cut root and soak root nodule replanting Rhizobium Inoculation bacterium liquid phase ratio, cut root nodule replanting and inoculate more multiple labeling rhizobium can be made to enter clover root.Illustrate that screening promotes that the method for clover body internal object rhizobium vegetative growth phase has certain guidance meaning to importing target rhizobium thus, but the clover whether inoculation method of screening is beneficial to reproductive stage equally need further checking.And the example of clover-rhizobium assembly breeding has also only tentatively been done to the research report of vegetative growth phase, the domestic and international research about this breeding method is less at present.The research utilizing the migration pathway of rhizobium to change seed endogenous rhizobium ratio still locates the elementary step, to the quantity how improving seed internal object rhizobium, how by target rhizobium and the accurate efficient combination of clover breeding, also still belongs to blank.Therefore explore raising alfalfa plants and seed endogenous rhizobium quantity, promote that a large amount of target rhizobium are migrated and surely grow in alfalfa plants and seed by the interference of allogenic material, the accurate efficient combination realizing excellent rhizobium and excellent alfalfa variety becomes inevitable.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, provides a kind of promotion method that target rhizobium are migrated and surely grow in alfalfa plants body.
To achieve these goals, technical scheme provided by the invention is: a kind of promotion method that target rhizobium are migrated and surely grow in alfalfa plants body, comprises the following steps:
1) bacterial strain is selected: the fluorescence labeling rhizobium Rhizobiummeliloti.GN5f (R.GN5f) chosen, its original strain Rhizobiummeliloti.GN5 (R.GN5) are separated the excellent Rhizobium strains from sweet agriculture No. 5 alfalfa (Medicagosativacv.GannongNo.5) root nodules; The Inner source fluorescence labeling rhizobium R.GN5f containing cyan fluorescent protein (CFP) is obtained by triparental mating;
(original and fluorescence labeling Rhizobium strains provides by key lab of the grass cultivation ecosystem Ministry of Education of Gansu Agriculture University, original strain is identified by DSMZ of institute of microbiology of the Chinese Academy of Sciences, full sweet clover sword bacterium (EnsifermelilotiLZgn5) by name, be numbered 101220,2013 No. 133rd, micro-searchings).
2) vegetable material is chosen: clover material is sweet agriculture No. 5 alfalfas;
3) bacterium liquid is prepared: will transfer into 50mlTY liquid nutrient medium after the activation of the bacterial strain TY of step 1) flat board, 28 DEG C, 180rpm/min shaken cultivation is to bacterium liquid optical density OD
600nmvalue is the centrifugal 5min of 0.5-1,4000rpm/min, with isopyknic aseptic water washing thalline after supernatant of skimming, and is broken up with whirlpool oscillator, is finally modulated into OD with sterile water
600nmbe the R.GN5f fluorescence labeling rhizobium liquid of 0.5;
4) matrine is added: the matrine chosen is 600mg/L, and content is 1.3%, and in aseptic operating platform, matrine solution is with after 0.22 μm of sterilised membrane filter and aseptic needle tubing filtration sterilization, is added in cultured bacterium liquid, to final concentration 600mg/L;
5) inoculate: respectively random selecting squaring period, initial bloom stage, the alfalfa plants of pod bearing period 5 strain, dig dark about 5cm within the scope of root 10cm to cheat, expose main root, side root, hair root, evenly watered in root by 50ml/ strain by the fluorescence labeling rhizobium liquid prepared, earthing buries root thereafter; The R.GN5f inoculation not adding matrine is contrast;
6) detect period and detect tissue:
A) squaring period ~ maturing stage: detect root, stem, leaf, flower, seed;
B) flowering stage ~ maturing stage: detect root, stem, leaf, flower gynoecium ovule, flower gynoecium style, flower gynoecium column cap, flower stamen flower pesticide, flower stamen filigree, petal;
C) pod bearing period ~ maturing stage: detect young tender seed, mature seed, pod skin;
7) detection method:
1. sample: adopt respectively at squaring period, initial bloom stage, pod bearing period the inoculation method adding the pouring of matrine bacterium liquid, postvaccinal 5d, 15d, 30d, 60d sampling detects to be analyzed, until seed harvest, not add the bacterium liquid pouring of matrine for contrast; Get clover individual plant, isolate the hair root of the dark soil layer of 5-10cm at random, stem, leaf take from same plant all at random, and are separated into top stem, bottom stem, upper leaf and inferior leads, filigree, petal, ovule, style, column cap, flower pesticide, pod skin and seed; Naturally dry after sampling tap water is clean;
the dull and stereotyped quantity of portable uviol lamp detects: root, stem, leaf respectively takes 1g, spend 5/inflorescence, pod skin 5/inflorescence, seed 5/inflorescence, put into the aseptic triangular flask of 50ml, adding available iodine concentration is that the disinfectant tamed iodine of 2500mg/L floods, aseptic water washing 5 times after concussion sterilization 4min, to triangular flask, non-foam shape liquid occurs, organizing of sterilizing completely is placed in sterile mortar respectively, adding 2ml sterile water fully grinds rear centrifugal, centrifugal rotational speed is 4000rpm/min, centrifugation time is 5min, absorption 0.2ml supernatant is spread evenly across root lapping liquid and dilutes 10 successively
1-10
3tY solid culture medium, repeat for three times, cultivate after 24-48h for 28 DEG C, record fluorescence labeling rhizobium quantity in every ware in dark under portable uviol lamp, fluorescence labeling rhizobium issue blue-green light at portable uviol lamp,
3. body formula fluorescence microscope detects: under the black and white body formula fluorescence microscope visual field, green glow excites cfp display white, and in the detection, the alfalfa tissue containing CFP mark rhizobia be white, is NFly organized as black.
Further, the promotion method that above-mentioned a kind of target rhizobium are migrated and surely grow in alfalfa plants body, the preparation method of TY medium is: tryptone 5g/L, dusty yeast 3g/L, CaC1
26H
2o1.3g/L, pH to 7.0,121 DEG C of sterilizing 26min.
Beneficial effect of the present invention is: the promotion method that a kind of target rhizobium provided by the invention are migrated and surely grow in alfalfa plants body, and its major advantage has:
1) the target rhizobium R.GN5f chosen is obtained by triparental mating, and the genetic stability of this bacterial strain is good, obtains easily, easy to use;
2) matrine chosen, to air-source and soil source Antimicrobial under 600mg/L concentration, to rhizobium unrestraint effect, promotes the migration of rhizobium in clover body on the contrary and surely grows; And matrine is cheap, nontoxic to environment;
3) the fluorescence labeling rhizobium liquid obtained containing matrine is easy, and inoculate simple to operate, consuming time shorter, suitable growth can be inoculated in period at any time, and inoculation is convenient;
4) obviously facilitate target rhizobium after adding matrine migrated in terrestrial stem, leaf, flower, seed by root and surely grow, lay the foundation for acquisition carries a large amount of target rhizobium seed.
Accompanying drawing explanation
Fig. 1 grows quantity for adding matrine inoculation R.GN5f seed is decided at the higher level but not officially announced: dull and stereotypedly under A-uviol lamp (336nm) detect; B-body formula fluorescence microscopy Microscopic observation.C-does not add in matrine inoculation R.GN5f seed and does not detect R.GN5f.
Fig. 2 detects R.GN5f quantity in florescence ovule under uviol lamp (336nm): A-inoculates separately; B-inoculates after adding matrine.
Fig. 3 is that body formula fluorescence microscope adds matrine inoculation R.GN5f and grows situation root is decided at the higher level but not officially announced: A-hair root; C-side root center pillar; E-side root cortex; And do not add matrine inoculation R.GN5f and grow situation root is decided at the higher level but not officially announced: B-hair root; D-side root center pillar; F-side root cortex.
Embodiment
Embodiment 1:
The promotion method that target rhizobium are migrated and surely grow in alfalfa plants body, comprises the following steps:
1) bacterial strain is selected: select to obtain by triparental mating the Inner source fluorescence labeling rhizobium R.GN5f containing cyan fluorescent protein (CFP).(original and fluorescence labeling bacterial strain provides by key lab of the grass cultivation ecosystem Ministry of Education of Gansu Agriculture University, and original strain has been identified biochemical characteristic by DSMZ of institute of microbiology of the Chinese Academy of Sciences and measured 16sRNA sequence)
2) vegetable material is chosen: vegetable material is sweet agriculture No. 5 alfalfas;
3) bacterium liquid is prepared: will transfer into 50mlTY liquid nutrient medium after the activation of the bacterial strain TY of step 1) flat board, 28 DEG C, 180rpm/min shaken cultivation is to bacterium liquid optical density OD
600nmvalue is the centrifugal 5min of 0.5-1,4000rpm/min, with isopyknic aseptic water washing thalline after supernatant of skimming, and is broken up with whirlpool oscillator, is finally modulated into OD with sterile water
600nmbe the R.GN5f fluorescence labeling rhizobium liquid of 0.5;
4) matrine is added: the matrine chosen is 600mg/L, and content is 1.3%, and in aseptic operating platform, matrine solution is with after 0.22 μm of sterilised membrane filter and aseptic needle tubing filtration sterilization, is added in cultured bacterium liquid, to final concentration 600mg/L;
5) inoculate: respectively random selecting squaring period, initial bloom stage, the alfalfa plants of pod bearing period 5 strain, dig dark about 5cm within the scope of root 10cm to cheat, expose main root, side root, hair root, evenly watered in root by 50ml/ strain by the fluorescence labeling rhizobium liquid prepared, earthing buries root thereafter;
6) detect period and detect tissue:
A) squaring period ~ maturing stage: detect root, stem, leaf, flower, seed;
B) flowering stage ~ maturing stage: detect root, stem, leaf, flower gynoecium ovule, flower gynoecium style, flower gynoecium column cap, flower stamen flower pesticide, flower stamen filigree, petal;
C) pod bearing period ~ maturing stage: detect young tender seed, mature seed, pod skin;
7) detection method:
1. sample: connect 5d, 15d, 30d, 60d after bacterium sampling detect analysis, until seed harvest respectively at squaring period, initial bloom stage, pod bearing period employing interpolation matrine bacterium liquid pouring inoculation method; Get clover individual plant, isolate the hair root of the dark soil layer of 5-10cm at random, stem, leaf take from same plant all at random, and are separated into top stem, bottom stem, upper leaf and inferior leads, filigree, petal, ovule, style, column cap, flower pesticide, pod skin and seed; Naturally dry after sampling tap water is clean;
the dull and stereotyped quantity of portable uviol lamp detects: root, stem, leaf respectively takes 1g, spend 5/inflorescence, pod skin 5/inflorescence, seed 5/inflorescence, put into the aseptic triangular flask of 50ml, adding available iodine concentration is that the disinfectant tamed iodine of 2500mg/L floods, aseptic water washing 5 times after concussion sterilization 4min, to triangular flask, non-foam shape liquid occurs, organizing of sterilizing completely is placed in sterile mortar respectively, adding 2ml sterile water fully grinds rear centrifugal, centrifugal rotational speed is 4000rpm/min, centrifugation time is 5min, absorption 0.2ml supernatant is spread evenly across root lapping liquid and dilutes 10 successively
1-10
3tY solid culture medium, repeat for three times, cultivate after 24-48h for 28 DEG C, record fluorescence labeling rhizobium quantity in every ware in dark under portable uviol lamp, fluorescence labeling rhizobium issue blue-green light at portable uviol lamp,
3. body formula fluorescence microscope detects: under the black and white body formula fluorescence microscope visual field, green glow excites cfp display white, and in the detection, the alfalfa tissue containing CFP mark rhizobia be white, is NFly organized as black.
The present invention adopts triparental mating method to obtain and carries hanced cyan fluorescent (CFP) characteristic, there is safety, stable and going down to posterity property is good and can the Inner source fluorescence labeling rhizobium R.GN5f bacterium liquid of real-time in-situ monitoring and other advantages, sweet agriculture No. 5 alfalfa (Medicagosativacv.GannongNo.5) roots of the natural plants based bacteriostat matrine combined inoculation reproductive stage of interpolation 600mg/L (1.3%); With do not add compared with matrine inoculation method, root, top stem, flower, seed, this fluorescence labeling rhizobium quantity of pod intracutaneous can be improved.Flower bud phase bacterium liquid waters separately, and in hair root, R.GN5f quantity is only up to 76.1943cfu/g, and can promote after adding matrine that R.GN5f migrates in hair root, and quantity is up to 1.29 × 10
5cfu/g, is significantly higher than bacterium liquid and inoculates separately process (p<0.05); During florescence 5d, in hair root, R.GN5f quantity is up to 34.30cfu/g, but late detection is less than fluorescence labeling rhizobium; And after adding matrine, can promote that R.GN5f migrates in hair root, 60d quantity, up to 488cfu/g, is significantly higher than bacterium liquid and inoculates separately process 13.23 times (p<0.05), and the time of growing of determining in hair root is longer than the time inoculating separately process.This period, R.GN5f mainly grew surely in the passage that floral organ is the formation of seed.Root is inoculated separately and is only detected in ovule, and quantity is 1.30/ inflorescence; Add the bacterium liquid inoculation after matrine, the position that floral organ Nei Keding grows and quantity are respectively ovule 1.60cfu/ inflorescence, style and column cap 1.50cfu/ inflorescence, flower pesticide 3.00cfu/ inflorescence, and after inoculation 60d, its quantity of pod intracutaneous is 1.20cfu/g.Pod bearing period seed and result intracutaneous R.GN5f quantity be respectively 2.10cfu/ grain and 2.00cfu/ grain, and separately inoculation does not detect.
Fig. 1-Fig. 3 be in uviol lamp (336nm) and body formula fluorescence microscopy Microscopic observation clover partial organ fluorescence labeling rhizobium R.GN5f determine the situation of growing.
Because used matrine bacteriostatic agent is cheap, noresidue after decomposing in the Nature, and easy, easy to detect with bacterium liquid combined inoculation technology, be easy to experimenter's operation.Therefore the present invention has good practicality and reliability.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. the target rhizobium promotion method of migrating in alfalfa plants body and surely growing, is characterized in that, comprise the following steps:
1) bacterial strain is selected: the fluorescence labeling rhizobium Rhizobiummeliloti.GN5f (R.GN5f) chosen, its original strain Rhizobiummeliloti.GN5 (R.GN5) are separated the excellent Rhizobium strains from sweet agriculture No. 5 alfalfa (Medicagosativacv.GannongNo.5) root nodules; The Inner source fluorescence labeling rhizobium R.GN5f containing cyan fluorescent protein (CFP) is obtained by triparental mating;
2) vegetable material is chosen: clover material is sweet agriculture No. 5 alfalfas;
3) bacterium liquid is prepared: will transfer into 50mlTY liquid nutrient medium after the activation of the bacterial strain TY of step 1) flat board, 28 DEG C, 180rpm/min shaken cultivation is to bacterium liquid optical density OD
600nmvalue is the centrifugal 5min of 0.5-1,4000rpm/min, with isopyknic aseptic water washing thalline after supernatant of skimming, and is broken up with whirlpool oscillator, is finally modulated into OD with sterile water
600nmbe the R.GN5f fluorescence labeling rhizobium liquid of 0.5;
4) matrine is added: the matrine chosen is 600mg/L, and content is 1.3%, and in aseptic operating platform, matrine solution is with after 0.22 μm of sterilised membrane filter and aseptic needle tubing filtration sterilization, is added in cultured bacterium liquid, to final concentration 600mg/L;
5) inoculate: respectively random selecting squaring period, initial bloom stage, the alfalfa plants of pod bearing period 5 strain, dig dark about 5cm within the scope of root 10cm to cheat, expose main root, side root, hair root, evenly watered in root by 50ml/ strain by the fluorescence labeling rhizobium liquid prepared, earthing buries root thereafter; The R.GN5f inoculation not adding matrine is contrast;
6) detect period and detect tissue:
A) squaring period ~ maturing stage: detect root, stem, leaf, flower, seed;
B) flowering stage ~ maturing stage: detect root, stem, leaf, flower gynoecium ovule, flower gynoecium style, flower gynoecium column cap, flower stamen flower pesticide, flower stamen filigree, petal;
C) pod bearing period ~ maturing stage: detect young tender seed, mature seed, pod skin;
7) detection method:
1. sample: adopt respectively at squaring period, initial bloom stage, pod bearing period the inoculation method adding the pouring of matrine bacterium liquid, postvaccinal 5d, 15d, 30d, 60d sampling detects to be analyzed, until seed harvest, not add the bacterium liquid pouring of matrine for contrast; Get clover individual plant, isolate the hair root of the dark soil layer of 5-10cm at random, stem, leaf take from same plant all at random, and are separated into top stem, bottom stem, upper leaf and inferior leads, filigree, petal, ovule, style, column cap, flower pesticide, pod skin and seed; Naturally dry after sampling tap water is clean;
the dull and stereotyped quantity of portable uviol lamp detects: root, stem, leaf respectively takes 1g, spend 5/inflorescence, pod skin 5/inflorescence, seed 5/inflorescence, put into the aseptic triangular flask of 50ml, adding available iodine concentration is that the disinfectant tamed iodine of 2500mg/L floods, aseptic water washing 5 times after concussion sterilization 4min, to triangular flask, non-foam shape liquid occurs, organizing of sterilizing completely is placed in sterile mortar respectively, adding 2ml sterile water fully grinds rear centrifugal, centrifugal rotational speed is 4000rpm/min, centrifugation time is 5min, absorption 0.2ml supernatant is spread evenly across root lapping liquid and dilutes 10 successively
1-10
3tY solid culture medium, repeat for three times, cultivate after 24-48h for 28 DEG C, record fluorescence labeling rhizobium quantity in every ware in dark under portable uviol lamp, fluorescence labeling rhizobium issue blue-green light at portable uviol lamp,
3. body formula fluorescence microscope detects: under the black and white body formula fluorescence microscope visual field, green glow excites cfp display white, and in the detection, the alfalfa tissue containing CFP mark rhizobia be white, is NFly organized as black.
2. a kind of target rhizobium according to claim 1 promotion method of migrating in alfalfa plants body and surely growing, it is characterized in that, the preparation method of TY medium is: tryptone 5g/L, dusty yeast 3g/L, CaC1
26H
2o1.3g/L, pH to 7.0,121 DEG C of sterilizing 26min.
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| CN108362514A (en) * | 2018-02-05 | 2018-08-03 | 湖南省农业生物技术研究所 | A kind of elegant jessamine root nodule quick sampling method |
| CN108770868A (en) * | 2018-05-18 | 2018-11-09 | 甘肃农业大学 | A method of promote rhizobium to migrate and colonize in alfalfa plants body |
| CN109679852A (en) * | 2019-02-21 | 2019-04-26 | 深圳市伊山万瑞科技有限公司 | A method of screening pulse family kuh-seng high-efficiency nitrogen-fixing rhizobium |
| CN109943496A (en) * | 2019-01-02 | 2019-06-28 | 甘肃农业大学 | One plant of rhizobium seeks the method with the efficient symbiosis alfalfa variety of specificity |
| CN111471702A (en) * | 2020-04-30 | 2020-07-31 | 福建农林大学 | Slow-growing rhizobium stable red fluorescent labeling vector and application thereof |
| CN113229091A (en) * | 2021-06-18 | 2021-08-10 | 黑龙江八一农垦大学 | Regulation and control method for alfalfa continuous cropping obstacle in grassland |
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| CN109943496A (en) * | 2019-01-02 | 2019-06-28 | 甘肃农业大学 | One plant of rhizobium seeks the method with the efficient symbiosis alfalfa variety of specificity |
| CN109679852A (en) * | 2019-02-21 | 2019-04-26 | 深圳市伊山万瑞科技有限公司 | A method of screening pulse family kuh-seng high-efficiency nitrogen-fixing rhizobium |
| CN111471702A (en) * | 2020-04-30 | 2020-07-31 | 福建农林大学 | Slow-growing rhizobium stable red fluorescent labeling vector and application thereof |
| CN111471702B (en) * | 2020-04-30 | 2022-07-26 | 福建农林大学 | Slow-growing rhizobium stable red fluorescent labeling vector and application thereof |
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