CN1682584A - Method for establishing symboitic relationship for arbuscular nycorrhizal fungi and tomato hairy root - Google Patents
Method for establishing symboitic relationship for arbuscular nycorrhizal fungi and tomato hairy root Download PDFInfo
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- CN1682584A CN1682584A CN 200410026858 CN200410026858A CN1682584A CN 1682584 A CN1682584 A CN 1682584A CN 200410026858 CN200410026858 CN 200410026858 CN 200410026858 A CN200410026858 A CN 200410026858A CN 1682584 A CN1682584 A CN 1682584A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Cultivation Of Plants (AREA)
- Pretreatment Of Seeds And Plants (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to the method of establishing symbiotic relationship between arbuscular nycorrhizal fungus and tomato hairy root. Germinated tomato seedling is first soaked with agrolacterium suspension and inoculated to sucrose-agar culture medium for culture to induce hairy root; the tomato hairy root is then transferred to M culture medium and AM fungi spore is inoculated for dark culture at 25 deg.c; and the germinated hypha invades into root system to establish the symbiotic relationship between arbuscular nycorrhizal fungus and tomato hairy root. The present invention induces tomato hairy root and establishes the symbiotic relationship between AM fungus and tomato hairy root on bacteria-free culture medium. The system of the present invention may be used in the amplification of AM fungus to obtain bacteria-free AM fungus material and in the relevant research of AM fungus under bacteria-free condition.
Description
[affiliated technical field]
The present invention relates to a kind of method of setting up arbuscular mycorrhizal fungi and tomato hairy root symbiotic relation.
[background technology]
Arbuscular mycorrhizal fungi (Arbuscular Mycorrhizal Fungi, AM fungi) is the soil fungi that a class can be set up symbiotic relation with 80% plant species.People have recognized that early the AM fungi can promote the absorption of root system of plant to mineral nutrition significantly, strengthen the resistance of plant to multiple soil-borne disease, thereby improve the growth of plant.Therefore, the consistent AM of the thinking fungi of various countries scientist has broad application prospects on most crops and horticultural crop, and be referred to as " bio-fertilizer " and " biopesticide " (Scientia Horticulturae, 1997,68:1-24).At present, for the research of AM fungi more and more widely, also more and more deep, many researchs need be carried out under aseptic condition, perhaps need to obtain aseptic AM fungal material.Yet up to the present, the AM fungi still can not pure culture, just can finish life cycle after must setting up symbiotic relation with host plant.Just because of this, the present AM fungus breeding of China mainly adopts the mode of substrate culture host plant to carry out, and these matrix mainly comprise soil, river sand, vermiculite, bead etc., under these condition of culture, can't obtain aseptic condition.For the biological property of arbuscular mycorrhizal fungi, need carry out under no matrix interference, aseptic condition with the research of the aspects such as interaction of edaphon, therefore former AM fungal culture method is difficult to be applied to this type of research.
[summary of the invention]
The objective of the invention is to limitation at present AM fungal culture method, provide a kind of method of on aseptic culture medium, setting up symbiotic relation, to obtain aseptic AM fungal material or under aseptic condition, to carry out the correlative study of AM fungi with arbuscular mycorrhizal fungi.
The method of on aseptic culture medium, setting up symbiotic relation provided by the present invention with arbuscular mycorrhizal fungi, mainly be to be material with tomato seeds, agrobacterium rhizogenes, AM fungi, form the tomato hairy root by inducing, on aseptic culture medium, inoculate the AM fungal spore then, promptly set up symbiotic relation between tomato hairy root and the AM fungi after to be formed the infecting, concrete operation method is as follows:
1, the tomato hairy root induces
After tomato seeds carries out surface sterilizing with 95% ethanol and 10% clorox, be seeded into after the washing on 2% the sucrose agar medium, after germinateing 7～10 days under 25 ℃ of black dull conditions, on superclean bench, seedling is cut into hypocotyl, mesocotyl and 3 parts of epicotyl, 10
7～10
8Soaked 20～30 minutes in the agrobacterium rhizogenes of cfu/ml (Agrobacterium rhizogenes) bacteria suspension, after in sterile water, washing 3-5 time, aseptic filter paper is inhaled and to be removed unnecessary bacterium liquid, is inoculated on 2% the sucrose agar medium, and the plant tissue surface begins to occur hairy root after about 7～15 days; After treating that hairy root grows to 2～5cm, cut hairy root, be placed on and contain on the antibiotic MS medium, through 3-5 successive transfer culture, until there not being the appearance of agrobacterium rhizogenes bacterium colony with the sterilization scissors;
2, the inoculation of AM fungi
The tomato hairy root is transferred on the M medium AM fungi (Gigaspora margarita) spore after inoculation sterilization on the surface of hairy root and the limit;
3, the foundation of syntaxial system
Postvaccinal culture dish is cultivated under 25 ℃ of black dull conditions, and the AM fungal spore can be sprouted after 1～5 day, and the germination mycelia can be invaded root system after 5-10 days, can see external mycelia after 3 weeks and grow in media surface, at this moment, shows that syntaxial system sets up;
4, cultivation and subculture
Can continue after syntaxial system is set up to cultivate 2-3 week under 25 ℃ of black dull conditions, if need carry out successive transfer culture, promptly one section root segment that contains external mycelia of clip is transferred on the new M medium later on, and the successive transfer culture time was advisable with 1 month.
The MS medium of mentioning in the said method is the medium commonly used of Plant Tissue Breeding, and the M medium is with reference to Becand G, the article that FortinJA 1988 delivers on New Phytologist (108:211-218) magazine, and M medium constituent is:
| Component | ????(mg/L) |
| ????MgSO 4.7H 2O ????KNO 3????KCL ????KH 2PO 4????Ca(NO 3) 2.4H 2O ????NaFe-EDTA ????KI ????MnCl 2.4H 2O ????ZnSO 4.7H 2O ????H 3BO 3????CuSO 4.5H 2O ????Na 2MnO 4.2H 2O glycine (amion acetic acid) thiamine hydrochloride Yan acid inositol Sugar agar pyridoxine hydrochloride pH | ????731.00 ????80.00 ????65.00 ????4.80 ????288.00 ????8.00 ????0.75 ????6.00 ????2.65 ????1.50 ????0.13 ????0.0024 ????3.00 ????0.10 ????0.50 ????50.00 ????10000.00 ????10000.00 ????0.10 ????5.5 |
[description of drawings]:
Fig. 1: tomato hairy root and AM mycosymbiosis system ideograph;
Fig. 2: the AM fungal spore infect (left side) and external mycelia (right side).
Among Fig. 1, the 1-culture dish; The external mycelia of 2-AM fungi; 3-tomato hairy root
[embodiment]
1, inducing of tomato hairy root:
Get each 10 of ruby and Fengshun tomato seeds, be placed in the water fully imbibition 12～24 hours,, after the sterile water washing 5 times, sow on 2% sucrose agar medium with 10% clorox sterilization 15～20 minutes; After culture dish is placed on and cultivates 7～10 days under 25 ℃ of black dull conditions, on superclean bench, seedling is cut into hypocotyl, mesocotyl and 3 parts of epicotyl, respectively 10
7～10
8Soaked 20～30 minutes in the agrobacterium rhizogenes of cfu/ml (Agrobacterium rhizogenes) the R1000 bacteria suspension, taking out the back washs 5 times with sterile water, wipe away the moisture on dried ground plant tissue surface again with aseptic filter paper, be inoculated on 2% the sucrose agar medium, place under 25 ℃ of black dull conditions and cultivate; Between culture period, often observe the formation of hairy root, the plant tissue surface begins to occur hairy root after about 7～15 days; After treating that hairy root grows to 2～5cm, cut hairy root, transfer on the MS medium of the ampicillin that adds 250mg/L, to remove agrobacterium rhizogenes with the sterilization scissors; Cultivate 2～3 all follow-up generations 1 time on the antibiotic MS medium containing, will not have the bacterium colony of agrobacterium rhizogenes this moment of degerming fully to occur around the hairy root behind the subculture 3 times; The hairy root of complete degerming is transferred to cultivate 7～10 days on the M medium of hairy root special use after, hairy root recovers growth;
2, the inoculation of AM fungi
Wet screening is collected the AM fungal spore: take by weighing the beaker that 20-50g AM fungal inoculant is put into 1L, carefully crumb with hand and to add the 0.7L running water behind the soil particle, leave standstill after stirring with glass bar is fierce after 1-3 minute and suspension to be poured into (aperture of upper screen is 1mm in the screen cloth that stacks, the aperture of lower screen is 100 μ m), remaining sediment adds the 0.5L running water again in beaker, glass bar leaves standstill about 1 minute after stirring, suspension is in pouring aforesaid screen cloth into, and repeated multiple times is till suspension is limpid; Fully wash mixture in the 1mm screen cloth with running water, remove the 1mm screen cloth then, carefully wash the mixture in the 100 μ m screen clothes again, and carefully mixture is all washed to one jiao, with wash bottle the whole flushings of mixture are transferred in the beaker of 100ml, again pour in the 100 μ m screen clothes after cleaning ultrasonically device concussion 30s, carefully transfer in the culture dish after running water carefully washes, under 20 times of stereomicroscopes, the spore in the culture dish is collected with sharp mouth suction pipe; In ultrasonic cleaner, handle 30 seconds to remove the dirt that spore shows, with in 2% chloramine-T solution, handling 30 minutes behind the distilled water wash spore, handled in 4 ℃ of refrigerators 12-18 hour with mixing antibiotic (0.02% streptomycin and 0.01% gentamicin) again; On superclean bench, the spore inoculating of sterilization is arrived the surface and the next door of the hairy root that recovers growth;
3, the foundation of syntaxial system
Postvaccinal culture dish is cultivated under 25 ℃ of black dull conditions, use the microscope tracing observation every day, generally inoculate 1～5 day after spore begin to sprout, can form hairy root after 5～10 days and infect, can see external mycelia after 3 weeks and come out, grow in media surface from hairy root; At this moment, the symbiotic relation of tomato hairy root and AM fungi is set up.
Claims (7)
1, a kind of method of setting up arbuscular mycorrhizal fungi and tomato hairy root symbiotic relation is characterized in that: be inoculated on the sucrose agar medium after soaking with the agrobacterium rhizogenes bacteria suspension by the tomato thin rice seedling after will germinateing and cultivate, induce to grow hairy root; Then the tomato hairy root is transferred on the M medium, the rearmounted dark condition of inoculation AM fungal spore is cultivated down, and the mycelia of waiting to germinate is invaded root system, promptly shows the symbiotic relation of having set up AM fungi and tomato hairy root.
2, method according to claim 1, operating process is as follows:
(1) inducing of tomato hairy root: tomato seeds is carried out surface sterilizing with 95% ethanol and 10% clorox, be seeded into after the washing on the sucrose agar medium, after dark condition germinateed 7～10 days down, on superclean bench, seedling cut into hypocotyl, mesocotyl and 3 parts of epicotyl, in agrobacterium rhizogenes (Agrobacterium rhizogenes) bacteria suspension, soaked 20～30 minutes, after in sterile water, washing 5 times, aseptic filter paper is inhaled and is removed unnecessary bacterium liquid, be inoculated on the sucrose agar medium, the plant tissue surface begins to occur hairy root after about 7～15 days; After treating that hairy root grows to 2～5cm, be placed on and contain on the antibiotic MS medium, through 3-5 successive transfer culture until there not being the appearance of agrobacterium rhizogenes bacterium colony;
(2) inoculation of AM fungi: the tomato hairy root is transferred on the M medium AM fungal spore after inoculation sterilization on the surface of hairy root and the limit;
(3) foundation of syntaxial system: postvaccinal culture dish is cultivated under black dull condition, the AM fungal spore can be sprouted after 1～5 day, the germination mycelia can be invaded root system after 5-10 days, can see external mycelia after 3 weeks and grow in media surface, and show that syntaxial system sets up this moment;
(4) cultivation and subculture: can continue after syntaxial system is set up to cultivate 2-3 week under black dull condition, one section root segment that contains external mycelia of clip is transferred on the new M medium when needing later on to carry out successive transfer culture.
3, the described method of claim 2, the concentration of the sucrose agar medium of use is 2%.
4, claim 1 or 2 described methods, the concentration of agrobacterium rhizogenes bacteria suspension is 10
7～10
8Cfu/ml.
5, claim 1 or 2 described methods, the temperature of cultivating under the black dull condition is 25 ℃.
6, the described method of claim 2, the sterilizing methods of AM fungal spore is: the AM fungal spore with handling 30 minutes in 2% the chloramine-T solution, was handled 12-18 minute for 4 ℃ with mixing antibiotic (0.02% streptomycin and 0.01% gentamicin) solution again.
7, the described method of claim 2, the time of successive transfer culture is 1 month.
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| CNB2004100268581A CN100558877C (en) | 2004-04-15 | 2004-04-15 | Set up the method for bush mycorrhizal fungi and tomato hairly root symbiotic relationship |
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|---|---|---|---|
| CNB2004100268581A CN100558877C (en) | 2004-04-15 | 2004-04-15 | Set up the method for bush mycorrhizal fungi and tomato hairly root symbiotic relationship |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101263778B (en) * | 2007-12-06 | 2010-08-18 | 云南大学 | Method for fast establishing DSE and plant symbiosis cultivation system and uses thereof |
| CN101595803B (en) * | 2008-06-03 | 2011-04-13 | 上海市农业科学院 | Method for producing edible fungus mycorrhizal seedling of mycorhiza |
| CN102166367A (en) * | 2010-12-30 | 2011-08-31 | 西北农林科技大学 | Method for AMF spore surface disinfection |
| CN105052499A (en) * | 2015-08-20 | 2015-11-18 | 云南大学 | Method for quickly establishing phialophora fungus and crop mutualism system |
| CN105063084A (en) * | 2015-09-02 | 2015-11-18 | 甘肃农业大学 | Method for obtaining hairy roots of broccoli |
| CN105532411A (en) * | 2016-01-28 | 2016-05-04 | 南京农业大学 | Method for storing arbuscular mycorrhizal fungi |
| CN105682449A (en) * | 2013-08-30 | 2016-06-15 | 赛姆普兰塔两合公司 | System and methods for continuous propagation and mass production of arbuscular mycorrhizal fungi in liquid culture |
| CN106857260A (en) * | 2017-03-06 | 2017-06-20 | 江门市鸿豪实友生物有限公司 | A kind of plant cultivation method |
| CN108444956A (en) * | 2018-01-26 | 2018-08-24 | 浙江师范大学 | With the method for laser confocal microscope and fluorescent dye observation AM fungi clump branch structures |
| CN111073842A (en) * | 2019-12-19 | 2020-04-28 | 华南农业大学 | Application of abscisic acid in promoting arbuscular mycorrhizal fungi spore production |
| CN113403204A (en) * | 2021-05-12 | 2021-09-17 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi and application of method |
-
2004
- 2004-04-15 CN CNB2004100268581A patent/CN100558877C/en not_active Expired - Lifetime
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101263778B (en) * | 2007-12-06 | 2010-08-18 | 云南大学 | Method for fast establishing DSE and plant symbiosis cultivation system and uses thereof |
| CN101595803B (en) * | 2008-06-03 | 2011-04-13 | 上海市农业科学院 | Method for producing edible fungus mycorrhizal seedling of mycorhiza |
| CN102166367A (en) * | 2010-12-30 | 2011-08-31 | 西北农林科技大学 | Method for AMF spore surface disinfection |
| CN102166367B (en) * | 2010-12-30 | 2013-07-10 | 西北农林科技大学 | Method for AMF spore surface disinfection |
| CN105682449A (en) * | 2013-08-30 | 2016-06-15 | 赛姆普兰塔两合公司 | System and methods for continuous propagation and mass production of arbuscular mycorrhizal fungi in liquid culture |
| CN105052499A (en) * | 2015-08-20 | 2015-11-18 | 云南大学 | Method for quickly establishing phialophora fungus and crop mutualism system |
| CN105063084A (en) * | 2015-09-02 | 2015-11-18 | 甘肃农业大学 | Method for obtaining hairy roots of broccoli |
| CN105532411A (en) * | 2016-01-28 | 2016-05-04 | 南京农业大学 | Method for storing arbuscular mycorrhizal fungi |
| CN106857260A (en) * | 2017-03-06 | 2017-06-20 | 江门市鸿豪实友生物有限公司 | A kind of plant cultivation method |
| CN108444956A (en) * | 2018-01-26 | 2018-08-24 | 浙江师范大学 | With the method for laser confocal microscope and fluorescent dye observation AM fungi clump branch structures |
| CN111073842A (en) * | 2019-12-19 | 2020-04-28 | 华南农业大学 | Application of abscisic acid in promoting arbuscular mycorrhizal fungi spore production |
| CN113403204A (en) * | 2021-05-12 | 2021-09-17 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi and application of method |
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Address after: 510070 No. 100 martyrs Middle Road, Guangdong, Guangzhou Patentee after: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) Address before: 510070 No. 100 martyrs Middle Road, Guangdong, Guangzhou Patentee before: Guangdong Institute of Microbiology |
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