CN100560717C - The method of agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm - Google Patents
The method of agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm Download PDFInfo
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- CN100560717C CN100560717C CNB2005100384125A CN200510038412A CN100560717C CN 100560717 C CN100560717 C CN 100560717C CN B2005100384125 A CNB2005100384125 A CN B2005100384125A CN 200510038412 A CN200510038412 A CN 200510038412A CN 100560717 C CN100560717 C CN 100560717C
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Abstract
The invention discloses the method for agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm, under aseptic condition, utilize Agrobacterium rhizogenes conversion plant rooting is grown fast, for plant nematode growth, growth and breeding provide enough nutrition, finish the cultured continuously of nematode.By this cultural method, can access a large amount of aseptic nematodes, can be directly used in inoculation and research application.
Description
Technical field
The invention belongs to the cultural method of a kind of plant nematode, a kind of specifically method of utilizing agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm.
Background technology
Plant nematode is the important causal organism of a class, wherein having many is internationally recognized crushing harmful organism such as pine wood nematode (Bursaphelenchus xylophilus), radopholus similes thorne (Radopholussimilis), potato white line worm (G.loboderapallida) and globodera rostochiensis (G.rostochiensis) etc., and the financial loss that whole world every year causes because of plant nematode harm is above 1,000 hundred million dollars.Plant nematode not only shows predation host plant nutrition and the physical abuse that feeding activity caused to the harm of plant, and its esophageal gland secretory product of what is more important causes host plant that a series of pathological changes take place and propagates other pathogen or the Secondary cases of stimulation and other pathogen of promotion infects harm.
With severe diseases sweep stem nematode on the northern China potato district sweet potato crop is example, after nematode is invaded sweet potato, the secretion of the back of the body esophageal gland polygalacturonase, amylase and proteolytic enzyme etc. rot sweet potato cell destruction, tissue, be accompanied by other fungi and bacterium aggravation harm, normal formation " the chaff heart " or be full of cracks, make sweet potato lose edibleness, the field piece underproduction 20%～50% of generally falling ill, grave illness field piece has no harvest basically not have and receives.
Because phase early 1940s is to the mid-50, the generation of the discovery of nematocides and use, some crushing plant nematode diseases and spreading and the announcement of Plant nematode and other plant pathogeny organism mutual relationship, people recognize that progressively plant nematode endangers the heavy economic losses that causes to agroforestry, thereby make plant nematode research obtain paying attention to and development.
For the generation and the harm of control line parasitosis well, must develop and use biochemistry and the biocontrol agent of nematode, development biochemistry and biocontrol agent at first will be understood biochemistry and the hereditary property of plant nematode.Accomplish this point, must cultivate and obtain the nematode population of a large amount of unanimities as research object.Relation between plant nematode, plant host and the environment is extremely complicated, and the variation that is difficult to be evaluated in the soil and is taken place.Thereby set up the important technological platform that an effective culture system of plant nematode is Plant nematode research.
Research around the plant nematode culture system has obtained some impressive progresses.The cultural method of current main plant parasitic nematode takes (1), host plant to cultivate; (2), single external source is cultivated on callus or the excised root; (3) on microorganism, cultivate; (4) not having external source on the substratum of special component cultivates.Show that the plant nematode cultivation by assorted external source cultivation beginning, carries out the transition to single external source and cultivates, develop into no external source now and cultivate.
Cultivate because most plant nematodes all can have external source, host plant is cultivated to separate and obtains a large amount of nematodes as research object, and shortcoming is that it is very difficult to study its biochemistry owing to the complicacy of plant nematode, plant host relation; Single external source is cultivated on callus or the excised root, is to study the normal method that adopts on the biochemical and hereditary developmental characteristic of plant nematode at present, and shortcoming is that the nematode population quantity of cultivating is little, can not succeeding transfer culture, and the continuity that influence is tested.This just requires to work out as early as possible single external source high-efficiency and continuous culture system of plant nematode.Soil is the home of most of nematodes.Soil has the moisture of nematode propagation and the root system of plant of nematode food source.Plant nematode has certain taxis to root system of plant, have greatly plant nematode with the feeding plant root system as hazard approach.
Agrobacterium rhizogenes (Agrobacterium rhizogenes) is the Gram-negative bacteria that belongs to Rhizobiaceae (Rhizobitaceae) Agrobacterium (Agrobacterium), can infect most dicotyledons and minority monocotyledons and discrete gymnosperm, the injury that brings out infected plant grows root of hair (hairy root).When Agrobacterium rhizogenes infection plant injury, the T-DNA fragment that the Ri plasmid is contained just changes the genome of plant over to.Infected vegetable cell can be bred fast in large quantities, grows root of hair thereby show on morphology at infected position.Stimulate the mechanism of taking root about Agrobacterium, the past is thought a kind of " irrelevant with plant hormone " always, thinks it may is the mechanism of action that causes similar growth hormone conduction mode by a kind of indirect gene interaction recently.Because each bar hairly root originates from a cell, cultivate so every YITIAOGEN is cloned in to contain on the antibiotic substratum as one, up to complete degerming.Transform root and can grow fast in the substratum that does not add exogenous hormone, branch is many, and geotropism is lost substantially.
Over nearly 20 years, people use the plant that Agrobacterium rhizogenes transforms higher economic worth, induce to produce root of hair (hairy root), utilize the root of hair culture technique to produce and extract useful secondary metabolite.The root of hair fast growth, tool hormone autonomy, the level of differentiation height, hereditary property is stable, and synthesis capability is stronger, has now developed into the another new culture technique after cell culture technology.
Summary of the invention
The present invention utilizes agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm, its major technique system is: under aseptic condition, utilize Agrobacterium rhizogenes conversion plant rooting is grown fast, for plant nematode growth, growth and breeding provide enough nutrition, finish the cultured continuously of nematode.
Technical scheme of the present invention is as follows:
The method of agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm is characterized in that may further comprise the steps:
(1), chooses plant tissue and sterilization;
(2), activation Agrobacterium rhizogenes;
(3), Agrobacterium rhizogenes is inoculated on the plant tissue inducing plant root of hair;
(4), the tip of a root vegetative point position of the root of hair of growing on the plant is transferred on the substratum grows; The tip of a root vegetative point position that intercepts root of hair on the substratum again is transferred on the substratum grows; So repeat,, obtain aseptic plant rooting through the number continuous tip of a root cultivation in generation;
(5), obtain nematode and the sterilization separate;
(6), plant nematode grows on the substratum of cultivating aseptic plant rooting under aseptic condition;
(7), substratum is placed in the clear water, nematode is free in clear water, realizes separating.
Described plant is a Radix Dauci Sativae, and substratum is the MSR substratum.
The method of described agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm is characterized in that may further comprise the steps:
(1), chooses clean peeling Radix Dauci Sativae, the clorox surface sterilization with 0.05%, and with sterile distilled water flushing, it is thick to be cut into 0.5-1cm, and carrot slice is inoculated in 1% water agar;
(2), with activatory Agrobacterium rhizogenes ATCC11325 bacterium liquid, get one in Radix Dauci Sativae section center, 20-25 ℃ of cultivation, on the Radix Dauci Sativae disk, the form layers place between xylem and phloem produces root of hair successively;
(3), select rapid, the sturdy root of growth, the tip of a root vegetative point of intercepting 1～3 cm long is transferred to MSR substratum growth root of hair;
(4), treat that the root of hair tip of a root is grown up after, intercept the tip of a root vegetative point of 2～3 cm long again, be transferred on the MSR substratum and grow; Repeated multiple times through number generation tip of a root cultivation continuously, obtains pure aseptic Radix Dauci Sativae root of hair;
(5), separate the sweet potato stem nematode that obtains on the host plant sweet potato morbidity potato piece;
(6), with nematode surface with the sterilization of 0.01-0.05% mercuric chloride after, use aseptic water washing, under sterile state, be inoculated into then in the MSR substratum of cultivating in the culture dish that root of hair is arranged, seal, be positioned over 20-25 ℃ of cultivation in the incubator of unglazed photograph then;
(7), substratum is put in the clear water, perhaps clear water is put in the substratum, nematode will swim in the clear water, separate.
Add fresh culture on the MSR substratum, root of hair just enters top substratum can obtain the required nutrient of growing again.
Accompanying drawing is an operational path of the present invention.
Advantage of the present invention:
(1), root of hair and nematode can grow simultaneously, the nematode population of cultivation is big;
(2), a large amount of lateral root and abundant root hair provide the agreeable to the taste site that takes food for nematode more on the root of hair; The heart
(3), cultivating system is transparent, the situation that takes food that can be by the microscopic examination nematode and growing Situation;
(4), the root of hair subculture is convenient, can Continuous Cultivation;
The nematode convenient separation of (5) cultivating.
Purposes of the present invention:
(1), utilize the relation that infects of this system's root system of plant and plant nematode, set up plant plant posted Give birth to the system that nematode resistance is identified. Root of hair has complete normal root institutional framework, meets the plant endoparasitism nematode Growing environment. Therefore more can reflect the interactional true feelings of plant nematode and host with this system Condition.
(2), by this cultural method, can access a large amount of aseptic nematodes, can be directly used in inoculation and research Use, avoided because the decline of the nematode vigor that sterilization causes. This monoxenic culture system also can simultaneously To be used for the preservation of nematode germplasm.
(3), provide a large amount of consistent nematode populations for research work. Be used for teaching department, be convenient to observation of students Understand the morphological feature of nematode, take food characteristic; Carry out the continuous of plant nematode biochemistry and hereditary developmental characteristic Journal of Sex Research;
(4), utilize this System For Screening, evaluation nematicide agent and the no external source of research to cultivate technical data is provided.
(5) root system can be used as genetically modified quick detection of expression experiment, and this is than sowing with real plants The cycle is short in basin or in the ground.
(6) key of research Plant nematode is the direct position that research nematode and host cell are related, Utilize the root of hair system to cultivate the method for nematode, can be a lot of high-tech experiments such as microchip research and make cDNA Libraries etc. provide the great many of experiments material.
Embodiment
Because Agrobacterium rhizogenes can be infected most dicotyledons and minority monocotyledons and discrete gymnosperm, the injury that brings out infected plant grows root of hair, thereby can set up important farm crop, cash crop and fruit tree that Agrobacterium rhizogenes can infect or the like root of hair system.And be applied to cultivation and the evaluation of plant nematode.Below to cultivate sweet potato stem nematode with the Radix Dauci Sativae root of hair be embodiment, its process is as follows:
One, the Radix Dauci Sativae root of hair induces
Radix Dauci Sativae is cleaned, the Radix Dauci Sativae exterior skin of pruning, clorox surface sterilization 20min with 0.05%, sterile distilled water flushing 5 times, it is thick to be cut into 0.5-1cm with scalpel then, carrot slice is inoculated in 1% water agar (be immersed in 1% water agar bottom the carrot slice, upper surface exposes), with activatory Agrobacterium rhizogenes ATCC11325 bacterium liquid, get one in Radix Dauci Sativae section center, 25 ℃ of cultivations.Through cultivation in 10 days, on the Radix Dauci Sativae disk, the form layers place between xylem and phloem produced root of hair successively.Root of hair is grown up about 3cm after 15 days.Select rapid, the sturdy root of growth, the tip of a root vegetative point of intercepting 2～3 cm long is transferred to MSR substratum (the root of hair cutting part is inserted on the MSR substratum); After treating that the tip of a root is grown up, intercept the tip of a root vegetative point of 2～3 cm long again, be transferred on the MSR substratum and grow; Repeated multiple times through number generation tip of a root cultivation continuously, so that remove the soil Agrobacterium that the root of hair surface may have, obtains pure aseptic Radix Dauci Sativae root of hair.
Two, the separation of sweet potato stem nematode
Separate the sweet potato stem nematode that obtains on the host plant sweet potato morbidity potato piece with the tray method.
Three, root of hair is cultivated nematode
Nematode is surperficial with behind the 0.01-0.05% mercuric chloride sterilization 20min, with aseptic water washing for several times, nematode amount by 100 in every ware is inoculated under sterile state in the MSR substratum of cultivating in the culture dish that root of hair is arranged then, with culture dish cap seal mouth, be positioned over 25 ℃ of cultivations in the incubator of unglazed photograph with sealing tape then.Can see the nematode that has bred in the culture dish more than several times of beginnings after one month.
Four, the renewal of root of hair substratum
Utilize the upwards characteristic of growth of root of hair, place fresh culture on old substratum, root of hair just enters top substratum can obtain the required nutrient of growing again, and it is convenient to make culturing base upgrade.
Five, the separation of nematode
A large amount of aseptic parasitic nematodes of on root of hair and root of hair substratum, growing.Nematode has hydrotaxis, and substratum is put in the clear water, perhaps clear water is put in the substratum, and nematode will swim in the clear water, separate.
MSR nutrient media components (mg/L) (Stephane Declerck 1989)
Stephane?Declerck；Desire?G.Strullu?and?Christian?Plenchette?Mycologia?90(4)1998，579
Claims (3)
1, the method for agro-bacteriumrhizogenes transferred plant rooting to cultivating plant parasite wireworm is characterized in that may further comprise the steps:
(1), chooses Radix Dauci Sativae tissue and sterilization;
(2), activation Agrobacterium rhizogenes;
(3), Agrobacterium rhizogenes is inoculated into the Radix Dauci Sativae tissue, bring out Radix Dauci Sativae and organize root of hair;
(4), the tip of a root vegetative point position of the root of hair that the Radix Dauci Sativae tissue is grown is transferred on the MSR substratum and grows; The tip of a root vegetative point position that intercepts root of hair on the MSR substratum again is transferred on the MSR substratum grows; So repeat,, obtain aseptic Radix Dauci Sativae root of hair through the number continuous tip of a root cultivation in generation;
(5), obtain sweet potato stem nematode and the sterilization separate;
(6), the Radix Dauci Sativae parasitic nematode is grown on the MSR substratum of cultivating aseptic Radix Dauci Sativae root of hair under aseptic condition;
(7), the MSR substratum is placed in the clear water, nematode is free in clear water, realizes separating.
2, method according to claim 1 is characterized in that may further comprise the steps:
(1), chooses clean peeling Radix Dauci Sativae, the clorox surface sterilization with 0.05%, and with sterile distilled water flushing, it is thick to be cut into 0.5～1cm, and carrot slice is inoculated in 1% water agar;
(2), with activatory Agrobacterium rhizogenes ATCC11325 bacterium liquid, get one in Radix Dauci Sativae section center, 20-25 ℃ of cultivation, on the Radix Dauci Sativae disk, the form layers place between xylem and phloem produces root of hair successively;
(3), select rapid, the sturdy root of growth, the tip of a root vegetative point of intercepting 1～3 cm long is transferred to MSR substratum growth root of hair;
(4), treat that the root of hair tip of a root is grown up after, intercept the tip of a root vegetative point of 2～3 cm long again, be transferred on the MSR substratum and grow; Repeated multiple times through number generation tip of a root cultivation continuously, obtains pure aseptic Radix Dauci Sativae root of hair;
(5), separate the sweet potato stem nematode that obtains on the host plant sweet potato morbidity potato piece;
(6), with nematode surface with the sterilization of 0.01-0.05% mercuric chloride after, use aseptic water washing, under sterile state, be inoculated into then in the MSR substratum of cultivating in the culture dish that root of hair is arranged, seal, be positioned over 20～25 ℃ of cultivations in the incubator of unglazed photograph then;
(7), substratum is put in the clear water, perhaps clear water is put in the substratum, nematode will swim in the clear water, separate.
3, method according to claim 3 is characterized in that adding fresh culture on the MSR substratum in (6) step, and root of hair just enters top substratum can obtain the required nutrient of growing again.
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| CN106135143B (en) * | 2016-08-18 | 2020-02-04 | 华南农业大学 | Method for culturing and propagating sterile radopholus similis by using pure carrot callus |
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| CN85107423A (en) * | 1985-09-29 | 1987-04-08 | 澳洲生物科技公司 | The liquid culture of nematode |
| CN1300817A (en) * | 1999-12-17 | 2001-06-27 | 广东省昆虫研究所 | Liquid culture method for pathogenic nematode of insect |
| WO2002038600A2 (en) * | 2000-11-09 | 2002-05-16 | Cenix Bioscience Gmbh | C. elegans genes involved in viability and/or reproduction and uses thereof |
| JP2004210645A (en) * | 2002-12-27 | 2004-07-29 | Sumitomo Chem Co Ltd | Method for controlling harmful plant-parasitic nematode |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85107423A (en) * | 1985-09-29 | 1987-04-08 | 澳洲生物科技公司 | The liquid culture of nematode |
| CN1300817A (en) * | 1999-12-17 | 2001-06-27 | 广东省昆虫研究所 | Liquid culture method for pathogenic nematode of insect |
| WO2002038600A2 (en) * | 2000-11-09 | 2002-05-16 | Cenix Bioscience Gmbh | C. elegans genes involved in viability and/or reproduction and uses thereof |
| JP2004210645A (en) * | 2002-12-27 | 2004-07-29 | Sumitomo Chem Co Ltd | Method for controlling harmful plant-parasitic nematode |
Non-Patent Citations (1)
| Title |
|---|
| 马铃薯茎线虫的培养. 徐进军等.西北农业学报，第13卷第4期. 2004 * |
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