CN106106168B - A method of explant endophyte when removal Plant Tissue Breeding - Google Patents
A method of explant endophyte when removal Plant Tissue Breeding Download PDFInfo
- Publication number
- CN106106168B CN106106168B CN201610576636.XA CN201610576636A CN106106168B CN 106106168 B CN106106168 B CN 106106168B CN 201610576636 A CN201610576636 A CN 201610576636A CN 106106168 B CN106106168 B CN 106106168B
- Authority
- CN
- China
- Prior art keywords
- explant
- cuttage
- branch
- amphotericin
- processing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G9/00—Cultivation in receptacles, forcing-frames or greenhouses; Edging for beds, lawn or the like
- A01G9/02—Receptacles, e.g. flower-pots or boxes; Glasses for cultivating flowers
- A01G9/029—Receptacles for seedlings
- A01G9/0299—Handling or transporting of soil blocks or seedlings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Soil Sciences (AREA)
- Botany (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention belongs to biotechnology, especially field of plant tissue culture technique, a kind of method of explant endophyte when disclosing removal Plant Tissue Breeding.The present invention carries out disinfection processing to cuttage soil using 50% carbendazim, it is carried out disinfection processing to cuttage branch using the aseptic aqueous solution containing 200mg/L amphotericin Bs and 100mg/L streptomycin sulphates, and the new hair branch of semi-lignified is pre-processed using the aseptic aqueous solution containing 100mg/L amphotericin Bs and 50mg/L streptomycin sulphates, so as to effectively remove the bacterium and fungi on the explants such as willow surface and inside, explant is made to keep sufficient vigor.
Description
Technical field
The present invention relates to biotechnologies, especially field of plant tissue culture technique, and in particular to a kind of removal plant
The method of explant endophyte when object tissue cultures.
Background technology
Willow belongs to Salicaceae (Salicacae) Populus (Populus L.) plant, is perennial woody arbor.Willow is not
Afforestation seeds and industrial cut stock seeds are only important, but also are the pattern seeds of perennial Forest-tree Gene Engineering.Cause
This, it is to carry out fast numerous and genetic engineering research the premise and basis of Tissues of Poplar Clones to obtain a large amount of sterile explants.
In conventional Plant Tissue Breeding, the sterilization method of explant is usually first to wash off surface mud with tap water
Soil disinfects in alcohol 30 seconds to 1 minute, then sterilizes a few minutes to more than ten minutes, nothing with the life mercury of 10% sodium hypochlorite or 0.1%
Bacterium water is inoculated with after rinsing.
Endophyte refers to that those are distributed in health plant body, and the bacterium of apparent Disease symptoms is not caused to plant tissue
Class, such as saprophytic bacteria, latent disease opportunistic pathogen, VA Mycorrhizal Fungi and nitrogen-fixing bacteria.Willow is perennial plant, and tree body inside Chang Jisheng has
Various bacteriums and fungi.Due to being protected by plant, conventional method for disinfection and sterilization is difficult to remove endophyte.Cut-off
Up to the present, seldom, conventional mode is killed due to being only capable of is reported for the mode of plant explant endophyte sterilization
Surface germ, and endophyte often causes the pollution again of culture after incubation.Therefore, the endophyte for developing highly effective and safe disappears
Malicious sterilizing methods are the key that one rings of Tissues of Poplar Clones culture, it would be highly desirable to develop a kind of side of the removal explant endophyte for willow
Method.
Invention content
The present invention can not remove willow in view of the above defects of the prior art, to improve routine disinfection sterilization method
The deficiency of equal explants endophyte provides a kind of new method of explant endophyte, the party when removing Plant Tissue Breeding
Method can effectively remove the bacterium and fungi on the explants such as willow surface and inside, and explant is made to keep sufficient vigor.
For this purpose, the present invention provides a kind of methods of explant endophyte when removal Plant Tissue Breeding comprising following
Step：
1, cuttage soil is disinfected：It is carried out disinfection processing to cuttage soil using 50% carbendazim.
2, cuttage branch is disinfected：Using the nothing containing 200mg/L amphotericin Bs and 100mg/L streptomycin sulphates
Bacterium aqueous solution carries out disinfection processing to cuttage branch, and after processing, top wax sealing processing is carried out to cuttage branch.
3, the selection and disinfection pretreatment of explant：Using containing 100mg/L amphotericin Bs and 50mg/L streptomycin sulphates
Aseptic aqueous solution the new hair branch of semi-lignified is pre-processed.
4, the alcohol sterilization treatment of explant.
5, the sodium hypochlorite sterilization treatment of explant.
The culture of 6 explants.
In a preferred embodiment of the present invention, cuttage soil is disinfected as with 50% carbendazim, with 30-40g/
m3Dosage mixed thoroughly with cuttage soil, be packed into polybag, it is closed, open after 5 days, dried 2-3 days under sunlight.
In a preferred embodiment of the present invention, cuttage branch disinfect for by the fringe bar of hibernation clean after, be cut into
15cm length is put into the aseptic aqueous solution containing 200mg/L amphotericin Bs and 100mg/L streptomycin sulphates, is handled 24 hours
Afterwards, top wax sealing processing is carried out, after processing in cuttage to nutritive cube, moves into greenhouse and is cultivated.
In a preferred embodiment of the present invention, explant select and sterilize pretreatment for clip semi-lignified new hair
Branch is inserted into containing in 100mg/L amphotericin Bs and 50mg/L streptomycin sulphate aseptic aqueous solutions, after handling 12 hours, cuts off
Blade is cleaned using 1% liquid detergent solution, after flowing water rinses, drains away the water, be put into superclean bench.
In a preferred embodiment of the present invention, the alcohol sterilization treatment of explant is the stem section of clip 5cm or so, is used
75% alcohol sterilizes 30 seconds, aseptic water washing 3 times.
In a preferred embodiment of the present invention, the sodium hypochlorite sterilizing of explant adds 0.1% polysorbas20 to take
10% hypochlorite disinfectant 8-10 minutes, with aseptic water washing explant 3 times, sterilizing filter paper blots.
In a preferred embodiment of the present invention, before cultivating explant, aseptically will further include
The step of interface contacted with disinfectant in explant material is cut off.
In a preferred embodiment of the present invention, the culture of explant is that the explant after sterilization is seeded in MS to open
In dynamic culture medium.
In a preferred embodiment of the present invention, the environmental condition of explant culture is photoperiod 16h/8h, intensity of illumination
27 DEG C/21 DEG C of 2500Lux, temperature.
In a preferred embodiment of the present invention, explant is the explant of willow, the preferably explant of eastern cottonwood.
Seen from the above description, compared with prior art, the present invention disappears to cuttage soil using 50% carbendazim
Poison processing carries out cuttage branch using the aseptic aqueous solution containing 200mg/L amphotericin Bs and 100mg/L streptomycin sulphates
It disinfects, and using the aseptic aqueous solution containing 100mg/L amphotericin Bs and 50mg/L streptomycin sulphates to semi-lignified
New hair branch pre-processed, so as to effectively remove the bacterium and fungi on the explants such as willow surface and inside, make outer
Implant keeps sufficient vigor.
Description of the drawings
Fig. 1：The upgrowth situation of the eastern cottonwood stem section explant handled through the method for the present invention.
Fig. 2：Using the upgrowth situation for the eastern cottonwood stem section explant that conventional disinfection method is handled.
A：Culture generates endogenetic bacteria pollution after 35 days；
B：Culture generates endogenetic fungus pollution after 35 days.
Specific implementation mode
Below by embodiment, the present invention is described in further detail, it is intended to limit this for illustrating rather than
Invention.It should be pointed out that those skilled in the art, it without departing from the principle of the present invention, can also be to this hair
Bright some improvement and modification can also be carried out, these improvement and modification are similarly fallen under the scope of the present invention.
Embodiment 1：The culture of eastern cottonwood explant
1, cuttage soil is disinfected
Turfy soil, perlite and river sand are pressed 6:2:2 ratio is sufficiently mixed as cuttage soil, applies 50% carbendazim
(30-40g/m3), it fully mixes thoroughly, is packed into polybag, is opened after closed 5 days, after being dried 2-3 days under sunlight, be packed into diameter 6cm's
In culturing pot.
2, cuttage branch is disinfected
The eastern cottonwood fringe bar of hibernation is cut into 15cm length, tap water rinses, and banister brush is used in combination to remove surface smut.It
Afterwards, by fringe bar be totally immersed into antibiotic disinfection mixed liquor (200mg/L amphotericin Bs and 100mg/L streptomycin sulphates are sterile water-soluble
Liquid) in, it handles 24 hours.Fringe bar surface moisture is blotted with aseptic filter paper, and fringe bar top 0.5cm is immersed to the paraffin melted
In, carry out top wax sealing processing.In treated tloig-cutlage to above-mentioned nutritive cube, it is positioned over temperature control automation heliogreenhouse
In, during which 25-28 DEG C of temperature carries out daily fertilizer and water management and the prevention and control of plant diseases, pest control.
3, the selection and disinfection pretreatment of explant
After cuttage 30 days, chooses the full eastern cottonwood semi-lignified of robust growth, no disease and pests harm, axillary bud and newly sends out branch,
Carry out antibiotic disinfection pretreatment.Method be semi-lignified is newly sent out branch bottom end be inserted into containing 100mg/L amphotericin Bs and
It is handled 12 hours in 50mg/L streptomycin sulphate aseptic aqueous solutions.
After new hair branch antibiotic disinfection pretreatment, is cut off with medical operation and remove tender tip and blade, retain 2mm's
Petiole is cleaned with 1% liquid detergent solution, is used in combination the tap water of flowing fully to rinse, is blotted explant surface with filter paper later
Moisture is put into super-clean bench.
4, the alcohol sterilization treatment of explant
Aseptically, explant is cut into the stem section of 2-3cm with the medical operation scissors after high-temperature sterilization, it is ensured that every
A stem section has 1-2 full axillary buds.Stem section is put into the glass culture dish after big sterilizing, pours into 75% alcohol, gently
It shakes, it is rapid to take out after about 30 seconds.
5, the sodium hypochlorite sterilization treatment of explant
Above-mentioned stem section is moved into sterile petri dish, 10% sodium hypochlorite that 30-50ml adds 0.1% polysorbas20 is poured into,
Disinfection 8-10 minutes, during which gently shakes 2-3 times.
Above-mentioned stem section is moved into sterile petri dish, with aseptic water washing explant 3 times, explant is blotted with the filter paper of sterilizing
The moisture in body surface face.
6, the culture of explant
The stem section of above-mentioned processing is placed in sterile petri dish, with the medical operation scissors to sterilize, will be connect with disinfectant
The stem section both ends interface excision 1-1.5mm touched, is seeded in later in MS primary culture mediums.
Culture bottle is moved into incubator for tissue culture, the condition of culture that incubator is arranged is photoperiod 16h/8h, intensity of illumination
2500Lux, cultivation temperature are 27 DEG C/21 DEG C.
7, the statistical analysis of explant cultivation results
After culture 5-10 days, eastern cottonwood explant surface contamination situation is counted；After culture 30-40 days, explant is counted
Endophytic bacterial contamination survives and upgrowth situation.The result of different sterilization methods compares specific as shown in table 1.
Under Different treatments, the growth of eastern cottonwood stem section explant and microbiological contamination situation are as shown in Fig. 2.
The different sterilization method Comparative results of table 1
Note：1：Conventional disinfection method；2：The sterilization method of the present invention.
Claims (9)
1. a kind of method of explant endophyte when removal eastern cottonwood tissue cultures comprising following steps：
(1) cuttage soil is disinfected：It is carried out disinfection processing to cuttage soil using 50% carbendazim；
(2) cuttage branch is disinfected：Using the sterile water containing 200mg/L amphotericin Bs and 100mg/L streptomycin sulphates
Solution carries out disinfection processing to cuttage branch, and after processing, top wax sealing processing is carried out to cuttage branch；
(3) selection and disinfection pretreatment of explant：Using containing 100mg/L amphotericin Bs and 50mg/L streptomycin sulphates
Aseptic aqueous solution pre-processes the new hair branch of semi-lignified；
(4) the alcohol sterilization treatment of explant；
(5) the sodium hypochlorite sterilization treatment of explant；
(6) culture of explant.
2. according to the method described in claim 1, wherein cuttage soil is disinfected as with 50% carbendazim, with 30-
40g/m3Dosage mixed thoroughly with cuttage soil, be packed into polybag, it is closed, open after 5 days, dried 2-3 days under sunlight.
3. method according to claim 1 or 2, wherein cuttage branch disinfect for by the fringe bar of hibernation clean after,
It is cut into 15cm length, is put into the aseptic aqueous solution containing 200mg/L amphotericin Bs and 100mg/L streptomycin sulphates, processing 24
After hour, top wax sealing processing is carried out, after processing in cuttage to nutritive cube, moves into greenhouse and is cultivated.
4. method according to claim 1 or 2, wherein explant selects and sterilizes pretreatment as clip semi-lignified
New hair branch, is inserted into and contains in 100mg/L amphotericin Bs and 50mg/L streptomycin sulphate aseptic aqueous solutions, after handling 12 hours,
Blade is cut off, is cleaned using 1% liquid detergent solution, after flowing water rinses, is drained away the water, be put into superclean bench.
5. the stem section that method according to claim 1 or 2, wherein the alcohol sterilization treatment of explant is clip 5cm or so,
It is sterilized 30 seconds using 75% alcohol, aseptic water washing 3 times.
6. method according to claim 1 or 2, the sodium hypochlorite sterilizing of wherein explant adds 0.1% tween to take
20 10% hypochlorite disinfectant 8-10 minutes, with aseptic water washing explant 3 times, sterilizing filter paper blots.
Further include in aseptic condition 7. method according to claim 1 or 2, wherein before cultivating explant
Lower the step of being cut off the interface contacted with disinfectant in explant material.
8. method according to claim 1 or 2, wherein the culture of the explant is to connect the explant after sterilization
Kind is in MS primary culture mediums.
9. method according to claim 1 or 2, wherein the environmental condition of the explant culture be photoperiod 16h/8h,
27 DEG C/21 DEG C of intensity of illumination 2500Lux, temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610576636.XA CN106106168B (en) | 2016-07-20 | 2016-07-20 | A method of explant endophyte when removal Plant Tissue Breeding |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610576636.XA CN106106168B (en) | 2016-07-20 | 2016-07-20 | A method of explant endophyte when removal Plant Tissue Breeding |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106106168A CN106106168A (en) | 2016-11-16 |
| CN106106168B true CN106106168B (en) | 2018-11-06 |
Family
ID=57290168
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610576636.XA Expired - Fee Related CN106106168B (en) | 2016-07-20 | 2016-07-20 | A method of explant endophyte when removal Plant Tissue Breeding |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106106168B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108834900B (en) * | 2018-07-31 | 2022-03-11 | 安徽农业大学 | Culture method for inhibiting explant oxidation browning and endophyte pollution in tea tree tissue culture |
| CN112655557B (en) * | 2020-12-22 | 2022-08-30 | 杭州师范大学 | Method for inhibiting endophytes of in vitro culture cucumber seedlings |
| CN113875584A (en) * | 2021-09-16 | 2022-01-04 | 台州学院 | Method for inducing tender shoots without endophyte by using plant branches |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101889551A (en) * | 2010-08-06 | 2010-11-24 | 合肥工业大学 | Tissue culture method of huperizia serrata |
| CN102550405A (en) * | 2011-12-26 | 2012-07-11 | 北京林业大学 | Breeding method of poplar haploid |
-
2016
- 2016-07-20 CN CN201610576636.XA patent/CN106106168B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101889551A (en) * | 2010-08-06 | 2010-11-24 | 合肥工业大学 | Tissue culture method of huperizia serrata |
| CN102550405A (en) * | 2011-12-26 | 2012-07-11 | 北京林业大学 | Breeding method of poplar haploid |
Non-Patent Citations (2)
| Title |
|---|
| 俄罗斯杨树组织培养中污染的解决办法;魏占才等;《防护林科技》;20091130(第6期);第103-105页，尤其是第4.1节 * |
| 毛果杨的组织培养与快速繁殖;张红梅等;《植物生理学通讯》;20090131;第45卷(第1期);第53页，尤其是第1-2，4.1节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106106168A (en) | 2016-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106106168B (en) | A method of explant endophyte when removal Plant Tissue Breeding | |
| CN103651124B (en) | A kind of abductive approach of plant regeneration of zingiber officinale | |
| CN104663439B (en) | Tissue culture and rapid propagation method of waterweed | |
| CN106171997B (en) | A kind of while explant surface and endophyte when removing Plant Tissue Breeding method | |
| CN105432473A (en) | Orchid seed surface sterilization method | |
| CN109452330A (en) | A kind of Plant Tissue Breeding bacteriostatic agent and its application in roxburgh anoectochilus terminal bud tissue cultures | |
| CN101933459A (en) | Method for disinfecting explant in drepanostachyum luodianense tissue culture process | |
| CN106106154A (en) | A kind of method of Oryza sativa L. Aseptic seedling culture | |
| CN103798139B (en) | A kind of is the method that outer implant sets up Bulbus Lilii embryo callus subculture regenerating system with petal | |
| CN105706924A (en) | Fast industrialized production method for ardisia violacea | |
| CN103283604B (en) | Method for removing endophytic bacteria of explants in lily tissue culture | |
| CN102232359A (en) | In-vitro rapid propagation method of double-petal Jasminum sambac | |
| CN102823446A (en) | Efficient method for sugarcane infection by smut spores | |
| CN107410033A (en) | The rapid propagation method of national spice berry snippings | |
| CN106106169B (en) | A kind of method of culture surface greasiness plant explant | |
| CN106386484A (en) | Anaphalis bulleyana cultivation and breeding method | |
| CN103598088B (en) | Chinaberry chromosome doubling method | |
| CN107494274B (en) | A kind of stripping bud sterilization method of alpine rose explant | |
| CN104221882B (en) | Explant sterilization method for ginkgo biloba tissue culture | |
| CN112425507A (en) | Efficient cultivation method of aseptic explants of Paeonia persica stems and explants | |
| CN109315292A (en) | A method of sweet potato explant is quickly bred using Shallow liquid culture method | |
| CN109329027B (en) | Efficient propagation method of sweet potatoes | |
| CN107114244B (en) | A kind of induction SiChuan is heralded spring aseptic seedling rapid propagation method | |
| CN105684906A (en) | Method for remedying mold contamination of tissue culture seedlings of Nakagawa-cho chrysanthemum indicum | |
| CN109997641A (en) | The method for culturing seedlings for preventing and treating potato soil-borne disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20181106 Termination date: 20190720 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |