CN109452330A - A kind of Plant Tissue Breeding bacteriostatic agent and its application in roxburgh anoectochilus terminal bud tissue cultures - Google Patents
A kind of Plant Tissue Breeding bacteriostatic agent and its application in roxburgh anoectochilus terminal bud tissue cultures Download PDFInfo
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- CN109452330A CN109452330A CN201711167809.3A CN201711167809A CN109452330A CN 109452330 A CN109452330 A CN 109452330A CN 201711167809 A CN201711167809 A CN 201711167809A CN 109452330 A CN109452330 A CN 109452330A
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- 241000719837 Anoectochilus Species 0.000 title claims abstract description 38
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- 210000003932 urinary bladder Anatomy 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
-
- 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
- A01H4/001—Culture apparatus for tissue culture
-
- 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
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
- A01N65/42—Aloeaceae [Aloe family] or Liliaceae [Lily family], e.g. aloe, veratrum, onion, garlic or chives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
- A01N65/48—Zingiberaceae [Ginger family], e.g. ginger or galangal
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of Plant Tissue Breeding bacteriostatic agent and its application in roxburgh anoectochilus terminal bud tissue cultures, the Plant Tissue Breeding bacteriostatic agent, in terms of 1L, the component including following dosage: 100~500ml of plant extraction liquid；5~20g of chitosan oligosaccharide；5~20ml of surfactant；Remaining is water；The plant extraction liquid is the alcohol extract of the mixture of garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati.The present invention prepares plant extraction liquid using garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati as raw material, and it is combined the plant extraction liquid and chitosan oligosaccharide to obtain bacteriostatic agent, the bacteriostatic agent can effectively reduce the pollution rate of bacterium and fungi in tissue cultures, and significantly improve the planting percent of roxburgh anoectochilus terminal bud tissue cultures, it reduces and generates cost, can be widely applied to the plant biotechnology fields such as quick breeding by group culture, seedling detoxification and the foundation of regeneration plant system.
Description
Technical field
The present invention relates to field of plant tissue culture technique more particularly to a kind of Plant Tissue Breeding bacteriostatic agent and its
Application in roxburgh anoectochilus terminal bud tissue cultures.
Background technique
Roxburgh anoectochilus terminal bud is China's tradition valuable ingredient of traditional Chinese medicine, also known as Shorthairy Antenoron, alias gold silkworm, metal and stone pine, Shu Caolian, bird ginseng, gold
Line taiwan anetochilus herb, gold thread disappear to the marrow, are orchid family (Orchidaceae) Anoectochilus Blume (Anoectochilus) herbaceos perennial.
" Fujian drug will " carries its sweet in flavor and neutral in nature, clearing heat and cooling blood, and expelling wind and removing dampness cures mainly hemoptysis, bronchitis, tubercular meningitis, kidney
Inflammation, cystitis, urinary stone, rheumatic arthritis, small two acute infantile convulsions, children's tetanus.It is sweet that " Chinese medicine sea " carries its nature and flavor
It is flat, enter lung, liver, kidney, bladder four and pass through, tool moistening lung to arrest cough, cool blood flat liver, it is clearing heat and detoxicating the effect of, it is frightened that lung heat hemoptysis, children can be controlled
Wind, difficult urination and odynuria etc..Modern medicine shows: roxburgh anoectochilus terminal bud mainly contains flavones, carbohydrate, volatile oil, alkaloid, has
Machine acid, amino acid, steroidal etc..The pharmacological action of roxburgh anoectochilus terminal bud and its extract be mainly shown as hypoglycemic, blood pressure lowering, it is antiviral,
The effects of anti-oxidant and other pharmacological effects are also constantly being found.With in-depth study, the fine quality day of roxburgh anoectochilus terminal bud
Benefit is prominent, by the favor of common people, has wide exploitation prospect.Roxburgh anoectochilus terminal bud China Guangxi, Guangdong, Hainan, Guizhou,
The provinces such as Sichuan, Yunnan are all distributed；But in recent years, people have caused its germ plasm resource rare the long-term excavation of roxburgh anoectochilus terminal bud.Mesh
Before, the large-scale production of roxburgh anoectochilus terminal bud may be implemented using plant tissue culture technique, meet the medicinal of people, research and ornamental need
It asks.
Roxburgh anoectochilus terminal bud tissue cultures (hereinafter referred to as " tissue culture ") large-scale production is quickly grown in China, and tissue culture technology is increasingly complete
It is kind, but different degrees of pollution is often presented in it in tissue culture.It is obtained in this stage of Initial culture due to pollution problem
The higher cost of aseptic seedling, although obtained aseptic seedling in Initial culture, occur a large amount of pollutions in squamous subculture, make
Tissue culture cost is high.
Therefore, reducing tissue culture pollution rate is sport technique segment important in large-scale production, while being also to measure tissue cultures
One of job costs, risk and important indicator of feasibility height.From the point of view of the overall process of tissue culture, each link is likely to occur
Different degrees of pollution, in addition to the standardization process of formulation sterile working during tissue culture, focuses on nothing to reduce pollution rate
Outside bacterium operation is realized, trains and supervised, while broad spectrum activity bacteriostatic agent etc. being added in the medium, and reduction bacterium and fungi dirt
The important method of dye.Effective bacteriostatic agent is studied, screened and promoted the use of, fungi and germ contamination can be reduced, simplifies sterile behaviour
Make, reduces tissue culture cost and risk.
Summary of the invention
The present invention provides a kind of Plant Tissue Breeding bacteriostatic agent and its application in roxburgh anoectochilus terminal bud tissue cultures, the plants
Object tissue cultures bacteriostatic agent can effectively reduce the pollution of bacterium and fungi in tissue cultures, and significantly improve roxburgh anoectochilus terminal bud tissue
The planting percent of culture reduces and generates cost, can be widely applied to quick breeding by group culture, seedling detoxification and the foundation of regeneration plant system etc.
Plant biotechnology field.
Technical solution provided by the invention, specific as follows:
A kind of Plant Tissue Breeding bacteriostatic agent, in terms of 1L, including following components:
100~500ml of plant extraction liquid；
5~20g of chitosan oligosaccharide；
5~20ml of surfactant；
Remaining is water；
The plant extraction liquid is the ethyl alcohol leaching of the mixture of garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati
Extract.
Preferably, the Plant Tissue Breeding bacteriostatic agent, in terms of 1L, including following components:
Plant extraction liquid 200ml；
Chitosan oligosaccharide 20g；
Surfactant 10ml；
Remaining is water；
The plant extraction liquid is the ethyl alcohol leaching of the mixture of garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati
Extract.
It is further preferred that the surfactant is tween 20.
It is found through experiment that be combined using above-mentioned plant extraction liquid with chitosan oligosaccharide, during capable of effectively inhibiting tissue culture
The bacterium and fungi of generation, especially bacillus, pseudomonad, chain spore fungi, rhodotorula mucilaginosa and Penicillium notatum.
Specifically, the preparation method of the plant extraction liquid, comprising:
(1) it after being mixed garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati, is added in ethyl alcohol and stirs powder
It is broken, obtain mixed liquor；
(2) after the mixed liquor being carried out ultrasonication, filtering removal solid impurity, re-evaporation concentration removal ethyl alcohol,
Obtain plant extraction liquid.
Preferably, in step (1), the garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati mass ratio be
1~3:1~2:1~2:1~2.
Preferably, the temperature of the ultrasonication is 40~70 DEG C in step (2), frequency is 30~50KHz.
The present invention also provides a kind of roxburgh anoectochilus terminal bud tissue cultures carried out using the Plant Tissue Breeding with bacteriostatic agent
Method, comprising:
(1) preparation of explant: taking the explant of roxburgh anoectochilus terminal bud, and cleaning is placed in the Plant Tissue Breeding bacteriostatic agent
Soaking disinfection takes out after rinsing, and obtains sterilizing explant；
(2) the sterilizing explant Fiber differentiation: is inoculated in the training of the induction containing the Plant Tissue Breeding bacteriostatic agent
It supports in base, carries out Fiber differentiation, obtain the callus containing Multiple Buds；
(3) proliferation and subculture culture: by the Multiple Buds be inoculated in the proliferation containing the Plant Tissue Breeding bacteriostatic agent after
For in culture medium, proliferation and subculture culture is carried out, tufted seedling is obtained；
(4) tufted seedling culture of rootage: is inoculated in the root media containing the Plant Tissue Breeding bacteriostatic agent
In, culture of rootage is carried out, tissue-cultured seedling is obtained；
(5) acclimatization and transplants.
The explant is the bulb of roxburgh anoectochilus terminal bud, seeds or leaves.
Preferably, the induced medium is MS+ sucrose 20-30gL-1+ agar 5-10gL-1+6-BA1.0-
2.0mg·L-1+NAA 0.2-1.0mg·L-1+GA 1.5mg·L-1+ bacteriostatic agent 200-300mlL-1。
Preferably, the proliferation and subculture culture medium is MS+ sucrose 20-30gL-1+ agar 5-10gL-1+6-BA
0.5-1.5mg·L-1+NAA 0.2-0.5mg·L-1+ bacteriostatic agent 300-400mlL-1。
Preferably, the root media is 1/2MS+ sucrose 20-30gL-1+ agar 5-10gL-1+NAA 0.3-
0.5mg·L-1+ bacteriostatic agent 100-150mlL-1。
Compared with prior art, the invention has the following advantages:
The present invention prepares plant extraction liquid by raw material of garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati, and will
The plant extraction liquid and chitosan oligosaccharide are combined to obtain bacteriostatic agent, which can effectively reduce in tissue cultures bacterium and true
The pollution of bacterium, and the planting percent of roxburgh anoectochilus terminal bud tissue cultures is significantly improved, it reduces and generates cost, can be widely applied to tissue culture quick propagation
Grow, seedling detoxification and regeneration plant system establish etc. plant biotechnology fields.
Detailed description of the invention
Fig. 1 is Plant Tissue Breeding bacteriostatic agent in embodiment 2 to the fungistatic effect of different bacterium；
Wherein, A is pseudomonad；B is bacillus；C is enterobacter amnigenus.
Fig. 2 is Plant Tissue Breeding bacteriostatic agent in embodiment 3 to the fungistatic effect of different fungies；
Wherein, A1For the experimental group for the spore fungi that interlinks；A2For the control group for the spore fungi that interlinks；B is rhodotorula mucilaginosa；C1For blueness
The experimental group of mould；C2For the control group of Penicillium notatum.
Fig. 3 is that Plant Tissue Breeding bacteriostatic agent tests the fungistatic effect of Penicillium notatum in embodiment 4；
Wherein, A and B is inoculation penicillin；C and D is the culture medium containing bacteriostatic agent+inoculation penicillin；E and F is not connect
Kind penicillin.
Specific embodiment
The present invention is further explained in the light of specific embodiments, but the invention is not limited to given implementations
Example.
The preparation of 1 Plant Tissue Breeding bacteriostatic agent of embodiment
1, the preparation of plant extraction liquid:
(1) 40g garlic bulb, 20g ginger rhizome, 20g balsam pear pericarp and 20g pericarpium granati are taken, after cleaning, is added
In 95% ethyl alcohol of 100ml, it is stirred and crushes, obtain mixed liquor；
(2) above-mentioned mixed liquor is subjected to ultrasonication, the temperature of ultrasonication is 60 DEG C, frequency 40KHz, the time
It will concentration using pure water after filtering removes solid impurity, then using Rotary Evaporators removal ethyl alcohol acquisition concentrate for 30min
Liquid is settled to 100ml, obtains plant extraction liquid.
2, the preparation of Plant Tissue Breeding bacteriostatic agent:
20ml plant extraction liquid, 2g chitosan oligosaccharide, 1ml cosolvent tween20 and 60ml pure water are taken, 100ml volumetric flask is put into
In, it is uniformly mixed, with deionized water constant volume in 100ml, pours into reagent bottle, be placed in 4 DEG C of refrigerators and save backup.
Comparative example 1
1, the preparation of plant extraction liquid:
(1) take 60g garlic bulb and 40g ginger rhizome, after cleaning, be added in 95% ethyl alcohol of 100ml, be stirred and
It crushes, obtains mixed liquor；
(2) above-mentioned mixed liquor is subjected to ultrasonication, the temperature of ultrasonication is 60 DEG C, frequency 40KHz, the time
It will concentration using pure water after filtering removes solid impurity, then using Rotary Evaporators removal ethyl alcohol acquisition concentrate for 30min
Liquid is settled to 100ml, obtains plant extraction liquid.
2, the preparation of Plant Tissue Breeding bacteriostatic agent:
20ml plant extraction liquid, 2g chitosan oligosaccharide, 1ml cosolvent tween20 and 60ml pure water are taken, 100ml volumetric flask is put into
In, it is uniformly mixed, with deionized water constant volume in 100ml, pours into reagent bottle, be placed in 4 DEG C of refrigerators and save backup.
Comparative example 2
1, the preparation of plant extraction liquid:
(1) take 40g garlic bulb, 20g ginger rhizome, 40g balsam pear pericarp, after cleaning, 95% ethyl alcohol of 100ml is added
In, it is stirred and crushes, obtain mixed liquor；
(2) above-mentioned mixed liquor is subjected to ultrasonication, the temperature of ultrasonication is 60 DEG C, frequency 40KHz, the time
It will concentration using pure water after filtering removes solid impurity, then using Rotary Evaporators removal ethyl alcohol acquisition concentrate for 30min
Liquid is settled to 100ml, obtains plant extraction liquid.
2, the preparation of Plant Tissue Breeding bacteriostatic agent:
20ml plant extraction liquid, 2g chitosan oligosaccharide, 1ml cosolvent tween20 and 60ml pure water are taken, 100ml volumetric flask is put into
In, it is uniformly mixed, with deionized water constant volume in 100ml, pours into reagent bottle, be placed in 4 DEG C of refrigerators and save backup.
Comparative example 3
1, the preparation of plant extraction liquid:
(1) take 40g garlic bulb, 20g ginger rhizome, 40g pericarpium granati, after cleaning, 95% ethyl alcohol of 100ml is added
In, it is stirred and crushes, obtain mixed liquor；
(2) above-mentioned mixed liquor is subjected to ultrasonication, the temperature of ultrasonication is 60 DEG C, frequency 40KHz, the time
It will concentration using pure water after filtering removes solid impurity, then using Rotary Evaporators removal ethyl alcohol acquisition concentrate for 30min
Liquid is settled to 100ml, obtains plant extraction liquid.
2, the preparation of Plant Tissue Breeding bacteriostatic agent:
20ml plant extraction liquid, 2g chitosan oligosaccharide, 1ml cosolvent tween20 and 60ml pure water are taken, 100ml volumetric flask is put into
In, it is uniformly mixed, with deionized water constant volume in 100ml, pours into reagent bottle, be placed in 4 DEG C of refrigerators and save backup.
Comparative example 4
1, the preparation of plant extraction liquid:
(1) 40g garlic bulb, 20g ginger rhizome, 20g balsam pear pericarp and 20g pericarpium granati are taken, after cleaning, is added
In 95% ethyl alcohol of 100ml, it is stirred and crushes, obtain mixed liquor；
(2) above-mentioned mixed liquor is subjected to ultrasonication, the temperature of ultrasonication is 60 DEG C, frequency 40KHz, the time
It will concentration using pure water after filtering removes solid impurity, then using Rotary Evaporators removal ethyl alcohol acquisition concentrate for 30min
Liquid is settled to 100ml, obtains plant extraction liquid.
2, the preparation of Plant Tissue Breeding bacteriostatic agent:
20ml plant extraction liquid, 2g chitosan, 1ml cosolvent tween20 and 60ml pure water are taken, 100ml volumetric flask is put into
In, it is uniformly mixed, with deionized water constant volume in 100ml, pours into reagent bottle, be placed in 4 DEG C of refrigerators and save backup.
Biocidal property of the 2 Plant Tissue Breeding bacteriostatic agent of embodiment to bacterium and fungi
The Plant Tissue Breeding bacteriostatic agent prepared using embodiment 1 and comparative example 1~4 is to 3 plants of bacteriums in tissue culture pollution
(pseudomonad, bacillus and enterobacter amnigenus) and 3 fungal strains (interlinkage spore fungi, rhodotorula mucilaginosa and Penicillium notatum) are pressed down
The research of bacterium effect.
Particular content is as follows:
The test of bacterium biocidal property: the Oxford cup of specification Φ 7.8mm × 6mm × 10mm (outer diameter × internal diameter × height) is taken, is placed in
At 121 DEG C after high pressure sterilization, Drying and cooling is spare.In in superclean bench by bacillus, pseudomonad and enterobacter amnigenus
It is coated on the surface LB plate (diameter 90mm), each plate contains 200 μ l concentration 106The test bacterium of CFU/ml.By Oxford cup
It is put into each dish surface, Plant Tissue Breeding prepared by extraction embodiment 1 is instilled in Oxford cup with 200 μ l of bacteriostatic agent, then is set
In 30 DEG C of 48~72h of culture, 3 repetitions of every group of carry out；The LB plate of bacteriostatic agent is not added with inoculated bacteria but as control.
The test of fungi biocidal property: the LB for the Plant Tissue Breeding bacteriostatic agent for taking the embodiment 1 containing 100 μ l to prepare is flat
Ware, in the bacterial plaque of fungi chain spore fungi, rhodotorula mucilaginosa and Penicillium notatum is inoculated in above-mentioned LB plate (diameter in superclean bench
60mm) surface is placed in 25 DEG C of culture 4-5d, every group of 3 repetitions；It is pair so that the LB plate of bacteriostatic agent but non-inoculated fungi is added
According to.
Test result is as follows:
Fig. 1 show containing embodiment 1 prepare Plant Tissue Breeding bacteriostatic agent Oxford cup around have obvious inhibition zone；
Fig. 2 shows that fungi growth is obviously pressed down in the plate of the Plant Tissue Breeding bacteriostatic agent prepared containing embodiment 1
System；Wherein, rhodotorula mucilaginosa antibacterial circle diameter reaches 16mm, be to bacteriostatic agent it is highly sensitive, chain spore fungi and Penicillium notatum it is antibacterial
Rate reaches 90% ((control bacterial plaque diameter-test organisms spot diameter)/control bacterial plaque diameter).
The data of specific fungistatic effect are shown in Table 1.
Table 1 presses down different strain with bacteriostatic agent using Plant Tissue Breeding prepared by embodiment 1 and comparative example 1~4
The result of bacterium processing
Note: "+" indicates: bacterium loop diameter 9-10mm, slight sensitive；" ++ " indicates: bacterium loop diameter 10-15mm, medium sensitivity；
" +++ " indicates: bacterium loop diameter 15-20mm, highly sensitive；" ++++" indicate: bacterium loop diameter > 20mm, it is extremely sensitive.
Inhibitory effect of the 3 Plant Tissue Breeding bacteriostatic agent of embodiment to Penicillium notatum
In Penicillium notatum is applied to seedlings of anoectochilus (tissue-cultured seedling that tissue culture obtains) surface in superclean bench, it is inoculated with respectively
In the culture medium of the Plant Tissue Breeding bacteriostatic agent prepared with or without embodiment 1, it is placed in 25 DEG C, illumination 12h/d, light intensity
2000lx cultivates 3-5d.
Test result: as shown in figure 3, the pollution level of the tissue-cultured seedling containing bacteriostatic agent is significantly relatively light.
Embodiment 4
A method of tissue cultures being carried out to roxburgh anoectochilus terminal bud with bacteriostatic agent using Plant Tissue Breeding, particular content is as follows:
(1) preparation of explant: the bulb of roxburgh anoectochilus terminal bud is chosen as explant, explant is rinsed with water 20 minutes, is put
Enter the Plant Tissue Breeding of the preparation of embodiment 1 with impregnating 20min in bacteriostatic agent, then with aseptic water washing 2 times, is filtered dry moisture, use
The scalpel impregnated through bacteriostatic agent cuts the explant after surface sterilizing, obtains explant to be seeded；
(2) preparation of culture medium: according to the ingredient and dosage of MS minimal medium, configuring corresponding mother liquor, successively takes a large amount of
Element mother liquor, microelement mother liquor, mother liquid of iron salt and organic principle, after mixing by induced medium, proliferation and subculture culture medium and
The component of root media is added Plant Tissue Breeding bacteriostatic agent prepared by hormone and embodiment 1.
The pH value that culture medium is measured with pH meter, with 1M NaOH solution or 1M hydrochloric acid tune pH to 5.8；Then fine jade is dissolved in heating
Rouge, finally plus water constant volume；The culture medium prepared is sub-packed in culture bottle, is sealed with bottle cap, 121 DEG C of high pressure sterilizations are cooled to
60 DEG C or so, Plant Tissue Breeding bacteriostatic agent prepared by embodiment 1 is aseptically added, it is standby as clean area after solidification
With.
(3) Fiber differentiation: sterile-processed roxburgh anoectochilus terminal bud explant is inoculated in induced medium, is placed in tissue culture room
Fiber differentiation is carried out, after cultivating 20d, obtains callus, the edge of bulb grows Multiple Buds；
Wherein, induced medium are as follows: sucrose 30gL-1+ agar 5gL-1+MS+6-BA 2.0mg·L-1+NAA
1.0mg·L-1+GA 1.5mg·L-1+ bacteriostatic agent (embodiment 1) 200mlL-1, pH to 5.8,121 DEG C of high pressure sterilizations；Induction training
Feeding condition are as follows: temperature is 25 DEG C, illumination 12h/d, light intensity 1500lx.
(4) proliferation and subculture culture: the Multiple Buds induced are cut, is seeded on proliferation and subculture culture medium, is proliferated
Squamous subculture, and switching in 25 days is primary, obtains tufted seedling；
Wherein, proliferation and subculture culture medium are as follows: sucrose 30gL-1+ agar 5gL-1+MS+6-BA 1.5mg·L-1+NAA
0.5mg·L-1+ bacteriostatic agent (embodiment 1) 300mlL-1, pH to 5.8,121 DEG C of high pressure sterilizations；The condition of proliferation and subculture culture
Are as follows: temperature is 25 DEG C, illumination 12h/d, light intensity 1500lx.
(5) culture of rootage: tufted seedling it is long to 2~4cm when, tufted seedling is cut into single plant, is inoculated on root media, into
Row culture of rootage obtains complete tissue-cultured seedling after culture 25 days；
Wherein, root media are as follows: sucrose 30gL-1+ agar 5gL-1+1/2MS+NAA 0.3mg·L-1+ bacteriostatic agent
(embodiment 1) 100mlL-1, pH to 5.8,121 DEG C of high pressure sterilizations；The condition of culture of rootage are as follows: temperature is 25 DEG C, illumination is
12h/d, light intensity 1500lx.
(6) acclimatization and transplants: the complete tissue-cultured seedling after taking out culture of rootage cleans the culture medium of root, transplants to hole tray,
Cultivation obtains roxburgh anoectochilus terminal bud；
Wherein, matrix is common detritus soil, perlite and pine bark；Greenhouse upper layer lid shading net, the environment of transplanting
Temperature is 25 DEG C.Finally, transplanting survival rate is up to 86%.
Embodiment 5
A method of tissue cultures being carried out to roxburgh anoectochilus terminal bud with bacteriostatic agent using Plant Tissue Breeding, particular content is as follows:
(1) preparation of explant: the blade of roxburgh anoectochilus terminal bud is chosen as explant, explant is rinsed with water 20 minutes, is put
Enter the Plant Tissue Breeding of the preparation of embodiment 1 with impregnating 20min in bacteriostatic agent, then with aseptic water washing 2 times, is filtered dry moisture, use
The scalpel impregnated through bacteriostatic agent cuts the explant after surface sterilizing, obtains explant to be seeded；
(2) preparation of culture medium: according to the ingredient and dosage of MS minimal medium, configuring corresponding mother liquor, successively takes a large amount of
Element mother liquor, microelement mother liquor, mother liquid of iron salt and organic principle, after mixing by induced medium, proliferation and subculture culture medium and
The component of root media is added Plant Tissue Breeding bacteriostatic agent prepared by hormone and embodiment 1.
The pH value that culture medium is measured with pH meter, with 1M NaOH solution or 1M hydrochloric acid tune pH to 5.8；Then fine jade is dissolved in heating
Rouge, finally plus water constant volume；The culture medium prepared is sub-packed in culture bottle, is sealed with bottle cap, 121 DEG C of high pressure sterilizations are cooled to
60 DEG C or so, Plant Tissue Breeding bacteriostatic agent prepared by embodiment 1 is aseptically added, it is standby as clean area after solidification
With.
(3) Fiber differentiation: sterile-processed roxburgh anoectochilus terminal bud explant is inoculated in induced medium, is placed in tissue culture room
Fiber differentiation is carried out, after cultivating 20d, obtains callus, the edge of blade grows Multiple Buds；
Wherein, induced medium are as follows: sucrose 30gL-1+ agar 5gL-1+MS+6-BA 2.0mg·L-1+NAA
1.0mg·L-1+GA 1.5mg·L-1+ bacteriostatic agent (embodiment 1) 200mlL-1, pH to 5.8,121 DEG C of high pressure sterilizations；Induction training
Feeding condition are as follows: temperature is 25 DEG C, illumination 12h/d, light intensity 1500lx.
(4) proliferation and subculture culture: the Multiple Buds induced are cut, is seeded on proliferation and subculture culture medium, is proliferated
Squamous subculture, and switching in 25 days is primary, obtains tufted seedling；
Wherein, proliferation and subculture culture medium are as follows: sucrose 30gL-1+ agar 5gL-1+MS+6-BA 1.5mg·L-1+NAA
0.5mg·L-1+ bacteriostatic agent (embodiment 1) 300mlL-1, pH to 5.8,121 DEG C of high pressure sterilizations；The condition of proliferation and subculture culture
Are as follows: temperature is 25 DEG C, illumination 12h/d, light intensity 1500lx.
(5) culture of rootage: tufted seedling it is long to 2~4cm when, tufted seedling is cut into single plant, is inoculated on root media, into
Row culture of rootage obtains complete tissue-cultured seedling after culture 25 days；
Wherein, root media are as follows: sucrose 30gL-1+ agar 5gL-1+1/2MS+NAA 0.3mg·L-1+ bacteriostatic agent
(embodiment 1) 100mlL-1, pH to 5.8,121 DEG C of high pressure sterilizations；The condition of culture of rootage are as follows: temperature is 25 DEG C, illumination is
12h/d, light intensity 1500lx.
(6) acclimatization and transplants: the complete tissue-cultured seedling after taking out culture of rootage cleans the culture medium of root, transplants to hole tray,
Cultivation obtains roxburgh anoectochilus terminal bud；
Wherein, matrix is common detritus soil, perlite and pine bark；Greenhouse upper layer lid shading net, the environment of transplanting
Temperature is 25 DEG C.Finally, transplanting survival rate is up to 83.5%.
Comparative example 5
A kind of method for tissue culture of roxburgh anoectochilus terminal bud, particular content are as follows:
(1) preparation of explant: the bulb of roxburgh anoectochilus terminal bud is chosen as explant, explant is rinsed with water 20 minutes, is put
Enter and sterilize 20min in sodium hypochlorite, then with aseptic water washing 2 times, be filtered dry moisture, with scalpel to the explant after surface sterilizing
It is cut, obtains explant to be seeded；
(2) preparation of culture medium: according to the ingredient and dosage of MS minimal medium, configuring corresponding mother liquor, successively takes a large amount of
Element mother liquor, microelement mother liquor, mother liquid of iron salt and organic principle, after mixing by induced medium, proliferation and subculture culture medium and
Hormone is added in the component of root media.
The pH value that culture medium is measured with pH meter, with 1M NaOH solution or 1M hydrochloric acid tune pH to 5.8；Then fine jade is dissolved in heating
Rouge, finally plus water constant volume；The culture medium prepared is sub-packed in culture bottle, is sealed with bottle cap, 121 DEG C of high pressure sterilizations, after solidification
It is spare as clean area.
(3) Fiber differentiation: sterile-processed roxburgh anoectochilus terminal bud explant is inoculated in induced medium, is placed in tissue culture room
Fiber differentiation is carried out, after cultivating 20d, obtains callus, the edge of bulb grows Multiple Buds；
Wherein, induced medium are as follows: sucrose 30gL-1+ agar 5gL-1+MS+6-BA 2.0mg·L-1+NAA
1.0mg·L-1+GA 1.5mg·L-1, pH to 5.8,121 DEG C of high pressure sterilizations；The condition of Fiber differentiation are as follows: temperature is 25 DEG C, light
According to for 12h/d, light intensity 1500lx.
(4) proliferation and subculture culture: the Multiple Buds induced are cut, is seeded on proliferation and subculture culture medium, is proliferated
Squamous subculture, and switching in 25 days is primary, obtains tufted seedling；
Wherein, proliferation and subculture culture medium are as follows: sucrose 30gL-1+ agar 5gL-1+MS+6-BA 1.5mg·L-1+NAA
0.5mg·L-1, pH to 5.8,121 DEG C of high pressure sterilizations；The condition of proliferation and subculture culture are as follows: temperature is 25 DEG C, illumination 12h/d,
Light intensity is 1500lx.
(5) culture of rootage: tufted seedling it is long to 2~4cm when, tufted seedling is cut into single plant, is inoculated on root media, into
Row culture of rootage obtains complete tissue-cultured seedling after culture 25 days；
Wherein, root media are as follows: sucrose 30gL-1+ agar 5gL-1+1/2MS+NAA 0.3mg·L-1, pH is extremely
5.8,121 DEG C of high pressure sterilizations；The condition of culture of rootage are as follows: temperature is 25 DEG C, illumination 12h/d, light intensity 1500lx.
(6) acclimatization and transplants: the complete tissue-cultured seedling after taking out culture of rootage cleans the culture medium of root, transplants to hole tray,
Cultivation obtains roxburgh anoectochilus terminal bud
Wherein, matrix is common detritus soil, perlite and pine bark.Greenhouse upper layer lid shading net, the environment of transplanting
Temperature is 25 DEG C.Finally, transplanting survival rate is only 72%.
Comparative example 6
In the roxburgh anoectochilus terminal bud tissue culture procedures of this comparative example, it is respectively adopted the bacteriostatic agent of comparative example 1~4, and remaining content
It is identical with embodiment 4.
Finally, transplanting survival rate is respectively 75% (comparative example 1), 73% (comparative example 2), 78% (comparative example 4), 80%
(comparative example 4)；In addition, the infected main bacteria seed of roxburgh anoectochilus terminal bud explant during tissue culture is bacillus and chain through detecting
Spore fungi (comparative example 1~3), pseudomonas and rhodotorula mucilaginosa (comparative example 4).
Claims (9)
1. a kind of Plant Tissue Breeding bacteriostatic agent, which is characterized in that in terms of 1L, the component including following dosage:
100~500ml of plant extraction liquid；
5~20g of chitosan oligosaccharide；
5~20ml of surfactant；
Remaining is water；
The plant extraction liquid is the alcohol extract of the mixture of garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati.
2. Plant Tissue Breeding bacteriostatic agent as described in claim 1, which is characterized in that in terms of 1L, including following dosage
Component:
Plant extraction liquid 200ml；
Chitosan oligosaccharide 20g；
Surfactant 10ml；
Remaining is water；
The plant extraction liquid is the alcohol extract of the mixture of garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati.
3. Plant Tissue Breeding bacteriostatic agent as described in claim 1 or 2 is any, which is characterized in that the plant extraction liquid
Preparation method, comprising:
(1) it after being mixed garlic bulb, ginger rhizome, balsam pear pericarp and pericarpium granati, is added in ethyl alcohol and stirs crushing, obtain
To mixed liquor；
(2) after the mixed liquor being carried out ultrasonication, filtering removal solid impurity, re-evaporation concentration removal ethyl alcohol is obtained
Plant extraction liquid.
4. Plant Tissue Breeding bacteriostatic agent as claimed in claim 3, which is characterized in that in step (1), the garlic squama
Stem, ginger rhizome, balsam pear pericarp and pericarpium granati mass ratio be 1~3:1~2:1~2:1~2.
5. Plant Tissue Breeding bacteriostatic agent as claimed in claim 3, which is characterized in that in step (2), at the ultrasonic wave
The temperature of reason is 40~70 DEG C, and frequency is 30~50KHz.
6. a kind of roxburgh anoectochilus terminal bud tissue carried out using Plant Tissue Breeding as claimed in any one of claims 1 to 5 with bacteriostatic agent
The method of culture characterized by comprising
(1) preparation of explant: taking the explant of roxburgh anoectochilus terminal bud, and cleaning is placed on the Plant Tissue Breeding and is soaked in bacteriostatic agent
Bubble takes out after rinsing, and obtains sterilizing explant；
(2) the sterilizing explant Fiber differentiation: is inoculated in the induced medium containing the Plant Tissue Breeding bacteriostatic agent
In, Fiber differentiation is carried out, the callus containing Multiple Buds is obtained；
(3) Multiple Buds proliferation and subculture culture: are inoculated in the training of the proliferation and subculture containing the Plant Tissue Breeding bacteriostatic agent
It supports in base, carries out proliferation and subculture culture, obtain tufted seedling；
(4) culture of rootage: the tufted seedling is inoculated in the root media containing the Plant Tissue Breeding bacteriostatic agent, into
Row culture of rootage, obtains tissue-cultured seedling；
(5) acclimatization and transplants.
7. method as claimed in claim 6, which is characterized in that the induced medium is MS+ sucrose 20-30gL-1+ agar
5-10g·L-1+6-BA 1.0-2.0mg·L-1+NAA 0.2-1.0mg·L-1+GA 1.5mg·L-1+ bacteriostatic agent 200-
300ml·L-1。
8. method as claimed in claim 6, which is characterized in that the proliferation and subculture culture medium is MS+ sucrose 20-30gL-1+
Agar 5-10gL-1+6-BA 0.5-1.5mg·L-1+NAA 0.2-0.5mg·L-1+ bacteriostatic agent 300-400mlL-1。
9. method as claimed in claim 6, which is characterized in that the root media is 1/2MS+ sucrose 20-30gL-1+
Agar 5-10gL-1+NAA 0.3-0.5mg·L-1+ bacteriostatic agent 100-150mlL-1。
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