CN109452330B - Bacteriostatic agent for plant tissue culture and application of bacteriostatic agent in anoectochilus formosanus tissue culture - Google Patents
Bacteriostatic agent for plant tissue culture and application of bacteriostatic agent in anoectochilus formosanus tissue culture Download PDFInfo
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- 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
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- 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
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- 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
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- 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]
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- 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
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- 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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Abstract
The invention discloses a bacteriostatic agent for plant tissue culture and application thereof in anoectochilus formosanus tissue culture, wherein the bacteriostatic agent for plant tissue culture comprises the following components in 1L: 100-500 ml of plant extract; 5-20 g of chitosan oligosaccharide; 5-20 ml of surfactant; the balance of water; the plant extract is ethanol extract of mixture of Bulbus Allii bulb, rhizoma Zingiberis recens rhizome, fructus Momordicae Charantiae pericarp and pericarpium Granati. The plant extract is prepared by taking the garlic bulbs, the ginger roots and rhizomes, the bitter gourd peel and the pomegranate peel as raw materials, and the plant extract and the chitosan oligosaccharide are combined to obtain the bacteriostatic agent, so that the bacteriostatic agent can effectively reduce the pollution rate of bacteria and fungi in tissue culture, remarkably improve the seedling rate of anoectochilus formosanus tissue culture, reduce the production cost, and can be widely applied to the plant biotechnology fields of tissue culture rapid propagation, seedling detoxification, regeneration plant line establishment and the like.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a bacteriostatic agent for plant tissue culture and application thereof in anoectochilus formosanus tissue culture.
Background
Anoectochilus roxburghii is a traditional precious medicinal material in China, also called Anoectochilus roxburghii, Japanese ardisia, caelia, Orthosiphon aristatus, Anoectochilus roxburghii and Anoectochilus roxburghii, and is a perennial herb plant of Orchidaceae (Orchidaceae) Kailium (Anoectochilus). The Fujian Zhi (Fujian medicine record) has sweet and mild nature and flavor, can clear heat and cool blood, dispel wind and remove dampness, and is mainly used for treating hemoptysis, bronchitis, tuberculous meningitis, nephritis, cystitis, urinary calculus, rheumatic arthritis, acute infantile convulsion and infantile tetanus. As recorded in Zhonghua Yao Hai (Chinese medicine), it has sweet and mild nature and flavor, enters lung, liver, kidney and bladder meridians, has the actions of moistening lung to arrest cough, cooling blood to calm liver, clearing heat and removing toxicity, and can be used for cough and bleeding due to lung heat, infantile convulsion, dysuria and urodynia. Modern medicine shows that: anoectochilus roxburghii mainly contains flavone, saccharide, volatile oil, alkaloid, organic acid, amino acid, steroid, etc. The pharmacological effects of Anoectochilus roxburghii and the extract thereof are mainly represented by the effects of reducing blood sugar, blood pressure, resisting viruses, resisting oxidation and the like, and other pharmacological effects are continuously discovered. With the intensive research, the excellent quality of the anoectochilus roxburghii is increasingly outstanding, is favored by people in the world, and has wide development and utilization prospects. Anoectochilus formosanus is distributed in Guangxi province, Guangdong province, Hainan province, Guizhou province, Sichuan province, Yunnan province and the like in China; however, in recent years, the long-term excavation of Anoectochilus roxburghii has led to the shortage of germplasm resources. At present, the plant tissue culture technology can be used for realizing the large-scale production of anoectochilus formosanus and meeting the medicinal, research and ornamental requirements of people.
The large-scale production of anoectochilus formosanus tissue culture (hereinafter referred to as 'tissue culture') is developed rapidly in China, the tissue culture technology is improved day by day, but the anoectochilus formosanus tissue culture often has pollution of different degrees in the tissue culture. In the stage of primary culture, the cost for obtaining aseptic seedlings is high due to the pollution problem, or although aseptic seedlings are obtained in the primary culture, a large amount of pollution is generated in the secondary culture, so that the tissue culture cost is high.
Therefore, the reduction of the tissue culture pollution rate is an important technical link in large-scale production, and is also one of important indexes for measuring the working cost, risk and feasibility of tissue culture. In order to reduce the pollution rate, in the tissue culture process, besides setting up a standardized flow of aseptic operation and paying attention to the consciousness, training and supervision of the aseptic operation, a broad-spectrum bacteriostatic agent and the like are added into a culture medium, so that the method is also an important method for reducing the pollution of bacteria and fungi. The effective bacteriostat is researched, screened and popularized, so that the pollution of fungi and bacteria can be reduced, the aseptic operation is simplified, and the tissue culture cost and risk are reduced.
Disclosure of Invention
The bacteriostatic agent for plant tissue culture can effectively reduce the pollution of bacteria and fungi in the tissue culture, obviously improve the seedling rate of the anoectochilus formosanus tissue culture, reduce the production cost, and can be widely applied to the plant biotechnology fields of tissue culture rapid propagation, germchit detoxification, regeneration plant line establishment and the like.
The technical scheme provided by the invention is as follows:
a bacteriostatic agent for plant tissue culture comprises the following components in 1L:
100-500 ml of plant extract;
5-20 g of chitosan oligosaccharide;
5-20 ml of surfactant;
the balance of water;
the plant extract is ethanol extract of mixture of Bulbus Allii bulb, rhizoma Zingiberis recens rhizome, fructus Momordicae Charantiae pericarp and pericarpium Granati.
Preferably, the bacteriostatic agent for plant tissue culture comprises the following components in 1L:
200ml of plant extract;
20g of chitosan oligosaccharide;
10ml of surfactant;
the balance of water;
the plant extract is ethanol extract of mixture of Bulbus Allii bulb, rhizoma Zingiberis recens rhizome, fructus Momordicae Charantiae pericarp and pericarpium Granati.
More preferably, the surfactant is tween 20.
Tests show that the combination of the plant extract and the chitosan oligosaccharide can effectively inhibit bacteria and fungi generated in the tissue culture process, particularly bacillus, pseudomonas, streptomyces, rhodotorula mucilaginosa and penicillium.
Specifically, the preparation method of the plant extract comprises the following steps:
(1) mixing garlic bulbs, ginger roots and stems, bitter gourd peel and pomegranate peel, adding ethanol, stirring and crushing to obtain a mixed solution;
(2) and (3) carrying out ultrasonic treatment on the mixed solution, filtering to remove solid impurities, and then evaporating and concentrating to remove ethanol to obtain a plant extracting solution.
Preferably, in the step (1), the mass ratio of the garlic bulbs to the ginger roots to the bitter gourd peel to the pomegranate peel is 1-3: 1-2.
Preferably, in the step (2), the temperature of the ultrasonic treatment is 40-70 ℃, and the frequency is 30-50 KHz.
The invention also provides a method for carrying out anoectochilus formosanus tissue culture by using the bacteriostatic agent for plant tissue culture, which comprises the following steps:
(1) preparing an explant: taking an explant of the clematis, cleaning, placing the explant into the bacteriostatic agent for plant tissue culture, soaking and disinfecting, taking out and washing to obtain a sterilized explant;
(2) and (3) induction culture: inoculating the sterilized explant into an induction culture medium containing the bacteriostatic agent for plant tissue culture, and carrying out induction culture to obtain callus containing cluster buds;
(3) proliferation and subculture: inoculating the callus containing the cluster buds into a proliferation subculture medium containing the bacteriostatic agent for plant tissue culture, and carrying out proliferation subculture to obtain cluster seedlings;
(4) rooting culture: inoculating the cluster seedlings into a rooting culture medium containing the bacteriostatic agent for plant tissue culture, and carrying out rooting culture to obtain tissue culture seedlings;
(5) hardening and transplanting the seedlings.
The explant is the bulb, seed or leaf of anoectochilus formosanus.
Preferably, the induction medium is MS + sucrose 20-30 g.L-1+ agar 5-10 g.L-1+6-BA 1.0-2.0mg·L-1+NAA 0.2-1.0mg·L-1+GA 1.5mg·L-1+ bacteriostatic agent 200-300 ml-L-1。
Preferably, the proliferation subculture medium is MS + sucrose 20-30 g.L-1+ agar 5-10 g.L-1+6-BA0.5-1.5mg·L-1+NAA 0.2-0.5mg·L-1+ bacteriostatic agent 300-400 ml-L-1。
Preferably, the rooting medium is 1/2MS + sucrose 20-30 g.L-1+ agar 5-10 g.L-1+NAA 0.3-0.5mg·L-1+ 100. sup. ml. L of bacteriostat-1。
Compared with the prior art, the invention has the following beneficial effects:
the plant extract is prepared by taking the garlic bulbs, the ginger roots and rhizomes, the bitter gourd peel and the pomegranate peel as raw materials, and the plant extract and the chitosan oligosaccharide are combined to obtain the bacteriostatic agent, so that the bacteriostatic agent can effectively reduce the pollution of bacteria and fungi in tissue culture, remarkably improve the seedling rate of anoectochilus formosanus tissue culture, reduce the production cost, and can be widely applied to the plant biotechnology fields of tissue culture rapid propagation, seedling detoxification, regeneration plant line establishment and the like.
Drawings
FIG. 1 shows the bacteriostatic effect of the bacteriostatic agent for plant tissue culture on different bacteria in example 2;
wherein A is pseudomonas; b is bacillus; c is Enterobacter aquaticus.
FIG. 2 shows the bacteriostatic effects of the bacteriostatic agent for plant tissue culture on different fungi in example 2;
wherein A is1Experimental group for alternaria alternata; a. the2Control group of alternaria alternata; b is Rhodotorula mucilaginosa; c1Is an experimental group of penicillium; c2Is a control group of penicillium.
FIG. 3 is a test of the bacteriostatic effect of the bacteriostatic agent for plant tissue culture on Penicillium in example 3;
wherein A and B are inoculated penicillin; c and D are culture medium containing bacteriostatic agent + inoculated penicillin; e and F are non-inoculated penicillins.
Detailed Description
The invention will now be further illustrated by reference to specific examples, but the invention is not limited to the examples given.
EXAMPLE 1 preparation of bacteriostatic agent for plant tissue culture
1. Preparing a plant extracting solution:
(1) taking 40g of garlic bulbs, 20g of ginger roots and stems, 20g of bitter gourd peel and 20g of pomegranate peel, cleaning, adding 100ml of 95% ethanol, stirring and crushing to obtain a mixed solution;
(2) and (3) carrying out ultrasonic treatment on the mixed solution at the temperature of 60 ℃, the frequency of 40KHz for 30min, filtering to remove solid impurities, removing ethanol by using a rotary evaporator to obtain a concentrated solution, and fixing the volume of the concentrated solution to 100ml by using pure water to obtain a plant extract.
2. Preparing a bacteriostatic agent for plant tissue culture:
taking 20ml of plant extract, 2g of chitosan oligosaccharide, 1ml of cosolvent tween20 and 60ml of pure water, putting into a 100ml volumetric flask, uniformly mixing, using deionized water to fix the volume to 100ml, pouring into a reagent bottle, and placing in a refrigerator at 4 ℃ for storage for later use.
Comparative example 1
1. Preparing a plant extracting solution:
(1) cleaning 60g of garlic bulb and 40g of ginger rhizome, adding 100ml of 95% ethanol, stirring and crushing to obtain a mixed solution;
(2) and (3) carrying out ultrasonic treatment on the mixed solution at the temperature of 60 ℃, the frequency of 40KHz for 30min, filtering to remove solid impurities, removing ethanol by using a rotary evaporator to obtain a concentrated solution, and fixing the volume of the concentrated solution to 100ml by using pure water to obtain a plant extract.
2. Preparing a bacteriostatic agent for plant tissue culture:
taking 20ml of plant extract, 2g of chitosan oligosaccharide, 1ml of cosolvent tween20 and 60ml of pure water, putting into a 100ml volumetric flask, uniformly mixing, using deionized water to fix the volume to 100ml, pouring into a reagent bottle, and placing in a refrigerator at 4 ℃ for storage for later use.
Comparative example 2
1. Preparing a plant extracting solution:
(1) taking 40g of garlic bulbs, 20g of ginger roots and stems and 40g of bitter gourd peels, cleaning, adding 100ml of 95% ethanol, stirring and crushing to obtain a mixed solution;
(2) and (3) carrying out ultrasonic treatment on the mixed solution at the temperature of 60 ℃, the frequency of 40KHz for 30min, filtering to remove solid impurities, removing ethanol by using a rotary evaporator to obtain a concentrated solution, and fixing the volume of the concentrated solution to 100ml by using pure water to obtain a plant extract.
2. Preparing a bacteriostatic agent for plant tissue culture:
taking 20ml of plant extract, 2g of chitosan oligosaccharide, 1ml of cosolvent tween20 and 60ml of pure water, putting into a 100ml volumetric flask, uniformly mixing, using deionized water to fix the volume to 100ml, pouring into a reagent bottle, and placing in a refrigerator at 4 ℃ for storage for later use.
Comparative example 3
1. Preparing a plant extracting solution:
(1) taking 40g of garlic bulbs, 20g of ginger roots and stems and 40g of pomegranate peels, cleaning, adding 100ml of 95% ethanol, stirring and crushing to obtain a mixed solution;
(2) and (3) carrying out ultrasonic treatment on the mixed solution at the temperature of 60 ℃, the frequency of 40KHz for 30min, filtering to remove solid impurities, removing ethanol by using a rotary evaporator to obtain a concentrated solution, and fixing the volume of the concentrated solution to 100ml by using pure water to obtain a plant extract.
2. Preparing a bacteriostatic agent for plant tissue culture:
taking 20ml of plant extract, 2g of chitosan oligosaccharide, 1ml of cosolvent tween20 and 60ml of pure water, putting into a 100ml volumetric flask, uniformly mixing, using deionized water to fix the volume to 100ml, pouring into a reagent bottle, and placing in a refrigerator at 4 ℃ for storage for later use.
Comparative example 4
1. Preparing a plant extracting solution:
(1) taking 40g of garlic bulbs, 20g of ginger roots and stems, 20g of bitter gourd peel and 20g of pomegranate peel, cleaning, adding 100ml of 95% ethanol, stirring and crushing to obtain a mixed solution;
(2) and (3) carrying out ultrasonic treatment on the mixed solution at the temperature of 60 ℃, the frequency of 40KHz for 30min, filtering to remove solid impurities, removing ethanol by using a rotary evaporator to obtain a concentrated solution, and fixing the volume of the concentrated solution to 100ml by using pure water to obtain a plant extract.
2. Preparing a bacteriostatic agent for plant tissue culture:
taking 20ml of plant extract, 2g of chitosan, 1ml of cosolvent tween20 and 60ml of pure water, putting into a 100ml volumetric flask, uniformly mixing, using deionized water to fix the volume to 100ml, pouring into a reagent bottle, and placing in a refrigerator at 4 ℃ for storage for later use.
Example 2 bacteriostatic Activity of bacteriostatic Agents for plant tissue culture against bacteria and fungi
The bacteriostatic effect of 3 strains of bacteria (pseudomonas, bacillus and enterobacter aquaticus) and 3 strains of fungi (alternaria, rhodotorula mucilaginosa and penicillium) in the culture contamination was investigated using the bacteriostatic agents for plant tissue culture prepared in example 1 and comparative examples 1 to 4.
The specific contents are as follows:
bacterial inhibition test: taking an Oxford cup with the specification of phi 7.8mm multiplied by 6mm multiplied by 10mm (outer diameter multiplied by inner diameter multiplied by height), placing the Oxford cup at 121 ℃ for high-pressure sterilization, drying and cooling for later use. Bacillus, Pseudomonas and Enterobacter aquaticus were spread on the surface of LB plates (diameter 90mm) each containing 200. mu.l of 10 concentration in a clean bench6CFU/ml test bacteria. Putting the oxford cups on the surfaces of the plates, sucking 200 mu l of the plant tissue culture bacteriostatic agent prepared in the embodiment 1, dripping the bacteriostatic agent into the oxford cups, and then culturing at 30 ℃ for 48-72 h, wherein each group is repeated for 3 times; LB plates inoculated with bacteria but without added bacteriostatic were used as controls.
And (3) testing the fungistatic activity: taking an LB plate containing 100 ul of the bacteriostatic agent for plant tissue culture prepared in example 1, inoculating bacterial plaques of fungal streptoverticillium, rhodotorula mucilaginosa and penicillium on the surface of the LB plate (diameter is 60mm) in a super clean bench, and culturing at 25 ℃ for 4-5d, wherein each group is repeated for 3 times; LB plates with added bacteriostatic but not inoculated with fungus were used as controls.
The test results are as follows:
FIG. 1 shows that there is a significant zone of inhibition around the oxford cup containing the bacteriostatic agent for plant tissue culture prepared in example 1;
FIG. 2 shows that the growth of fungi was significantly inhibited in a dish containing the bacteriostatic for plant tissue culture prepared in example 1; wherein, the diameter of the rhodotorula mucilaginosa inhibition zone reaches 16mm, the rhodotorula mucilaginosa inhibition zone is highly sensitive to the bacteriostatic agent, and the inhibition rate of the streptomycete fungi and the penicillium reaches 90 percent (the diameter of a control bacterial plaque-the diameter of a test bacterial plaque)/the diameter of the control bacterial plaque).
The data of the specific bacteriostatic effect are shown in table 1.
TABLE 1 results of bacteriostasis treatment of various bacterial species using the bacteriostats for plant tissue culture prepared in example 1 and comparative examples 1 to 4
Note: "+" indicates: the diameter of the fungus ring is 9-10mm, and the fungus ring is slightly sensitive; "+ +" indicates: the diameter of the fungus ring is 10-15mm, and the fungus ring is moderately sensitive; "+ + + +" indicates: the diameter of the fungus ring is 15-20mm, and the fungus ring is highly sensitive; "+ ++" indicates: the diameter of the fungus ring is more than 20mm, and the fungus ring is extremely sensitive.
Example 3 inhibitory Effect of bacteriostatic agent for plant tissue culture on Penicillium
The penicillium is smeared on the surface of anoectochilus roxburghii seedlings (tissue culture seedlings obtained by tissue culture) in a superclean bench, and is respectively inoculated in a culture medium containing or not containing the bacteriostatic agent for plant tissue culture prepared in the example 1, and the culture is carried out for 3-5 days at the temperature of 25 ℃ under the illumination of 12h/d and the light intensity of 2000 lx.
And (3) test results: as shown in fig. 3, the degree of contamination of the tissue culture seedlings containing the bacteriostatic agent was significantly less.
Example 4
A method for carrying out tissue culture on anoectochilus formosanus by utilizing a bacteriostatic agent for plant tissue culture comprises the following specific contents:
(1) preparing an explant: selecting the bulbs of anoectochilus formosanus as explants, washing the explants with water for 20 minutes, putting the explants into the bacteriostatic agent for plant tissue culture prepared in the embodiment 1, soaking for 20 minutes, washing with sterile water for 2 times, draining the water, and cutting the explants with sterilized surfaces by using a scalpel soaked with the bacteriostatic agent to obtain the explants to be inoculated;
(2) preparation of a culture medium: preparing corresponding mother liquor according to the components and the dosage of the MS basic culture medium, sequentially taking a macroelement mother liquor, a microelement mother liquor, an iron salt mother liquor and organic components, mixing, and adding hormone and the bacteriostatic agent for plant tissue culture prepared in the embodiment 1 according to the components of an induction culture medium, a proliferation subculture medium and a rooting culture medium.
Measuring the pH value of the culture medium by using a pH meter, and adjusting the pH value to 5.8 by using 1M NaOH solution or 1M hydrochloric acid; heating to melt agar, and adding water to constant volume; the prepared culture medium is subpackaged in a culture bottle, the bottle cap is used for sealing, the high-pressure sterilization is carried out at the temperature of 121 ℃, the temperature is cooled to about 60 ℃, the bacteriostatic agent for plant tissue culture prepared in the embodiment 1 is added under the aseptic condition, and the bacteriostatic agent is solidified and placed in a clean area for standby.
(3) And (3) induction culture: inoculating the sterilized anoectochilus roxburghii explant on an induction culture medium, placing the culture medium in a tissue culture chamber for induction culture, and culturing for 20 days to obtain a callus, wherein cluster buds grow on the edge of a bulb;
wherein, the induction culture medium is: sucrose 30 g.L-1+ agar 5 g. L-1+MS+6-BA 2.0mg·L-1+NAA1.0mg·L-1+GA 1.5mg·L-1+ bacteriostatic agent (example 1)200 ml. L-1Sterilizing at 121 deg.C under high pressure with pH of 5.8; the conditions of the induction culture are as follows: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(4) Proliferation and subculture: cutting the induced cluster buds, inoculating the cut cluster buds to a proliferation subculture medium, performing proliferation subculture, and performing grafting once every 25 days to obtain cluster seedlings;
wherein, the proliferation subculture medium comprises: sucrose 30 g.L-1+ agar 5 g. L-1+MS+6-BA 1.5mg·L-1+NAA0.5mg·L-1+ bacteriostatic agent (example 1)300 ml. L-1Sterilizing at 121 deg.C under high pressure with pH of 5.8; the conditions of proliferation subculture were: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(5) Rooting culture: when the cluster seedlings grow to 2-4 cm, cutting the cluster seedlings into single plants, inoculating the single plants to a rooting culture medium, carrying out rooting culture, and culturing for 25 days to obtain complete tissue culture seedlings;
wherein, the rooting culture medium is: sucrose 30 g.L-1+ agar 5 g. L-1+1/2MS+NAA 0.3mg·L-1+ bacteriostatic agent (example 1)100 ml. L-1Sterilizing at 121 deg.C under high pressure with pH of 5.8; the rooting culture conditions are as follows: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(6) Hardening and transplanting seedlings: taking out the complete tissue culture seedlings after rooting culture, cleaning the culture medium at the root, transplanting the complete tissue culture seedlings into a hole tray, and culturing to obtain anoectochilus formosanus;
wherein the matrix is common humus soil, perlite and pine bark; the greenhouse is covered with a shading net on the upper layer, and the transplanting environment temperature is 25 ℃. Finally, the transplanting survival rate reaches 86%.
Example 5
A method for carrying out tissue culture on anoectochilus formosanus by utilizing a bacteriostatic agent for plant tissue culture comprises the following specific contents:
(1) preparing an explant: selecting leaves of anoectochilus formosanus as explants, washing the explants with water for 20 minutes, soaking the explants in the bacteriostatic agent for plant tissue culture prepared in the embodiment 1 for 20 minutes, washing with sterile water for 2 times, draining the water, and cutting the explants with sterilized surfaces by using a scalpel soaked with the bacteriostatic agent to obtain the explants to be inoculated;
(2) preparation of a culture medium: preparing corresponding mother liquor according to the components and the dosage of the MS basic culture medium, sequentially taking a macroelement mother liquor, a microelement mother liquor, an iron salt mother liquor and organic components, mixing, and adding hormone and the bacteriostatic agent for plant tissue culture prepared in the embodiment 1 according to the components of an induction culture medium, a proliferation subculture medium and a rooting culture medium.
Measuring the pH value of the culture medium by using a pH meter, and adjusting the pH value to 5.8 by using 1M NaOH solution or 1M hydrochloric acid; heating to melt agar, and adding water to constant volume; the prepared culture medium is subpackaged in a culture bottle, the bottle cap is used for sealing, the high-pressure sterilization is carried out at the temperature of 121 ℃, the temperature is cooled to about 60 ℃, the bacteriostatic agent for plant tissue culture prepared in the embodiment 1 is added under the aseptic condition, and the bacteriostatic agent is solidified and placed in a clean area for standby.
(3) And (3) induction culture: inoculating the sterilized anoectochilus roxburghii explant on an induction culture medium, placing the culture medium in a tissue culture chamber for induction culture, and culturing for 20 days to obtain callus, wherein cluster buds grow on the edge of a leaf;
wherein, the induction culture medium is: sucrose 30 g.L-1+ agar 5 g. L-1+MS+6-BA 2.0mg·L-1+NAA1.0mg·L-1+GA1.5mg·L-1+ bacteriostatic agent (example 1)200 ml. L-1pH to 5.8, high pressure at 121 deg.CSterilizing; the conditions of the induction culture are as follows: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(4) Proliferation and subculture: cutting the induced cluster buds, inoculating the cut cluster buds to a proliferation subculture medium, performing proliferation subculture, and performing grafting once every 25 days to obtain cluster seedlings;
wherein, the proliferation subculture medium comprises: sucrose 30 g.L-1+ agar 5 g. L-1+MS+6-BA 1.5mg·L-1+NAA0.5mg·L-1+ bacteriostatic agent (example 1)300 ml. L-1Sterilizing at 121 deg.C under high pressure with pH of 5.8; the conditions of proliferation subculture were: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(5) Rooting culture: when the cluster seedlings grow to 2-4 cm, cutting the cluster seedlings into single plants, inoculating the single plants to a rooting culture medium, carrying out rooting culture, and culturing for 25 days to obtain complete tissue culture seedlings;
wherein, the rooting culture medium is: sucrose 30 g.L-1+ agar 5 g. L-1+1/2MS+NAA 0.3mg·L-1+ bacteriostatic agent (example 1)100 ml. L-1Sterilizing at 121 deg.C under high pressure with pH of 5.8; the rooting culture conditions are as follows: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(6) Hardening and transplanting seedlings: taking out the complete tissue culture seedlings after rooting culture, cleaning the culture medium at the root, transplanting the complete tissue culture seedlings into a hole tray, and culturing to obtain anoectochilus formosanus;
wherein the matrix is common humus soil, perlite and pine bark; the greenhouse is covered with a shading net on the upper layer, and the transplanting environment temperature is 25 ℃. Finally, the transplanting survival rate reaches 83.5 percent.
Comparative example 5
A tissue culture method of Anoectochilus roxburghii comprises the following specific contents:
(1) preparing an explant: selecting the bulbs of anoectochilus formosanus as explants, washing the explants with water for 20 minutes, putting the explants into sodium hypochlorite for disinfection for 20 minutes, washing with sterile water for 2 times, draining water, and cutting the explants with sterilized surfaces by using a scalpel to obtain explants to be inoculated;
(2) preparation of a culture medium: preparing corresponding mother liquor according to the components and the dosage of the MS basic culture medium, sequentially taking a macroelement mother liquor, a microelement mother liquor, an iron salt mother liquor and organic components, mixing, and adding hormone according to the components of an induction culture medium, a proliferation subculture medium and a rooting culture medium.
Measuring the pH value of the culture medium by using a pH meter, and adjusting the pH value to 5.8 by using 1M NaOH solution or 1M hydrochloric acid; heating to melt agar, and adding water to constant volume; and (3) subpackaging the prepared culture medium into culture bottles, sealing the culture bottles by using bottle caps, sterilizing at the high pressure of 121 ℃, and placing the culture medium in a clean area for later use after solidification.
(3) And (3) induction culture: inoculating the sterilized anoectochilus roxburghii explant on an induction culture medium, placing the culture medium in a tissue culture chamber for induction culture, and culturing for 20 days to obtain a callus, wherein cluster buds grow on the edge of a bulb;
wherein, the induction culture medium is: sucrose 30 g.L-1+ agar 5 g. L-1+MS+6-BA 2.0mg·L-1+NAA1.0mg·L-1+GA 1.5mg·L-1Sterilizing at 121 deg.C under high pressure with pH of 5.8; the conditions of the induction culture are as follows: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(4) Proliferation and subculture: cutting the induced cluster buds, inoculating the cut cluster buds to a proliferation subculture medium, performing proliferation subculture, and performing grafting once every 25 days to obtain cluster seedlings;
wherein, the proliferation subculture medium comprises: sucrose 30 g.L-1+ agar 5 g. L-1+MS+6-BA 1.5mg·L-1+NAA0.5mg·L-1Sterilizing at 121 deg.C under high pressure with pH of 5.8; the conditions of proliferation subculture were: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(5) Rooting culture: when the cluster seedlings grow to 2-4 cm, cutting the cluster seedlings into single plants, inoculating the single plants to a rooting culture medium, carrying out rooting culture, and culturing for 25 days to obtain complete tissue culture seedlings;
wherein, the rooting culture medium is: sucrose 30 g.L-1+ agar 5 g. L-1+1/2MS+NAA 0.3mg·L-1Sterilizing at 121 deg.C under high pressure with pH of 5.8; the rooting culture conditions are as follows: the temperature is 25 ℃, the illumination is 12h/d, and the light intensity is 1500 lx.
(6) Hardening and transplanting seedlings: taking out the complete tissue culture seedling after rooting culture, cleaning the culture medium at the root, transplanting the seedling into a hole tray, and culturing to obtain the anoectochilus formosanus.
Wherein the matrix is common humus soil, perlite and pine bark. The greenhouse is covered with a shading net on the upper layer, and the transplanting environment temperature is 25 ℃. Finally, the survival rate of transplantation is only 72%.
Comparative example 6
In the tissue culture process of anoectochilus formosanus of the comparative example, the bacteriostatic agents of the comparative examples 1-4 are respectively adopted, and the rest contents are completely the same as those of the example 4.
Finally, the transplanting survival rates were 75% (comparative example 1), 73% (comparative example 2), 78% (comparative example 4), and 80% (comparative example 4), respectively; in addition, the main strains infected by the anoectochilus formosanus explants in the tissue culture process are bacillus and streptomyces (comparative examples 1-3), pseudomonas and rhodotorula mucilaginosa (comparative example 4).
Claims (9)
1. The bacteriostatic agent for plant tissue culture is characterized by comprising the following components in 1L:
100-500 ml of plant extract;
5-20 g of chitosan oligosaccharide;
5-20 ml of surfactant;
the balance of water;
the plant extract is ethanol extract of mixture of Bulbus Allii bulb, rhizoma Zingiberis recens rhizome, fructus Momordicae Charantiae pericarp and pericarpium Granati.
2. The bacteriostatic agent for plant tissue culture according to claim 1, comprising the following components in an amount of 1L:
200ml of plant extract;
20g of chitosan oligosaccharide;
10ml of surfactant;
the balance of water;
the plant extract is ethanol extract of mixture of Bulbus Allii bulb, rhizoma Zingiberis recens rhizome, fructus Momordicae Charantiae pericarp and pericarpium Granati.
3. The bacteriostatic agent for plant tissue culture according to claim 1 or 2, wherein the preparation method of the plant extract comprises:
(1) mixing garlic bulbs, ginger roots and stems, bitter gourd peel and pomegranate peel, adding ethanol, stirring and crushing to obtain a mixed solution;
(2) and (3) carrying out ultrasonic treatment on the mixed solution, filtering to remove solid impurities, and then evaporating and concentrating to remove ethanol to obtain a plant extracting solution.
4. The bacteriostatic agent for plant tissue culture according to claim 3, wherein in the step (1), the mass ratio of the garlic bulbs to the ginger roots to the bitter gourd peel to the pomegranate peel is 1-3: 1-2.
5. The bacteriostatic agent for plant tissue culture according to claim 3, wherein the temperature of the ultrasonic treatment in step (2) is 40-70 ℃ and the frequency is 30-50 KHz.
6. A method for culturing Anoectochilus roxburghii tissue by using the bacteriostatic agent for plant tissue culture as defined in any one of claims 1-5, which comprises the following steps:
(1) preparing an explant: taking an explant of the clematis, cleaning, soaking in the bacteriostatic agent for plant tissue culture, taking out, and washing to obtain a sterilized explant;
(2) and (3) induction culture: inoculating the sterilized explant into an induction culture medium containing the bacteriostatic agent for plant tissue culture, and carrying out induction culture to obtain callus containing cluster buds;
(3) proliferation and subculture: inoculating the callus containing the cluster buds into a proliferation subculture medium containing the bacteriostatic agent for plant tissue culture, and carrying out proliferation subculture to obtain cluster seedlings;
(4) rooting culture: inoculating the cluster seedlings into a rooting culture medium containing the bacteriostatic agent for plant tissue culture, and carrying out rooting culture to obtain tissue culture seedlings;
(5) hardening and transplanting the seedlings.
7. The method of claim 6, wherein the induction medium is MS + sucrose 20-30 g-L-1+ agar 5-10 g.L-1+6-BA 1.0-2.0mg·L-1+NAA 0.2-1.0mg·L-1+GA1.5mg·L-1+ bacteriostatic agent 200-300 ml-L-1。
8. The method of claim 6, wherein the proliferation sub-culture medium is MS + sucrose 20-30 g-L-1+ agar 5-10 g.L-1+6-BA 0.5-1.5mg·L-1+NAA 0.2-0.5mg·L-1+ bacteriostatic agent 300-400 ml-L-1。
9. The method of claim 6, wherein said rooting medium is 1/2MS + sucrose 20-30 g-L-1+ agar 5-10 g.L-1+NAA 0.3-0.5mg·L-1+ 100. sup. ml. L of bacteriostat-1。
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