CN103250644B - Method for reducing tissue culture rate of anoectochilus roxburghii and culture medium formula thereof - Google Patents
Method for reducing tissue culture rate of anoectochilus roxburghii and culture medium formula thereof Download PDFInfo
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- CN103250644B CN103250644B CN201310184747.2A CN201310184747A CN103250644B CN 103250644 B CN103250644 B CN 103250644B CN 201310184747 A CN201310184747 A CN 201310184747A CN 103250644 B CN103250644 B CN 103250644B
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Abstract
The invention relates to a method for reducing the tissue culture rate of anoectochilus roxburghii and a culture medium formula thereof. The technical scheme is as follows: the culture medium formula is characterized by comprising the following components: 1/2 of MS, 40-60g/L of banana, 0.8-1.2g/L of active carbon, 0.8-1.2g/L of NAA, 0.4-0.8mg/L of 6-BA, 22-30g/L of cane sugar, 5.5-6.5g/L of agar, 40-60g/L of onion and 10-20g/L of crab shell powder. The method is characterized by comprising the following steps of: 1) disinfection of an inoculation chamber; 2) disinfection of a culture chamber; 3) preparation of culture medium; 4) sterilization of the culture medium and inoculation instruments; 5) inoculation of explants; and 6) culture of tissue culture seedlings.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of method and culture medium prescription thereof that reduces Herba Anoectochili roxburghii group training pollution rate.
Background technology
Herba Anoectochili roxburghii (Anoectochilus roxburghii) has another name called Rough Melic, for the orchid family is opened lip Cymbidium per nnial herb, it is China's tradition rare Chinese medicine, there is the effects such as clearing heat and cooling blood, removing dampness and detoxicating, be used for the treatment of the diseases such as diabetes, ephritis, urocystitis, in the laudatory title of among the people enjoying " king of medicine ".Zhe Nan, Min Tai and Guangdong and Guangxi Provinces one band long-term to boil water with Herba Anoectochili roxburghii, make tea, Baoshang, for improving the health, nourishing, take good care of.Modern study shows to contain the Multiple components such as polysaccharide, flavonoid, cardiac glycoside in Herba Anoectochili roxburghii, can strengthen body immunity, improves Abwehrkraft des Koepers, prevents the invasion and attack of disease, strengthens and improve the effects such as physique.
Herba Anoectochili roxburghii relies on for a long time excavates collection wild resource, and wild Herba Anoectochili roxburghii breeding potential is extremely low, causes resource standing stock to decline rapidly, and parts of traditional producing region can be adopted without medicine.Herba Anoectochili roxburghii seedling is the key link of its cultivation, and the quality of seedling is directly connected to the growing of Herba Anoectochili roxburghii, yield and quality.Because Herba Anoectochili roxburghii seed is not had an endosperm, without germinating power, only, under mycosymbiosis, just can impel its seed germination growth, so produce the upper main stem section that adopts, breed seedling.But the pollution problem (pollution rate is generally more than 20%) causing due to microorganisms such as bacterium, fungies in group training production process is the major obstacle of restricting current Herba Anoectochili roxburghii seedling breeding always.The pollution initial stage can cause the reduction of proliferate efficiency, cultivated material delayed growth; The pollution later stage can cause tissue cultured seedling to transplant difficulty and dead, and the meeting even having causes the heritable variation of cultivated material.Therefore find a kind of method that reduces Herba Anoectochili roxburghii group training pollution rate and substratum thereof for raising group training success ratio, reduce costs, increase economic efficiency etc. all significant.
Summary of the invention
The object of the invention is to overcome the deficiency that above-mentioned background technology exists, a kind of Herba Anoectochili roxburghii group training method of pollution rate and technical scheme of culture medium prescription thereof of reducing is provided.
Technical scheme provided by the invention is:
Reduce a culture medium prescription for Herba Anoectochili roxburghii group training pollution rate, it is characterized in that described culture medium prescription is 1/2MS, banana 40～60g/L, gac 0.8～1.2g/L, NAA0.8～1.2mg/L, 6-BA0.4～0.8mg/L, sucrose 22～30g/L, agar powder 5.5～6.5g/L, onion 40～60g/L, Carapax Eriocheir sinensis powder 10～20g/L.
Described a kind of culture medium prescription that reduces Herba Anoectochili roxburghii group training pollution rate, is characterized in that described culture medium prescription is 1/2MS, banana 50g/L, gac 1.0g/L, NAA1.0mg/L, 6-BA0.6mg/L, sucrose 26g/L, agar powder 6.0g/L, onion 50g/L, Carapax Eriocheir sinensis powder 15g/L.
Culture medium prescription reduces the method for Herba Anoectochili roxburghii group training pollution rate, it is characterized in that comprising the following steps:
1) transfer room sterilization: with tarragon and trypterygine, transfer room fumigation is once carried out to sterilization monthly; Weekly, with ozonizer, transfer room is carried out to sterilization one time;
2) culturing room's sterilization: monthly with tarragon and trypterygine, culturing room's fumigation is once carried out to sterilization; With ozonizer, culturing room is carried out to sterilization one time weekly;
3) substratum preparation: take 1/2MS as basic medium, prepare according to formula, after mixing, the pH value of substratum is adjusted to 5.8～6.0; The substratum preparing is heated to agar and dissolves completely, add a cover after being sub-packed in while hot in clean culturing bottle;
4) substratum and the sterilizing of inoculation utensil: adopt high pressure steam process to carry out sterilizing to substratum and inoculation utensil.
5) explant inoculation:
When the Herba Anoectochili roxburghii plant that adopts self-sow is during as explant, with tap water, clean Herba Anoectochili roxburghii, proceed to 70% ethanol rinsing 30s, use 0.1% mercuric chloride sterilizing 10min, sterilized water rinses 5～6 times repeatedly, drains away the water again, be cut into and be with 1 stem segment, be inoculated on ready substratum;
When adopting Herba Anoectochili roxburghii group training aseptic seedling as explant, Herba Anoectochili roxburghii is cut into and is with 1 stem segment, be inoculated on ready substratum;
6) tissue cultured seedling is cultivated: the culturing bottle of having inoculated is moved into culturing room, culture condition: 24 ± 2 ℃ of temperature, light application time 12h/d, intensity of illumination 1800～2000lx, incubation time four months.
Described culturing room should regularly sweep, and sprays 75% alcohol dedusting every day, and the overcast and rainy dehumidifying of turning on the aircondition, to keep clean and room air to be dried; Find the culturing bottle that there is a pollution in culturing room sterilising treatment immediately, with non-proliferation.Before inoculation, transfer room and Bechtop are opened ultra violet lamp sterilization 30min, then open Bechtop blowing 20min, with using after bromogeramine wiping table top; The culturing bottle polluting must can be used after autoclave sterilization.
During described step 4) sterilizing, will guarantee Autoclave cleaning inside, inner cold air drains, and the sterilizing thing of filling can not be stacked overfill, keeps Autoclave temperature at 121 ℃, 105kPa, 20min; After medium sterilization, first put between buffering, draw samples is placed on culturing room and cultivates 2～3d, if media surface and inner all without any bacterium vestige and fungi bacterial plaque can be used; Cutter, cut, the metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, then in the sterilizer with quartz sand high-temperature sterilization 5～10min, cooling rear use.
The invention has the beneficial effects as follows: method and the substratum thereof that reduces Herba Anoectochili roxburghii group training pollution rate that provide, rationally novel in design; In the present invention, substratum contains 1/2MS, banana, gac, NAA, 6-BA, sucrose, agar powder, onion, Carapax Eriocheir sinensis powder.In onion, sulfocompound can suppress the growth of the multiple-microorganisms such as Staphylococcus, streptococcus, vibrios, genus bacillus.In Carapax Eriocheir sinensis powder, contain abundant chitin and chitosan, intestinal bacteria, streptococcus aureus, yeast, Bacillus subtilus, aspergillus niger etc. are had to obvious restraining effect; Adopt tarragon and trypterygine fumigation to carry out sterilization, sterilising effect good (Herba Anoectochili roxburghii group training pollution rate is down to 5% below) not only, and there is no environmental pollution, cost is significantly reduction also.The culture medium prescription providing is reasonable, successful; The method providing is simple to operate, is easy to grasp, reproducible, is applicable to applying on a large scale.
Embodiment
Described with tarragon and trypterygine to transfer room and culturing room's fumigation, refer to tarragon after drying (water ratio is less than 5% conventionally) directly light, after trypterygine is dry, first use 95% alcohol-pickled 24h, the alcohol of take when stifling is lighted as ignition dope.By 3 points, be uniformly distributed, light rear airtight stifling 1h.
Adopt ozonizer to carry out the time of sterilization culturing room, transfer room, should select to determine according to ozonizer power, careat size.
It is ordinary method that substratum and the sterilizing of inoculation utensil adopt high pressure steam process.
The culture medium prescription of reduction Herba Anoectochili roxburghii group training pollution rate of the present invention, comprises 1/2MS, banana 40～60g/L, gac 0.8～1.2g/L, NAA0.8～1.2mg/L, 6-BA0.4～0.8mg/L, sucrose 22～30g/L, agar powder 5.5～6.5g/L, onion 40～60g/L, Carapax Eriocheir sinensis powder 10～20g/L.
Preferred: 1/2MS, banana 50g/L, gac 1.0g/L, NAA1.0mg/L, 6-BA0.6mg/L, sucrose 26g/L, agar powder 6.0g/L, onion 50g/L, Carapax Eriocheir sinensis powder 15g/L.
Further illustrate by the following examples the present invention.
Embodiment 1
1) explant is chosen: adopt the Herba Anoectochili roxburghii plant of self-sow as explant, with tap water, clean Herba Anoectochili roxburghii plant, proceed to 70% ethanol rinsing 30s, use 0.1% mercuric chloride sterilizing 10min, sterilized water rinses 5～6 times repeatedly, drains away the water again, be cut into and be with 1 stem segment, standby.
2) substratum preparation: take 1/2MS as basic medium, add banana 60g/L, gac 1.0g/L, NAA1.0mg/L, 6-BA0.6mg/L, sucrose 26g/L, agar powder 6.0g/L, onion 50g/L, Carapax Eriocheir sinensis powder 15g/L simultaneously, after mixing, the pH value of substratum is adjusted to 6.0.The substratum preparing is heated to agar and dissolves completely, add a cover after being sub-packed in while hot in clean culturing bottle.
3) substratum and the sterilizing of inoculation utensil: adopt high pressure steam process to carry out sterilizing to substratum and inoculation utensil.Keep Autoclave temperature at 121 ℃, 105kPa, 20min.Cutter, cut, the metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, then in the sterilizer with quartz sand high-temperature sterilization 10min, cooling rear use.
4) inoculation: regularly (month) carries out fumigation with tarragon and trypterygine to transfer room, regularly (one week) carries out sterilization with ozonizer to transfer room.Before inoculation, transfer room and Bechtop are opened ultra violet lamp sterilization 30min, then open Bechtop blowing 20min, with using after bromogeramine wiping table top.The stem section of handling well is laid on substratum.
5) tissue cultured seedling is cultivated: the culturing bottle of having inoculated is moved into culturing room, culture condition: 24 ℃ of temperature, light application time 12h/d, intensity of illumination 1800lx, incubation time four months.Every day is sprayed 75% alcohol dedusting by culturing room, and maintenance is clean and room air is dry, and regularly (one month) carries out fumigation with tarragon and trypterygine to culturing room, and regularly (one week) carries out sterilization with ozonizer to culturing room.
Result of implementation: in culturing process, Herba Anoectochili roxburghii tissue culture seedling pollution rate is 4.8%.After incubation time four months, the average plant height 6.8cm of tissue cultured seedling, 2.5 of average root of hair numbers, average leaf has a net increase of 3.5.
Embodiment 2
1) explant is chosen: adopt Herba Anoectochili roxburghii group training aseptic seedling as explant, by Herba Anoectochili roxburghii defoliation, stem is cut into is with 1 stem segment, standby.
2) substratum preparation: take 1/2MS as basic medium, add banana 50g/L, gac 0.8g/L, NAA1.2mg/L, 6-BA0.4mg/L, sucrose 22g/L, agar powder 5.5g/L, onion 60g/L, Carapax Eriocheir sinensis powder 20g/L simultaneously, after mixing, the pH value of substratum is adjusted to 5.8.The substratum preparing is heated to agar and dissolves completely, add a cover after being sub-packed in while hot in clean culturing bottle.
3) substratum and the sterilizing of inoculation utensil: adopt high pressure steam process to carry out sterilizing to substratum and inoculation utensil.Keep Autoclave temperature at 121 ℃, 105kPa, 20min.Cutter, cut, the metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, then in the sterilizer with quartz sand high-temperature sterilization 8min, cooling rear use.
4) inoculation: regularly (month) carries out fumigation with tarragon and trypterygine to transfer room, regularly (one week) carries out sterilization with ozonizer to transfer room.Before inoculation, transfer room and Bechtop are opened ultra violet lamp sterilization 30min, then open Bechtop blowing 20min, with using after bromogeramine wiping table top.The stem section of handling well is laid on substratum.
5) tissue cultured seedling is cultivated: the culturing bottle of having inoculated is moved into culturing room, culture condition: 24 ℃ of temperature, light application time 12h/d, intensity of illumination 2000lx, incubation time four months.Every day is sprayed 75% alcohol dedusting by culturing room, and maintenance is clean and room air is dry, and regularly (one month) carries out fumigation with tarragon and trypterygine to culturing room, and regularly (one week) carries out sterilization with ozonizer to culturing room.
Result of implementation: in culturing process, Herba Anoectochili roxburghii tissue culture seedling pollution rate is 3.2%.After incubation time four months, the average plant height 6.3cm of tissue cultured seedling, 2.6 of average root of hair numbers, average leaf has a net increase of 3.4.
Embodiment 3
1) explant is chosen: adopt Herba Anoectochili roxburghii group training aseptic seedling as explant, and the terminal bud of 1 joint of clip Herba Anoectochili roxburghii band, standby.
2) substratum preparation: take 1/2MS as basic medium, add banana 50g/L, gac 1.2g/L, NAA0.8mg/L, 6-BA0.8mg/L, sucrose 30g/L, agar powder 6.5g/L, onion 60g/L, Carapax Eriocheir sinensis powder 20g/L simultaneously, after mixing, the pH value of substratum is adjusted to 5.8.The substratum preparing is heated to agar and dissolves completely, add a cover after being sub-packed in while hot in clean culturing bottle.
3) substratum and the sterilizing of inoculation utensil: adopt high pressure steam process to carry out sterilizing to substratum and inoculation utensil.Keep Autoclave temperature at 121 ℃, 105kPa, 20min.Cutter, cut, the metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, then in the sterilizer with quartz sand high-temperature sterilization 8min, cooling rear use.
4) inoculation: regularly (month) carries out fumigation with tarragon and trypterygine to transfer room, regularly (one week) carries out sterilization with ozonizer to transfer room.Before inoculation, transfer room and Bechtop are opened ultra violet lamp sterilization 30min, then open Bechtop blowing 20min, with using after bromogeramine wiping table top.The terminal bud of 1 joint of Herba Anoectochili roxburghii band of handling well is inserted in substratum.
5) tissue cultured seedling is cultivated: the culturing bottle of having inoculated is moved into culturing room, culture condition: 24 ℃ of temperature, light application time 12h/d, intensity of illumination 2000lx, 3 first quarter moons of incubation time.Every day is sprayed 75% alcohol dedusting by culturing room, and maintenance is clean and room air is dry, and regularly (one month) carries out fumigation with tarragon and trypterygine to culturing room, and regularly (one week) carries out sterilization with ozonizer to culturing room.
Result of implementation: in culturing process, Herba Anoectochili roxburghii tissue culture seedling pollution rate is 3.4%.After 3 first quarter moons of incubation time, the average plant height 6.6cm of tissue cultured seedling, 2.3 of average root of hair numbers, average leaf has a net increase of 3.1.
Claims (5)
1. reduce a substratum for Herba Anoectochili roxburghii group training pollution rate, it is characterized in that described culture medium prescription is 1/2MS, banana 40～60g/L, gac 0.8～1.2g/L, NAA0.8～1.2mg/L, 6-BA0.4～0.8mg/L, sucrose 22～30g/L, agar powder 5.5～6.5g/L, onion 40～60g/L, Carapax Eriocheir sinensis powder 10～20g/L.
2. a kind of substratum that reduces Herba Anoectochili roxburghii group training pollution rate as claimed in claim 1, is characterized in that described culture medium prescription is 1/2MS, banana 50g/L, gac 1.0g/L, NAA1.0mg/L, 6-BA0.6mg/L, sucrose 26g/L, agar powder 6.0g/L, onion 50g/L, Carapax Eriocheir sinensis powder 15g/L.
3. utilize substratum described in claim 1 or 2 to reduce a method for Herba Anoectochili roxburghii group training pollution rate, it is characterized in that comprising the following steps:
1) transfer room sterilization: monthly with tarragon and trypterygine, transfer room fumigation is once carried out to sterilization; With ozonizer, transfer room is carried out to sterilization one time weekly;
2) culturing room's sterilization: monthly with tarragon and trypterygine, culturing room's fumigation is once carried out to sterilization; Monthly with ozonizer, culturing room is carried out to sterilization one time;
3) substratum preparation: prepare according to claim 1 or culture medium prescription claimed in claim 2, after mixing, the pH value of substratum is adjusted to 5.8～6.0; The substratum preparing is heated to agar and dissolves completely, add a cover after being sub-packed in while hot in clean culturing bottle;
4) substratum and the sterilizing of inoculation utensil: adopt high pressure steam process to carry out sterilizing to substratum and inoculation utensil;
5) explant inoculation:
When the Herba Anoectochili roxburghii plant that adopts self-sow is during as explant, with tap water, clean Herba Anoectochili roxburghii, proceed to 70% ethanol rinsing 30s, use 0.1% mercuric chloride sterilizing 10min, sterilized water rinses 5～6 times repeatedly, drains away the water again, be cut into and be with 1 stem segment, be inoculated on ready substratum;
When adopting Herba Anoectochili roxburghii group training aseptic seedling as explant, Herba Anoectochili roxburghii is cut into and is with 1 stem segment, be inoculated on ready substratum;
6) tissue cultured seedling is cultivated: the culturing bottle of having inoculated is moved into culturing room, culture condition: 24 ± 2 ℃ of temperature, light application time 12h/d, intensity of illumination 1800～2000lx, incubation time four months;
Before inoculation, transfer room and Bechtop are opened ultra violet lamp sterilization 30min, then open Bechtop blowing 20min, with using after bromogeramine wiping table top; The culturing bottle polluting must can be used after autoclave sterilization.
4. a kind of method of utilizing substratum described in claim 1 or 2 to reduce Herba Anoectochili roxburghii group training pollution rate according to claim 3, it is characterized in that: described culturing room should regularly sweep, spray 75% alcohol dedusting every day, the overcast and rainy dehumidifying of turning on the aircondition, to keep clean and room air to be dried; Find the culturing bottle that there is a pollution in culturing room sterilising treatment immediately, with non-proliferation.
5. a kind of method of utilizing substratum described in claim 1 or 2 to reduce Herba Anoectochili roxburghii group training pollution rate according to claim 4, it is characterized in that: described step 4) will guarantee Autoclave cleaning inside during sterilizing, inner cold air drains, the sterilizing thing of filling can not be stacked overfill, keeps Autoclave temperature at 121 ℃, 105kPa, 20min; After medium sterilization, first put between buffering, draw samples is placed on culturing room and cultivates 2～3d, if media surface and inner all without any bacterium vestige and fungi bacterial plaque can be used; Cutter, cut, tweezers metal apparatus before inoculation with 75% cotton ball soaked in alcohol wiping, then in the sterilizer with quartz sand high-temperature sterilization 5～10min, cooling rear use.
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| CN105230496A (en) * | 2015-11-17 | 2016-01-13 | 广西荷松农业发展有限公司 | Tissue culture seedling raising method for Hesong grass |
| CN105684908A (en) * | 2016-02-05 | 2016-06-22 | 王少荣 | Method for remedying mold contamination of tissue culture seedlings of Stevia rebaudiana |
| CN106212281B (en) * | 2016-07-28 | 2017-10-27 | 广西陆川县乌坭坡珍珠番石榴专业合作社 | A kind of method for tissue culture for improving banana survival rate |
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