CN103250644A - 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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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 roxburgh anoectochilus terminal bud group training pollution rate.
Background technology
Roxburgh anoectochilus terminal bud (Anoectochilus roxburghii) has another name called rough melic herb, for the orchid family is opened lip Cymbidium herbaceos perennial, it is China's tradition rare Chinese medicine, have effects such as clearing heat and cooling blood, removing dampness and detoxicating, be used for the treatment of diseases such as diabetes, ephritis, cystitis, in the laudatory title of among the people enjoying " king of medicine ".Zhe Nan, Min Tai and one belt length phase of Guangdong and Guangxi Provinces be to boil water with roxburgh anoectochilus terminal bud, make tea, Baoshang, is used for improving the health, nourishing and take good care of.Modern study shows can strengthen body immunity by multiple compositions such as containing polysaccharide, flavonoids, cardiac glycosides in the roxburgh anoectochilus terminal bud, and the invasion and attack that improve Abwehrkraft des Koepers, ward off disease strengthen and improve effects such as physique.
Roxburgh anoectochilus terminal bud relies on for a long time excavates the collection wild resource, and wild roxburgh anoectochilus terminal bud reproduction rate is extremely low, causes the resource reserves to descend rapidly, and the parts of traditional producing region has not had medicine and can adopt.The roxburgh anoectochilus terminal bud seedling is the key link of its cultivation, and the quality of seedling is directly connected to the growing of roxburgh anoectochilus terminal bud, yield and quality.Because the roxburgh anoectochilus terminal bud seed is not had an endosperm, no germination ability only under mycosymbiosis, just can be impelled its seed germination growth, goes up main employing stem section and breeds seedling so produce.But the pollution problem (pollution rate is generally more than 20%) that causes owing to microorganisms such as bacterium, fungies in group training production process is the major obstacle of restricting current roxburgh anoectochilus terminal bud seedling breeding always.The pollution initial stage can cause the reduction of proliferate efficiency, the culture materials delayed growth; The pollution later stage can cause group training transplantation of seedlings difficulty and dead, even the meeting that has causes the genetic variation of culture materials.Therefore seek a kind of method that reduces roxburgh anoectochilus terminal bud group training pollution rate and medium thereof for raising group training success rate, reduce cost, increase economic efficiency etc. all significant.
Summary of the invention
The objective of the invention is to overcome the deficiency that the above-mentioned background technology exists, a kind of method of roxburgh anoectochilus terminal bud group training pollution rate and technical scheme of culture medium prescription thereof of reducing is provided.
Technical scheme provided by the invention is:
A kind of culture medium prescription that reduces roxburgh anoectochilus terminal bud group training pollution rate is characterized in that described culture medium prescription is 1/2MS, banana 40～60g/L, active carbon 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 roxburgh anoectochilus terminal bud group training pollution rate is characterized in that described culture medium prescription is 1/2MS, banana 50g/L, active carbon 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 roxburgh anoectochilus terminal bud group training pollution rate, it is characterized in that may further comprise the steps:
1) transfer room sterilization: with tarragon and thunder godvine the transfer room fumigation is once carried out sterilization in every month; With ozone generator transfer room is carried out sterilization one time weekly;
2) culturing room sterilization: with tarragon and thunder godvine culturing room's fumigation was once carried out sterilization in every month; With ozone generator culturing room is carried out sterilization one time weekly;
3) medium preparation: be basal medium with 1/2MS, prepare according to prescription that the pH value with medium behind the mixing is adjusted to 5.8～6.0; Prepared culture medium is heated to agar dissolves fully, add a cover after being sub-packed in the clean blake bottle while hot;
4) medium and the sterilization of inoculation utensil: adopt high pressure steam process that medium and inoculation utensil are sterilized.
5) explant inoculation:
When the roxburgh anoectochilus terminal bud plant of adopting self-sow during as explant, clean roxburgh anoectochilus terminal bud with running water, change 70% ethanol rinsing 30s over to, with 0.1% mercuric chloride sterilization 10min, sterile water washes 5～6 times repeatedly, drains away the water again, be cut into and be with 1 stem segment, be inoculated on the ready medium;
When adopting roxburgh anoectochilus terminal bud group training aseptic seedling as explant, roxburgh anoectochilus terminal bud is cut into is with 1 stem segment, be inoculated on the ready medium;
6) group training seedling is cultivated: will inoculate good blake bottle and move into culturing room, condition of culture: 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 is to keep clean and room air drying; Find the blake bottle that there is a pollution in culturing room sterilization treatment immediately, with non-proliferation.Before the inoculation, transfer room and superclean bench are opened ultra violet lamp sterilization 30min, open superclean bench blowing 20min then, with using behind the bromogeramine wiping table top; The blake bottle that pollutes must can use through behind the autoclave sterilization.
Described step 4) will guarantee the autoclave cleaning inside when sterilizing, and inner cold air drains, and the sterilization thing of filling can not be stacked overfill, keeps the autoclave temperature at 121 ℃, 105kPa, 20min; Behind the medium sterilization, put into earlier between buffering, draw samples is placed on culturing room and cultivates 2～3d, if media surface and inside all do not have any bacterium vestige and fungi bacterial plaque, can use; Cutter, cut, metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, high-temperature sterilization 5～10min in having the sterilizer of quartz sand again, use the cooling back.
The invention has the beneficial effects as follows: method and the medium thereof that reduces roxburgh anoectochilus terminal bud group training pollution rate that provide, rationally novel in design; Medium contains 1/2MS, banana, active carbon, NAA, 6-BA, sucrose, agar powder, onion, Carapax Eriocheir sinensis powder among the present invention.Sulfur-containing compound can suppress multiple microbial growths such as staphylococcus, streptococcus, vibrios, bacillus in the onion.Contain abundant chitin and shitosan in the Carapax Eriocheir sinensis powder, Escherichia coli, staphylococcus aureus, saccharomycete, hay bacillus, aspergillus niger etc. are had the obvious suppression effect; Adopt tarragon and thunder godvine fumigation to carry out sterilization, sterilization effect good (roxburgh anoectochilus terminal bud group training pollution rate is down to 5% below) not only, and do not have environmental pollution, cost is significantly reduction also.The culture medium prescription that provides is reasonable, and effect is obvious; The method that provides is simple to operate, is easy to grasp, and good reproducibility is fit to apply on a large scale.
Embodiment
Described with tarragon and thunder godvine to transfer room and culturing room's fumigation, refer to tarragon after drying (moisture content is usually less than 5%) directly light, earlier with 95% alcohol-pickled 24h, be that combustion adjuvant is lighted with alcohol when fumigating after the thunder godvine drying.Evenly distribute by 3 points, light the airtight stifling 1h in back.
Adopt ozone generator that culturing room, transfer room are carried out time of sterilization, should select to determine according to ozone generator power, careat size.
It is conventional method that high pressure steam process is adopted in medium and the sterilization of inoculation utensil.
The culture medium prescription of reduction roxburgh anoectochilus terminal bud group training pollution rate of the present invention comprises 1/2MS, banana 40～60g/L, active carbon 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.
Preferably: 1/2MS, banana 50g/L, active carbon 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 specify the present invention by the following examples.
Embodiment 1
1) explant is chosen: adopt the roxburgh anoectochilus terminal bud plant of self-sow as explant, clean the roxburgh anoectochilus terminal bud plant with running water, change 70% ethanol rinsing 30s over to, with 0.1% mercuric chloride sterilization 10min, sterile water washes 5～6 times repeatedly, drains away the water again, be cut into and be with 1 stem segment, standby.
2) medium preparation: be basal medium with 1/2MS, the pH value that adds simultaneously behind banana 60g/L, active carbon 1.0g/L, NAA1.0mg/L, 6-BA0.6mg/L, sucrose 26g/L, agar powder 6.0g/L, onion 50g/L, the Carapax Eriocheir sinensis powder 15g/L mixing medium is adjusted to 6.0.Prepared culture medium is heated to agar dissolves fully, add a cover after being sub-packed in the clean blake bottle while hot.
3) medium and the sterilization of inoculation utensil: adopt high pressure steam process that medium and inoculation utensil are sterilized.Keep the autoclave temperature at 121 ℃, 105kPa, 20min.Cutter, cut, metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, high-temperature sterilization 10min in having the sterilizer of quartz sand again, use the cooling back.
4) inoculation: regularly (one month) carries out fumigation with tarragon and thunder godvine to transfer room, and regular (week) carries out sterilization with ozone generator to transfer room.Before the inoculation, transfer room and superclean bench are opened ultra violet lamp sterilization 30min, open superclean bench blowing 20min then, with using behind the bromogeramine wiping table top.The stem section of handling well is tiled on the medium.
5) group training seedling is cultivated: will inoculate good blake bottle and move into culturing room, condition of culture: 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, keeps clean and room air drying, and regularly (one month) carries out fumigation with tarragon and thunder godvine to culturing room, and regular (week) carries out sterilization with ozone generator to culturing room.
Result of implementation: in the incubation, roxburgh anoectochilus terminal bud tissue culture seedling pollution rate is 4.8%.Behind the incubation time four months, the average plant height 6.8cm of group training seedling, several 2.5 of average root of hair, average leaf has a net increase of 3.5.
Embodiment 2
1) explant is chosen: adopt roxburgh anoectochilus terminal bud group training aseptic seedling as explant, with the roxburgh anoectochilus terminal bud defoliation, stem is cut into is with 1 stem segment, standby.
2) medium preparation: be basal medium with 1/2MS, the pH value that adds simultaneously behind banana 50g/L, active carbon 0.8g/L, NAA1.2mg/L, 6-BA0.4mg/L, sucrose 22g/L, agar powder 5.5g/L, onion 60g/L, the Carapax Eriocheir sinensis powder 20g/L mixing medium is adjusted to 5.8.Prepared culture medium is heated to agar dissolves fully, add a cover after being sub-packed in the clean blake bottle while hot.
3) medium and the sterilization of inoculation utensil: adopt high pressure steam process that medium and inoculation utensil are sterilized.Keep the autoclave temperature at 121 ℃, 105kPa, 20min.Cutter, cut, metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, high-temperature sterilization 8min in having the sterilizer of quartz sand again, use the cooling back.
4) inoculation: regularly (one month) carries out fumigation with tarragon and thunder godvine to transfer room, and regular (week) carries out sterilization with ozone generator to transfer room.Before the inoculation, transfer room and superclean bench are opened ultra violet lamp sterilization 30min, open superclean bench blowing 20min then, with using behind the bromogeramine wiping table top.The stem section of handling well is tiled on the medium.
5) group training seedling is cultivated: will inoculate good blake bottle and move into culturing room, condition of culture: 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, keeps clean and room air drying, and regularly (one month) carries out fumigation with tarragon and thunder godvine to culturing room, and regular (week) carries out sterilization with ozone generator to culturing room.
Result of implementation: in the incubation, roxburgh anoectochilus terminal bud tissue culture seedling pollution rate is 3.2%.Behind the incubation time four months, the average plant height 6.3cm of group training seedling, several 2.6 of average root of hair, average leaf has a net increase of 3.4.
Embodiment 3
1) explant is chosen: adopt roxburgh anoectochilus terminal bud group training aseptic seedling as explant, and the terminal bud of 1 joint of clip roxburgh anoectochilus terminal bud band, standby.
2) medium preparation: be basal medium with 1/2MS, the pH value that adds simultaneously behind banana 50g/L, active carbon 1.2g/L, NAA0.8mg/L, 6-BA0.8mg/L, sucrose 30g/L, agar powder 6.5g/L, onion 60g/L, the Carapax Eriocheir sinensis powder 20g/L mixing medium is adjusted to 5.8.Prepared culture medium is heated to agar dissolves fully, add a cover after being sub-packed in the clean blake bottle while hot.
3) medium and the sterilization of inoculation utensil: adopt high pressure steam process that medium and inoculation utensil are sterilized.Keep the autoclave temperature at 121 ℃, 105kPa, 20min.Cutter, cut, metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, high-temperature sterilization 8min in having the sterilizer of quartz sand again, use the cooling back.
4) inoculation: regularly (one month) carries out fumigation with tarragon and thunder godvine to transfer room, and regular (week) carries out sterilization with ozone generator to transfer room.Before the inoculation, transfer room and superclean bench are opened ultra violet lamp sterilization 30min, open superclean bench blowing 20min then, with using behind the bromogeramine wiping table top.The terminal bud of 1 joint of roxburgh anoectochilus terminal bud band of handling well is inserted in the medium.
5) group training seedling is cultivated: will inoculate good blake bottle and move into culturing room, condition of culture: 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, keeps clean and room air drying, and regularly (one month) carries out fumigation with tarragon and thunder godvine to culturing room, and regular (week) carries out sterilization with ozone generator to culturing room.
Result of implementation: in the incubation, roxburgh anoectochilus terminal bud tissue culture seedling pollution rate is 3.4%.Behind 3 first quarter moons of incubation time, the average plant height 6.6cm of group training seedling, several 2.3 of average root of hair, average leaf has a net increase of 3.1.
Claims (6)
1. a culture medium prescription that reduces roxburgh anoectochilus terminal bud group training pollution rate is characterized in that described culture medium prescription is 1/2MS, banana 40～60g/L, active carbon 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 culture medium prescription that reduces roxburgh anoectochilus terminal bud group training pollution rate as claimed in claim 1 is characterized in that described culture medium prescription is 1/2MS, banana 50g/L, active carbon 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. one kind is utilized claim 1 or 2 described culture medium prescriptions to reduce the method that the roxburgh anoectochilus terminal bud group is trained pollution rate, it is characterized in that may further comprise the steps:
1) transfer room sterilization: with tarragon and thunder godvine the transfer room fumigation was once carried out sterilization in every month; With ozone generator transfer room is carried out sterilization one time weekly;
2) culturing room sterilization: with tarragon and thunder godvine culturing room's fumigation was once carried out sterilization in every month; With ozone generator culturing room was carried out sterilization one time in every month;
3) medium preparation: be basal medium with 1/2MS, prepare according to prescription that the pH value with medium behind the mixing is adjusted to 5.8～6.0; Prepared culture medium is heated to agar dissolves fully, add a cover after being sub-packed in the clean blake bottle while hot;
4) medium and the sterilization of inoculation utensil: adopt high pressure steam process that medium and inoculation utensil are sterilized.
5) explant inoculation:
When the roxburgh anoectochilus terminal bud plant of adopting self-sow during as explant, clean roxburgh anoectochilus terminal bud with running water, change 70% ethanol rinsing 30s over to, with 0.1% mercuric chloride sterilization 10min, sterile water washes 5～6 times repeatedly, drains away the water again, be cut into and be with 1 stem segment, be inoculated on the ready medium;
When adopting roxburgh anoectochilus terminal bud group training aseptic seedling as explant, roxburgh anoectochilus terminal bud is cut into is with 1 stem segment, be inoculated on the ready medium;
6) group training seedling is cultivated: will inoculate good blake bottle and move into culturing room, condition of culture: 24 ± 2 ℃ of temperature, light application time 12h/d, intensity of illumination 1800～2000lx, incubation time four months.
4. a kind of method that reduces roxburgh anoectochilus terminal bud group training pollution rate according to claim 3, it is characterized in that: before the inoculation, transfer room and superclean bench are opened ultra violet lamp sterilization 30min, open superclean bench blowing 20min then, with using behind the bromogeramine wiping table top; The blake bottle that pollutes must can use through behind the autoclave sterilization.
5. a kind of method that reduces roxburgh anoectochilus terminal bud group training pollution rate according to claim 4, it is characterized in that: described culturing room should regularly sweep, and sprays 75% alcohol dedusting every day, and the overcast and rainy dehumidifying of turning on the aircondition is to keep totally and the room air drying; Find the blake bottle that there is a pollution in culturing room sterilization treatment immediately, with non-proliferation.
6. a kind of method that reduces roxburgh anoectochilus terminal bud group training pollution rate according to claim 5, it is characterized in that: described step 4) will guarantee the autoclave cleaning inside when sterilizing, inner cold air drains, the sterilization thing of filling can not be stacked overfill, keeps the autoclave temperature at 121 ℃, 105kPa, 20min; Behind the medium sterilization, put into earlier between buffering, draw samples is placed on culturing room and cultivates 2～3d, if media surface and inside all do not have any bacterium vestige and fungi bacterial plaque, can use; Cutter, cut, metal apparatus such as tweezers before inoculation with 75% cotton ball soaked in alcohol wiping, high-temperature sterilization 5～10min in having the sterilizer of quartz sand again, use the cooling back.
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| CN104692956A (en) * | 2015-01-31 | 2015-06-10 | 浙江农林大学 | Special fertilizer for anoectochilus roxburghii and preparation method of special fertilizer |
| 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 |
| CN106212281A (en) * | 2016-07-28 | 2016-12-14 | 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 improving Fructus Musae survival rate |
| CN111471618A (en) * | 2020-04-13 | 2020-07-31 | 厦门德运科技有限公司 | Extraction and purification method of anoectochilus formosanus symbiotic bacteria |
| CN112167064A (en) * | 2020-11-11 | 2021-01-05 | 山东农业大学 | Tissue culture and rapid propagation method for salvia miltiorrhiza |
| CN113598049A (en) * | 2021-08-12 | 2021-11-05 | 福建省中科生物股份有限公司 | Chinese herbal medicine pre-cooking liquid for inhibiting tissue culture plant endophyte pollution and application method |
| CN114223544A (en) * | 2021-12-31 | 2022-03-25 | 泉州景圃生物科技股份有限公司 | Cultivation method of sunlight seedlings |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104692956A (en) * | 2015-01-31 | 2015-06-10 | 浙江农林大学 | Special fertilizer for anoectochilus roxburghii and preparation method of special fertilizer |
| 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 |
| CN106212281A (en) * | 2016-07-28 | 2016-12-14 | 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 improving Fructus Musae survival rate |
| 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 |
| CN111471618A (en) * | 2020-04-13 | 2020-07-31 | 厦门德运科技有限公司 | Extraction and purification method of anoectochilus formosanus symbiotic bacteria |
| CN112167064A (en) * | 2020-11-11 | 2021-01-05 | 山东农业大学 | Tissue culture and rapid propagation method for salvia miltiorrhiza |
| CN113598049A (en) * | 2021-08-12 | 2021-11-05 | 福建省中科生物股份有限公司 | Chinese herbal medicine pre-cooking liquid for inhibiting tissue culture plant endophyte pollution and application method |
| CN114223544A (en) * | 2021-12-31 | 2022-03-25 | 泉州景圃生物科技股份有限公司 | Cultivation method of sunlight seedlings |
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