CN100382681C - Artificial quick propagation method for hemsley rockvine root - Google Patents
Artificial quick propagation method for hemsley rockvine root Download PDFInfo
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Abstract
The present invention relates to an artificial fast breeding method for spun gold suspended gourd, which comprises the following special steps: pretreating explants, sterilizing the explants, inoculation, inducing culture, differentiation culture for calluses, enrichment culture, strengthening sprout culture, rooting culture, sprout hadening and transplanting, transplanting sprout bed management, provisional planting tissue culture sprouts, shearing cutting slips, treating the cutting slips with medical agents, cottage and cuttage sprout bed management. The present invention has the advantages that a large quantity of spun gold suspended gourd tissue culture sprout are bred in test tubes at a high speed in a short period by utilizing the technical advantages of plant tissue culture, the monthly breeding rate reaches 3 to 5 times, and the yearly tissue culture sprout producing ability reaches more than ten thousand sprouts per square meter. The prevent invention solves the problem of the shortage of the spun gold suspended gourd resource. The provided artificial fast breeding method uses few explant materials, which can effectively save rare endangered plants, realize the mass production, and provide seed and sprout foundation for the further series development of the products of the spun gold suspended gourd.
Description
Technical field
The present invention relates to the non-test tube breeding of plant tissue culture breeding and plant combination technique (the artificial quick propagating technology of wild medicinal plant), mainly is a kind of artificial quick breeding method of hemsley rockvine root.
Background technology
Hemsley rockvine root (Tetrastigma hemsleyanum Diels et Gilg) has another name called gold thread hoist, radix tetrastigme, tetratigma hemsleyanum, it is the perennial liane that overgrows of Vitaceae, mainly be used as medicine with the piece root, cool in nature, the mildly bitter flavor suffering, the function of tool heat-clearing antispastic, dispelling pathogenic wind and eliminating phlegm, promoting blood circulation and stopping pain clinically is used for the treatment of diseases such as infantile hyperpyrexia convulsions, stomachache, pneumonia, asthma, hepatitis.Prove that through modern medicine study its effective ingredient is a Flavonoid substances, can act on the topology isomerase, suppress the amplification of tumour, be used for the treatment of multiple malignant tumour.Because hemsley rockvine root is very peculiar for the environmental requirement of oneself growth, it only in Guangxi, there is a spot of distribution in some mountain areas of province such as Jiangxi, Zhejiang, it must be grown in the remote, thickly forested mountains of 700 meters of height above sea level and in the shade of overhanging cliff, above scattered light to be arranged, the next door will have thin water to ooze out, will have leaf to cover below, temperature remains on about 18 ℃ throughout the year.It 1 year only long 1 cun, just can grow up to 7 cucurbits in 7 years,, find hemsley rockvine root for local medicinal herb growers, just look like to have found gold, for these years, some the little mountain villages in the Pan'an have the people once to fall to death in remote, thickly forested mountains for hemsley rockvine root.Because mad the adopting of having no to control excessively dug, and adds the harsh requirement of hemsley rockvine root to growing environment, so the herbal medicine of this preciousness is now endangered.
Hemsley rockvine root mainly produces offspring with the rattan branch of overgrowing under wild state, but reproduction rate is very low.Since the Ming Dynasty, some medicinal herb growers of Pan'an constantly attempt cultivating hemsley rockvine root.They support hemsley rockvine root in sandstone or the black earth in land for growing field crops in, but the result is " ending without result ".Hong Kong one tame biological R﹠D institution was that matrix was cultivated hemsley rockvine root more than 10 year with ash, perlite, putty soil once, and its hemsley rockvine root that survives is done scientific research not enough and used.1994, Jinhua, Zhejiang Ai Ke wild plant Co., Ltd finally finished the hemsley rockvine root of artificial imitative natural green cultivation, and has proved that its effect equates with wild hemsley rockvine root from the transplanting wild hemsley rockvine root.It is " spun gold ground first " capsule of main component, anti-gene mutation (tumour causes owing to gene mutation) with the hemsley rockvine root that Zhejiang Ai Ke wild plant Co., Ltd associating Hong Kong Ai Ke bio tech ltd, Zhejiang College Of Traditional Chinese Medicine have been developed jointly, obtained the approval of hygiene department, begun to produce in batches.But existing resources can not satisfy the clinical treatment needs far away.Do not see the report that artificial breeding fast of hemsley rockvine root and the research of GAP culture technique are arranged at home as yet.So be badly in need of solving the problem of a large amount of seedling quick propagating technologies that are used for the large-scale artificial cultivation.
Summary of the invention
The present invention will solve the defective of above-mentioned prior art, and a kind of artificial quick breeding method of hemsley rockvine root is provided.
The technical scheme that the present invention solves its technical problem employing is: the artificial quick breeding method of this hemsley rockvine root, and concrete steps are as follows:
1) explant preliminary treatment: the rattan that clip 10～15cm is long, rinsing in being added with the running water that volumn concentration is 1% liquid detergent, and then with running water flushing 20～40min;
2) explant sterilization: the material that preliminary treatment is good carries out the material sterilization, elder generation is alcohol-pickled 0.5～1.0min of 75% with volumn concentration, with aseptic water washing 1 time, be 0.1% mercuric chloride solution with weight percentage again or be aqueous sodium hypochlorite solution sterilization 10～15min of 2%, use aseptic water washing at last 3～5 times with volumn concentration;
3) inoculation: the material that will sterilize is placed on suck dry moisture on the filter paper of sterilizing, and cuts the stem section of the long band axillalry bud of the long terminal bud of 0.5～1.0cm and 0.5～1.0cm, is seeded in the inducing culture;
4) inducing culture: induce terminal bud and axillalry bud, stem segment base portion has callus to take place, and continues inducing culture, until dissolving the bud of growing thickly in the callus punishment of stem segment base portion;
5) callus differentiation culture: with the callus that stem segment base portion induces, be cut into the stripping and slicing of 0.5cm * 0.5cm size, be inoculated in the callus differential medium; Condition of culture is: 25 ± 2 ℃ of temperature, and illumination 10～12h/d, intensity of illumination is 1500～2000lx; Cultivation differentiates the bud of growing thickly until the callus increase and on the callus surface;
6) enrichment culture: have the callus of the bud of growing thickly to be cut into two parts with long earlier: top stem and the callus that has the bottom stem; Behind the callus of undercut stem bottom, the fritter that is cut into 0.5～1.0cm size is inoculated in the proliferated culture medium again; Base portion at bud induces callus earlier, constantly differentiates the bud of growing thickly on the base portion callus surface of bud subsequently, continues to cultivate until young shoot elongation, mounted blade; Month rate of increase reaches 3～5 times;
7) the top stem of strong seedling culture: 2cm is inoculated in and carries out strong seedling culture in the strong seedling culture base;
8) the top stem that culture of rootage: 3cm is above, or the tissue cultivating seedling after the process strong seedling culture more than the 3cm are inoculated in and carry out culture of rootage in the root media, and culture of rootage induces root until the base portion at tissue cultivating seedling;
9) hardening and transplanting: when culture of rootage in the time of 30 days, after blake bottle moved into intermediate house and carry out hardening from the constant temperature culture chamber earlier, take out test-tube plantlet and be placed on flush away agar in the warm water, be transplanted into then in the dish of seedbed or cave; Matrix in seedbed or the cave dish is peat, perlite and vermiculite, and three's volume ratio is 3: 1: 1; Water once permeable at last;
10) transplant seedbed management: come the humidity of perception environment, temperature, illumination, carbonic acid gas, soil humidity and the nutrient solution EC value of environment by the electronics blade, spray, heat, light filling, increase gas and increase liquid by computer control seedbed facility;
11) tissue cultivating seedling is heeled in: the tissue cultivating seedling in the dish of seedbed or cave is heeled in the nutritive cube, watered once permeablely, according to the wet situation of doing of nutritive cube mesostroma, water afterwards, execute 2000 times of soilless culture nutrient fluids one time every watering in a week;
12) clip plugged ear: clip contains the stem section of a leaf one bud as plugged ear, and the plugged ear that shears can not be air-dry, in time enters next step processing or water spray and keeps moistening;
13) plugged ear chemicals treatment: the mixed aqueous solution of preparing the ABT root-inducing powder of 700 times carbendazim and 50mg/L; Or the mixed aqueous solution of the indolebutyric acid of 700 times carbendazim and 500mg/L; Plugged ear was soaked in above-mentioned solution 15 seconds～60 minutes;
14) cuttage: plugged ear is inserted the peat of packing into: perlite: the volume ratio of vermiculite is the seedbed or the peat of 3: 1: 1 matrix: perlite: the volume ratio of vermiculite is in the cave dish of 3: 1: 1 matrix; 1000～1500 strains are inserted for every square metre in the seedbed, and blade is not overlapped;
15) cuttage seedbed management: come the humidity of perception environment, temperature, illumination, carbonic acid gas, soil humidity and the nutrient solution EC value of environment by the electronics blade, spray, heat, light filling, increase gas and increase liquid by computer control seedbed facility;
Wherein, the content of additive is in minimal medium and each the stage medium:
(1) minimal medium: MS minimal medium, sucrose or white sugar are 20～30g/L, and agar is 7～9g/L, and pH is 5.6～5.8;
(2) inducing culture: MS+6-BA0.5～3.0mg/L+IBA0.1～0.5mg/L or MS+6-BA0.5～3.0mg/L+NAA0.1～0.5mg/L;
(3) callus differential medium: MS+6-BA2.0～5.0mg/L+IBA0.1～0.5mg/L or MS+6-BA2.0～5.0mg/L+NAA0.1～0.5mg/L;
(4) proliferated culture medium: MS+6-BA0.5～3.0mg/L+IBA0.1～0.5mg/L or MS+6-BA0.5～3.0mg/L+NAA0.1～0.5mg/L;
(5) strong seedling culture base: MS+6-BA0.01～0.1mg/L+IBA0.01～0.1mg/L or MS+6-BA0.01～0.1mg/L+NAA0.01～0.1mg/L;
(6) root media: 1/2MS+IBA1.5mg/L+IAA0.1mg/L+ active carbon 100～1000mg/L or MS+IBA0.25mg/L+NAA0.1mg/L.
Wherein, the content of additive is preferably in minimal medium and each the stage medium:
1) minimal medium: select the MS minimal medium for use, white sugar is 30g/L, and agar is 7g/L, and pH is 5.8;
2) inducing culture: MS+6-BA2mg/L+IBA0.2mg/L;
3) callus differential medium: MS+6-BA3.0mg/L+IBA0.2mg/L;
4) proliferated culture medium: MS+6-BA1.5mg/L+IBA0.15mg/L;
5) strong seedling culture base: MS+6-BA0.05mg/L+IBA0.01mg/L;
6) root media: 1/2MS+IBA1.5mg/L+IAA0.1mg/L+ active carbon 100～1000mg/L or MS+IBA0.25mg/L+NAA0.1mg/L.
Wherein, described transplanting seedbed and cuttage seedbed management are the regulation and control of temperature, illumination, carbonic acid gas, soil humidity and nutrient solution EC value of humidity, the environment of environment, are specially:
1) humidity control: after transplanting or the cuttage is 95%～90%, reduces to 80%～60% after taking root; Or use the electronic auto-control instrument of growing seedlings to make it to enter automatic interval atomize;
2) temperature control: 20～25 ℃;
3) illumination control: 5000～6000lx;
4) carbonic acid gas: CO
2Concentration is 1000～1500ppm;
5) soil humidity: 70%～80%;
6) nutrient solution EC value: the electronics blade is measured when the EC value is lower than 1/2 normal concentration in the matrix, and computer system will be opened the soilless culture nutrient fluid magnetic valve, carries out replenishing of soilless culture nutrient fluid in good time.
The effect that the present invention is useful is:
1, utilizes the plant tissue culture technique advantage, a large amount of fast in vitro breeding hemsley rockvine root tissue cultivating seedling in a short time,, the moon, the rate of increase reached 3～5 times, and a year tissue cultivating seedling production capacity culturing room area reaches more than 10,000 seedlings for every square metre, has solved the problem of the inadequate resource of hemsley rockvine root.
2, when tissue cultivating seedling is transplanted, utilize the take root advantage of fast and well developed root system, survival rate height, seedling stalwartness of the non-tube rapid propagation technology of plant, by computer control system is that tissue cultivating seedling is transplanted the environment of taking root of creating the best, solved the low difficult problem of transplanting survival rate of tissue cultivating seedling, what make tissue cultivating seedling plants survival rate from original about 60%, brings up to more than 95%.
3, when non-test tube is bred, utilizing the take root advantage of fast and well developed root system, survival rate height, seedling stalwartness of the non-tube rapid propagation technology of plant, is that tissue cultivating seedling is transplanted and created best cuttage environment by computer control system, and rooting rate reaches 100%, can promote 14 days root of hairs early, increase by 2/plugged ear of root amount.
4, utilize the low characteristics of the non-tube rapid propagation technology of plant seedling cost, breed the hemsley rockvine root seedling with tissue cultivating seedling more in a large number as female parent, solved the difficult problem that group is cultivated seedling cost height, is difficult to promote, the cost that makes seedling is reduced to 0.2 yuan/strain from 1.0 yuan original/strain.
5, the artificial quick-breeding method that proposes adopts explant material few, can save rare or endangered species effectively, and practicable large-scale production is for the product depth series exploitation of hemsley rockvine root provides the seedling basis.
Embodiment
The present invention is described in further detail by following examples, but content of the present invention is not limited thereto.
Embodiment 1
1) explant preliminary treatment: the rattan that clip 10～15cm is long, after the rinsing, wash about 30min with running water in being added with the running water that volume ratio is 1% liquid detergent.
2) explant sterilization: the material that preliminary treatment is good, on superclean bench, carry out the material sterilization, elder generation is alcohol-pickled 0.5～1.0min of 75% with volume ratio, with aseptic water washing 1 time, be 0.1% mercuric chloride solution sterilization, 10～15min with weight ratio again, or be 2% aqueous sodium hypochlorite solution sterilization, 10～15min with volume ratio, use aseptic water washing at last 3～5 times.
3) inoculation: the material that will sterilize is placed on suck dry moisture on the filter paper of sterilizing, and cuts long terminal bud of about 0.5～1.0cm and the stem section of being with axillalry bud with scalpel, is seeded in the inducing culture.
4) inducing culture: inducing culture is until inducing terminal bud and axillalry bud, and stem segment base portion has callus to take place, and continues inducing culture until dissolving the bud of growing thickly in the callus punishment of stem segment base portion;
5) callus differentiation culture: with the callus that stem segment base portion induces, be cut into the stripping and slicing of 0.5cm * 0.5cm size, be inoculated in the callus differential medium; Condition of culture is: 25 ± 2 ℃ of temperature, illumination 10～12h/d, intensity of illumination are 1500～2000lx; Cultivation differentiates the bud of growing thickly until the callus increase and on the callus surface;
6) enrichment culture: the callus of the bud of growing thickly will long be arranged, be cut into the top stem, have the callus of bottom stem, cut the bottom callus after, the fritter that is cut into 0.5～1.0cm size is inoculated in the proliferated culture medium; Cultivation induces callus until the base portion at bud, constantly differentiates the bud of growing thickly on the base portion callus surface of bud subsequently, continue to cultivate until the young shoot elongation, mounted blade, its month the rate of increase reach 3～5 times;
7) the above top stem of strong seedling culture: 2cm is inoculated in and carries out strong seedling culture in the strong seedling culture base;
8) tissue culture plant inoculation after top stem that culture of rootage: 3cm is above or the process strong seedling culture carries out culture of rootage in root media, culture of rootage induces root until the base portion at tissue cultivating seedling;
9) hardening and transplanting: when 30 days left and right sides of culture of rootage, after blake bottle earlier moved into intermediate house and carry out hardening from the constant temperature culture chamber, take out test-tube plantlet and be placed on flush away agar in the warm water, transplant then peat is being housed: perlite: the volume ratio of vermiculite is in the seedbed or cave dish of 3: 1: 1 matrix, waters once permeable at last;
10) transplant seedbed management: the environmental parameter in seedbed is by intelligent plant, and the humidity of perception environment, temperature, illumination, carbonic acid gas, soil humidity, nutrient solution EC value are sprayed, heated, light filling, increase gas, increase liquid by computer control seedbed facility;
11) tissue cultivating seedling is heeled in: the tissue cultivating seedling in the dish of seedbed or cave is heeled in the nutritive cube, watered once permeablely, according to the wet situation of doing of nutritive cube mesostroma, water afterwards, execute 2000 times of thin soilless culture nutrient fluids one time every watering in a week;
12) clip plugged ear: the stem section of clip one leaf one bud, the plugged ear that shears can not be air-dry, in time enter next step processing or water spray and keep moistening;
13) plugged ear chemicals treatment: the mixed aqueous solution of preparing the ABT root-inducing powder of 700 times carbendazim and 50mg/L; Or the mixed aqueous solution of the indolebutyric acid of 700 times carbendazim and 500mg/L; Plugged ear was soaked in above-mentioned solution 15 seconds～60 minutes;
14) cuttage: plugged ear is inserted the peat of packing into: perlite: the volume ratio of vermiculite is in the seedbed or cave dish of 3: 1: 1 matrix; 1000～1500 strains can be inserted for every square metre in the seedbed, are not advisable so that blade is overlapped;
15) cuttage seedbed management: the environmental parameter in seedbed is by intelligent plant (electronics leaf), the humidity of perception environment, temperature, illumination, carbonic acid gas, soil humidity, nutrient solution EC value are sprayed, are heated, light filling, increase gas, increase liquid by computer control seedbed facility.
The medium of tissue culture propagation in vitro, comprise minimal medium and be applicable to that respectively the medium of not training the stage is on the same group formed and content is: minimal medium: select the MS minimal medium for use, 20～30g/L that sucrose or white sugar are, agar are 7～9g/L, and pH is 5.6～5.8; Inducing culture: MS+6-BA0.5～3.0mg/L+IBA0.1～0.5mg/L or NAA0.1～0.5mg/L; Callus differentiation culture: MS+6-BA2.0～5.0mg/L+IBA0.1～0.5mg/L or NAA0.1～0.5mg/L; Proliferated culture medium: MS+6-BA0.5～3.0mg/L+IBA0.1～0.5mg/L or NAA0.1～0.5mg/L; Strong seedling culture: MS+6-BA0.01～0.1mg/L+IBA0.01～0.1mg/L or NAA0.01～0.1mg/L; Root media: 1/2MS+IBA1.5mg/L+IAA0.1mg/L+ active carbon 100～1000mg/L or MS+IBA0.25mg/L+NAA0.1mg/L.
Transplant seedbed and cuttage seedbed supervisory packet and draw together the regulation and control of humidity, temperature, illumination, carbonic acid gas, soil humidity, nutrient solution EC value.Humidity control: 1 week was 95%～90% after the cuttage, reduced to 80%～60% after taking root; Or use the electronic auto-control instrument of growing seedlings, make it to enter automatic interval atomize.Temperature control: 20～25 ℃.Illumination control: about 35%, 5000～6000lx.Carbonic acid gas: CO
2Concentration is 1000～1500ppm.Soil humidity: 70%～80%.Nutrient solution EC value: intelligent blade is measured when the EC value is lower than 1/2 normal concentration in the matrix, and computer system will be in conjunction with expert system, and intelligent opening soilless culture nutrient fluid magnetic valve carries out replenishing of soilless culture nutrient fluid in good time.After the cuttage 14 days, rooting percent reaches 100%.
Embodiment 2
1) explant preliminary treatment: the rattan that clip 10～15cm is long, after the rinsing, wash about 30min with running water in the running water that is added with 1% liquid detergent.
2) explant sterilization: the material that preliminary treatment is good, on superclean bench, carry out the material sterilization, earlier with alcohol-pickled 0.5～1.0min of 75%, with aseptic water washing 1 time, again with 0.1% mercuric chloride solution sterilization, 10～15min, or, use aseptic water washing at last 3～5 times with 2% aqueous sodium hypochlorite solution sterilization, 10～15min.
3) inoculation: the material that will sterilize is placed on suck dry moisture on the filter paper of sterilizing, and cuts long terminal bud of about 0.5～1.0cm and the stem section of being with axillalry bud with scalpel, is seeded in the inducing culture.
4) inducing culture: the stem section with terminal bud and band axillalry bud is seeded in the bud inducing culture of MS+6-BA2mg/L+IBA0.2mg/L+ white sugar 30g/L+ agar 7g/L.Behind the inducing culture 10 days, induce successively and sprout and axillalry bud; Stem segment base portion has callus that about 0.5～1.0cm size takes place; Behind the inducing culture 20 days, callus punishment dissolves the bud of growing thickly in stem segment base portion.
5) callus differentiation culture: the callus that the young shoot base portion is induced, be cut into the stripping and slicing of 0.5cm * 0.5cm size, be inoculated in the callus differential medium of MS+6-BA3.0mg/L+IBA0.2mg/L+ white sugar 30g/L+ agar 7g/L, 25 ± 2 ℃ of temperature, illumination 12 hours/day, intensity of illumination are about 2000lx, cultivate after 10 days, callus begins to increase successively, and at callus surface differentiation young shoot.
6) enrichment culture: with the long callus that clump bud is arranged, be cut into the top stem, have the callus of bottom stem or young shoot, after cutting the bottom callus, the fritter that is cut into 0.5～1.0cm size is inoculated in the proliferated culture medium of MS+6-BA1.5mg/L+IBA0.15mg/L+ white sugar 30g/L+ agar 7g/L.Cultivate after 10 days, induce callus at the base portion of bud, about 1.0～1.5cm size constantly differentiates the bud of growing thickly on the base portion callus surface of bud subsequently, cultivate after 20 days, and the young shoot elongation, blade launches successively.Its month, the rate of increase reached 3～5 times.
7) the above top stem of strong seedling culture: 2cm is inoculated in the strong seedling culture base of MS+6-BA0.05mg/L+IBA0.01mg/L+ white sugar 30g/L+ agar 7g/L and carries out strong seedling culture.Result of the test shows that tissue cultivating seedling stem chap after strong seedling culture, mounted blade are also constantly grown up.
8) tissue culture plant inoculation after top stem that culture of rootage: 2cm is above or the process strong seedling culture carries out culture of rootage in the root media of 1/2MS+IBA1.5mg/L+IAA0.1mg/L+ white sugar 20g/L+ agar 7g/L+ active carbon 100～1000mg/L or MS+IBA0.25mg/L+NAA0.1mg/L+ white sugar 30g/L+ agar 7g/L.After the culture of rootage 10 days, induce root successively at the base portion of tissue cultivating seedling, about 5 of every strains, many every strains reach about 10, and rooting rate can reach more than 95%.
9) hardening and transplanting: when 30 days left and right sides of culture of rootage, blake bottle is moved into intermediate house from the constant temperature culture chamber earlier, hardening is about 1 week, take out test-tube plantlet gently with tweezers, be placed on flush away agar in the water, transplant then peat is being housed: perlite: in the seedbed of vermiculite=3: 1: 1 matrix or the cave dish, water once permeable at last.
10) transplant seedbed management: the environmental parameter in seedbed is by intelligent plant (electronics leaf), the humidity of perception environment, temperature, illumination, carbonic acid gas, soil humidity, nutrient solution EC value are sprayed, are heated, light filling, increase gas, increase liquid by computer control seedbed facility.After transplanting 20, the transplanting survival rate of tissue cultivating seedling reaches more than 95%.
11) tissue cultivating seedling is heeled in: after tissue cultivating seedling is transplanted 30 days, constantly grow young leaves, when sending new root, tissue cultivating seedling in the dish of cave can be heeled in the nutritive cube of diameter 8cm, water once permeable, afterwards according to the wet situation of doing of nutritive cube mesostroma, water, execute 2000 times of thin soilless culture nutrient fluids one time every watering in a week.After heeling in 30 days, the survival rate of heeling in of tissue cultivating seedling reaches more than 98%.Tissue cultivating seedling is heeled in seedling and is constantly grown young leaves afterwards, takes out living shoot.
12) clip plugged ear: the stem section of clip one leaf one bud, the stem section of the following preferably 1～2cm of joint is so that cuttage.The plugged ear that shears can not be air-dry, in time enter next step processing or water spray and keep moistening.
13) plugged ear chemicals treatment: the mixed aqueous solution of preparing the IBA of 700 times carbendazim, 200～1000 times root-growing agent or 500mg/L.Plugged ear was soaked in above-mentioned solution 15 seconds～60 minutes.
14) cuttage: plugged ear is inserted the good peat of infiltration permeability of packing into: perlite: in the seedbed or cave dish of vermiculite=3: 1: 1 matrix; Need not too dark in the plugged ear insertion matrix; 1000～1500 strains can be inserted for every square metre in general seedbed, are not advisable so that blade is overlapped.
15) cuttage seedbed management: the environmental parameter in seedbed is by intelligent plant (electronics leaf), the humidity of perception environment, temperature, illumination, carbonic acid gas, soil humidity, nutrient solution EC value are sprayed, are heated, light filling, increase gas, increase liquid by computer control seedbed facility.
Transplant seedbed and cuttage seedbed supervisory packet and draw together the regulation and control of humidity, temperature, illumination, carbonic acid gas, soil humidity, nutrient solution EC value.Humidity control: 1 week was about 95% after the cuttage, reduced to about 70% after taking root; Or use the electronic auto-control instrument of growing seedlings, make it to enter automatic interval atomize.Temperature control: 20～25 ℃.Illumination control: about 35%, 5000～6000lx.Carbonic acid gas: CO
2Concentration is with 1200ppm.Soil humidity: 70%～80%.Nutrient solution EC value: intelligent blade is measured when the EC value is lower than 1/2 normal concentration in the matrix, and computer system will be in conjunction with expert system, and intelligent opening soilless culture nutrient fluid magnetic valve carries out replenishing of soilless culture nutrient fluid in good time.After the cuttage 14 days, rooting percent reaches 100%.
Claims (3)
1. the artificial quick breeding method of a hemsley rockvine root is characterized in that:
1) explant preliminary treatment: the rattan that clip 10～15cm is long, rinsing in being added with the running water that volumn concentration is 1% liquid detergent, and then with running water flushing 20～40min;
2) explant sterilization: the material that preliminary treatment is good carries out the material sterilization, elder generation is alcohol-pickled 0.5～1.0min of 75% with volumn concentration, with aseptic water washing 1 time, be 0.1% mercuric chloride solution with weight percentage again or be aqueous sodium hypochlorite solution sterilization 10～15min of 2%, use aseptic water washing at last 3～5 times with volumn concentration;
3) inoculation: the material that will sterilize is placed on suck dry moisture on the filter paper of sterilizing, and cuts the stem section of the long band axillalry bud of the long terminal bud of 0.5～1.0cm and 0.5～1.0cm, is seeded in the inducing culture;
4) inducing culture: induce terminal bud and axillalry bud, stem segment base portion has callus to take place, and continues inducing culture, until dissolving the bud of growing thickly in the callus punishment of stem segment base portion;
5) callus differentiation culture: with the callus that stem segment base portion induces, be cut into the stripping and slicing of 0.5cm * 0.5cm size, be inoculated in the callus differential medium; Condition of culture is: 25 ± 2 ℃ of temperature, and illumination 10～12h/d, intensity of illumination is 1500～2000lx; Cultivation differentiates the bud of growing thickly until the callus increase and on the callus surface;
6) enrichment culture: have the callus of the bud of growing thickly to be cut into two parts with long earlier: top stem and the callus that has the bottom stem; Behind the callus of undercut stem bottom, the fritter that is cut into 0.5～1.0cm size is inoculated in the proliferated culture medium again; Base portion at bud induces callus earlier, constantly differentiates the bud of growing thickly on the base portion callus surface of bud subsequently, continues to cultivate until young shoot elongation, mounted blade; Month rate of increase reaches 3～5 times;
7) the top stem of strong seedling culture: 2cm is inoculated in and carries out strong seedling culture in the strong seedling culture base;
8) the top stem that culture of rootage: 3cm is above, or the tissue cultivating seedling after the process strong seedling culture more than the 3cm are inoculated in and carry out culture of rootage in the root media, and culture of rootage induces root until the base portion at tissue cultivating seedling;
9) hardening and transplanting: when culture of rootage in the time of 30 days, after blake bottle moved into intermediate house and carry out hardening from the constant temperature culture chamber earlier, take out test-tube plantlet and be placed on flush away agar in the warm water, be transplanted into then in the dish of seedbed or cave; Matrix in seedbed or the cave dish is peat, perlite and vermiculite, and three's volume ratio is 3: 1: 1; Water once permeable at last;
10) transplant seedbed management: come the humidity of perception environment, temperature, illumination, carbonic acid gas, soil humidity and the nutrient solution EC value of environment by the electronics blade, spray, heat, light filling, increase gas and increase liquid by computer control seedbed facility;
11) tissue cultivating seedling is heeled in: the tissue cultivating seedling in the dish of seedbed or cave is heeled in the nutritive cube, watered once permeablely, according to the wet situation of doing of nutritive cube mesostroma, water afterwards, execute 2000 times of soilless culture nutrient fluids one time every watering in a week;
12) clip plugged ear: clip contains the stem section of a leaf one bud as plugged ear, and the plugged ear that shears can not be air-dry, in time enters next step processing or water spray and keeps moistening;
13) plugged ear chemicals treatment: the mixed aqueous solution of preparing the ABT root-inducing powder of 700 times carbendazim and 50mg/L; Or the mixed aqueous solution of the indolebutyric acid of 700 times carbendazim and 500mg/L; Plugged ear was soaked in above-mentioned solution 15 seconds～60 minutes;
14) cuttage: plugged ear is inserted the peat of packing into: perlite: the volume ratio of vermiculite is the seedbed or the peat of 3: 1: 1 matrix: perlite: the volume ratio of vermiculite is in the cave dish of 3: 1: 1 matrix; 1000～1500 strains are inserted for every square metre in the seedbed, and blade is not overlapped;
15) cuttage seedbed management: come the humidity of perception environment, temperature, illumination, carbonic acid gas, soil humidity and the nutrient solution EC value of environment by the electronics blade, spray, heat, light filling, increase gas and increase liquid by computer control seedbed facility;
Wherein, the content of additive is in minimal medium and each the stage medium:
(1) minimal medium: MS minimal medium, sucrose or white sugar are 20～30g/L, and agar is 7～9g/L, and pH is 5.6～5.8;
(2) inducing culture: MS+6-BA0.5～3.0mg/L+IBA0.1～0.5mg/L or MS+6-BA0.5～3.0mg/L+NAA0.1～0.5mg/L;
(3) callus differential medium: MS+6-BA2.0～5.0mg/L+IBA0.1～0.5mg/L or MS+6-BA2.0～5.0mg/L+NAA0.1～0.5mg/L;
(4) proliferated culture medium: MS+6-BA0.5～3.0mg/L+IBA0.1～0.5mg/L or MS+6-BA0.5～3.0mg/L+NAA0.1～0.5mg/L;
(5) strong seedling culture base: MS+6-BA0.01～0.1mg/L+IBA0.01～0.1mg/L or MS+6-BA0.01～0.1mg/L+NAA0.01～0.1mg/L;
(6) root media: 1/2MS+IBA1.5mg/L+IAA0.1mg/L+ active carbon 100～1000mg/L or MS+IBA0.25mg/L+NAA0.1mg/L.
2. the artificial quick breeding method of hemsley rockvine root according to claim 1 is characterized in that, the content of additive is in minimal medium and each the stage medium:
1) minimal medium: select the MS minimal medium for use, white sugar is 30g/L, and agar is 7g/L, and pH is 5.8;
2) inducing culture: MS+6-BA2mg/L+IBA0.2mg/L;
3) callus differential medium: MS+6-BA3.0mg/L+IBA0.2mg/L;
4) proliferated culture medium: MS+6-BA1.5mg/L+IBA0.15mg/L;
5) strong seedling culture base: MS+6-BA0.05mg/L+IBA0.01mg/L;
6) root media: 1/2MS+IBA1.5mg/L+IAA0.1mg/L+ active carbon 100～1000mg/L or MS+IBA0.25mg/L+NAA0.1mg/L.
3. the artificial quick breeding method of hemsley rockvine root according to claim 1 is characterized in that: described transplanting seedbed and cuttage seedbed management are the regulation and control of temperature, illumination, carbonic acid gas, soil humidity and nutrient solution EC value of humidity, the environment of environment:
1) humidity control: after transplanting or the cuttage is 95%～90%, reduces to 80%～60% after taking root; Or use the electronic auto-control instrument of growing seedlings to make it to enter automatic interval atomize;
2) temperature control: 20～25 ℃;
3) illumination control: 5000～6000lx;
4) carbonic acid gas: CO
2Concentration is 1000～1500ppm;
5) soil humidity: 70%～80%;
6) nutrient solution EC value: the electronics blade is measured when the EC value is lower than 1/2 normal concentration in the matrix, and computer system will be opened the soilless culture nutrient fluid magnetic valve, carries out replenishing of soilless culture nutrient fluid in good time.
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| CN101103700B (en) * | 2007-08-06 | 2010-08-04 | 浙江大学 | Tissue culture quick reproduction method of radix tetrastigmae |
| CN102296083B (en) * | 2011-09-05 | 2013-04-24 | 杭州师范大学 | Method for inducing and propagating transgenic hairy roots of tetrastigma hemsleyanum dielset gilg |
| CN102895422B (en) * | 2012-11-15 | 2014-11-26 | 陈前岭 | Application of tetrastlgma hemsleyanum in preparing drug for treating livestock viral infectious diseases |
| CN103548656A (en) * | 2013-11-15 | 2014-02-05 | 宁夏天地缘锦绣园林花卉有限公司 | Matrix for dianthus caryophylus seed and seedling tray cutting and breeding |
| CN104737770B (en) * | 2015-04-22 | 2017-05-31 | 詹月星 | The cultural method that a kind of shoulder pole rattan quickly breeds |
| CN105248073A (en) * | 2015-09-22 | 2016-01-20 | 江苏农林职业技术学院 | Cutting propagation method of Zoysia matrella |
| CN105165616B (en) * | 2015-09-30 | 2017-10-03 | 江苏农林职业技术学院 | A kind of method of efficiently quick breeding radix tetrastigme test tube seedling |
| CN106718925B (en) * | 2017-01-04 | 2019-04-02 | 广州甘蔗糖业研究所湛江甘蔗研究中心 | A kind of roxburgh anoectochilus terminal bud field rapid propagation method |
| CN108496777A (en) * | 2018-03-09 | 2018-09-07 | 西藏藏药集团股份有限公司 | A kind of soilless culture method of Tibetan medicine material convolvulate asiabell root in imminent danger |
| CN108887181A (en) * | 2018-06-27 | 2018-11-27 | 上饶师范学院 | A method of it improving the transplantation of seedlings of radix tetrastigme test tube and tames effect |
| CN108887120A (en) * | 2018-06-27 | 2018-11-27 | 上饶师范学院 | A method of it improving radix tetrastigme hayashishita container and plants root tuber inducing effect |
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