CN104686357A - Establishment method of rapid polygonum capitatum in-vitro propagation system - Google Patents
Establishment method of rapid polygonum capitatum in-vitro propagation system Download PDFInfo
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Abstract
The invention discloses an establishment method of a rapid polygonum capitatum in-vitro propagation system. The polygonum capitatum is a polygonaceous polygonum plant, is used in miao minority in guizhou province, can be used as medicine wholly and can be commonly used for treating urinary system infection, rheumatalgia and other symptoms. The wild resource of polygonum capitatum is distributed rarely and dispersedly, and is in shortage severely due to excessive industrial consumption and deterioration of the ecological environment, and therefore, large-scale production is seriously hindered. According to the method, the vane of the polygonum capitatum is used as an explant, the callus is subjected to induction culture, multiplication culture, differentiation, rooting culture, acclimatization and transplantation to acquire the polygonum capitatum in-vitro regeneration plant, the rapid propagation technology system for polygonum capitatum tissue culture is established and scale development is promoted.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to a kind of method for building up of polygonum capitatum rapid propagation in vitro system.
Background technology
Polygonum capitatum (
polygonum capitatum) have another name called solar grass, the four seasons red, capitate knotweed herb etc., be polygonaceae arsesmart, be mainly distributed in the provinces such as Guizhou, Chongqing, Sichuan, Hubei, Hunan and Guangxi.For Miao in Guizhou medication, its applicating history is long, acrial part or all herbal medicine, has pungent, the micro-feature puckery, cool in nature of taste; Among the people being usually used in treats the diseases such as urinary system infection contamination, blood urine, cystitis, rheumatalgia, diarrhoea, dysentery.At present, the exploitation of polygonum capitatum mainly concentrate on Guizhou Province of China, are that one of six seedlings medicine kinds are given priority in the modernization of Chinese medicine.In addition, polygonum capitatum relies on seminal propagation, and germination rate is higher, well developed root system, and adsorption capacity is very strong, and each stipes can grow adventive root, has good fixation to soil, is desirable wild cover plant.The stem, leaf, flower etc. of polygonum capitatum plant have higher ornamental value, are a kind of wild ground cover plants, in conjunction with the garden landscape that shrub Plantlet formation is high and low layered, can be used on the greening treatment aspects such as vertical greening, side slope nursing and land virescence.
At present, the technology such as polygonum capitatum many employings seed breeding, cuttage, plant division and tissue cultures are bred.Because features such as seed harvest amount are large, germination rate is high, storage is convenient, adopt uniform broadcasting effectively can form Land cover, make seed seedling-raising become the main method of main breeding; Cuttage adopts base to insert technology, coordinates Corticosteroids better can reach the object of Fast-propagation; Division propagation aspect, the stolon due to polygonum capitatum produces aerial root and easily forms plantlet, and propagation method is simple, easily very fast seedling.But, the distribution of polygonum capitatum wild resource rare loose, industry consumes excessively and going from bad to worse of ecotope causes polygonum capitatum resource seriously deficient, seriously hinders the needs of its large-scale production.Therefore, aseptic plant is obtained by plant tissue culture technique and the high quality seedling breeding of method setting up complete set becomes the task of top priority.The present invention with polygonum capitatum blade for explant; the in vitro plant again of polygonum capitatum is have successfully been obtained by processes such as induction of callus, Multiplying culture, differentiation, culture of rootage, acclimatization and transplantses; set up polygonum capitatum tissue culture rapid propagation technique system, to promote its large-scale development.
Summary of the invention
The object of the present invention is to provide out a kind of method for building up of polygonum capitatum rapid propagation in vitro system, the present invention with polygonum capitatum blade for explant, the in vitro plant again of polygonum capitatum is have successfully been obtained by processes such as induction of callus, Multiplying culture, differentiation, culture of rootage, acclimatization and transplantses, set up polygonum capitatum tissue culture rapid propagation technique system, thus achieve object of the present invention.
The method for building up of a kind of polygonum capitatum rapid propagation in vitro system of the present invention, comprises the following steps:
(1) explant sterilization: win polygonum capitatum and just launch blade, rinses 60 ~ 90min with the form of dripping in running water.Aseptically, with 70% ~ 80% alcohol solution dipping 20s ~ 60s, aseptic water washing 4 ~ 6 times, then with 0.1% ~ 0.2% mercuric chloride solution sterilization 5 ~ 15min, then use aseptic water washing explant to non-foam.For subsequent use blot the moisture on surface with aseptic filter paper after.
(2) callus induction: the blade that step (1) disinfects is cut 0.5cm
2fritter, slight cutting 2 cuttves on master pulse, vacuum side of blade is inoculated into inducing culture down and carries out induction of callus.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 1000 ~ 2000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up induction situation afterwards in 30 days.
(3) Multiplying culture: the eugonic embryo callus that selecting step (2) Fiber differentiation obtains, cuts block that diameter is about 0.3cm and is seeded to proliferated culture medium and carries out squamous subculture.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 1000 ~ 2000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% after 45 days to add up proliferative conditions.
(4) differentiation is cultivated: the callus of step (3) being bred takes out and is cut into block that diameter is about 0.5cm and is seeded to differential medium and carries out differentiation cultivation.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up differentiation situation afterwards in 45 days.
(5) culture of rootage: from base portion cut step (4) differentiation cultivate the healthy and strong test-tube plantlet that obtains be seeded in root media and carry out root induction.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up situation of taking root afterwards in 30 days.
(6) acclimatization and transplants: the bottle seedling possessing transplanting condition that root media obtains is carried out hardening, bottle seedling to move under normal temperature after 4 ~ 5 days, open bottle cap 2 ~ 3 days, then the medium being attached to shoot root and fastening is washed away, transplanting to filling by peat soil: cultivate in the container bag of the transplanting culture matrix that vermiculite=1:1 forms, transplanting and with 0.3% potassium permanganate solution, matrix spray sterilized and added a cover film for first 5 days, after 3 days, opening film, stir matrix, transplant water of matrix being drenched for first 1 day.By matrix compacting, and individual plant bagging need be carried out during transplanting, sooner or later spraying, ensure that growing environment is moist.Transplant and cut off plastic sack two jiaos after 7 days, after 14 days, completely de-bag, transplants and adds up survival rate after 30 days.
Inducing culture described in above-mentioned steps (2) is: MS+0.1 ~ 1.0mg/L NAA+1.0 ~ 2.0mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (3) is: MS+2.0 ~ 5.0mg/L 6-BA+0.1 ~ 0.5mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Differential medium described in above-mentioned steps (4) is: MS+4.0 ~ 8.0mg/L 6-BA+0.1 ~ 0.5mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Root media described in above-mentioned steps (5) is: 1/2MS+1.0 ~ 2.0mg/L ABT
5+ 0.1 ~ 0.5mg/L NAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Advantage of the present invention is: polygonum capitatum (
polygonum capitatum) be polygonaceae arsesmart, be Miao in Guizhou medication, herb can be used as medicine, and is usually used in illness such as treatment urinary system infection contamination, rheumatalgia etc.The distribution of polygonum capitatum wild resource rare loose, industry consumes excessively and going from bad to worse of ecotope causes polygonum capitatum resource seriously deficient, seriously hinders the needs of its large-scale production.Therefore, aseptic plant is obtained by plant tissue culture technique and the high quality seedling breeding of method setting up complete set becomes the task of top priority.The present invention with polygonum capitatum blade for explant; the in vitro plant again of polygonum capitatum is have successfully been obtained by processes such as induction of callus, Multiplying culture, differentiation, culture of rootage, acclimatization and transplantses; set up polygonum capitatum tissue culture rapid propagation technique system, to promote its large-scale development.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
embodiment 1
(1) explant sterilization: win polygonum capitatum and just launch blade, rinses 60min with the form of dripping in running water.Aseptically, with 70% alcohol solution dipping 60s, aseptic water washing 4 times, then with 0.1% mercuric chloride solution sterilization 10min, then use aseptic water washing explant to non-foam.For subsequent use blot the moisture on surface with aseptic filter paper after.
(2) callus induction: the blade that step (1) disinfects is cut 0.5cm
2fritter, slight cutting 2 cuttves on master pulse, vacuum side of blade is inoculated into inducing culture down and carries out induction of callus.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 1000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate inductivity after 30 days under the condition of 75% be 78.9%.Described inducing culture is: MS+0.5mg/L NAA+1.5mg/L 6-BA+25g/L sucrose+3.8g/L agar, pH is 5.5.
(3) Multiplying culture: the eugonic embryo callus that selecting step (2) Fiber differentiation obtains, cuts block that diameter is about 0.3cm and is seeded to proliferated culture medium and carries out squamous subculture.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 1000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate callus recruitment after 45 days under the condition of 75% be 86.9%.Described proliferated culture medium is: MS+3.0mg/L 6-BA+0.2mg/L IBA+25g/L sucrose+4.5g/L agar, pH is 5.5.
(4) differentiation is cultivated: the callus of step (3) being bred takes out and is cut into block that diameter is about 0.5cm and is seeded to differential medium and carries out differentiation cultivation.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate differentiation rate after 45 days under the condition of 75% be 93.3%, and indefinite bud mean is 5.3.Described differential medium is: MS+5.0mg/L 6-BA+0.3mg/L IBA+18g/L sucrose+5.5g/L agar, pH is 5.5.
(5) culture of rootage: from base portion cut step (4) differentiation cultivate the healthy and strong test-tube plantlet that obtains be seeded in root media and carry out root induction.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate rooting rate after 30 days under the condition of 75% be 91.6%.Described root media is: 1/2MS+1.5mg/L ABT
5+ 0.2mg/L NAA+24g/L sucrose+4.6g/L agar, pH is 5.5.
(6) acclimatization and transplants: the bottle seedling possessing transplanting condition that root media obtains is carried out hardening, bottle seedling moves to normal temperature after lower 4 days, open bottle cap 2 days, then the medium being attached to shoot root and fastening is washed away, transplanting to filling by peat soil: cultivate in the container bag of the transplanting culture matrix that vermiculite=1:1 forms, transplanting and with 0.3% potassium permanganate solution, matrix spray sterilized and added a cover film for first 5 days, after 3 days, opening film, stir matrix, transplant water of matrix being drenched for first 1 day.By matrix compacting, and individual plant bagging need be carried out during transplanting, sooner or later spraying, ensure that growing environment is moist.Transplant and cut off plastic sack two jiaos after 7 days, completely de-bag after 14 days, transplanting survival rate after 30 days is 92.4%.
Embodiment 2
(1) explant sterilization: win polygonum capitatum and just launch blade, rinses 60min with the form of dripping in running water.Aseptically, with 75% alcohol solution dipping 40s, aseptic water washing 5 times, then with 0.15% mercuric chloride solution sterilization 6min, then use aseptic water washing explant to non-foam.For subsequent use blot the moisture on surface with aseptic filter paper after.
(2) callus induction: the blade that step (1) disinfects is cut 0.5cm
2fritter, slight cutting 2 cuttves on master pulse, vacuum side of blade is inoculated into inducing culture down and carries out induction of callus.Inoculation is placed on illumination every day 14 hours, and intensity of illumination is 1500lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is that to cultivate inductivity after 30 days under the condition of 75% be 83.1%.Described inducing culture is: MS+0.8mg/L NAA+2.0mg/L 6-BA+25g/L sucrose+3.8g/L agar, pH is 5.7.
(3) Multiplying culture: the eugonic embryo callus that selecting step (2) Fiber differentiation obtains, cuts block that diameter is about 0.3cm and is seeded to proliferated culture medium and carries out squamous subculture.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 1500lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is that to cultivate callus recruitment after 45 days under the condition of 75% be 93.6%.Described proliferated culture medium is: MS+5.0mg/L 6-BA+0.5mg/L IBA+28g/L sucrose+4.8g/L agar, pH is 5.7.
(4) differentiation is cultivated: the callus of step (3) being bred takes out and is cut into block that diameter is about 0.5cm and is seeded to differential medium and carries out differentiation cultivation.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 2500lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is that to cultivate differentiation rate after 45 days under the condition of 75% be 90.5%, and indefinite bud mean is 5.5.Described differential medium is: MS+5.0mg/L 6-BA+0.5mg/L IBA+24g/L sucrose+5.5g/L agar, pH is 5.7.
(5) culture of rootage: from base portion cut step (4) differentiation cultivate the healthy and strong test-tube plantlet that obtains be seeded in root media and carry out root induction.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is that to cultivate rooting rate after 30 days under the condition of 75% be 91.6%.Described root media is: 1/2MS+2.0mg/L ABT
5+ 0.5mg/L NAA+28g/L sucrose+5.3g/L agar, pH is 5.5.
(6) acclimatization and transplants: the bottle seedling possessing transplanting condition that root media obtains is carried out hardening, bottle seedling moves to normal temperature after lower 4 days, open bottle cap 2 days, then the medium being attached to shoot root and fastening is washed away, transplanting to filling by peat soil: cultivate in the container bag of the transplanting culture matrix that vermiculite=1:1 forms, transplanting and with 0.3% potassium permanganate solution, matrix spray sterilized and added a cover film for first 5 days, after 3 days, opening film, stir matrix, transplant water of matrix being drenched for first 1 day.By matrix compacting, and individual plant bagging need be carried out during transplanting, sooner or later spraying, ensure that growing environment is moist.Transplant and cut off plastic sack two jiaos after 7 days, completely de-bag after 14 days, transplanting survival rate after 30 days is 93.7%.
Claims (5)
1. a method for building up for polygonum capitatum rapid propagation in vitro system, is characterized in that comprising the following steps:
(1) explant sterilization: win polygonum capitatum and just launch blade; 60 ~ 90min is rinsed with the form of dripping in running water; aseptically; with 70% ~ 80% alcohol solution dipping 20s ~ 60s; aseptic water washing 4 ~ 6 times; again with 0.1% ~ 0.2% mercuric chloride solution sterilization 5 ~ 15min, then use aseptic water washing explant to non-foam, for subsequent use blot the moisture on surface with aseptic filter paper after;
(2) callus induction: the blade that step (1) disinfects is cut 0.5cm
2fritter; slight cutting 2 cuttves on master pulse; vacuum side of blade is inoculated into inducing culture down and carries out induction of callus; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 1000 ~ 2000lx; being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up induction situation afterwards in 30 days;
(3) Multiplying culture: the eugonic embryo callus that selecting step (2) Fiber differentiation obtains; cut block that diameter is about 0.3cm to be seeded to proliferated culture medium and to carry out squamous subculture; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 1000 ~ 2000lx; being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% after 45 days to add up proliferative conditions;
(4) differentiation is cultivated: the callus of step (3) being bred takes out and is cut into block that diameter is about 0.5cm and is seeded to differential medium and carries out differentiation cultivation; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 2000 ~ 3000lx; being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up differentiation situation afterwards in 45 days;
(5) culture of rootage: from base portion cut step (4) differentiation cultivate the healthy and strong test-tube plantlet that obtains be seeded in root media and carry out root induction; inoculation is placed on illumination every day 12 ~ 14 hours; intensity of illumination is 2000 ~ 3000lx; being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% ~ 80% to add up situation of taking root afterwards in 30 days;
(6) acclimatization and transplants: the bottle seedling possessing transplanting condition that root media obtains is carried out hardening, bottle seedling to move under normal temperature after 4 ~ 5 days, open bottle cap 2 ~ 3 days, then the medium being attached to shoot root and fastening is washed away, transplant to filling by peat soil: cultivate in the container bag of the transplanting culture matrix that vermiculite=1:1 forms, transplant and with 0.3% potassium permanganate solution, matrix spray is sterilized and added a cover film for first 5 days, film is opened after 3 days, stir matrix, transplant water of matrix being drenched for first 1 day, need by matrix compacting during transplanting, and carry out individual plant bagging, sooner or later spraying, guarantee growing environment is moist, transplant and cut off plastic sack two jiaos after 7 days, completely de-bag after 14 days, transplant and add up survival rate after 30 days.
2. the method for building up of a kind of polygonum capitatum rapid propagation in vitro system according to claim 1, it is characterized in that the inducing culture described in step (2) is: MS+0.1 ~ 1.0mg/L NAA+1.0 ~ 2.0mg/L 6-BA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
3. the method for building up of a kind of polygonum capitatum rapid propagation in vitro system according to claim 1, it is characterized in that the proliferated culture medium described in step (3) is: MS+2.0 ~ 5.0mg/L 6-BA+0.1 ~ 0.5mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
4. the method for building up of a kind of polygonum capitatum rapid propagation in vitro system according to claim 1, it is characterized in that the differential medium described in step (4) is: MS+4.0 ~ 8.0mg/L 6-BA+0.1 ~ 0.5mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
5. the method for building up of a kind of polygonum capitatum rapid propagation in vitro system according to claim 1, is characterized in that the root media described in step (5) is: 1/2MS+1.0 ~ 2.0mg/L ABT
5+ 0.1 ~ 0.5mg/L NAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
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| CN109392719A (en) * | 2018-11-25 | 2019-03-01 | 钟天路 | A kind of method for building up of madder regenerating system |
| WO2020127888A3 (en) * | 2018-12-21 | 2020-08-13 | Oriflame Cosmetics Ag | Composition and formulation for preventing and/or reducing urban darkening of the skin |
| CN116058281A (en) * | 2022-12-02 | 2023-05-05 | 甘肃农业大学 | Method for rapid propagation of polygonum mongolicum tissue |
| CN116439131A (en) * | 2023-04-03 | 2023-07-18 | 内蒙古库布其沙漠技术研究院 | Tissue culture seedling raising method for sand-fixing plant polygonum sapidum |
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| CN117426304A (en) * | 2023-12-20 | 2024-01-23 | 北京花乡花木集团有限公司 | Tissue culture method of polygonum giganteum |
| CN117426304B (en) * | 2023-12-20 | 2024-03-12 | 北京花乡花木集团有限公司 | Tissue culture method of polygonum giganteum |
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Application publication date: 20150610 |