CN102124952B - Method for fast propagating hydrilla varticillata through tissue culture - Google Patents
Method for fast propagating hydrilla varticillata through tissue culture Download PDFInfo
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Abstract
The invention relates to a method for fast propagating hydrilla varticillata through tissue culture, comprising the following steps of: (1) collecting stem segments of the hydrilla varticillata; (2) detoxifying: detoxifying the stem segments of the hydrilla varticillata; (3) performing induced culture: inoculating the stem segments of the hydrilla varticillata into an induction culture medium for the induced culture so as to obtain adventitious buds of the hydrilla varticillata; (4) performing propagation culture: inoculating the adventitious buds of the hydrilla varticillata onto a propagation culture medium for the propagation culture so as to further culture a large quantity of the adventitious buds; (5) performing rooting culture: inoculating the adventitious buds obtained through the propagation culture onto a rooting culture medium for the rooting culture; and (6) opening a culture bottle of the hydrilla varticillata with root hairs, which is subjected to the rooting culture, placing under an indoor natural condition for seedling domestication, taking out tissue culture seedlings inside the culture bottle, cleaning, and transplanting into a pond for culture. The method provided by the invention meets the demands of ecological water body restoration and purification, fishery culture, organic fertilizer composting, and the like and can be used for sustainably and massively supplying the tissue culture seedlings of the hydrilla varticillata all the year round.
Description
Technical field
The present invention relates to a kind of Plant Tissue Breeding method for quickly breeding, especially a kind of hydrilla verticillata quick breeding method for tissue culture belongs to biological technical field.
Background technology
Hydrilla verticillata (Hydrilla verticillata) is a kind of perennial heavy this plant of pasture and water of water huge legendary turtle section (Hydrocharitaceae); Generally be grown in China everywhere in the waters, its resistance to soiling is strong, and the eutrophication of water body is had stronger adaptive capacity; In contaminated serious water body, also can grow; Can play the effect of purifying water body, in addition, this plant is generally planted in the fish pond; For shrimps, fish, poultry etc. provide bait, extensive, this water plants of establishing in large scale is that agriculture composting fertilizer provides organic matter.
In the market the hydrilla verticillata seedling is in great demand, the research of relevant hydrilla verticillata seldom only concentrates on aspects such as purifying water body, raising fish and shrimp application.The general salvaging of required hydrilla verticillata comes from natural environment, and destroy hydrilla verticillata in the salvaging process easily and distribute, and original wild environment, and the hydrilla verticillata of natural distribution is limited, is difficult to satisfy the wilderness demand on the engineering.Quick, a large amount of propagation techniques to hydrilla verticillata very need.Report about hydrilla verticillata only has one piece at present, and the cultivation composition base of selecting for use is comparatively single, and the growth coefficient of acquisition is lower.
Summary of the invention
The objective of the invention is to overcome the deficiency that exists in the prior art; A kind of hydrilla verticillata quick breeding method for tissue culture is provided; Obtain a large amount of indoor aseptic hydrilla verticillata engineering seedlings; For restoration of the ecosystem purifying water body, fishery cultivating, fertilizer composting etc. provide needs, accomplish the whole year, sustainable, supply the hydrilla verticillata tissue cultivating seedling on a large scale.
According to technical scheme provided by the invention, said hydrilla verticillata quick breeding method for tissue culture may further comprise the steps:
(1) from open-air lake, gather hydrilla verticillata stem section spring, in running water, wash 2～4h after, remove impurity, put into sterile water and soak 20～30min, through aseptic water washing 3～5 times, subsequent use again;
(2) detoxification: the hydrilla verticillata stem section that will handle through step (1) is with the alcohol disinfecting 4～8s of concentration 70%～75%; After using aseptic water washing 4～6 times again; Putting into concentration and be 0.1%～0.15% mercuric chloride sterilizes behind 3～5min with aseptic water washing 4～6 times; Place on the aseptic paper and dry, cut into 1～2cm;
(3) inducing culture: the hydrilla verticillata stem section after will cutting is inoculated and was carried out inducing culture on the inducing culture 25～30 days, and inducing culture goes out the hydrilla verticillata indefinite bud; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200～1600Lux, and periodicity of illumination is 10～12h/d;
(4) enrichment culture: after the hydrilla verticillata indefinite bud is cut into into 1～2cm, is seeded in and carries out enrichment culture 25～35d on the proliferated culture medium, further turn out a large amount of indefinite buds; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200～1600Lux, and periodicity of illumination is 10～12h/d;
(5) culture of rootage: the indefinite bud that will behind enrichment culture, obtain cuts into 1～2cm, and has 2～3 little sections, is seeded in to carry out culture of rootage 30～35d on the root media; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200～1600Lux, and periodicity of illumination is 10～12h/d;
The blake bottle of the hydrilla verticillata that has coring that (6) will be after culture of rootage is opened; Be positioned over that 2～3d refines seedling under the indoor natural conditions; Refining seedling temperature is at 10～30 ℃, and indoor intensity of illumination is 1000～1200Lux, takes out the tissue cultivating seedling in the blake bottle; Clean, go in the pond under the natural conditions and culture.
H wherein is hour that d is the sky.
Said inducing culture is to add the 6-BA of 0.1～0.3mg/L or the NAA of 0.1～0.3mg/L in the 1/2MS medium; Said proliferated culture medium adds the 6-BA of 0.5～1.0mg/L and the NAA of 0.1～0.3mg/L for the MS medium; Said root media adds 6-BA, the NAA of 0.1～0.3mg/L and the IBA of 0.2～0.4mg/L of 0.2～0.4mg/L for the MS medium; The composition of said MS medium is made up of macroelement, trace element, organic principle, molysite, sucrose and agar.
The composition of said MS medium comprises:
Said macroelement is: (1) NH
4NO
3, 1650gm/L; (2) KNO
3, 1900mg/L; (3) CaCl2H
2O, 440mg/L; (4) MgSO
47H
2O, 370mg/L; (5) KH
2PO
4, 170mg/L;
Said trace element is: (1) MnSO
4H
2O, 22.3mg/L; (2) ZnSO
47H
2O, 8.6mg/L; (3) CoCl6H
2O, 0.025mg/L; (4) CuSO
45H
2O, 0.02mg/L; (5) H
3BO
3, 6.2mg/L; (6) Na
2MO
42H
2O, 0.25mg/L; (7) KI, 0.83mg/L;
Said organic principle is: (1) nicotinic acid, 0.5mg/L; (2) puridoxine hydrochloride, 0.5mg/L; (3) thiamine hydrochloride, 0.1mg/L; (4) inositol, 100mg/L; (5) glycine, 2mg/L;
Said molysite is: (1) FeSO
47H
2O, 28.7mg/L; (2) Na
2-EDTA, 37.3mg/L;
Said sucrose is 30000mg/L;
Said agar is 5800～6200mg/L.
The composition of said 1/2MS medium is the half the of MS medium.
Said inducing culture, proliferated culture medium and root media are that 120～121 ℃, pressure are the 18～20min that sterilizes under the condition of 0.103～0.105MPa in temperature.
The present invention utilizes plant tissue culture technique, obtains a large amount of indoor aseptic hydrilla verticillata engineering seedlings, for restoration of the ecosystem purifying water body, fishery cultivating, fertilizer composting etc. provide needs, accomplish the whole year, sustainable, supply the hydrilla verticillata tissue cultivating seedling on a large scale; Sterilization method when the present invention has improved the hydrilla verticillata initial culture, the pollution when having reduced initial culture improves just for the survival rate of inducing; Survival rate is reached more than 70%, and improved minimal medium, and in medium, add different hormone; Add certain density hormone and make that evoking adventive bud is more in incubation, when enrichment culture, each budlet can be induced 16 more than the bud; In culture of rootage, add the finite concentration of different hormones, make that the number of taking root increases more in the rooting process; Each blastogenesis root is more than 10, and root is sturdy.
Embodiment
Below in conjunction with specific embodiment the present invention is described further.
The inducing culture that the present invention uses is as adding the 6-BA of 0.1～0.3mg/L or the NAA of 0.1～0.3mg/L in the 1/2MS medium; Proliferated culture medium adds the 6-BA of 0.5～1.0mg/L and the NAA of 0.1～0.3mg/L for the MS medium; Root media adds 6-BA, the NAA of 0.1～0.3mg/L and the IBA of 0.2～0.4mg/L of 0.2～0.4mg/L for the MS medium; The composition of said MS medium is made up of macroelement, trace element, organic principle, molysite, sucrose and agar.
The composition of said MS medium comprises:
Said macroelement is: (1) NH
4NO
3, 1650gm/L; (2) KNO
3, 1900mg/L; (3) CaCl2H
2O, 440mg/L; (4) MgSO
47H
2O, 370mg/L; (5) KH
2PO
4, 170mg/L;
Said trace element is: (1) MnSO
4H
2O, 22.3mg/L; (2) ZnSO
47H
2O, 8.6mg/L; (3) CoCl6H
2O, 0.025mg/L; (4) CuSO
45H
2O, 0.02mg/L; (5) H
3BO
3, 6.2mg/L; (6) Na
2MO
42H
2O, 0.25mg/L; (7) KI, 0.83mg/L;
Said organic principle is: (1) nicotinic acid, 0.5mg/L; (2) puridoxine hydrochloride, 0.5mg/L; (3) thiamine hydrochloride, 0.1mg/L; (4) inositol, 100mg/L; (5) glycine, 2mg/L;
Said molysite is: (1) FeSO
47H
2O, 28.7mg/L; (2) Na
2-EDTA, 37.3mg/L;
Said sucrose is 30000mg/L;
Said agar is 5800～6200mg/L.
The composition of said 1/2MS medium is the half the of MS medium.
Said inducing culture, proliferated culture medium and root media are that 120～121 ℃, pressure are the 18～20min that sterilizes under the condition of 0.103～0.105MPa in temperature all.
Instance one: a kind of hydrilla verticillata quick breeding method for tissue culture may further comprise the steps:
(1) from open-air lake, gather hydrilla verticillata stem section spring, in running water, behind the flushing 2h, remove impurity, put into sterile water and soak 20min, through aseptic water washing 3 times, subsequent use again;
(2) detoxification: the hydrilla verticillata stem section that will handle through step (1) is with the alcohol disinfecting 4s of concentration 70%; After using aseptic water washing 4 times again; Put into concentration and be behind 0.1% the mercuric chloride sterilization 3min with aseptic water washing 4 times, place on the aseptic paper and dry, cut into 1cm;
(3) inducing culture: the hydrilla verticillata stem section after will cutting is inoculated and was carried out inducing culture on the inducing culture 25 days, and inducing culture goes out the hydrilla verticillata indefinite bud; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200Lux, and periodicity of illumination is 10h/d; Pollution rate is in 10%, and just generation induces inductivity to reach more than 80%;
(4) enrichment culture: after the hydrilla verticillata indefinite bud is cut into into 1cm, is seeded in and carries out enrichment culture 25d on the proliferated culture medium, further turn out a large amount of indefinite buds; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200Lux, and periodicity of illumination is 10h/d;
(5) culture of rootage: the indefinite bud that will behind enrichment culture, obtain cuts into 1cm, and has 2～3 little sections, is seeded in to carry out culture of rootage 30d on the root media; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200Lux, and periodicity of illumination is 10h/d;
(6) hydrilla verticillata stem section goes out a large amount of roots through culture of rootage; Take root at 10 above well developed root systems, and growing way is also relatively good, hydrilla verticillata length is about 5cm; The blake bottle of the hydrilla verticillata that has coring that will be after culture of rootage is opened; Be positioned over that 2d refines seedling under the indoor natural conditions, refining seedling temperature is at 10 ℃, and indoor intensity of illumination is 1000Lux; Take out the tissue cultivating seedling in the blake bottle, clean, go in the pond under the natural conditions and culture.
Instance two: a kind of hydrilla verticillata quick breeding method for tissue culture may further comprise the steps:
(1) from open-air lake, gather hydrilla verticillata stem section spring, in running water, behind the flushing 4h, remove impurity, put into sterile water and soak 30min, through aseptic water washing 5 times, subsequent use again;
(2) detoxification: the hydrilla verticillata stem section that will handle through step (1) is with the alcohol disinfecting 8s of concentration 75%; After using aseptic water washing 6 times again; Put into concentration and be behind 0.15% the mercuric chloride sterilization 5min with aseptic water washing 6 times, place on the aseptic paper and dry, cut into 2cm;
(3) inducing culture: the hydrilla verticillata stem section after will cutting is inoculated and was carried out inducing culture on the inducing culture 30 days, and inducing culture goes out the hydrilla verticillata indefinite bud; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1600Lux, and periodicity of illumination is 12h/d; Pollution rate is in 20%, and just generation induces inductivity to reach more than 90%;
(4) enrichment culture: after the hydrilla verticillata indefinite bud is cut into into 2cm, is seeded in and carries out enrichment culture 35d on the proliferated culture medium, further turn out a large amount of indefinite buds; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1600Lux, and periodicity of illumination is 12h/d;
(5) culture of rootage: the indefinite bud that will behind enrichment culture, obtain cuts into 2cm, and has 3 little sections, is seeded in to carry out culture of rootage 35d on the root media; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1600Lux, and periodicity of illumination is 12h/d;
(6) hydrilla verticillata stem section goes out a large amount of roots through culture of rootage; Take root at 10 above well developed root systems, and growing way is also relatively good, hydrilla verticillata length is about 5cm; The blake bottle of the hydrilla verticillata that has coring that will be after culture of rootage is opened; Be positioned over that 3d refines seedling under the indoor natural conditions, refining seedling temperature is at 30 ℃, and indoor intensity of illumination is 1200Lux; Take out the tissue cultivating seedling in the blake bottle, clean, go in the pond under the natural conditions and culture.
Instance three: a kind of hydrilla verticillata quick breeding method for tissue culture may further comprise the steps:
(1) from open-air lake, gather hydrilla verticillata stem section spring, in running water, behind the flushing 3h, remove impurity, put into sterile water and soak 25min, through aseptic water washing 4 times, subsequent use again;
(2) detoxification: the hydrilla verticillata stem section that will handle through step (1) is with the alcohol disinfecting 6s of concentration 75%; After using aseptic water washing 5 times again; Put into concentration and be behind 0.15% the mercuric chloride sterilization 4min with aseptic water washing 5 times, place on the aseptic paper and dry, cut into 1～2cm;
(3) inducing culture: the hydrilla verticillata stem section after will cutting is inoculated and was carried out inducing culture on the inducing culture 25 days, and inducing culture goes out the hydrilla verticillata indefinite bud; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1500Lux, and periodicity of illumination is 12h/d; Pollution rate is in 15%, and just generation induces inductivity to reach more than 85%;
(4) enrichment culture: after the hydrilla verticillata indefinite bud is cut into into 1～2cm, is seeded in and carries out enrichment culture 30d on the proliferated culture medium, further turn out a large amount of indefinite buds; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1400Lux, and periodicity of illumination is 12h/d;
(5) culture of rootage: the indefinite bud that will behind enrichment culture, obtain cuts into 1～2cm, and has 2～3 little sections, is seeded in to carry out culture of rootage 30d on the root media; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1400Lux, and periodicity of illumination is 12h/d;
(6) hydrilla verticillata stem section goes out a large amount of roots through culture of rootage; Take root at 10 above well developed root systems, and growing way is also relatively good, hydrilla verticillata length is about 5cm; The blake bottle of the hydrilla verticillata that has coring that will be after culture of rootage is opened; Be positioned over that 3d refines seedling under the indoor natural conditions, refining seedling temperature is at 20 ℃, and indoor intensity of illumination is 1200Lux; Take out the tissue cultivating seedling in the blake bottle, clean, go in the pond under the natural conditions and culture.
Claims (4)
1. a hydrilla verticillata quick breeding method for tissue culture is characterized in that, may further comprise the steps:
(1) from open-air lake, gather hydrilla verticillata stem section spring, in running water, wash 2 ~ 4h after, remove impurity, put into sterile water and soak 20 ~ 30min, through aseptic water washing 3 ~ 5 times, subsequent use again;
(2) detoxification: the hydrilla verticillata stem section that will handle through step (1) is with the alcohol disinfecting 4 ~ 8s of concentration 70% ~ 75%; After using aseptic water washing 4 ~ 6 times again; Putting into concentration and be 0.1% ~ 0.15% mercuric chloride sterilizes behind 3 ~ 5min with aseptic water washing 4 ~ 6 times; Place on the aseptic paper and dry, cut into 1 ~ 2cm;
(3) inducing culture: the hydrilla verticillata stem section after will cutting is inoculated and was carried out inducing culture on the inducing culture 25 ~ 30 days, and inducing culture goes out the hydrilla verticillata indefinite bud; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200 ~ 1600Lux, and periodicity of illumination is 10 ~ 12h/d;
(4) enrichment culture: after the hydrilla verticillata indefinite bud is cut into 1 ~ 2cm, is seeded in and carries out enrichment culture 25 ~ 35d on the proliferated culture medium, further turn out a large amount of indefinite buds; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200 ~ 1600Lux, and periodicity of illumination is 10 ~ 12h/d;
(5) culture of rootage: the indefinite bud that will behind enrichment culture, obtain cuts into 1 ~ 2cm, and has 2 ~ 3 little sections, is seeded in to carry out culture of rootage 30 ~ 35d on the root media; Culturing room's temperature is 25 ± 2 ℃, and intensity of illumination is 1200 ~ 1600Lux, and periodicity of illumination is 10 ~ 12h/d;
The blake bottle of the hydrilla verticillata that has coring that (6) will be after culture of rootage is opened; Be positioned over that 2 ~ 3d refines seedling under the indoor natural conditions; Refining seedling temperature is at 10 ~ 30 ℃, and indoor intensity of illumination is 1000 ~ 1200 Lux, takes out the tissue cultivating seedling in the blake bottle; Clean, go in the pond under the natural conditions and culture;
Said inducing culture is to add the 6-BA of 0.1 ~ 0.3mg/L or the NAA of 0.1 ~ 0.3mg/L in the 1/2MS medium; Said proliferated culture medium adds the 6-BA of 0.5 ~ 1.0mg/L and the NAA of 0.1 ~ 0.3mg/L for the MS medium; Said root media adds 6-BA, the NAA of 0.1 ~ 0.3mg/L and the IBA of 0.2 ~ 0.4mg/L of 0.2 ~ 0.4mg/L for the MS medium; The composition of said MS medium is made up of macroelement, trace element, organic principle, molysite, sucrose and agar.
2. hydrilla verticillata quick breeding method for tissue culture as claimed in claim 1 is characterized in that: the composition of said MS medium comprises:
Said macroelement is: (1) NH
4NO
3, 1650mg/L; (2) KNO
3, 1900mg/L; (3) CaCl2H
2O, 440mg/L; (4) MgSO
47H
2O, 370mg/L and (5) KH
2PO
4, 170mg/L;
Said trace element is: (1) MnSO
4H
2O, 22.3mg/L; (2) ZnSO
47H
2O, 8.6mg/L; (3) CoCl6H
2O, 0.025mg/L; (4) CuSO
45H
2O, 0.02mg/L; (5) H
3BO
3, 6.2mg/L; (6) Na
2MO
42H
2O, 0. 25mg/L and (7) KI, 0.83mg/L;
Said organic principle is: (1) nicotinic acid, 0. 5mg/L; (2) puridoxine hydrochloride, 0.5mg/L; (3) thiamine hydrochloride, 0.1mg/L; (4) inositol, 100mg/L and (5) glycine, 2mg/L;
Said molysite is: (1) FeSO
47H
2O, 28.7mg/L and (2) Na
2-EDTA, 37.3mg/L;
Said sucrose is 30000mg/L;
Said agar is 5800 ~ 6200mg/L.
3. hydrilla verticillata quick breeding method for tissue culture as claimed in claim 1 is characterized in that: the composition of said 1/2MS medium is the half the of MS medium.
4. hydrilla verticillata quick breeding method for tissue culture as claimed in claim 1 is characterized in that: said inducing culture, proliferated culture medium and root media are that 120 ~ 121 ℃, pressure are the 18 ~ 20min that sterilizes under the condition of 0.103 ~ 0.105MPa in temperature.
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| CN102511389A (en) * | 2011-11-28 | 2012-06-27 | 宁波大学 | Induction method of grateloupia filicina filament |
| CN105393908B (en) * | 2015-11-16 | 2018-07-31 | 水生藻安生物科技（武汉）有限公司 | The method and modular combination of the quick modularization plantation of submerged plant tissue-cultured seedling |
| CN108901849B (en) * | 2018-07-26 | 2022-03-08 | 水生藻安生物科技（武汉）有限公司 | Tissue culture method of aseptic seedlings of hydrilla verticillata |
| CN109220769B (en) * | 2018-11-15 | 2020-09-22 | 中国水产科学研究院黄海水产研究所 | Method for improving commodity yield and product quality of caulerpa lentillifera |
| CN111543327A (en) * | 2020-06-11 | 2020-08-18 | 南大（常熟）研究院有限公司 | Production method of detoxified seedlings suitable for rapid propagation of submerged plants |
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| 蒋金辉等.工具种轮叶黑藻的组织培养与快速繁殖.《湖泊科学》.2008,第20卷(第2期),第215-220页. * |
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