CN104012411B - The tissue culture method of a kind of cowberry - Google Patents
The tissue culture method of a kind of cowberry Download PDFInfo
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- CN104012411B CN104012411B CN201410250457.8A CN201410250457A CN104012411B CN 104012411 B CN104012411 B CN 104012411B CN 201410250457 A CN201410250457 A CN 201410250457A CN 104012411 B CN104012411 B CN 104012411B
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Abstract
The present invention discloses the tissue culture method of a kind of cowberry, comprises the following steps: (1) selection; (2) bud Primary culture; (3) proliferation and subculture is cultivated; (4) root culture; (5) acclimatization and transplants. Cowberry of the present invention organize compounding method by improveing the composition of minimum medium and content, auxiliary rational cultural method simultaneously, make that the explant reproduction speed of cowberry is fast, the breeding cycle is short and breeding potential height, surviving rate height, not easily fungal infection venereal disease evil, improved variety degree height, so that finally cultivating the cowberry obtained there is good promotion and application value.
Description
Technical field
The present invention relates to the tissue culture method of a kind of cowberry, belong to phytology technical field.
Background technology
The mode being suitable for cowberry breeding has a variety of, e.g., and cottage propagation, root stock cottage propagation, division propagation and seed breeding etc. Above-mentioned propagation method has breeding cycle length, easy infection fungal disease, the reduction of improved variety degree, and the good characteristic that the use of seedling inferior causes cowberry intrinsic is lost day by day, and this constrains the development of these seeds to a certain extent and applies.
Summary of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide the probability of catching an illness that a kind of cultivation period is short, can effectively reduce cowberry, it is to increase the tissue culture method of the cowberry of its using value.
In order to realize foregoing invention object, the present invention is achieved through the following technical solutions:
The tissue culture method of a kind of cowberry, it is characterised in that, comprise the following steps:
(1) selection: in the 3-5 month, choose robust growth semi-lignified new slightly, after clear water rinses 3-5 time, use filter paper suck dry moisture, then, on Bechtop, by new slightly with after the mercuric chloride sterilizing 5-10min of 0.1%, by sterile water wash 3-5 time, more for subsequent use after filter paper suck dry moisture;
(2) bud Primary culture: from step (1) processed new slightly cut the stem section getting 1-2cm with a full bud, form explant, by in the base portion of described explant insertion bud Primary culture base, it is 25-30 DEG C in temperature, intensity of illumination 2000 2500lx, when light application time 12h/d, carry out the cultivation of 30-40d, form shoot;
(3) proliferation and subculture is cultivated: is seeded in proliferation and subculture substratum by the shoot described in step (2), is 25-30 DEG C in temperature, intensity of illumination 2000 2500lx, when light application time 12-16h/d, every 40-50d subculture once, obtains a large amount of shoot;
(4) root culture: the shoot after being bred in a large number by explant transfers to root induction in root media, carries out acclimatization and transplants after cultivating 40-50d;
(5) acclimatization and transplants: first shoot step (4) obtained adapts to 7-15d in culturing room, then at ambient temperature, clear water washing 3-5 time, remove the substratum in base portion, then the Miao Panzhong being transplanted in booth, keep relative air humidity more than 80%, after then taming hardening 30-35d, carry out Routine Management.
Further, described bud Primary culture base comprises: improvement MS+0.05��0.1mgL-1BA+1.0��2.0mgL-1ZT+1.0��2.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2.
Described proliferation and subculture is cultivated and is comprised improvement MS+1.0��2.0mgL-1BA+1.0��2.0mgL-1ZT+0.1��0.2mgL-1NAA+1.0��2.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2.
And described root media comprises 1/2 improvement MS+0.05��0.1mgL-1IBA+0��0.01mgL-1NAA+0.6��1.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2.
And, above-mentioned improvement MS comprises grand nutrition element, micronutrient element and organic reagent.
Wherein, the component of described grand nutrition element and the concentration of its correspondence is: saltpetre 190mg/L; Ammonium sulfate 165mg/L; Magnesium sulfate heptahydrate 350mg/L; Four water-calcium nitrate 680mg/L; Anhydrous potassium dihydrogenphosphate 100mg/L; Disodium ethylene diamine tetraacetate 73.4mg/L; Iron vitriol 18.5mg/L.
The component of described micronutrient element and the concentration of its correspondence is: four water manganous sulfate 15.6mg/L; Zinc sulfate 2.0mg/L; Boric acid 3.5mg/L; Potassiumiodide 0.5mg/L; Sodium orthomolybdate 0.3mg/L; Cobalt chloride 0.05mg/L, copper sulfate 0.02mg/L,
The component of described organic reagent and the concentration of its correspondence is: vitamin 0.1mg/L; Nicotinic acid 1.5mg/L; Pyridoxine hydrochloride 1.5mg/L; Inositol 100.0mg/L.
The invention has the beneficial effects as follows: cowberry of the present invention organize compounding method by improveing the composition of minimum medium and content, auxiliary rational cultural method simultaneously, make that the explant reproduction speed of cowberry is fast, the breeding cycle is short and breeding potential height, surviving rate height, not easily fungal infection venereal disease evil, improved variety degree height, so that finally cultivating the cowberry obtained there is good promotion and application value.
Embodiment
Below in conjunction with specific embodiment, the specific embodiment of the present invention is described in detail:
In the present embodiment, the improvement MS adopted in all embodiments includes grand nutrition element, micronutrient element and organic reagent,
Wherein, the component of described grand nutrition element and the concentration of its correspondence is: saltpetre 190mg/L; Ammonium sulfate 165mg/L; Magnesium sulfate heptahydrate 350mg/L; Four water-calcium nitrate 680mg/L; Anhydrous potassium dihydrogenphosphate 100mg/L; Disodium ethylene diamine tetraacetate 73.4mg/L; Iron vitriol 18.5mg/L.
The component of described micronutrient element and the concentration of its correspondence is: four water manganous sulfate 15.6mg/L; Zinc sulfate 2.0mg/L; Boric acid 3.5mg/L; Potassiumiodide 0.5mg/L; Sodium orthomolybdate 0.3mg/L; Cobalt chloride 0.05mg/L, copper sulfate 0.02mg/L,
The component of described organic reagent and the concentration of its correspondence is: vitamin 0.1mg/L; Nicotinic acid 1.5mg/L; Pyridoxine hydrochloride 1.5mg/L; Inositol 100.0mg/L.
Embodiment 1:
A tissue culture method for cowberry, comprises the following steps:
(1) selection: in March, choose robust growth semi-lignified new slightly, after clear water rinses 3 times, use filter paper suck dry moisture, then, on Bechtop, by new slightly with after the mercuric chloride sterilizing 5min of 0.1%, by sterile water wash 3 times, more for subsequent use after filter paper suck dry moisture;
(2) bud Primary culture: from step (1) processed new slightly cut the stem section getting 1-2cm with a full bud, form explant, by the base portion of described explant insertion bud Primary culture base, it is 25-30 DEG C in temperature, intensity of illumination 2000-2500lx, when light application time 12h/d, carry out the cultivation of 30d, form shoot, stem segment with axillary buds starts to sprout, stem point obviously extends, and about 50d bud point grows up to the new tip of about 3cm, and described bud Primary culture base comprises: improvement MS+0.05mgL- 1BA+1.0mgL-1ZT+1.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(3) proliferation and subculture is cultivated: be seeded in proliferation and subculture substratum by the shoot described in step (2), it is 25-30 DEG C in temperature, intensity of illumination 2000-2500lx, when light application time 12-16h/d, every 40d subculture once, obtaining a large amount of shoot, set up the regeneration system of cowberry shoot, described proliferation and subculture is cultivated and is comprised improvement MS+1.0mgL-1BA+1.0mgL-1ZT+0.1mgL-1NAA+1.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(4) root culture: the shoot after being bred in a large number by explant transfers to root induction in root media, after cultivating 40d, stem leaf development is normal, and root growth is healthy and strong, then carries out acclimatization and transplants, and described root media comprises 1/2 improvement MS+0.05mgL-1IBA+0.6gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(5) acclimatization and transplants: first shoot step (4) obtained adapts to 7d in culturing room, then at ambient temperature, clear water washs 3 times, remove the substratum in base portion, then the Miao Panzhong being transplanted in booth, keep relative air humidity more than 80%, after then taming hardening 30d, carry out Routine Management.
Embodiment 2:
A tissue culture method for cowberry, comprises the following steps:
(1) selection: in May, choose robust growth semi-lignified new slightly, after clear water rinses 5 times, use filter paper suck dry moisture, then, on Bechtop, by new slightly with after the mercuric chloride sterilizing 10min of 0.1%, by sterile water wash 5 times, more for subsequent use after filter paper suck dry moisture;
(2) bud Primary culture: from step (1) processed new slightly cut the stem section getting 1-2cm with a full bud, form explant, by in the base portion of described explant insertion bud Primary culture base, it is 25-30 DEG C in temperature, intensity of illumination 2000-2500lx, when light application time 12h/d, carries out the cultivation of 40d, forming shoot, described bud Primary culture base comprises: improvement MS+0.1mgL-1BA+2.0mgL-1ZT+2.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(3) proliferation and subculture is cultivated: be seeded in proliferation and subculture substratum by the shoot described in step (2), it is 25-30 DEG C in temperature, intensity of illumination 2000-2500lx, when light application time 12-16h/d, every 50d subculture once, obtaining a large amount of shoot, described proliferation and subculture is cultivated and is comprised improvement MS+2.0mgL-1BA+2.0mgL-1ZT+0.2mgL-1NAA+2.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(4) root culture: the shoot after being bred in a large number by explant transfers to root induction in root media, carries out acclimatization and transplants after cultivating 40-50d, and described root media comprises 1/2 improvement MS+0.1mgL-1IBA+0.01mgL- 1NAA+1.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(5) acclimatization and transplants: first shoot step (4) obtained adapts to 15d in culturing room, then at ambient temperature, clear water washs 5 times, remove the substratum in base portion, then the Miao Panzhong being transplanted in booth, keep relative air humidity more than 80%, after then taming hardening 35d, carry out Routine Management.
Embodiment 3:
A tissue culture method for cowberry, comprises the following steps:
(1) selection: in April, choose robust growth semi-lignified new slightly, after clear water rinses 4 times, use filter paper suck dry moisture, then, on Bechtop, by new slightly with after the mercuric chloride sterilizing 8min of 0.1%, by sterile water wash 4 times, more for subsequent use after filter paper suck dry moisture;
(2) bud Primary culture: from step (1) processed new slightly cut the stem section getting 1-2cm with a full bud, form explant, by in the base portion of described explant insertion bud Primary culture base, it is 25-30 DEG C in temperature, intensity of illumination 2000-2500lx, when light application time 12h/d, carries out the cultivation of 35d, forming shoot, described bud Primary culture base comprises: improvement MS+0.08mgL-1BA+1.5mgL-1ZT+1.5gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(3) proliferation and subculture is cultivated: be seeded in proliferation and subculture substratum by the shoot described in step (2), it is 25-30 DEG C in temperature, intensity of illumination 2000-2500lx, when light application time 12-16h/d, every 45d subculture once, obtaining a large amount of shoot, described proliferation and subculture is cultivated and is comprised improvement MS+1.5mgL-1BA+1.5mgL-1ZT+0.15mgL-1NAA+1.5gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(4) root culture: the shoot after being bred in a large number by explant transfers to root induction in root media, carries out acclimatization and transplants after cultivating 45d, and described root media comprises 1/2 improvement MS+0.08mgL-1IBA+0.005mgL-1NAA+0.8gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(5) acclimatization and transplants: first shoot step (4) obtained adapts to 12d in culturing room, then at ambient temperature, clear water washs 4 times, remove the substratum in base portion, then the Miao Panzhong being transplanted in booth, keep relative air humidity more than 80%, after then taming hardening 32d, carry out Routine Management.
The present invention not only can solve the problem that cowberry kind matter is degenerated, reach the object of good seed detoxification, purification and rejuvenation and breeding fast, and be the important channel of highly efficient regeneration seed source, still genetic transformation or the desirable acceptor material of selection by mutation seed selection new variety is carried out, so that finally cultivating the cowberry obtained there is good promotion and application value.
Below disclosing the present invention with better embodiment, so it is not intended to limiting the invention, and all employings are equal to replacement or the technical scheme that obtains of equivalent transformation mode, all drop within protection scope of the present invention.
Claims (1)
1. the tissue culture method of a cowberry, it is characterised in that, comprise the following steps:
(1) selection: in the 3-5 month, choose robust growth semi-lignified new slightly, after clear water rinses 3-5 time, use filter paper suck dry moisture, then, on Bechtop, by new slightly with after the mercuric chloride sterilizing 5-10min of 0.1%, by sterile water wash 3-5 time, more for subsequent use after filter paper suck dry moisture;
(2) bud Primary culture: from step (1) processed new slightly cut the stem section getting 1-2cm with a full bud, form explant, by in the base portion of described explant insertion bud Primary culture base, it is 25-30 DEG C in temperature, intensity of illumination 2000 2500lx, when light application time 12h/d, carry out the cultivation of 30-40d, form shoot; Described bud Primary culture base comprises: improvement MS+0.05��0.1mgL-1BA+1.0��2.0mgL-1ZT+1.0��2.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(3) proliferation and subculture is cultivated: is seeded in proliferation and subculture substratum by the shoot described in step (2), is 25-30 DEG C in temperature, intensity of illumination 2000 2500lx, when light application time 12-16h/d, every 40-50d subculture once, obtains a large amount of shoot; Described proliferation and subculture is cultivated and is comprised improvement MS+1.0��2.0mgL-1BA+1.0��2.0mgL-1ZT+0.1��0.2mgL-1NAA+1.0��2.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(4) root culture: the shoot after being bred in a large number by explant transfers to root induction in root media, carries out acclimatization and transplants after cultivating 40-50d; Described root media comprises 1/2 improvement MS+0.05��0.1mgL-1IBA+0��0.01mgL-1NAA+0.6��1.0gL-1Agar+10gL-1Sucrose, and pH value is 5.2;
(5) acclimatization and transplants: first shoot step (4) obtained adapts to 7-15d in culturing room, then at ambient temperature, clear water washing 3-5 time, remove the substratum in base portion, then the Miao Panzhong being transplanted in booth, keep relative air humidity more than 80%, after then taming hardening 30-35d, carry out Routine Management;
Described improvement MS comprises grand nutrition element, micronutrient element and organic reagent;
The component of described grand nutrition element and the concentration of its correspondence is: saltpetre 190mg/L; Ammonium sulfate 165mg/L; Magnesium sulfate heptahydrate 350mg/L; Four water-calcium nitrate 680mg/L; Anhydrous potassium dihydrogenphosphate 100mg/L; Disodium ethylene diamine tetraacetate 73.4mg/L; Iron vitriol 18.5mg/L;
The component of described micronutrient element and the concentration of its correspondence is: four water manganous sulfate 15.6mg/L; Zinc sulfate 2.0mg/L; Boric acid 3.5mg/L; Potassiumiodide 0.5mg/L; Sodium orthomolybdate 0.3mg/L; Cobalt chloride 0.05mg/L, copper sulfate 0.02mg/L;
The component of described organic reagent and the concentration of its correspondence is: vitamin 0.1mg/L; Nicotinic acid 1.5mg/L; Pyridoxine hydrochloride 1.5mg/L; Inositol 100.0mg/L.
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| CN104719157A (en) * | 2015-03-06 | 2015-06-24 | 朱远星 | Vaccinium australe tissue culture and rapid propagation method |
| CN106258434A (en) * | 2016-08-19 | 2017-01-04 | 江苏艺轩园林景观工程有限公司 | Blue berry booth Seedling inserts rear management method |
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| RU1824115C (en) * | 1991-02-12 | 1993-06-30 | Центральный Ботанический Сад Ан Бсср | Method of multiplication of viccinium vitis-idaea l in vitro |
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