CN104082142B - A kind of happiness carrys out blueberry tissue and cultivates proliferated culture medium and preparation method thereof - Google Patents
A kind of happiness carrys out blueberry tissue and cultivates proliferated culture medium and preparation method thereof Download PDFInfo
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- CN104082142B CN104082142B CN201410309154.9A CN201410309154A CN104082142B CN 104082142 B CN104082142 B CN 104082142B CN 201410309154 A CN201410309154 A CN 201410309154A CN 104082142 B CN104082142 B CN 104082142B
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Abstract
The invention discloses the proliferated culture medium that a kind of happiness carrys out blueberry tissue cultivation: improvement WPM substratum+NAA 0.5 mg/L+GA 0.2 mg/L+ZT 2-3mg/L+ sucrose 30g/L+ agar 7 g/L, pH is 5.2; Containing macroelement in often liter of improvement WPM substratum: 893mg saltpetre, 119 mg ammonium sulfate, 370mg magnesium sulfate, 278mg calcium nitrate tetrahydrate, 170mg potassium primary phosphate; Molysite: 74.6mg disodium ethylene diamine tetraacetate, 55.6mg ferrous sulfate; Trace element: 22.3mg manganous sulfate, 8.6mg zinc sulfate, 0.025mg copper sulfate, 0.25mg Sodium orthomolybdate, 6.2mg boric acid; 0.415mg potassiumiodide; Organism: 0.5mg nicotinic acid, 0.5mg vitamin, 0.5mg hydrochloric acid adjoin alcohol of trembling, l00mg inositol, 2mg glycine, adopts this substratum 30d proliferation times to reach more than 12 times.
Description
Technical field
The present invention relates to plant tissue culture fast breeding technique field, mainly a kind of suitable happiness carrys out the substratum of blueberry tissue culture seedling proliferation.
Background technology
Cowberry is Ericaceae (Ericaceae) Vaccinium (Vaccinium) shrub small berries fruit tree.Its fruit is blue or red, sour-sweet moderate, and fruit is low-yield and containing multiple anti-oxidant composition, have high nutritive value and medical care effect.Biologically active substance in cranberry has the function of improving eyesight, anti-eye strain, vision enhancing.At present, R&D institution in all parts of the country and enterprise have carried out the research of the malicious tissue culture fast-propagation aspect of a large amount of indigo plant, can promote the main nursery means that new quality product kind becomes blueberry seedling rapidly.Because blueberry kind is more, the proliferated culture medium that is suitable for also be not quite similar, what a lot of factorial praluction adopted at present is all unified culture medium prescription, specific aim is not strong, cause the fast numerous speed of different varieties different, greatly have impact on the factorial praluction of blueberry, thus add production cost.Happiness comes to be the kind of cowberry, and wide adaptability is the main breed of northern area, usually adopts the method for tissue culture to carry out Fast-propagation.But owing to adopting existing conventional group culturation rapid propagating technology, it is low that happiness carrys out proliferation times, and the speed of growth is slow, have impact on the fast development that happiness carrys out cowberry, optimization happiness carrys out cowberry culture medium prescription and seems of crucial importance.
Summary of the invention
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide and a kind ofly can improve happiness and carry out the proliferation times of cowberry, can proliferating cycle be shortened again, and the happiness of propagation seedling robust growth carrying out the culture medium prescription of cowberry propagation.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of happiness carrys out the proliferated culture medium that blueberry tissue is cultivated, and comprises following component: improvement WPM substratum+NAA (naphthylacetic acid) 0.5 mg/L+GA (Plant hormones regulators,gibberellins) 0.2 mg/L+ zeatin (ZT) 2-3mgL
-1+ sucrose 30 gL
-1+ agar 7 gL
-1, pH is 5.2.
Above-mentioned improvement WPM minimum medium comprises following component:
Macroelement: 893mgL
-1saltpetre, 119 mgL
-1ammonium sulfate, 370 mgL
-1magnesium sulfate, 96 mgL
-1calcium nitrate tetrahydrate, 170mg potassium primary phosphate;
Trace element: 22.3 mgL
-1manganous sulfate, 8.6 mgL
-1zinc sulfate, 0.025 mgL
-1copper sulfate, 0.25mgL
-1sodium orthomolybdate, 6.2 mgL
-1boric acid;
Molysite: 74.6mgL
-1disodium ethylene diamine tetraacetate, 55.6 mgL
-1ferrous sulfate;
Organism: 0.5 mgL
-1nicotinic acid, 0.5 mgL
-1vitamin, 0.5 mgL
-1hydrochloric acid adjoins alcohol of trembling, l00 mgL
-1inositol, 2 mgL
-1glycine.
Above-mentioned happiness carrys out the preparation method of the proliferated culture medium that blueberry tissue is cultivated, and the method comprises the steps:
(1) preparation improvement WPM substratum:
The preparation of (11) 50 times of macroelement mother liquors: take 44.65g saltpetre, 5.95g ammonium sulfate, 18.5g magnesium sulfate respectively, be then settled to 1000ml with after distilled water dissolving, be made into mother liquor I;
Take 13.9g calcium nitrate tetrahydrate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor II;
Take 8.5g potassium primary phosphate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor III;
Prepare often liter of proliferated culture medium during use and get mother liquor I, mother liquor II, each 20ml of mother liquor III;
The preparation of the micro-mother liquor of (12) 250 times: take 5.575g manganous sulfate, 2.15g zinc sulfate, 0.00625g copper sulfate, 0.0625g Sodium orthomolybdate, 1.55g boric acid, 0.10375 g potassiumiodide, is settled to 1000ml after dissolving with distilled water, be made into mother liquor IV, prepare often liter of proliferated culture medium during use and get 4ml mother liquor IV;
The preparation of the EDTA-mother liquid of iron salt of (13) 100 times: take disodium ethylene diamine tetraacetate, 7.46g; Ferrous sulfate 5.56g, dissolves respectively, and mixing is settled to 1000mL, is made into mother liquor V, prepares often liter of proliferated culture medium and get 10ml mother liquor V during use;
The preparation of the organic composition mother liquor of (14) 100 times: take 0.2g glycine, 0.005g vitamin, 0.005g pyridoxine hydrochloride, 0.005g nicotinic acid, 10g inositol, after dissolving with distilled water, mixing is settled to 1000ml, is made into mother liquor VI, prepares often liter of proliferated culture medium and get 10ml mother liquor VI during use;
(2) preparation of hormone mother liquor: take 50mg NAA, first dissolves with 5ml volumetric concentration 95% alcohol, then adds water and be settled to 100ml, be made into 0.5mgmL
-1nAA mother liquor, during use prepare often liter of proliferated culture medium get 1mlNAA mother liquor; Take 5mg GA, first dissolve with 5ml volumetric concentration 95% alcohol, then add water and be settled to 100ml, be made into 0.05mgmL
-1gA mother liquor, during use prepare often liter of proliferated culture medium get 4mlGA mother liquor; Take 50mg zeatin, first use a small amount of 0.1molL
-1naOH dissolves, and then adds water and is settled to 100ml, be made into 0.5mgmL
-1zeatin mother liquor, during use prepare often liter of proliferated culture medium get 4-6ml zeatin mother liquor;
(3) after adding by the consumption preparing often liter of proliferated culture medium in above-mentioned steps (11) to step (14), then add agar 7g, sucrose 30g, NAA mother liquor 1ml, GA mother liquor 4ml, is settled to 1L after heating for dissolving, uses 0.lmolL
-1hydrochloric acid adjusts pH to 5.2, sterilizing; When substratum temperature is down to 60-70 DEG C, inoculation platform adopts sterilization by filtration to add the zeatin mother liquor of preparation in step (2), and often liter of proliferated culture medium adds 4ml-6ml zeatin mother liquor, then packing substratum on inoculation platform.
The using method of proliferated culture medium of the present invention is: aseptic seedling be seeded on the proliferated culture medium of the sterilizing prepared on super clean bench, cultivate under the condition of intensity of illumination 2000Lux, culture condition is: temperature 25-27 DEG C, and light application time is 14 hours.
Beneficial effect: compared with prior art, advantage of the present invention is:
1. form substratum comparatively conventional medium nitrate nitrogen content increase;
2. adopt this substratum to breed, proliferating cycle is short, and proliferation times is high;
3. breed seedling healthy and strong.
embodiment:
Below in conjunction with specific embodiment, concrete introduction is done to the present invention.
embodiment 1:
Happiness carrys out the proliferated culture medium that blueberry tissue is cultivated, and comprises following component: improvement WPM substratum+NAA 0.5 mg/L+GA 0.2 mg/L+ZT 2mgL
-1+ sucrose 30 gL
-1+ agar 7 gL
-1, all the other are distilled water, and pH is 5.2;
Wherein improve WPM substratum and comprise following component:
Macroelement: 893mgL
-1saltpetre, 119 mgL
-1ammonium sulfate, 370 mgL
-1magnesium sulfate, 278 mgL
-1nitrocalcite, 170mgL
-1potassium primary phosphate;
Trace element: 22.3 mgL
-1manganous sulfate, 8.6 mgL
-1zinc sulfate, 0.025 mgL
-1copper sulfate, 0.25mgL
-1sodium orthomolybdate, 6.2 mgL
-1boric acid; 0.415 mgL
-1potassiumiodide;
Molysite: 74.6mgL
-1disodium ethylene diamine tetraacetate, 55.6 mgL
-1ferrous sulfate;
Organism: 0.5 mgL
-1nicotinic acid, 0.5 mgL
-1vitamin, 0.5 mgL
-1hydrochloric acid adjoins alcohol of trembling, l00 mgL
-1inositol, 2 mgL
-1glycine.
Above-mentioned proliferated culture medium preparation method is as follows:
(1) preparation improvement WPM substratum:
The preparation of (11) 50 times of macroelement mother liquors: take 44.65g saltpetre, 5.95g ammonium sulfate, 18.5g magnesium sulfate respectively, be then settled to 1000ml with after distilled water dissolving, be made into mother liquor I;
Take 13.9g calcium nitrate tetrahydrate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor II;
Take 8.5g potassium primary phosphate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor III;
Prepare often liter of proliferated culture medium during use and get mother liquor I, mother liquor II, each 20ml of mother liquor III;
The preparation of the micro-mother liquor of (12) 250 times: take 5.575g manganous sulfate, 2.15g zinc sulfate, 0.00625g copper sulfate, 0.0625g Sodium orthomolybdate, 1.55g boric acid, 0.10375 g potassiumiodide, is settled to 1000ml after dissolving with distilled water, be made into mother liquor IV, prepare often liter of proliferated culture medium during use and get 4ml mother liquor IV;
The preparation of the EDTA-mother liquid of iron salt of (13) 100 times: take disodium ethylene diamine tetraacetate, 7.46g; Ferrous sulfate 5.56g, dissolves respectively, and mixing is settled to 1000mL, is made into mother liquor V, prepares often liter of proliferated culture medium and get 10ml mother liquor V during use;
The preparation of the organic composition mother liquor of (14) 100 times: take 0.2g glycine, 0.005g vitamin, 0.005g pyridoxine hydrochloride, 0.005g nicotinic acid, 10g inositol, after dissolving with distilled water, mixing is settled to 1000ml, is made into mother liquor VI, prepares often liter of proliferated culture medium and get 10ml mother liquor VI during use;
(2) preparation of hormone mother liquor: take 50mg NAA, first dissolves with 5ml volumetric concentration 95% alcohol, then adds water and be settled to 100ml, be made into 0.5mgmL
-1nAA mother liquor, during use prepare often liter of proliferated culture medium get 1mlNAA mother liquor; Take 5mg GA, first dissolve with 5ml volumetric concentration 95% alcohol, then add water and be settled to 100ml, be made into 0.05mgmL
-1gA mother liquor, during use prepare often liter of proliferated culture medium get 4mlGA mother liquor; Take 50mg zeatin, first use a small amount of 0.1molL
-1naOH dissolves, and then adds water and is settled to 100ml, be made into 0.5mgmL
-1zeatin mother liquor, during use prepare often liter of proliferated culture medium get 4ml zeatin mother liquor;
(3) after adding by the consumption preparing often liter of proliferated culture medium in above-mentioned steps (11) to step (14), then add agar 7g, sucrose 30g, NAA mother liquor 1ml, GA mother liquor 4ml, is settled to 1L after heating for dissolving, uses 0.lmolL
-1hydrochloric acid adjusts pH to 5.2, sterilizing; When substratum temperature is down to 60-70 DEG C, inoculation platform adopts sterilization by filtration to add the zeatin mother liquor of preparation in step (2), and often liter of proliferated culture medium adds 4ml zeatin mother liquor, then packing substratum on inoculation platform.
The using method of proliferated culture medium of the present invention is: aseptic seedling be seeded on the proliferated culture medium of the sterilizing prepared on super clean bench, cultivate under the condition of intensity of illumination 2000Lux, culture condition is: temperature 25-27 DEG C, and light application time is 14 hours.
Within 30 days, statistics discovery plant strain growth is healthy and strong afterwards, and blade is in green, and plant proliferation times reaches 12.8.
embodiment 2:
Happiness carrys out the proliferated culture medium that blueberry tissue is cultivated, and comprises following component: improvement WPM substratum+NAA 0.5 mg/L+GA 0.2 mg/L+ZT 3mgL
-1+ sucrose 30 gL
-1+ agar 7 gL
-1, all the other are distilled water, and pH is 5.2;
Wherein improve WPM substratum and comprise following component:
Macroelement: 893mgL
-1saltpetre, 119 mgL
-1ammonium sulfate, 370 mgL
-1magnesium sulfate, 278 mgL
-1nitrocalcite, 170mgL
-1potassium primary phosphate;
Trace element: 22.3 mgL
-1manganous sulfate, 8.6 mgL
-1zinc sulfate, 0.025 mgL
-1copper sulfate, 0.25mgL
-1sodium orthomolybdate, 6.2 mgL
-1boric acid; 0.415 mgL
-1potassiumiodide;
Molysite: 74.6mgL
-1disodium ethylene diamine tetraacetate, 55.6 mgL
-1ferrous sulfate;
Organism: 0.5 mgL
-1nicotinic acid, 0.5 mgL
-1vitamin, 0.5 mgL
-1hydrochloric acid adjoins alcohol of trembling, l00 mgL
-1inositol, 2 mgL
-1glycine.
Proliferated culture medium preparation method is as follows:
(1) preparation improvement WPM substratum:
The preparation of (11) 50 times of macroelement mother liquors: take 44.65g saltpetre, 5.95g ammonium sulfate, 18.5g magnesium sulfate respectively, be then settled to 1000ml with after distilled water dissolving, be made into mother liquor I;
Take 13.9g calcium nitrate tetrahydrate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor II;
Take 8.5g potassium primary phosphate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor III;
Prepare often liter of proliferated culture medium during use and get mother liquor I, mother liquor II, each 20ml of mother liquor III;
The preparation of the micro-mother liquor of (12) 250 times: take 5.575g manganous sulfate, 2.15g zinc sulfate, 0.00625g copper sulfate, 0.0625g Sodium orthomolybdate, 1.55g boric acid, 0.10375 g potassiumiodide, is settled to 1000ml after dissolving with distilled water, be made into mother liquor IV, prepare often liter of proliferated culture medium during use and get 4ml mother liquor IV;
The preparation of the EDTA-mother liquid of iron salt of (13) 100 times: take disodium ethylene diamine tetraacetate, 7.46g; Ferrous sulfate 5.56g, dissolves respectively, and mixing is settled to 1000mL, is made into mother liquor V, prepares often liter of proliferated culture medium and get 10ml mother liquor V during use;
The preparation of the organic composition mother liquor of (14) 100 times: take 0.2g glycine, 0.005g vitamin, 0.005g pyridoxine hydrochloride, 0.005g nicotinic acid, 10g inositol, after dissolving with distilled water, mixing is settled to 1000ml, is made into mother liquor VI, prepares often liter of proliferated culture medium and get 10ml mother liquor VI during use;
(2) preparation of hormone mother liquor: take 50mg NAA, first dissolves with 5ml volumetric concentration 95% alcohol, then adds water and be settled to 100ml, be made into 0.5mgmL
-1nAA mother liquor, during use prepare often liter of proliferated culture medium get 1mlNAA mother liquor; Take 5mg GA, first dissolve with 5ml volumetric concentration 95% alcohol, then add water and be settled to 100ml, be made into 0.05mgmL
-1gA mother liquor, during use prepare often liter of proliferated culture medium get 4mlGA mother liquor; Take 50mg zeatin, first use a small amount of 0.1molL
-1naOH dissolves, and then adds water and is settled to 100ml, be made into 0.5mgmL
-1zeatin mother liquor, during use prepare often liter of proliferated culture medium get 4ml zeatin mother liquor;
(3) after adding by the consumption preparing often liter of proliferated culture medium in above-mentioned steps (11) to step (14), then add agar 7g, sucrose 30g, NAA mother liquor 1ml, GA mother liquor 4ml, is settled to 1L after heating for dissolving, uses 0.lmolL
-1hydrochloric acid adjusts pH to 5.2, sterilizing; When substratum temperature is down to 60-70 DEG C, inoculation platform adopts sterilization by filtration to add the zeatin mother liquor of preparation in step (2), and often liter of proliferated culture medium adds 6ml zeatin mother liquor, then packing substratum on inoculation platform.
Aseptic seedling be seeded on super clean bench on the proliferated culture medium of the sterilizing prepared, cultivate under the condition of intensity of illumination 2000Lux, culture condition is: temperature 25-27 DEG C, and light application time is 14 hours.
Within 30 days, statistics discovery plant strain growth is healthy and strong afterwards, and blade is in green, and plant proliferation times reaches 13.2.
reference examples 1:
Happiness is carried out cowberry to be inoculated in the proliferated culture medium of traditional WPM, and its medium component and content comprise:
Macroelement: 400mgL
-1saltpetre, 900mgL
-1potassium sulfate, 370mgL
-1magnesium sulfate, 96 mgL
-1cALCIUM CHLORIDE DIHYDRATE, 556 mgL
-1nitrocalcite, 170 mgL
-1potassium primary phosphate;
Trace element: 22.3 mgL
-1manganous sulfate, 8.6 mgL
-1zinc sulfate, 0.025 mgL
-1copper sulfate, 0.25 mgL
-1sodium orthomolybdate, 6.2mgL
-1boric acid;
Molysite: 37.3 mgL
-1disodium ethylene diamine tetraacetate, 27.8 mgL
-1ferrous sulfate;
Organism: 0.5 mgL
-1nicotinic acid, 0.5 mgL
-1vitamin, 0.5 mgL
-1hydrochloric acid adjoins alcohol of trembling, l00 mgL
-1inositol, 2 mgL
-1glycine;
Sucrose 30 gL
-1, agar 7 gL
-1
Zeatin 2mgL
-1;
All the other are distilled water, and pH is 5.2;
Preparation method is as follows:
(1) preparation improvement WPM substratum:
The preparation of (11) 50 times of macroelement mother liquors: take 20g saltpetre, 45g potassium sulfate, 18.5g magnesium sulfate respectively, be then settled to 1000ml with after distilled water dissolving, be made into mother liquor I;
Take 4.8g CALCIUM CHLORIDE DIHYDRATE, 27.8g calcium nitrate tetrahydrate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor II;
Take 8.5g potassium primary phosphate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor III;
Prepare often liter of proliferated culture medium during use and get mother liquor I, mother liquor II, each 20ml of mother liquor III;
The preparation of the micro-mother liquor of (12) 250 times: take 5.575g manganous sulfate, 2.15g zinc sulfate, 0.00625g copper sulfate, 0.0625g Sodium orthomolybdate, 1.55g boric acid, is settled to 1000ml after dissolving with distilled water, be made into mother liquor IV, prepare often liter of proliferated culture medium during use and get 4ml;
The preparation of the EDTA-mother liquid of iron salt of (13) 100 times: take 3.73g disodium ethylene diamine tetraacetate, 2.78g ferrous sulfate, dissolves respectively, mixing is settled to 1000mL, is made into mother liquor V, prepares often liter of proliferated culture medium and get 10ml during use;
The preparation of the organic composition mother liquor of (14) 100 times: take 0.2g glycine, 0.005g vitamin, 0.005g pyridoxine hydrochloride, 0.005g nicotinic acid, after 10g inositol dissolves, mixing is settled to 1000ml, is made into mother liquor VI, prepares often liter of proliferated culture medium and get 10ml during use;
(15) preparation of hormone mother liquor: take 50mg zeatin, first use 5ml0.1molL
-1naOH dissolves, and then adds water and is settled to 100ml, be made into 0.5mgmL
-1mother liquor, during use prepare often liter of proliferated culture medium get 4ml;
(2) after adding by the consumption preparing often liter of proliferated culture medium in above-mentioned steps (11) to step (15), then add agar 7g, sucrose 30g, is settled to 1L after heating for dissolving, uses 0.lmolL
-1hydrochloric acid adjusts pH to 5.2, sterilizing; When substratum temperature is down to 60-70 DEG C, inoculation platform adopts sterilization by filtration to add zeatin mother liquor, and zeatin mother liquid concentration is 0.5 mgmL
-1, often liter of substratum adds 4ml zeatin mother liquor, then packing substratum on inoculation platform.
Using method: aseptic seedling be seeded on the proliferated culture medium of the sterilizing prepared on super clean bench, cultivate under the condition of intensity of illumination 2000Lux, culture condition is: temperature 25-27 DEG C, every day, light application time was 14 hours.
Within 30 days, statistics discovery plant strain growth is slow afterwards, and blade is partially yellow, and plant proliferation times is 4.4, is starkly lower than proliferation times of the present invention.
The present invention is illustrated according to above-described embodiment and should be appreciated that above-described embodiment does not limit the present invention in any form, 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 (3)
1. happiness carrys out the proliferated culture medium that blueberry tissue is cultivated, and it is characterized in that, comprises following component: improvement WPM substratum+NAA 0.5 mg/L+GA 0.2 mg/L+ZT 2-3mgL
-1+ sucrose 30 gL
-1+ agar 7 gL
-1, pH is 5.2;
Described improvement WPM substratum comprises following component:
Macroelement: 893mgL
-1saltpetre, 119 mgL
-1ammonium sulfate, 370 mgL
-1magnesium sulfate, 278 mgL
-1nitrocalcite, 170mgL
-1potassium primary phosphate;
Trace element: 22.3 mgL
-1manganous sulfate, 8.6 mgL
-1zinc sulfate, 0.025 mgL
-1copper sulfate, 0.25mgL
-1sodium orthomolybdate, 6.2 mgL
-1boric acid; 0.415 mgL
-1potassiumiodide;
Molysite: 74.6mgL
-1disodium ethylene diamine tetraacetate, 55.6 mgL
-1ferrous sulfate;
Organism: 0.5 mgL
-1nicotinic acid, 0.5 mgL
-1vitamin, 0.5 mgL
-1hydrochloric acid adjoins alcohol of trembling, l00 mgL
-1inositol, 2 mgL
-1glycine.
2. happiness according to claim 1 carrys out the preparation method of the proliferated culture medium that blueberry tissue is cultivated, and it is characterized in that: the method comprises the steps:
(1) preparation improvement WPM substratum:
The preparation of (11) 50 times of macroelement mother liquors: take 44.65g saltpetre, 5.95g ammonium sulfate, 18.5g magnesium sulfate respectively, be then settled to 1000ml with after distilled water dissolving, be made into mother liquor I;
Take 13.9g calcium nitrate tetrahydrate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor II;
Take 8.5g potassium primary phosphate, be settled to 1000ml after dissolving with distilled water, be made into mother liquor III;
Prepare often liter of proliferated culture medium during use and get mother liquor I, mother liquor II, each 20ml of mother liquor III;
The preparation of the micro-mother liquor of (12) 250 times: take 5.575g manganous sulfate, 2.15g zinc sulfate, 0.00625g copper sulfate, 0.0625g Sodium orthomolybdate, 1.55g boric acid, 0.10375 g potassiumiodide, is settled to 1000ml after dissolving with distilled water, be made into mother liquor IV, prepare often liter of proliferated culture medium during use and get 4ml mother liquor IV;
The preparation of the EDTA-mother liquid of iron salt of (13) 100 times: take disodium ethylene diamine tetraacetate, 7.46g; Ferrous sulfate 5.56g, dissolves respectively, and mixing is settled to 1000mL, is made into mother liquor V, prepares often liter of proliferated culture medium and get 10ml mother liquor V during use;
The preparation of the organic composition mother liquor of (14) 100 times: take 0.2g glycine, 0.005g vitamin, 0.005g pyridoxine hydrochloride, 0.005g nicotinic acid, 10g inositol, after dissolving with distilled water, mixing is settled to 1000ml, is made into mother liquor VI, prepares often liter of proliferated culture medium and get 10ml mother liquor VI during use;
(2) preparation of hormone mother liquor: take 50mg NAA, first dissolves with 5ml volumetric concentration 95% alcohol, then adds water and be settled to 100ml, be made into 0.5mgmL
-1nAA mother liquor, during use prepare often liter of proliferated culture medium get 1mlNAA mother liquor; Take 5mg GA, first dissolve with 5ml volumetric concentration 95% alcohol, then add water and be settled to 100ml, be made into 0.05mgmL
-1gA mother liquor, during use prepare often liter of proliferated culture medium get 4mlGA mother liquor; Take 50mg zeatin, first use a small amount of 0.1molL
-1naOH dissolves, and then adds water and is settled to 100ml, be made into 0.5mgmL
-1zeatin mother liquor, during use prepare often liter of proliferated culture medium get 4-6ml zeatin mother liquor;
(3) after adding by the consumption preparing often liter of proliferated culture medium in above-mentioned steps (11) to step (14), then add agar 7g, sucrose 30g, NAA mother liquor 1ml, GA mother liquor 4ml, is settled to 1L after heating for dissolving, uses 0.lmolL
-1hydrochloric acid adjusts pH to 5.2, sterilizing; When substratum temperature is down to 60-70 DEG C, inoculation platform adopts sterilization by filtration to add the zeatin mother liquor of preparation in step (2), and often liter of proliferated culture medium adds 4ml-6ml zeatin mother liquor, then packing substratum on inoculation platform.
3. described in claim 1, happiness carrys out the using method of the proliferated culture medium that blueberry tissue is cultivated, it is characterized in that, aseptic seedling is seeded on super clean bench on the proliferated culture medium of the sterilizing prepared, cultivate under the condition of intensity of illumination 2000Lux, culture condition is: temperature 25-27 DEG C, and every day, light application time was 14 hours.
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| CN105454049B (en) * | 2016-01-04 | 2017-11-10 | 江苏农林职业技术学院 | A kind of Kiwi berry proliferated culture medium and its preparation method and application |
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| CN106508681B (en) * | 2016-11-14 | 2018-07-20 | 黑龙江省林业科学研究所 | A kind of cowberry detoxification mating system and its virus-free culture base |
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