CN103636506B - method for performing plant culture by utilizing shepherdia argentea caulicle regenerated plant induction culture medium and - Google Patents

method for performing plant culture by utilizing shepherdia argentea caulicle regenerated plant induction culture medium and Download PDF

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CN103636506B
CN103636506B CN201310721553.1A CN201310721553A CN103636506B CN 103636506 B CN103636506 B CN 103636506B CN 201310721553 A CN201310721553 A CN 201310721553A CN 103636506 B CN103636506 B CN 103636506B
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silver
colored
culture
young stem
illumination
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CN103636506A (en
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王承义
李晶
宋国华
孙一萌
王丹
徐永波
梁鸣
林玉秋
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FORESTRY RESEARCH INSTITUTE OF HEILONGJIANG PROVINCE
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FORESTRY RESEARCH INSTITUTE OF HEILONGJIANG PROVINCE
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Abstract

The invention discloses a shepherdia argentea caulicle regenerated plant induction culture medium and a method for performing plant culture by utilizing the culture medium, relates to a regenerated plant induction culture medium and a culture method, and aims to solve the technical problems of low rooting rate in a conventional shepherdia argentea clonal cutting culture method and low differentiation rate of plants cultured by an MS (murashige-skoog) culture medium. The culture medium consists of macroelement, trace element, iron salt, organic matters, hormones, sucrose and agar, wherein the hormones are 6-benzylaminopurine, zeatin and naphthalene acetic acid. The method comprises the following steps of collecting annual dormant shoots of shepherdia argentea from the second third of April and the first third of May, and performing hydroponic culture to obtain caulicles; washing and sterilizing the caulicles, inoculating the caulicles to the shepherdia argentea caulicle regenerated plant induction culture medium, placing the inoculated caulicles under a dark condition for culture after inoculation, and transferring the caulicles to a light condition for culture to obtain shepherdia argentea regenerated plants. According to the culture method, the tufted seedling inductivity reaches over 80 percent, meanwhile, the cost is lowered by 50 percent, and the seed nature purity is over 98 percent.

Description

The method of plant cultivation is carried out with the young stem regeneration plant inducing culture of silver-colored buffalo fruit
Technical field
The invention belongs to field of plant tissue culture technique, is a kind of silver-colored buffalo fruit young stem regeneration plant induction special culture media.
Background technology
Silver buffalo fruit calendar year 2001 introduces the plantation of Daxing ' anling, heilongjiang area from Canada, after the cultivation of Daxinganling District, and can safe overwintering normal growth and solid.
Fruit belongs to silver buffalo fruit (Shepherdia canadensis) Elaeangnaceae buffalo, and perennial shrub, originates in northern US, plant height 5.5 meters, spends little, yellow, midspring at florescence, food value of leaf ground closely, has the silver color of Elaeagnus, narrow leaf, long 2 ~ 6cm, fall foliage, leaf look constant, and fruit is red to orange red.Silver buffalo fruit has the good characteristics such as resistance to atmospheric drought, Salt And Alkali Tolerance, the poor carbuncle of resistance to soil, disease and insect resistance, area minimum temperature-40 DEG C.This seeds well developed root system, nitrogen fixing capacity is strong.Silver buffalo fruit at sand ground, hilly ground, grow saline and alkaline drought and barren, and can be used for abandoned mine land revegetation, improve region environment and have very high using value, is excellent water and soil conservation and seeds of checking winds and fixing drifting sand.Silver buffalo fruits general flavone content up to 4.02mg/g, Vitamin C content 208.20mg/100g, and be rich in the elements such as potassium, calcium, magnesium, zinc; Containing 18 seed amino acids, wherein containing necessary 8 kinds of human body, total amount reaches 8.20%.Young stem and leaf crude protein content reaches 16.34%, is greater than crude protein content index (15%) in roughage; Crude fiber content 6.34%, crude fiber content index (18%) in roughage; Nitrogen free extract is up to 58.77%.Silver buffalo fruits is red or orange red, nutritious, delicious flavour, edible, and hip number is large, also can be used as nonwood forest trees, has certain potentiality to be exploited.And silver-colored buffalo fruit compared with sea-buckthorn without quil, being convenient to harvesting, is a kind of novel economizer woods seeds.
Silver 9 ~ October of buffalo fruits maturing stage, fruit does not fall for a long time, and plucking seed time is longer, and plants skin depth and hard, and with grease and brown glued membrane, cause resting stage long, seed treatment is more difficult, and nursery difficulty is large.Because the heterozygosis rate of seed is higher, seedling quality is difficult to control, and can be solved the problem by tissue-culturing quick-propagation, ensures the uniformity of seedling quality and descendant inheritting, can keep the merit of original kind, and to guaranteeing the quality, pure keeping has special role.The current domestic report about silver-colored buffalo fruit group training aspect is very few, and asexual cottage method is mainly taked in the breeding of relevant silver-colored buffalo fruit, but rooting rate is lower, only has about 60%, does not still have the report that tufted seedling is induced at present, and has no application in production.Carry out regeneration plant Fiber differentiation with MS medium to silver-colored buffalo fruit, its differentiation rate is 30% ~ 50%, and differentiation rate is low.
Summary of the invention
The rooting rate that the present invention seeks to solve the breeding of existing silver-colored buffalo fruit asexual cuttage is low, by the technical problem that MS medium culture Plantlet Differentiation rate is low, the invention provides the young stem regeneration plant inducing culture of silver-colored buffalo fruit and (is designated as SN 1) and silver-colored buffalo fruit plant cultur method.
The young stem regeneration plant inducing culture (SN of silver-colored buffalo fruit of the present invention 1) often liter of NH by 400 ~ 450mg 4nO 3, 900 ~ 920mg KNO 3, 170 ~ 190mg KH 2pO 4, 370 ~ 395mg MgSO 47H 2the CaCl of O, 100 ~ 110mg 22H 2the H of O, 6.2 ~ 6.5mg 3bO 3, 22.3 ~ 24.3mg MnSO 44H 2the ZnSO of O, 8.6 ~ 9.2mg 47H 2the Na of O, 0.25 ~ 0.3mg 2moO 42H 2the CuSO of O, 0.025 ~ 0.03mg 45H 2the CoCl of O, 0.025 ~ 0.03mg 26H 2the Na of O, 37.3 ~ 37.9mg 2the FeSO of-EDTA, 27.8 ~ 28.2mg 47H 2the C of O, 100 ~ 105mg 6h 12o 6, 0.5 ~ 0.7mg C 6h 5nO 2, 0.1 ~ 0.15mg C 12h 17clN 4the C of OSHCl, 0.5 ~ 0.6mg 8h 11o 3the C of NHCl, 2 ~ 2.5mg 2h 5nO 2, the 6-benzylaminopurine (6-BA) of 0.5 ~ 2.0mg, 0.05 ~ 0.5mg zeatin (ZT) and 0.05 ~ 0.5mg methyl α-naphthyl acetate (NAA), the sucrose of 20 ~ 25g, the agar of 6 ~ 7g and surplus distilled water composition.
Ammonium nitrate (NH in present embodiment 4nO 3), potassium nitrate (KNO 3), potassium dihydrogen phosphate (KH 2pO 4), magnesium sulfate (MgSO 47H 2and calcium chloride dihydrate (CaCl O) 22H 2o) be the macroelement in medium; Boric acid (H 3bO 3), manganese sulphate (MnSO 44H 2o), zinc sulphate (ZnSO 47H 2o), sodium molybdate (Na 2moO 42H 2o), copper sulphate (CuSO 45H 2and cobalt chloride (CoCl O) 26H 2o) be the trace element in medium; Disodium ethylene diamine tetraacetate (Na 2-EDTA) and ferrous sulfate (FeSO 47H 2o) be the molysite in medium; Inositol (C 6h 12o 6), nicotinic acid (C 6h 5nO 2), thiamine hydrochloride (C 12h 17clN 4oSHCl), puridoxine hydrochloride (C 8h 11o 3and glycine (C NHCl) 2h 5nO 2) be the organic matter in medium; 6-benzylaminopurine (6-BA), zeatin (ZT) and methyl α-naphthyl acetate (NAA) are the hormone in medium.
Utilize the above-mentioned young stem regeneration plant inducing culture of silver-colored buffalo fruit to carry out the method for silver-colored buffalo fruit plant cultivation, carry out according to the following steps:
One, in 4 the middle ten days ~ the first tenday period of a month in May, gather the raw then resting shoot of silver-colored buffalo fruit, be placed on water planting vernalization in illumination box, illumination box changes 1 water every day, when young stem grows to 2 ~ 3cm, shears off for subsequent use.
The young stem cleaning of silver-colored buffalo fruit of two, step one being cultivated, sterilization;
Three, the young stem through step 2 process is cut on super-clean bench the stem section of 1 ~ 2cm, be inoculated on the above-mentioned young stem regeneration plant inducing culture of silver-colored buffalo fruit, after inoculation, cultivate 6 ~ 7 days under first putting into dark condition, then proceed to illumination cultivation 15 ~ 20 days, obtain silver-colored buffalo fruit regeneration plant.
Medium of the present invention is adjusted by the consumption of macroelement on the basis of MS medium, reduces the NO in macroelement 3 -, Ca 2+concentration of element, reduces to 0 by trace element by KI content, is conducive to the Organ Differentiation of silver-colored buffalo fruit.Utilize 6-benzylaminopurine, zeatin and methyl α-naphthyl acetate as plant growth regulator, the young stem obtained through water planting with the raw then resting shoot of silver-colored buffalo fruit is for culture materials regeneration induction plant, medium of the present invention is utilized to carry out the cultivation of the young stem regeneration plant of silver-colored buffalo fruit, tufted seedling inductivity reaches more than 80%, more than 30% is improved, simultaneously cost-saving about 50% than the differentiation-inducing rate of existing MS medium.Experiment shows, medium Shoot propagation of the present invention is obvious, easy regeneration induction plant.Induce indefinite bud and regeneration plant by young stem culture, and kind property uniformity is strong, transplants plant strain growth and property determination through plantlet in vitro, kind property purity more than 98%, shows high consistency and yielding ability, and can reach the object of Fast-propagation good seed.
Embodiment
Embodiment one: the young stem regeneration plant inducing culture of silver-colored buffalo fruit of present embodiment (is designated as SN 1) often liter of NH by 400 ~ 450mg 4nO 3, 900 ~ 920mg KNO 3, 170 ~ 190mg KH 2pO 4, 370 ~ 395mg MgSO 47H 2the CaCl of O, 100 ~ 110mg 22H 2the H of O, 6.2 ~ 6.5mg 3bO 3, 22.3 ~ 24.3mg MnSO 44H 2the ZnSO of O, 8.6 ~ 9.2mg 47H 2the Na of O, 0.25 ~ 0.3mg 2moO 42H 2the CuSO of O, 0.025 ~ 0.03mg 45H 2the CoCl of O, 0.025 ~ 0.03mg 26H 2the Na of O, 37.3 ~ 37.9mg 2the FeSO of-EDTA, 27.8 ~ 28.2mg 47H 2the C of O, 100 ~ 105mg 6h 12o 6, 0.5 ~ 0.7mg C 6h 5nO 2, 0.1 ~ 0.15mg C 12h 17clN 4the C of OSHCl, 0.5 ~ 0.6mg 8h 11o 3the C of NHCl, 2 ~ 2.5mg 2h 5nO 2, the 6-benzylaminopurine (6-BA) of 0.5 ~ 2.0mg, 0.05 ~ 0.5mg zeatin (ZT) and 0.05 ~ 0.5mg methyl α-naphthyl acetate (NAA), the sucrose of 20 ~ 25g, the agar of 6 ~ 7g and surplus distilled water composition.
Ammonium nitrate (NH in present embodiment 4nO 3), potassium nitrate (KNO 3), potassium dihydrogen phosphate (KH 2pO 4), magnesium sulfate (MgSO 47H 2and calcium chloride dihydrate (CaCl O) 22H 2o) be the macroelement in medium; Boric acid (H 3bO 3), manganese sulphate (MnSO 44H 2o), zinc sulphate (ZnSO 47H 2o), sodium molybdate (Na 2moO 42H 2o), copper sulphate (CuSO 45H 2and cobalt chloride (CoCl O) 26H 2o) be the trace element in medium; Disodium ethylene diamine tetraacetate (Na 2-EDTA) and ferrous sulfate (FeSO 47H 2o) be the molysite in medium; Inositol (C 6h 12o 6), nicotinic acid (C 6h 5nO 2), thiamine hydrochloride (C 12h 17clN 4oSHCl), puridoxine hydrochloride (C 8h 11o 3and glycine (C NHCl) 2h 5nO 2) be the organic matter in medium; 6-benzylaminopurine (6-BA), zeatin (ZT) and methyl α-naphthyl acetate (NAA) are the hormone in medium.
The consumption of macroelement, compared with existing MS medium, adjusts, reduces the NO in macroelement by the medium of present embodiment 3 -, Ca 2+concentration of element; In trace element, KI content is reduced to 0, the present invention (SN 1) concentration of mineral salt, lower than inorganic salt concentration contained by existing MS medium, is conducive to the Organ Differentiation of silver-colored buffalo fruit in medium.Utilize the cultivation carrying out the young stem regeneration plant of silver-colored buffalo fruit, tufted seedling inductivity reaches more than 80%, improves more than 30% than the differentiation-inducing rate of existing MS medium, and simultaneously cost-saving about 50%.And induce indefinite bud and regeneration plant by young stem culture, and kind property uniformity is strong, transplants plant strain growth and property determination through plantlet in vitro, kind property purity more than 98%, show high consistency and yielding ability, and the object of Fast-propagation good seed can be reached.
Embodiment two: present embodiment and embodiment one often rise by the NH of 400mg unlike the young stem regeneration plant inducing culture of silver-colored buffalo fruit 4nO 3, 900mg KNO 3, 170mg KH 2pO 4, 370mg MgSO 47H 2the CaCl of O, 100mg 22H 2the H of O, 6.2mg 3bO 3, 22.3mg MnSO 44H 2the ZnSO of O, 8.6mg 47H 2the Na of O, 0.25mg 2moO 42H 2the CuSO of O, 0.025mg 45H 2the CoCl of O, 0.025mg 26H 2the Na of O, 37.3mg 2the FeSO of-EDTA, 27.8mg 47H 2the C of O, 100mg 6h 12o 6, 0.5mg C 6h 5nO 2, 0.1mg C 12h 17clN 4the C of OSHCl, 0.5mg 8h 11o 3the C of NHCl, 2mg 2h 5nO 2, the 6-benzylaminopurine of 1.0mg, 0.1mg zeatin and 0.05mg methyl α-naphthyl acetate, the sucrose of 20g, the agar of 6g and surplus distilled water composition.
Utilize the young stem regeneration plant inducing culture of silver-colored buffalo fruit of present embodiment to carry out the young stem regenerated plant culture of silver-colored buffalo fruit, regeneration plant inductivity (i.e. Plantlet Differentiation rate) can reach 80% ~ 85%, saves input cost 50% than contrast MS medium.
Embodiment three: present embodiment and embodiment one or two often rise by the NH of 400mg unlike the young stem regeneration plant inducing culture of silver-colored buffalo fruit 4nO 3, 900mg KNO 3, 170mg KH 2pO 4, 370mg MgSO 47H 2the CaCl of O, 100mg 22H 2the H of O, 6.2mg 3bO 3, 22.3mg MnSO 44H 2the ZnSO of O, 8.6mg 47H 2the Na of O, 0.25mg 2moO 42H 2the CuSO of O, 0.025mg 45H 2the CoCl of O, 0.025mg 26H 2the Na of O, 37.3mg 2the FeSO of-EDTA, 27.8mg 47H 2the C of O, 100mg 6h 12o 6, 0.5mg C 6h 5nO 2, 0.1mg C 12h 17clN 4the C of OSHCl, 0.5mg 8h 11o 3the C of NHCl, 2mg 2h 5nO 2, the 6-benzylaminopurine of 2.0mg, 0.5mg zeatin and 0.1mg methyl α-naphthyl acetate, the sucrose of 20g, the agar of 6g and surplus distilled water composition.
Utilize the young stem regeneration plant inducing culture of silver-colored buffalo fruit of present embodiment to carry out the young stem regenerated plant culture of silver-colored buffalo fruit, regeneration plant inductivity (i.e. Plantlet Differentiation rate) can reach 80% ~ 82%, saves input cost 50% than contrast MS medium.
Embodiment four: utilize the young stem regeneration plant inducing culture of silver-colored buffalo fruit described in embodiment one to carry out the method for silver-colored buffalo fruit plant cultivation, carry out according to the following steps:
One, in 4 the middle ten days ~ the first tenday period of a month in May, gather the raw then resting shoot of silver-colored buffalo fruit, be placed on water planting vernalization in illumination box, illumination box changes 1 water every day, when young stem grows to 2 ~ 3cm, shears off for subsequent use;
The young stem cleaning of silver-colored buffalo fruit of two, step one being cultivated, sterilization;
Three, the young stem through step 2 process is cut into the stem section of 1 ~ 2cm, be seeded on the young stem regeneration plant inducing culture of silver-colored buffalo fruit described in embodiment one, after inoculation, cultivate 6 ~ 7 days under first putting into dark condition, then proceed to illumination cultivation 15 ~ 20 days, obtain silver-colored buffalo fruit regeneration plant.
Embodiment five: when present embodiment and embodiment four carry out light culture unlike illumination box in step one, the temperature of illumination box is 24 ~ 26 DEG C.Other is identical with embodiment four.
Embodiment six: present embodiment and embodiment four or five are unlike when carrying out illumination cultivation in step one, adopt fluorescent lamp to do light source, illumination every day 12 ~ 14h, intensity of illumination is 15 μm of olm- 2s- 1-20 μm of olm- 2s- 1, the temperature of incubator is 24 ~ 26 DEG C.Other is identical with embodiment four or five.
Embodiment seven: one of present embodiment and embodiment four to six unlike the young stem cleaning of silver-colored buffalo fruit, disinfecting process are: first make surface clean with washing agent, remove surface dirt, clear water rinses again, then the young stem of silver-colored buffalo fruit is placed on superclean bench, with 75% ethanol sterilizing 10s, aseptic water washing 3 ~ 5 times, then use 0.1% mercuric chloride liquid disinfectant 5min, aseptic water washing 3 ~ 5 times, is placed on filter paper and blots surface moisture.Other is identical with one of embodiment four to six.
Embodiment eight: one of present embodiment and embodiment four to seven are 24 ~ 26 DEG C unlike the Conditions Temperature cultivated under the dark condition in step 3, intensity of illumination is 0.Other is identical with one of embodiment four to seven.
Embodiment nine: one of present embodiment and embodiment four to eight unlike the condition of the illumination cultivation in step 3 are: fluorescent lamp does light source, illumination every day 12 ~ 14h, intensity of illumination is 15 μm of olm -2s -1~ 20 μm of olm -2s -1, temperature is 24 ~ 26 DEG C.Other is identical with one of embodiment four to eight.
With following verification experimental verification beneficial effect of the present invention:
Test 1: utilize the young stem regeneration plant inducing culture of silver-colored buffalo fruit to carry out the method for silver-colored buffalo fruit plant cultivation, carry out according to the following steps:
One, at point in the morning 9 ~ 10 on April 20th, 2012, fine, gather the raw then resting shoot of the silver-colored buffalo fruit of Daxing ' anling, heilongjiang, elite stand is life in 10 years, robust growth, the resting shoot of collection is placed on water planting vernalization in illumination box, and illumination box changes 1 water every day, when young stem grows to 2 ~ 3cm, young stem is sheared off; Wherein during illumination cultivation, adopt fluorescent lamp to do light source, every day, light application time was 12h, and intensity of illumination is 15 μm of olm -2s -1, the temperature of incubator is 24 ~ 26 DEG C.
Two, the young stem of silver-colored buffalo fruit step one cultivated first makes surface clean with washing agent, remove surface dirt, clear water rinses again, then the young stem of silver-colored buffalo fruit is placed on superclean bench, with 75% ethanol sterilizing 10s, aseptic water washing 5 times, then use 0.1% mercuric chloride liquid disinfectant 5min, aseptic water washing 5 times, is placed on filter paper and blots surface moisture;
Three, often rise by the NH of 400mg by the young stem regeneration plant inducing culture of silver-colored buffalo fruit 4nO 3, 900mg KNO 3, 170mg KH 2pO 4, 370mg MgSO 47H 2the CaCl of O, 100mg 22H 2the H of O, 6.2mg 3bO 3, 22.3mg MnSO 44H 2the ZnSO of O, 8.6mg 47H 2the Na of O, 0.25mg 2moO 42H 2the CuSO of O, 0.025mg 45H 2the CoCl of O, 0.025mg 26H 2the Na of O, 37.3mg 2the FeSO of-EDTA, 27.8mg 47H 2the C of O, 100mg 6h 12o 6, 0.5mg C 6h 5nO 2, 0.1mg C 12h 17clN 4the C of OSHCl, 0.5mg 8h 11o 3the C of NHCl, 2mg 2h 5nO 2, the 6-benzylaminopurine (6-BA) of 1.0mg, the methyl α-naphthyl acetate (NAA) of 0.05mg zeatin (ZT) and 0.1mg, the sucrose of 20g, the agar of 6g and surplus distilled water composition, be prepared into the young stem regeneration plant inducing culture of solid-state silver-colored buffalo fruit;
Four, by after being cut into the stem section of 1.5cm through the young stem scalpel of step 2 process, being inoculated into step 3 prepares on the young stem regeneration plant inducing culture of silver-colored buffalo fruit, every bottle graft kind 3 ~ 5 young stems, after inoculation, cultivate 7 days under first putting into dark condition, then proceed to illumination cultivation 18 days, obtain silver-colored buffalo fruit regeneration plant; The condition of cultivating under the dark condition wherein in step 4 is temperature is 24 ~ 26 DEG C, and intensity of illumination is 0; The condition of illumination cultivation is: temperature is 24 ~ 26 DEG C, does light source with fluorescent lamp, illumination every day 12 ~ 14h, and intensity of illumination is 18 μm of olm -2s -1.
In the medium that this test adopts, macroelement and micro-summation, namely the content of mineral salt is 1977.4mg/L, and existing MS medium inorganic salt content is 4568.23mg/L, and in the medium of existing MS medium inorganic salt content and this test 1, the ratio of inorganic salt content is 2.31:1.Relative to existing MS medium, the consumption of macroelement is adjusted, reduce the NO in macroelement 3 -, Ca 2+concentration of element; In trace element, KI content is reduced to 0, these means are all conducive to the Organ Differentiation of silver-colored buffalo fruit.In this test 1, the bud induction rate of the young stem of silver-colored buffalo fruit is 89%, and tufted seedling inductivity is 81%.Rate raising 33% more differentiation-inducing than existing MS medium.Experiment shows, the medium Shoot propagation of this test 1 is obvious, easy regeneration induction plant plant.This test 1 induces indefinite bud and regeneration plant by young stem culture, its kind of property uniformity is strong, measure through plantlet in vitro transplanting plant strain growth and character of fruitage, planting property purity is 99%, the object of Fast-propagation good seed can be reached, be widely applied aborning, show high consistency and yielding ability.
Test 2: this test replaces unlike the operation below of: step one with test 1, other is identical with testing 1.
One, at point in the morning 9 ~ 10 on January 20th, 2012, fine, gather the raw then resting shoot of the silver-colored buffalo fruit of Daxing ' anling, heilongjiang, elite stand is life in 10 years, robust growth, the branch of collection is placed on water planting vernalization in illumination box, and illumination box changes 1 water every day, when young stem grows to 2 ~ 3cm, young stem is sheared off; Wherein during illumination cultivation, adopt fluorescent lamp to do light source, every day, light application time was 12h, and intensity of illumination is 15 μm of olm -2s -1, the temperature of incubator is 24 ~ 26 DEG C.
This test 2 is different unlike the time of the branch for the silver-colored buffalo fruit gathered from test 1, result of the test: be pollution rate to the cultivation results of the resting shoot of collection in February be 5%, and bud induction rate is 30%, and tufted seedling inductivity is 27%.Inductivity is lower.The similar results that pollution rate is low, inductivity is low is all there is in Harvest time in the test in 1 ~ March.
Test 3: this test replaces unlike the operation below of: step one with test 1, other is identical with testing 1.
One, at point in the morning 9 ~ 10 on June 20th, 2012, fine, gather the green branch of the silver-colored buffalo fruit of Daxing ' anling, heilongjiang, elite stand is life in 10 years, robust growth, using the green branch gathered as young stem;
The green branch that this test 3 gathers June is cultivated, and result is pollution rate is 35%, and inductivity is 81%.Its inductivity is higher, but pollution rate is also higher, the high cultivation survival rate being unfavorable for the later stage of pollution rate.In the test in 6 ~ July, Harvest time all occurs that inductivity is high, the similar results that pollution rate is high.
Comparative trial 1,2 and 3 known, the acquisition time of branch is cultivated very important for seedling.
Test 4: this test replaces unlike the operation below of: step 3 with test 1, other is identical with testing 1.
Often rise by the NH of 400mg by the young stem regeneration plant inducing culture of silver-colored buffalo fruit 4nO 3, 900mg KNO 3, 170mg KH 2pO 4, 370mg MgSO 47H 2the CaCl of O, 100mg 22H 2the H of O, 6.2mg 3bO 3, 22.3mg MnSO 44H 2the ZnSO of O, 8.6mg 47H 2the Na of O, 0.25mg 2moO 42H 2the CuSO of O, 0.025mg 45H 2the CoCl of O, 0.025mg 26H 2the Na of O, 37.3mg 2the FeSO of-EDTA, 27.8mg 47H 2the C of O, 100mg 6h 12o 6, 0.5mg C 6h 5nO 2, 0.1mg C 12h 17clN 4the C of OSHCl, 0.5mg 8h 11o 3the C of NHCl, 2mg 2h 5nO 2, the 6-benzylaminopurine (6-BA) of 2.0mg, the methyl α-naphthyl acetate (NAA) of 0.1mg zeatin (ZT) and 0.2mg, the sucrose of 20g, the agar of 6g and surplus distilled water composition, be prepared into the young stem regeneration plant inducing culture of solid-state silver-colored buffalo fruit.
In this test 4, the bud induction rate of the young stem of silver-colored buffalo fruit is 90%, and tufted seedling inductivity is 85%.Rate raising 36% more differentiation-inducing than existing MS medium.Experiment shows, the medium Shoot propagation of this test is obvious, easy regeneration induction plant plant.This test 4 induces indefinite bud and regeneration plant by young stem culture, its kind of property uniformity is strong, measure through plantlet in vitro transplanting plant strain growth and character of fruitage, planting property purity is 99%, the object of Fast-propagation good seed can be reached, be widely applied aborning, show high consistency and yielding ability.

Claims (6)

1. carry out the method for plant cultivation with the young stem regeneration plant inducing culture of silver-colored buffalo fruit, it is characterized in that the method is carried out according to the following steps:
One, in 4 the middle ten days ~ the first tenday period of a month in May, gather the raw then resting shoot of silver-colored buffalo fruit, be placed on water planting vernalization in illumination box, illumination box changes 1 water every day, when young stem grows to 2 ~ 3cm, shears off for subsequent use;
The young stem cleaning of silver-colored buffalo fruit of two, step one being cultivated, sterilization;
Three, the young stem through step 2 process is cut into the stem section of 1 ~ 2cm, is seeded on the young stem regeneration plant inducing culture of silver-colored buffalo fruit, after inoculation, cultivates 7 days under first putting into dark condition, then proceed to illumination cultivation 15 ~ 20 days, obtain silver-colored buffalo fruit regeneration plant, the wherein said young stem regeneration plant inducing culture of silver-colored buffalo fruit often rises by the ammonium nitrate of 400 ~ 450mg, the potassium nitrate of 900 ~ 920mg, the potassium dihydrogen phosphate of 170 ~ 190mg, the epsom salt of 370 ~ 395mg, the calcium chloride dihydrate of 100 ~ 110mg, the boric acid of 6.2 ~ 6.5mg, the four water manganese sulphates of 22.3 ~ 24.3mg, the white vitriol of 8.6 ~ 9.2mg, the Sodium Molybdate Dihydrate of 0.25 ~ 0.3mg, the cupric sulfate pentahydrate of 0.025 ~ 0.03mg, the CoCL2 6H2O of 0.025 ~ 0.03mg, the disodium ethylene diamine tetraacetate of 37.3 ~ 37.9mg, the ferrous sulfate heptahydrate of 27.8 ~ 28.2mg, the inositol of 100 ~ 105mg, the nicotinic acid of 0.5 ~ 0.7mg, the thiamine hydrochloride of 0.1 ~ 0.15mg, the puridoxine hydrochloride of 0.5 ~ 0.6mg, the glycine of 2 ~ 2.5mg, the 6-benzylaminopurine of 0.5 ~ 2.0mg, 0.05 ~ 0.5mg zeatin and 0.05 ~ 0.5mg methyl α-naphthyl acetate, the sucrose of 20 ~ 25g, the agar of 6 ~ 7g and the distilled water composition of surplus.
2. the young stem regeneration plant inducing culture of use according to claim 1 silver-colored buffalo fruit carries out the method for plant cultivation, and when it is characterized in that in step one, illumination box carries out light culture, the temperature of illumination box is 24 ~ 26 DEG C.
3. the young stem regeneration plant inducing culture of use according to claim 1 and 2 silver-colored buffalo fruit carries out the method for plant cultivation, and when it is characterized in that carrying out illumination cultivation in step one, adopt fluorescent lamp to do light source, illumination every day 12 ~ 14h, intensity of illumination is 15 μm of olm -2s -1~ 20 μm of olm -2s -1, the temperature of incubator is 24 ~ 26 DEG C.
4. the young stem regeneration plant inducing culture of use according to claim 1 and 2 silver-colored buffalo fruit carries out the method for plant cultivation, it is characterized in that the young stem of silver-colored buffalo fruit cleans, disinfecting process is: first make surface clean with washing agent, remove surface dirt, clear water rinses again, is then placed on superclean bench, with 75% ethanol sterilizing 10s by the young stem of silver-colored buffalo fruit, aseptic water washing 3 ~ 5 times, use 0.1% mercuric chloride liquid disinfectant 5min again, aseptic water washing 3 ~ 5 times, is placed on filter paper and blots surface moisture.
5. the young stem regeneration plant inducing culture of use according to claim 1 and 2 silver-colored buffalo fruit carries out the method for plant cultivation, and the condition of cultivating under it is characterized in that the dark condition in step 3 is temperature is 24 ~ 26 DEG C, and intensity of illumination is 0.
6. the young stem regeneration plant inducing culture of use according to claim 1 and 2 silver-colored buffalo fruit carries out the method for plant cultivation, it is characterized in that the condition of the illumination cultivation in step 3 is: do light source with fluorescent lamp, illumination every day 12 ~ 14h, intensity of illumination is 15 μm of olm -2s -1~ 20 μm of olm -2s -1, temperature is 24 ~ 26 DEG C.
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