CN107372118B - A kind of golden larch test tube seeding cultivating method - Google Patents
A kind of golden larch test tube seeding cultivating method Download PDFInfo
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- CN107372118B CN107372118B CN201710757963.XA CN201710757963A CN107372118B CN 107372118 B CN107372118 B CN 107372118B CN 201710757963 A CN201710757963 A CN 201710757963A CN 107372118 B CN107372118 B CN 107372118B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The present invention provides a kind of golden larch test tube seeding cultivating method, belongs to rare Naked plant tissue culture seedlings-raising technical field.1) golden larch test tube seeding cultivating method, which includes the following steps, draws materials；2) paper bridge Liquid Culture；3) intermediate brood body proliferation；4) culture of rootage；5) acclimatization and transplants.Above-mentioned golden larch test tube seeding cultivating method makes the germination rate, planting percent and high survival rate of golden larch seed, can produce golden larch on a large scale.
Description
Technical field
The invention belongs to rare Naked plant tissue culture seedlings-raising technical fields, specifically, being related to a kind of golden larch examination
Pipe seeding cultivating method.
Background technique
Money is Gymnospermae Coniferales Pinaceae, rare species.Golden larch economic, medicinal, ornamental and section with higher
Learn researching value.Since its trunk is tall and straight, tree crown is roomy, and tree performance is dignified, beautiful, and autumn leaves is golden yellow, can express aspect better
Variation is widely introduced a fine variety for countries in the world botanical garden, has wide application market prospect.Pseudolarix plant is famous ancient
Relict plant, golden larch are the distinctive single platymisciums in China, precious rare tree species, due to its special classification position, money
Pine becomes botanical system development important research to picture.This valuable plant legacy is decided to be Chinese Second Class Key Protected Plant, to grinding
The systematic growth for studying carefully Pinaceae has certain scientific meaning.Golden larch is classified as 1500 meters of In Middle And Lower Reaches of Changjiang River height above sea level by forest department
The excellent reproducting tree species of following mountain and hill, many cities and botanical garden introducing and planting.
The main cuttage of the breeding of golden larch and seeding and seedling raising, because golden larch distribution is fragmentary, individual is rare, solid to have significantly
Intermittence, seed is in great shortage at present, and import needs a large amount of foreign exchange, this is to restrict golden larch to develop key factor.Cuttage and seedling culture, by
In resinous, pine tar in golden larch body, the cuttings notch during cuttage, which is easily taken root by secretion pollution effect, to be survived.With plant
In terms of object clone technology is used in landscape flower plant cultivating seedlings, more and more successes, gram of many magaphanerophytes are obtained
Grand technology also reaches its maturity, and still, the test tube seedling cultivation of golden larch is nearly at blank and starting stage.
Summary of the invention
For deficiency above-mentioned in the prior art, the present invention provides a kind of golden larch test tube seeding cultivating method, passes through the party
Method makes the germination rate, planting percent and high survival rate of golden larch seed, can produce golden larch on a large scale.
In order to achieve the above object, the solution that the present invention uses is:
A kind of golden larch test tube seeding cultivating method, comprising the following steps: 1) draw materials: taking annual golden larch seedling 2.0-
Spray of 3.0 ㎝ with terminal bud is cleaned after cutting off blade and sterilizes to obtain culture medium seedling；
2) circular filter paper: being converted into goblet shape and filled in test tube by paper bridge Liquid Culture, one end direction examination for making filter paper be open
The half that fluid nutrient medium reaches filter paper in test tube is poured into the bottom of pipe, and the closed one end of filter paper is made to expose Liquid Culture
Culture medium seedling, is stripped that long 2mm, that the stem apex with 1-2 root axillary bud is placed in filter paper in test tube is closed by base on superclean bench
On one end, tampon carries out illumination cultivation beyond the Great Wall on test tube mouth, and intermediate brood body proliferation is carried out after differentiation is formed；Liquid
Body culture medium is by minimal medium+zeatin 0.5-1 ㎎/L+ diethyl aminoethyl hexanoate 0.1-0.5 ㎎/L+ thiocarbamide 5-10 ㎎/L+ glutamic acid 50
㎎/L+ caseinhydrolysate matter 50-100 ㎎/L+ glucose 2-5 ㎎/L composition；
3) intermediate brood body proliferation: after differentiation is formed, Multiple Buds are cut into single plant, breaks up in liquid and cultivates
Illumination Multiplying culture is carried out on base, and grows into test tube seedling, and liquid differential medium is identical as the composition of fluid nutrient medium；
4) culture of rootage: when test tube seedling grows into 2-3 ㎝, being transferred in root media, is 23-25 DEG C in temperature
Under conditions of, carry out culture of rootage；Root media is by+No. 2 ABT root-inducing powder 30-50 ㎎/L+ glutamic acid of 1/2 minimal medium
50-100mg/L composition；
5) acclimatization and transplants: when test tube seedling forms the root of 2-3 ㎝ long, being transferred to room temperature lower refining seedling 3-5 days, cleans root training
Support base, then impregnate 1-2min in 20 ㎎/L agricultural streptomycin solution, transplanting is in the matrix to after sterilizing, by the area Lian Miao with
Other zone isolations, temperature are controlled at 23-25 DEG C, and humid control is in 80%-90%.
The beneficial effect of golden larch test tube seeding cultivating method provided by the invention is, according to golden larch growth characteristics and cell
Institutional framework feature and its contain the substances such as resin, natural gum in vivo, lead to the easy brown stain of culture and difficult feature of taking root, system
For the formula of fluid nutrient medium and root media, fluid nutrient medium and root media contain lower ammonium salt, higher nitre
Hydrochlorate and thiamine hydrochloride, mineral salinity is higher, is suitble to the culture growth of this kind of resinous class xylophyta of golden larch, simultaneously
And cooperate golden larch test tube seeding cultivating method, efficiently solve pollution, brown stain and difficulty in golden larch test tube seedling production process
Break up the problems such as difficult to take root, the germination rate, planting percent and survival rate of golden larch seed are made by golden larch test tube seeding cultivating method
Height can produce golden larch on a large scale.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Golden larch test tube seeding cultivating method provided by the invention is further described:
A kind of golden larch test tube seeding cultivating method the following steps are included:
1) it draws materials: taking annual spray of the golden larch seedling 2.0-3.0 ㎝ with terminal bud, clean and sterilize after cutting off blade
Culture medium seedling is obtained, the spray after the specific steps of disinfection will be cleaned rinses 30-38s with 70% alcohol, with aseptic water washing 3-5
It is secondary, in order to clean alcohol, wherein alcohol concentration cannot be too high or too low, and the effect of disinfection and sterilization is all bad, then exists
5-10min is impregnated in 10-12% hydrogenperoxide steam generator, with aseptic water washing 3-5 times, in order to clean hydrogen peroxide；So
It is impregnated 20-30 minutes with 20 ㎎/L agricultural streptomycin afterwards, wherein agricultural streptomycin is a kind of antibiotic medicament, and wide sterilization spectrum is right
Various bacteria, fungi have bactericidal effect, are in order to clean agricultural streptomycin, using alcohol, mistake with 5 purposes of aseptic water washing
Hydrogen oxide and agricultural streptomycin gradually sterilize spray, reach good sterilization effect, take out spray and are done with sterile
Culture medium seedling is obtained after Adsorption of Filter Paper, main purpose is to suck residual moisture on spray by sterile dry filter paper.
2) circular filter paper: being converted into goblet shape and filled in test tube by paper bridge Liquid Culture, one end direction examination for making filter paper be open
The half that fluid nutrient medium reaches filter paper in test tube is poured into the bottom of pipe, and the closed one end of filter paper is made to expose the liquid
Culture medium seedling is stripped long 2mm by culture medium on superclean bench, the stem apex with 1-2 root axillary bud is placed in filter paper in test tube and seals
On the one end closed, tampon carries out illumination cultivation beyond the Great Wall on test tube mouth, and intermediate brood body is carried out after differentiation is formed and is increased
It grows, wherein illumination uses fluorescent lamp plus incandescent lamp for mixing light source, illumination condition are as follows: 1000-2000Lx of intensity of illumination, light
According to time 16-18h/d.
Fluid nutrient medium is by minimal medium+zeatin 0.5-1 ㎎/L+ diethyl aminoethyl hexanoate 0.1-0.5 ㎎/L+ thiocarbamide 5-10 ㎎/L
50 ㎎ of+glutamic acid/L+ caseinhydrolysate matter 50-100 ㎎/L+ glucose 2-5 ㎎/L composition；Wherein, zeatin is to be present in height
A kind of natural plant cells mitogen substance of equal plants, is formed with well the division, growth and callus of cell
Facilitation, diethyl aminoethyl hexanoate are auxin substances, can promote cell division elongation, the growth of tachyauxesis point, differentiation, can activate
Excellent genes play one's part to the full, and thiocarbamide is a kind of weak cytokinin, have and delay cell tissue aging, and breaking dormancy promotees
Many-sided effect such as characteristic of disease is done into sprouting, sterilization, raising.
Minimal medium is by 2600 ㎎ of potassium nitrate/150 ㎎ of L+ amine sulfate/250 ㎎ of L+ U-Ramin MC/L+ epsom salt
400 ㎎/300 ㎎ of L+ dipotassium hydrogen phosphate/15 ㎎ of L+ ferrous sulfate/25 ㎎ of L+ sodium ethylene diamine tetracetate/10 ㎎ of L+ zinc sulfate/L+ bis-
1.0 ㎎ of titanium oxide/5.0 ㎎ of L+ potassium iodide/5.0 ㎎ of L+ niacin/0.5 ㎎ of L+ ascorbic acid/1.0 ㎎ of L+ thiamine hydrochloride/L+ flesh
150 ㎎ of alcohol/L composition.
Using paper bridge Liquid Culture, nutriment can be reduced pollution, be had by the balanced lasting supply culture of filter paper
Sharp culture health differential growth can effectively solve pollution, brown stain and difficult differentiation in golden larch test tube seedling production process
The problems such as difficult to take root.
3) intermediate brood body proliferation: after differentiation is formed, Multiple Buds are cut into single plant, breaks up in liquid and cultivates
Illumination Multiplying culture is carried out on base, and grows into test tube seedling, liquid differential medium i.e. liquid identical as the composition of fluid nutrient medium
Body differential medium is by minimal medium+zeatin 0.5-1 ㎎/L+ diethyl aminoethyl hexanoate 0.1-0.5 ㎎/L+ thiocarbamide 5-10 ㎎/L+ paddy ammonia
50 ㎎ of acid/L+ caseinhydrolysate matter 50-100 ㎎/L+ glucose 2-5 ㎎/L composition；Illumination in the condition and step 2) of illumination
Condition is identical.
4) culture of rootage: when test tube seedling grows into 2-3 ㎝, being transferred in root media, is 23-25 DEG C in temperature
Under conditions of, carry out culture of rootage；Root media is by+No. 2 ABT root-inducing powder 30-50 ㎎/L+ glutamic acid of 1/2 minimal medium
50-100mg/L composition, it is preferred that root media is by+No. 2 40 ㎎ of ABT root-inducing powder/L+ glutamic acid of 1/2 minimal medium
75mg/L composition, 1/2 minimal medium is by 1300 ㎎ of potassium nitrate/85 ㎎ of L+ amine sulfate/125 ㎎ of L+ U-Ramin MC/L+, seven water sulphur
Sour 200 ㎎ of magnesium/150 ㎎ of L+ dipotassium hydrogen phosphate/7.5 ㎎ of L+ ferrous sulfate/17.5 ㎎ of L+ sodium ethylene diamine tetracetate/L+ zinc sulfate 5
0.5 ㎎ of ㎎/L+ titanium dioxide/2.5 ㎎ of L+ potassium iodide/2.5 ㎎ of L+ niacin/0.25 ㎎ of L+ ascorbic acid/L+ thiamine hydrochloride 0.5
75 ㎎ of ㎎/L+ inositol/L composition.
5) acclimatization and transplants: when test tube seedling forms the root of 2-3 ㎝ long, being transferred to room temperature lower refining seedling 3-5 days, cleans root training
Support base, then impregnate 1-2min in 20 ㎎/L agricultural streptomycin solution, transplanting is in the matrix to after sterilizing, by the area Lian Miao with
Other zone isolations, temperature are controlled at 23-25 DEG C, and humid control is in 80%-90%, wherein matrix is 1:1 vermiculite and fertile soil
Mixture, and the matrix is sterilized with carbendazim.
Embodiment 1
A kind of golden larch test tube seeding cultivating method, comprising the following steps:
1) it draws materials: taking annual spray of 2.0 ㎝ of golden larch seedling with terminal bud, cleaned after cutting off blade, after cleaning
Spray rinse 30s with 70% alcohol and with aseptic water washing 3 times then 5min impregnated in 10% hydrogenperoxide steam generator, with nothing
Bacterium water rinses 3 times, is then impregnated 20 minutes with 20 ㎎/L agricultural streptomycin, with aseptic water washing 3 times, takes out spray and is done with sterile
Culture medium seedling is obtained after Adsorption of Filter Paper；
2) circular filter paper: being converted into goblet shape and filled in test tube by paper bridge Liquid Culture, one end direction examination for making filter paper be open
The half that fluid nutrient medium reaches filter paper in test tube is poured into the bottom of pipe, and the closed one end of filter paper is made to expose Liquid Culture
Culture medium seedling is stripped long 2mm by base on superclean bench, the stem apex of 1 axillary bud of band is placed in filter paper in test tube closed one
On end, fluid nutrient medium is by 2600 ㎎ of potassium nitrate/150 ㎎ of L+ amine sulfate/250 ㎎ of L+ U-Ramin MC/L+ epsom salt 400
300 ㎎ of ㎎/L+ dipotassium hydrogen phosphate/15 ㎎ of L+ ferrous sulfate/25 ㎎ of L+ sodium ethylene diamine tetracetate/10 ㎎ of L+ zinc sulfate/L+ dioxy
Change 1.0 ㎎ of titanium/5.0 ㎎ of L+ potassium iodide/5.0 ㎎ of L+ niacin/0.5 ㎎ of L+ ascorbic acid/1.0 ㎎ of L+ thiamine hydrochloride/L+ inositol
150 ㎎/0.5 ㎎ of L+ zeatin/0.1 ㎎ of L+ diethyl aminoethyl hexanoate/5 ㎎ of L+ thiocarbamide/50 ㎎ of L+ glutamic acid/50 ㎎ of L+ caseinhydrolysate matter/L
2 ㎎ of+glucose/L composition, the illumination cultivation of tampon progress beyond the Great Wall on test tube mouth, intensity of illumination 1000Lx, light application time 16h/d,
Intermediate brood body proliferation is carried out after differentiation is formed；
3) intermediate brood body proliferation: after differentiation is formed, Multiple Buds are cut into single plant, breaks up in liquid and cultivates
Illumination Multiplying culture, intensity of illumination 1000Lx, light application time 16h/d are carried out on base, and grow into test tube seedling, liquid differentiation culture
Base is by 2600 ㎎ of potassium nitrate/150 ㎎ of L+ amine sulfate/250 ㎎ of L+ U-Ramin MC/400 ㎎ of L+ epsom salt/L+ dipotassium hydrogen phosphate
300 ㎎/15 ㎎ of L+ ferrous sulfate/25 ㎎ of L+ sodium ethylene diamine tetracetate/10 ㎎ of L+ zinc sulfate/1.0 ㎎ of L+ titanium dioxide/L+ iodate
5.0 ㎎ of potassium/5.0 ㎎ of L+ niacin/0.5 ㎎ of L+ ascorbic acid/1.0 ㎎ of L+ thiamine hydrochloride/150 ㎎ of L+ inositol/L+ zeatin 0.5
0.1 ㎎ of ㎎/L+ diethyl aminoethyl hexanoate/5 ㎎ of L+ thiocarbamide/50 ㎎ of L+ glutamic acid/50 ㎎ of L+ caseinhydrolysate matter/2 ㎎ of L+ glucose/L composition；
4) culture of rootage: when test tube seedling grows into 2 ㎝, being transferred in root media, the item for being 23 DEG C in temperature
Under part, culture of rootage is carried out；Root media is by 1300 ㎎ of potassium nitrate/85 ㎎ of L+ amine sulfate/125 ㎎ of L+ U-Ramin MC/L+ seven
200 ㎎ of water magnesium sulfate/150 ㎎ of L+ dipotassium hydrogen phosphate/7.5 ㎎ of L+ ferrous sulfate/17.5 ㎎ of L+ sodium ethylene diamine tetracetate/L+ sulfuric acid
5 ㎎ of zinc/0.5 ㎎ of L+ titanium dioxide/2.5 ㎎ of L+ potassium iodide/2.5 ㎎ of L+ niacin/0.25 ㎎ of L+ ascorbic acid/L+ thiamine hydrochloride
0.5 ㎎/75 ㎎ of L+ inositol/30 ㎎ of L+2 ABT root-inducing powder/L+ glutamic acid 50mg/L composition；
5) acclimatization and transplants: when test tube seedling forms the root of 2 ㎝ long, being transferred to room temperature lower refining seedling 3 days, cleans root culture
Then base impregnates 1min in 20 ㎎/L agricultural streptomycin solution, in the matrix to after sterilizing, matrix is 1:1 vermiculite for transplanting
It with humic earth mixtures, and is sterilized with carbendazim, by the area Lian Miao and other zone isolations, temperature control is at 23 DEG C, humid control
80%.
Embodiment 2
A kind of golden larch test tube seeding cultivating method, comprising the following steps:
1) it draws materials: taking annual spray of 3.0 ㎝ of golden larch seedling with terminal bud, cleaned after cutting off blade, after cleaning
Spray rinse 38s with 70% alcohol and with aseptic water washing 5 times then impregnate 10min in 12% hydrogenperoxide steam generator, use
It aseptic water washing 5 times, is then impregnated 30 minutes with 20 ㎎/L agricultural streptomycin, with aseptic water washing 5 times, takes out spray with sterile
Culture medium seedling is obtained after dry Adsorption of Filter Paper；
2) circular filter paper: being converted into goblet shape and filled in test tube by paper bridge Liquid Culture, one end direction examination for making filter paper be open
The half that fluid nutrient medium reaches filter paper in test tube is poured into the bottom of pipe, and the closed one end of filter paper is made to expose Liquid Culture
Culture medium seedling is stripped long 2mm by base on superclean bench, the stem apex of 2 axillary buds of band is placed in filter paper in test tube closed one
On end, fluid nutrient medium is by 2600 ㎎ of potassium nitrate/150 ㎎ of L+ amine sulfate/250 ㎎ of L+ U-Ramin MC/L+ epsom salt 400
300 ㎎ of ㎎/L+ dipotassium hydrogen phosphate/15 ㎎ of L+ ferrous sulfate/25 ㎎ of L+ sodium ethylene diamine tetracetate/10 ㎎ of L+ zinc sulfate/L+ dioxy
Change 1.0 ㎎ of titanium/5.0 ㎎ of L+ potassium iodide/5.0 ㎎ of L+ niacin/0.5 ㎎ of L+ ascorbic acid/1.0 ㎎ of L+ thiamine hydrochloride/L+ inositol
150 ㎎/1 ㎎ of L+ zeatin/0.5 ㎎ of L+ diethyl aminoethyl hexanoate/10 ㎎ of L+ thiocarbamide/50 ㎎ of L+ glutamic acid/100 ㎎ of L+ caseinhydrolysate matter/L
5 ㎎ of+glucose/L composition, the illumination cultivation of tampon progress beyond the Great Wall on test tube mouth, intensity of illumination 2000Lx, light application time 18h/d,
Intermediate brood body proliferation is carried out after differentiation is formed；
3) intermediate brood body proliferation: after differentiation is formed, Multiple Buds are cut into single plant, breaks up in liquid and cultivates
Illumination Multiplying culture, intensity of illumination 2000Lx, light application time 18h/d are carried out on base, and grow into test tube seedling, liquid differentiation culture
Base is by 2600 ㎎ of potassium nitrate/150 ㎎ of L+ amine sulfate/250 ㎎ of L+ U-Ramin MC/400 ㎎ of L+ epsom salt/L+ dipotassium hydrogen phosphate
300 ㎎/15 ㎎ of L+ ferrous sulfate/25 ㎎ of L+ sodium ethylene diamine tetracetate/10 ㎎ of L+ zinc sulfate/1.0 ㎎ of L+ titanium dioxide/L+ iodate
5.0 ㎎ of potassium/5.0 ㎎ of L+ niacin/0.5 ㎎ of L+ ascorbic acid/1.0 ㎎ of L+ thiamine hydrochloride/150 ㎎ of L+ inositol/L+ zeatin 1
0.5 ㎎ of ㎎/L+ diethyl aminoethyl hexanoate/10 ㎎ of L+ thiocarbamide/50 ㎎ of L+ glutamic acid/100 ㎎ of L+ caseinhydrolysate matter/5 ㎎ of L+ glucose/L group
At；
4) culture of rootage: when test tube seedling grows into 3 ㎝, being transferred in root media, the item for being 25 DEG C in temperature
Under part, culture of rootage is carried out；Root media is by 1300 ㎎ of potassium nitrate/85 ㎎ of L+ amine sulfate/125 ㎎ of L+ U-Ramin MC/L+ seven
200 ㎎ of water magnesium sulfate/150 ㎎ of L+ dipotassium hydrogen phosphate/7.5 ㎎ of L+ ferrous sulfate/17.5 ㎎ of L+ sodium ethylene diamine tetracetate/L+ sulfuric acid
5 ㎎ of zinc/0.5 ㎎ of L+ titanium dioxide/2.5 ㎎ of L+ potassium iodide/2.5 ㎎ of L+ niacin/0.25 ㎎ of L+ ascorbic acid/L+ thiamine hydrochloride
0.5 ㎎/75 ㎎ of L+ inositol/50 ㎎ of L+2 ABT root-inducing powder/L+ glutamic acid 100mg/L composition；
5) acclimatization and transplants: when test tube seedling forms the root of 3 ㎝ long, being transferred to room temperature lower refining seedling 5 days, cleans root culture
Then base impregnates 2min in 20 ㎎/L agricultural streptomycin solution, in the matrix to after sterilizing, matrix is 1:1 vermiculite for transplanting
It with humic earth mixtures, and is sterilized with carbendazim, by the area Lian Miao and other zone isolations, temperature control is at 25 DEG C, humid control
90%.
Embodiment 3
A kind of golden larch test tube seeding cultivating method, comprising the following steps:
1) it draws materials: taking annual spray of 3.5 ㎝ of golden larch seedling with terminal bud, cleaned after cutting off blade, after cleaning
Spray rinse 35s with 70% alcohol and with aseptic water washing 4 times then 8min impregnated in 11% hydrogenperoxide steam generator, with nothing
Bacterium water rinses 4 times, is then impregnated 35 minutes with 20 ㎎/L agricultural streptomycin, with aseptic water washing 4 times, takes out spray and is done with sterile
Culture medium seedling is obtained after Adsorption of Filter Paper；
2) circular filter paper: being converted into goblet shape and filled in test tube by paper bridge Liquid Culture, one end direction examination for making filter paper be open
The half that fluid nutrient medium reaches filter paper in test tube is poured into the bottom of pipe, and the closed one end of filter paper is made to expose Liquid Culture
Culture medium seedling is stripped long 2mm by base on superclean bench, the stem apex of 2 axillary buds of band is placed in filter paper in test tube closed one
On end, fluid nutrient medium is by 2600 ㎎ of potassium nitrate/150 ㎎ of L+ amine sulfate/250 ㎎ of L+ U-Ramin MC/L+ epsom salt 400
300 ㎎ of ㎎/L+ dipotassium hydrogen phosphate/15 ㎎ of L+ ferrous sulfate/25 ㎎ of L+ sodium ethylene diamine tetracetate/10 ㎎ of L+ zinc sulfate/L+ dioxy
Change 1.0 ㎎ of titanium/5.0 ㎎ of L+ potassium iodide/5.0 ㎎ of L+ niacin/0.5 ㎎ of L+ ascorbic acid/1.0 ㎎ of L+ thiamine hydrochloride/L+ inositol
150 ㎎/0.8 ㎎ of L+ zeatin/0.3 ㎎ of L+ diethyl aminoethyl hexanoate/8 ㎎ of L+ thiocarbamide/50 ㎎ of L+ glutamic acid/80 ㎎ of L+ caseinhydrolysate matter/L
3 ㎎ of+glucose/L composition, the illumination cultivation of tampon progress beyond the Great Wall on test tube mouth, intensity of illumination 1500Lx, light application time 17h/d,
Intermediate brood body proliferation is carried out after differentiation is formed；
3) intermediate brood body proliferation: after differentiation is formed, Multiple Buds are cut into single plant, breaks up in liquid and cultivates
Illumination Multiplying culture, intensity of illumination 1500Lx, light application time 17h/d are carried out on base, and grow into test tube seedling, liquid differentiation culture
Base is by 2600 ㎎ of potassium nitrate/150 ㎎ of L+ amine sulfate/250 ㎎ of L+ U-Ramin MC/400 ㎎ of L+ epsom salt/L+ dipotassium hydrogen phosphate
300 ㎎/15 ㎎ of L+ ferrous sulfate/25 ㎎ of L+ sodium ethylene diamine tetracetate/10 ㎎ of L+ zinc sulfate/1.0 ㎎ of L+ titanium dioxide/L+ iodate
5.0 ㎎ of potassium/5.0 ㎎ of L+ niacin/0.5 ㎎ of L+ ascorbic acid/1.0 ㎎ of L+ thiamine hydrochloride/150 ㎎ of L+ inositol/L+ zeatin 0.8
0.3 ㎎ of ㎎/L+ diethyl aminoethyl hexanoate/8 ㎎ of L+ thiocarbamide/50 ㎎ of L+ glutamic acid/80 ㎎ of L+ caseinhydrolysate matter/3 ㎎ of L+ glucose/L composition；
4) culture of rootage: when test tube seedling grows into 3 ㎝, being transferred in root media, the item for being 24 DEG C in temperature
Under part, culture of rootage is carried out；Root media is by 1300 ㎎ of potassium nitrate/85 ㎎ of L+ amine sulfate/125 ㎎ of L+ U-Ramin MC/L+ seven
200 ㎎ of water magnesium sulfate/150 ㎎ of L+ dipotassium hydrogen phosphate/7.5 ㎎ of L+ ferrous sulfate/17.5 ㎎ of L+ sodium ethylene diamine tetracetate/L+ sulfuric acid
5 ㎎ of zinc/0.5 ㎎ of L+ titanium dioxide/2.5 ㎎ of L+ potassium iodide/2.5 ㎎ of L+ niacin/0.25 ㎎ of L+ ascorbic acid/L+ thiamine hydrochloride
0.5 ㎎/75 ㎎ of L+ inositol/40 ㎎ of L+2 ABT root-inducing powder/L+ glutamic acid 75mg/L composition；
5) acclimatization and transplants: when test tube seedling forms the root of 3 ㎝ long, being transferred to room temperature lower refining seedling 4 days, cleans root culture
Then base impregnates 2min in 20 ㎎/L agricultural streptomycin solution, in the matrix to after sterilizing, matrix is 1:1 vermiculite for transplanting
It with humic earth mixtures, and is sterilized with carbendazim, by the area Lian Miao and other zone isolations, temperature control is at 25 DEG C, humid control
90%.
Comparative example 1
A kind of golden larch test tube seeding cultivating method, comprising the following steps:
1) it draws materials: taking annual spray of 3.5 ㎝ of golden larch seedling with terminal bud, cleaned after cutting off blade, after cleaning
Spray rinse 35s with 70% alcohol and with aseptic water washing 4 times then 8min impregnated in 11% hydrogenperoxide steam generator, with nothing
Bacterium water rinses 4 times, is then impregnated 35 minutes with 20 ㎎/L agricultural streptomycin, with aseptic water washing 4 times, takes out spray and is done with sterile
Culture medium seedling is obtained after Adsorption of Filter Paper；
2) solid culture: culture medium seedling is stripped into long 2mm on superclean bench, the stem apex of 2 axillary buds of band is placed in admittedly
On body culture medium, solid medium is by MS culture medium+0.8 ㎎ of zeatin/0.3 ㎎ of L+ diethyl aminoethyl hexanoate/8 ㎎ of L+ thiocarbamide/L+ glutamic acid
50 ㎎/80 ㎎ of L+ caseinhydrolysate matter/3 ㎎ of L+ glucose/L composition, tampon carries out illumination cultivation, illumination beyond the Great Wall on test tube mouth
Intensity 1500Lx, light application time 17h/d carry out intermediate brood body proliferation after differentiation is formed；
3) intermediate brood body proliferation: after differentiation is formed, Multiple Buds are cut into single plant, on differential medium
Illumination Multiplying culture, intensity of illumination 1500Lx, light application time 17h/d are carried out, and grows into test tube seedling, differential medium is trained by MS
Support base+0.8 ㎎ of zeatin/0.3 ㎎ of L+ diethyl aminoethyl hexanoate/8 ㎎ of L+ thiocarbamide/50 ㎎ of L+ glutamic acid/80 ㎎ of L+ caseinhydrolysate matter/Portugal L+
3 ㎎ of grape sugar/L composition；
4) culture of rootage: when test tube seedling grows into 3 ㎝, being transferred in root media, the item for being 24 DEG C in temperature
Under part, culture of rootage is carried out；Root media is by 1/2MS culture medium+2.5 ㎎ of potassium iodide/2.5 ㎎ of L+ niacin/L+ ascorbic acid
0.25 ㎎/0.5 ㎎ of L+ thiamine hydrochloride/75 ㎎ of L+ inositol/40 ㎎ of L+2 ABT root-inducing powder/L+ glutamic acid 75mg/L composition；
5) acclimatization and transplants: when test tube seedling forms the root of 3 ㎝ long, being transferred to room temperature lower refining seedling 4 days, cleans root culture
Then base impregnates 2min in 20 ㎎/L agricultural streptomycin solution, in the matrix to after sterilizing, matrix is 1:1 vermiculite for transplanting
It with humic earth mixtures, and is sterilized with carbendazim, by the area Lian Miao and other zone isolations, temperature control is at 25 DEG C, humid control
90%.
Experimental example 1
The case where growing to golden larch test tube seedling in embodiment 1-3 and comparative example 1 observes and records, as a result such as 1 institute of table
Show:
Table 1
As shown in Table 1, using the formula of the present processes and culture medium be most suitable for golden larch take root, by this method with
The formula of culture medium makes the germination rate, planting percent and high survival rate of golden larch seed, can produce golden larch on a large scale.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of golden larch test tube seeding cultivating method, it is characterised in that: the following steps are included:
1) it draws materials: taking annual spray of the golden larch seedling 2.0-3.0 ㎝ with terminal bud, clean and sterilize and to obtain training after cutting off blade
Support base seedling；
2) circular filter paper: being converted into goblet shape and filled in test tube by paper bridge Liquid Culture, makes one end of filter paper opening towards test tube
The half that fluid nutrient medium reaches filter paper in test tube is poured into bottom, and the closed one end of filter paper is made to expose the Liquid Culture
The culture medium seedling is stripped long 2mm by base on superclean bench, the stem apex with 1-2 root axillary bud is placed in filter paper in test tube and seals
On the one end closed, tampon carries out illumination cultivation beyond the Great Wall on test tube mouth, and intermediate brood body is carried out after differentiation is formed and is increased
It grows；The fluid nutrient medium is by minimal medium+zeatin 0.5-1 ㎎/L+ diethyl aminoethyl hexanoate 0.1-0.5 ㎎/L+ thiocarbamide 5-10 ㎎/L+
50 ㎎ of glutamic acid/L+ caseinhydrolysate matter 50-100 ㎎/L+ glucose 2-5 ㎎/L composition；The minimal medium is by potassium nitrate
2600 ㎎/150 ㎎ of L+ amine sulfate/250 ㎎ of L+ U-Ramin MC/400 ㎎ of L+ epsom salt/300 ㎎ of L+ dipotassium hydrogen phosphate/L+ sulphur
15 ㎎ of sour ferrous iron/25 ㎎ of L+ sodium ethylene diamine tetracetate/10 ㎎ of L+ zinc sulfate/1.0 ㎎ of L+ titanium dioxide/5.0 ㎎ of L+ potassium iodide/L+
5.0 ㎎ of niacin/0.5 ㎎ of L+ ascorbic acid/1.0 ㎎ of L+ thiamine hydrochloride/150 ㎎ of L+ inositol/L composition；
3) intermediate brood body proliferation: after differentiation is formed, Multiple Buds are cut into single plant, on liquid differential medium
Illumination Multiplying culture is carried out, and grows into test tube seedling, the liquid differential medium is identical as the composition of the fluid nutrient medium；
4) culture of rootage: when test tube seedling grows into 2-3 ㎝, being transferred in root media, the item for being 23-25 DEG C in temperature
Under part, culture of rootage is carried out；The root media is by+No. 2 ABT root-inducing powder 30-50 ㎎/L+ glutamic acid of 1/2 minimal medium
50-100mg/L composition；1/2 minimal medium is by 1300 ㎎ of potassium nitrate/85 ㎎ of L+ amine sulfate/125 ㎎ of L+ U-Ramin MC/L
200 ㎎ of+epsom salt/150 ㎎ of L+ dipotassium hydrogen phosphate/7.5 ㎎ of L+ ferrous sulfate/17.5 ㎎ of L+ sodium ethylene diamine tetracetate/L+
5 ㎎ of zinc sulfate/0.5 ㎎ of L+ titanium dioxide/2.5 ㎎ of L+ potassium iodide/2.5 ㎎ of L+ niacin/0.25 ㎎ of L+ ascorbic acid/L+ hydrochloric acid sulphur
0.5 ㎎ of amine element/75 ㎎ of L+ inositol/L composition；
5) acclimatization and transplants: when test tube seedling forms the root of 2-3 ㎝ long, being transferred to room temperature lower refining seedling 3-5 days, cleans root culture
Then base impregnates 1-2min in 20 ㎎/L agricultural streptomycin solution, transplanting is in the matrix to after sterilizing, by Lian Miaoqu and its
His zone isolation, temperature are controlled at 23-25 DEG C, and humid control is in 80%-90%.
2. golden larch test tube seeding cultivating method according to claim 1, it is characterised in that: in step 1), the disinfection is
70% alcohol of the spray after cleaning rinses 30-38s, with aseptic water washing 3-5 times, then in 10-12% peroxidating
5-10min is impregnated in hydrogen solution, with aseptic water washing 3-5 times, is then impregnated 20-30 minutes, is used with 20 ㎎/L agricultural streptomycin
Aseptic water washing 3-5 times obtains the culture medium seedling after taking out the sterile dry Adsorption of Filter Paper of spray.
3. golden larch test tube seeding cultivating method according to claim 2, it is characterised in that: the light in step 2) and step 3)
According to condition are as follows: 1000-2000Lx of intensity of illumination, light application time 16-18h/d.
4. golden larch test tube seeding cultivating method according to claim 3, it is characterised in that: the illumination is added with fluorescent lamp
Incandescent lamp is mixing light source.
5. golden larch test tube seeding cultivating method according to claim 1, it is characterised in that: in step 5), the matrix is
1:1 vermiculite and humic earth mixtures.
6. golden larch test tube seeding cultivating method according to claim 1, it is characterised in that: the root media is by 1/2
+ No. 2 40 ㎎ of ABT root-inducing powder of minimal medium/L+ glutamic acid 75mg/L composition.
7. golden larch test tube seeding cultivating method according to claim 1, it is characterised in that: matrix described in step 5) uses
Carbendazim disinfection.
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