CN107711517A - A kind of candidum tissue culturing seedling breeding method of high-survival rate - Google Patents
A kind of candidum tissue culturing seedling breeding method of high-survival rate Download PDFInfo
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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 invention discloses a kind of candidum tissue culturing seedling breeding method of high-survival rate, step are as follows：Pretreatment dendrobium candidum capsule is cut, by seed uniformly dispersing on germination medium, is cultivated in tissue culture room, obtains protocorm；Protocorm is forwarded in proliferated culture medium and cultivated, obtains protocorms；Protocorms are forwarded in differential medium and cultivated, obtain seedling；Seedling is forwarded in the tissue culture bottle containing seedling culture medium and cultivated, obtains strong sprout；Tissue culture bottle containing strong sprout is sent into hardening room, carries out hardening, seedling after being refined；Seedling after refining is taken out from tissue culture bottle, the culture medium of root carrying is removed using clear water, you can obtains tissue-cultured seedling.Candidum tissue culturing seedling breeding method disclosed by the invention, the tissue-cultured seedling cultivated is healthy and strong, and transplanting survival rate is high, steady quality, may advantageously facilitate the production-scale expansion of dendrobium candidum, has wide market prospects.
Description
Technical field
The present invention relates to dendrobe cultivation technical field, the candidum tissue culturing seedling cultivation side of specifically a kind of high-survival rate
Method.
Background technology
As Chinese Famous traditional medicine, dendrobium candidum have tonifying-Yin and nourishing-stomach, moisten the lung and relieve the cough, heat-clearing improving eyesight the effect of, separately
Outside, also strengthen body immunity, eliminate tumour, suppress the effect such as cancer.Early in《Compendium of Materia Medica》Dendrobium candidum is just listed in
It is medicinal,《Sheng Nong's herbal classic》In be listed in top grade,《Taoist Scriptures》Neutralize Herba Saussureae Involueratae, three double ginsengs, cycle of sixty years Fu from, hundred
20 years tubers of multiflower knotweed, remote mountains ganoderma lucidum, cordyceps sinensis etc. are listed in " Chinese nine big celestial grass " together, and dendrobium candidum is located at " nine big celestial grass "
First of.Because dendrobium candidum is very rare, in recent years, dendrobium candidum artificial cultivation industry develops more fast in China
Speed.However, dendrobium candidum requires harsh to growing environment, seedling is difficult, using artificial fecundation methods such as traditional plant division, cuttages
To be bred, growth rate is very low, meanwhile, the problems such as tissue-cultured seedling quality is difficult to ensure that also be present, greatly constrain selenium-rich
The production-scale expansion of dendrobium candidum.
The content of the invention
It is an object of the invention to provide a kind of candidum tissue culturing seedling breeding method of high-survival rate, to solve the above-mentioned back of the body
The problem of being proposed in scape technology.
To achieve the above object, the present invention provides following technical scheme：
A kind of candidum tissue culturing seedling breeding method of high-survival rate, step are as follows：1）Choose ripe uncracked dendrobium candidum
Capsule, after being rinsed using clear water, it is put into 0.1% mercuric chloride solution and soaks 3-4min, after taking-up, using aseptic water washing 2-3 times,
It is then placed in pretreatment fluid and soaks 7-10min, after taking-up, using aseptic water washing 4-5 times, blots surface moisture, obtain pre-
Dendrobium candidum capsule is handled, the pretreatment fluid is made up of citric acid and alcohol；2）Pretreatment dendrobium candidum capsule is cut, will
Seed uniformly dispersing is cultivated on germination medium in tissue culture room, temperature be 22-24 DEG C, light application time 8h/d,
Intensity of illumination is cultivated 19-22 days under conditions of being 1000-1200lux, obtains protocorm；3）Protocorm is forwarded to Multiplying culture
In base, 24-25 is cultivated under conditions of temperature is 23-25 DEG C, light application time 8h/d, intensity of illumination are 2000-2500lux
My god, obtain protocorms；4）Protocorms are forwarded in differential medium, temperature be 23-25 DEG C, light application time 12h/
D, intensity of illumination is cultivated 26-28 days under conditions of being 2000-2500lux, obtains seedling；5）Seedling is forwarded to and trained containing nursery
In the tissue culture bottle for supporting base, under conditions of temperature is 23-25 DEG C, light application time 15h/d, intensity of illumination are 2800-3000lux
Culture 80-90 days, obtain strong sprout；6）Tissue culture bottle containing strong sprout is sent into hardening room, carries out the hardening of 12-15 days, is obtained
Seedling after refining；7）Seedling after refining is taken out from tissue culture bottle, the culture medium of root carrying is removed using clear water, you can obtains tissue-cultured seedling.
As the further scheme of the present invention：The preparation method of the pretreatment fluid is：10g citric acids are taken, input is extremely
In 100mL 85% alcohol, after being uniformly mixed, you can.
As further scheme of the invention：The germination medium promotes liquid to form by 1/2MS culture mediums and sprouting.
As further scheme of the invention：The preparation method of the germination medium is as follows：Eupatorium lindleynun var. trifoliolatum is taken, is cleaned,
After drying, crush, put into percolator, diacolation processing is carried out using 70% alcohol of 12-15 times of weight, obtains seepage pressure effects
Liquid and diacolation residue, diacolation residue is added to 70% alcohol of 4-5 times of weight, then ultrasonication 40-50min, filtering, obtained
Ultrasonic extract and ultrasonic wave extraction residue are obtained, percolate and ultrasonic extract are merged, adds gross weight 3%
Chitosan oligosaccharide, after being uniformly mixed, be concentrated by evaporation as the 20% of original volume, produce sprouting and promote liquid, will sprout promote liquid according to
30g/L amount is added into 1/2MS culture mediums, you can.
As further scheme of the invention：The proliferated culture medium is using MS culture mediums and adds 10g/L bananas juices
Formed with 2g/L disodium ethylene diamine tetraacetates.
As further scheme of the invention：The differential medium is using MS culture mediums and adds 15g/L Succus Rhizoma Dioscoreaes
Formed with 0.4g/L methyl α-naphthyl acetates.
As further scheme of the invention：The seedling culture medium promotes liquid to form by MS culture mediums and nursery.
As further scheme of the invention：The preparation method of the seedling culture medium is as follows：Japanese Cayratia Herb hay is taken,
Cleaning, after drying, crush, put into percolator, diacolation processing is carried out using 70% alcohol of 10-12 times of weight, obtains diacolation
Extract solution and diacolation residue, diacolation residue is added to 70% alcohol of 6-8 times of weight, then ultrasonication 40-50min, mistake
Filter, ultrasonic extract and ultrasonic wave extraction residue are obtained, percolate and ultrasonic extract are merged, add gross weight
5% chitosan oligosaccharide, after being uniformly mixed, it is concentrated by evaporation as the 15% of original volume, produces nursery and promote liquid, nursery promotion liquid is pressed
Added according to 20g/L amount into MS culture mediums, you can.
Compared with prior art, the beneficial effects of the invention are as follows：
Candidum tissue culturing seedling breeding method disclosed by the invention, the tissue-cultured seedling cultivated is healthy and strong, and transplanting survival rate is high, and quality is steady
It is fixed, the production-scale expansion of dendrobium candidum is may advantageously facilitate, there are wide market prospects.Dendrobium candidum group disclosed by the invention
Seeding cultivating method is trained, is bred using tissue culture technology, propagation efficiency is very high, can realize the extensive of selenium-enriched officinal dendrobium stem
Production, it is simple to operate, it is easy to spread.
Embodiment
Technical scheme is described in more detail with reference to embodiment.
Embodiment 1
A kind of candidum tissue culturing seedling breeding method of high-survival rate, step are as follows：
1）Ripe uncracked dendrobium candidum capsule is chosen, after being rinsed using clear water, is put into 0.1% mercuric chloride solution and soaks 3min,
After taking-up, using aseptic water washing 2 times, it is then placed in pretreatment fluid and soaks 7min, after taking-up, using aseptic water washing 4 times,
Surface moisture is blotted, obtains pretreatment dendrobium candidum capsule, the pretreatment fluid is made up of citric acid and alcohol, the pretreatment
The preparation method of liquid is：10g citric acids are taken, are put into 100mL 85% alcohol, after being uniformly mixed, you can；
2）Pretreatment dendrobium candidum capsule is cut, by seed uniformly dispersing on germination medium, trained in tissue culture room
Support, cultivated 19 days under conditions of temperature is 22 DEG C, light application time 8h/d, intensity of illumination are 1000lux, obtain protocorm,
The germination medium promotes liquid to form by 1/2MS culture mediums and sprouting, and the preparation method of the germination medium is as follows：Take open country
Horse chases after, cleaning, after drying, crushes, puts into percolator, carries out diacolation processing using 70% alcohol of 12 times of weight, is oozed
Filter extract solution and diacolation residue, diacolation residue is added to 70% alcohol of 4 times of weight, then ultrasonication 40min, filtering, obtain
Ultrasonic extract and ultrasonic wave extraction residue are obtained, percolate and ultrasonic extract are merged, adds gross weight 3%
Chitosan oligosaccharide, after being uniformly mixed, be concentrated by evaporation as the 20% of original volume, produce sprouting and promote liquid, will sprout promote liquid according to
30g/L amount is added into 1/2MS culture mediums, you can；
3）Protocorm is forwarded in proliferated culture medium, temperature be 23 DEG C, light application time 8h/d, intensity of illumination be
Cultivated 24 days under conditions of 2000lux, obtain protocorms, the proliferated culture medium is using MS culture mediums and adds 10g/L perfume
Any of several broadleaf plants juice and 2g/L disodium ethylene diamine tetraacetates composition；
4）Protocorms are forwarded in differential medium, temperature be 23 DEG C, light application time 12h/d, intensity of illumination be
Cultivated 26 days under conditions of 2000lux, obtain seedling, the differential medium is using MS culture mediums and adds 15g/L Succus Rhizoma Dioscoreaes
Formed with 0.4g/L methyl α-naphthyl acetates；
5）Seedling is forwarded in the tissue culture bottle containing seedling culture medium, temperature be 23 DEG C, light application time 15h/d, illumination
Intensity is cultivated 80 days under conditions of being 2800lux, obtains strong sprout, and the seedling culture medium promotes liquid group by MS culture mediums and nursery
Into the preparation method of the seedling culture medium is as follows：Japanese Cayratia Herb hay is taken, is cleaned, after drying, crushes, puts into percolator
In, diacolation processing is carried out using 70% alcohol of 10 times of weight, obtains percolate and diacolation residue, diacolation residue is added 6
70% alcohol of times weight, then ultrasonication 40min, filtering, obtains ultrasonic extract and ultrasonic wave extraction residue, will
Percolate and ultrasonic extract merge, and add the chitosan oligosaccharide of gross weight 5%, after being uniformly mixed, are concentrated by evaporation as original
The 15% of volume, produce nursery and promote liquid, nursery promotion liquid is added into MS culture mediums according to 20g/L amount, you can；
6）Tissue culture bottle containing strong sprout is sent into hardening room, carries out the hardening of 12 days, seedling after being refined；
7）Seedling after refining is taken out from tissue culture bottle, the culture medium of root carrying is removed using clear water, you can obtains tissue-cultured seedling.
Embodiment 2
A kind of candidum tissue culturing seedling breeding method of high-survival rate, step are as follows：
1）Ripe uncracked dendrobium candidum capsule is chosen, after being rinsed using clear water, is put into 0.1% mercuric chloride solution and soaks 4min,
After taking-up, using aseptic water washing 2 times, it is then placed in pretreatment fluid and soaks 8min, after taking-up, using aseptic water washing 4 times,
Surface moisture is blotted, obtains pretreatment dendrobium candidum capsule, the pretreatment fluid is made up of citric acid and alcohol, the pretreatment
The preparation method of liquid is：10g citric acids are taken, are put into 100mL 85% alcohol, after being uniformly mixed, you can；
2）Pretreatment dendrobium candidum capsule is cut, by seed uniformly dispersing on germination medium, trained in tissue culture room
Support, cultivated 20 days under conditions of temperature is 22 DEG C, light application time 8h/d, intensity of illumination are 1100lux, obtain protocorm,
The germination medium promotes liquid to form by 1/2MS culture mediums and sprouting, and the preparation method of the germination medium is as follows：Take open country
Horse chases after, cleaning, after drying, crushes, puts into percolator, carries out diacolation processing using 70% alcohol of 13 times of weight, is oozed
Filter extract solution and diacolation residue, diacolation residue is added to 70% alcohol of 4 times of weight, then ultrasonication 42min, filtering, obtain
Ultrasonic extract and ultrasonic wave extraction residue are obtained, percolate and ultrasonic extract are merged, adds gross weight 3%
Chitosan oligosaccharide, after being uniformly mixed, be concentrated by evaporation as the 20% of original volume, produce sprouting and promote liquid, will sprout promote liquid according to
30g/L amount is added into 1/2MS culture mediums, you can；
3）Protocorm is forwarded in proliferated culture medium, temperature be 23 DEG C, light application time 8h/d, intensity of illumination be
Cultivated 24 days under conditions of 2200lux, obtain protocorms, the proliferated culture medium is using MS culture mediums and adds 10g/L perfume
Any of several broadleaf plants juice and 2g/L disodium ethylene diamine tetraacetates composition；
4）Protocorms are forwarded in differential medium, temperature be 23 DEG C, light application time 12h/d, intensity of illumination be
Cultivated 27 days under conditions of 2200lux, obtain seedling, the differential medium is using MS culture mediums and adds 15g/L Succus Rhizoma Dioscoreaes
Formed with 0.4g/L methyl α-naphthyl acetates；
5）Seedling is forwarded in the tissue culture bottle containing seedling culture medium, temperature be 25 DEG C, light application time 15h/d, illumination
Intensity is cultivated 83 days under conditions of being 2800lux, obtains strong sprout, and the seedling culture medium promotes liquid group by MS culture mediums and nursery
Into the preparation method of the seedling culture medium is as follows：Japanese Cayratia Herb hay is taken, is cleaned, after drying, crushes, puts into percolator
In, diacolation processing is carried out using 70% alcohol of 11 times of weight, obtains percolate and diacolation residue, diacolation residue is added 6
70% alcohol of times weight, then ultrasonication 45min, filtering, obtains ultrasonic extract and ultrasonic wave extraction residue, will
Percolate and ultrasonic extract merge, and add the chitosan oligosaccharide of gross weight 5%, after being uniformly mixed, are concentrated by evaporation as original
The 15% of volume, produce nursery and promote liquid, nursery promotion liquid is added into MS culture mediums according to 20g/L amount, you can；
6）Tissue culture bottle containing strong sprout is sent into hardening room, carries out the hardening of 13 days, seedling after being refined；
7）Seedling after refining is taken out from tissue culture bottle, the culture medium of root carrying is removed using clear water, you can obtains tissue-cultured seedling.
Embodiment 3
A kind of candidum tissue culturing seedling breeding method of high-survival rate, step are as follows：
1）Ripe uncracked dendrobium candidum capsule is chosen, after being rinsed using clear water, is put into 0.1% mercuric chloride solution and soaks 4min,
After taking-up, using aseptic water washing 3 times, it is then placed in pretreatment fluid and soaks 9min, after taking-up, using aseptic water washing 4 times,
Surface moisture is blotted, obtains pretreatment dendrobium candidum capsule, the pretreatment fluid is made up of citric acid and alcohol, the pretreatment
The preparation method of liquid is：10g citric acids are taken, are put into 100mL 85% alcohol, after being uniformly mixed, you can；
2）Pretreatment dendrobium candidum capsule is cut, by seed uniformly dispersing on germination medium, trained in tissue culture room
Support, cultivated 21 days under conditions of temperature is 23 DEG C, light application time 8h/d, intensity of illumination are 1100lux, obtain protocorm,
The germination medium promotes liquid to form by 1/2MS culture mediums and sprouting, and the preparation method of the germination medium is as follows：Take open country
Horse chases after, cleaning, after drying, crushes, puts into percolator, carries out diacolation processing using 70% alcohol of 14 times of weight, is oozed
Filter extract solution and diacolation residue, diacolation residue is added to 70% alcohol of 5 times of weight, then ultrasonication 45min, filtering, obtain
Ultrasonic extract and ultrasonic wave extraction residue are obtained, percolate and ultrasonic extract are merged, adds gross weight 3%
Chitosan oligosaccharide, after being uniformly mixed, be concentrated by evaporation as the 20% of original volume, produce sprouting and promote liquid, will sprout promote liquid according to
30g/L amount is added into 1/2MS culture mediums, you can；
3）Protocorm is forwarded in proliferated culture medium, temperature be 24 DEG C, light application time 8h/d, intensity of illumination be
Cultivated 24 days under conditions of 2300lux, obtain protocorms, the proliferated culture medium is using MS culture mediums and adds 10g/L perfume
Any of several broadleaf plants juice and 2g/L disodium ethylene diamine tetraacetates composition；
4）Protocorms are forwarded in differential medium, temperature be 24 DEG C, light application time 12h/d, intensity of illumination be
Cultivated 27 days under conditions of 2300lux, obtain seedling, the differential medium is using MS culture mediums and adds 15g/L Succus Rhizoma Dioscoreaes
Formed with 0.4g/L methyl α-naphthyl acetates；
5）Seedling is forwarded in the tissue culture bottle containing seedling culture medium, temperature be 24 DEG C, light application time 15h/d, illumination
Intensity is cultivated 85 days under conditions of being 2900lux, obtains strong sprout, and the seedling culture medium promotes liquid group by MS culture mediums and nursery
Into the preparation method of the seedling culture medium is as follows：Japanese Cayratia Herb hay is taken, is cleaned, after drying, crushes, puts into percolator
In, diacolation processing is carried out using 70% alcohol of 11 times of weight, obtains percolate and diacolation residue, diacolation residue is added 7
70% alcohol of times weight, then ultrasonication 45min, filtering, obtains ultrasonic extract and ultrasonic wave extraction residue, will
Percolate and ultrasonic extract merge, and add the chitosan oligosaccharide of gross weight 5%, after being uniformly mixed, are concentrated by evaporation as original
The 15% of volume, produce nursery and promote liquid, nursery promotion liquid is added into MS culture mediums according to 20g/L amount, you can；
6）Tissue culture bottle containing strong sprout is sent into hardening room, carries out the hardening of 14 days, seedling after being refined；
7）Seedling after refining is taken out from tissue culture bottle, the culture medium of root carrying is removed using clear water, you can obtains tissue-cultured seedling.
Embodiment 4
A kind of candidum tissue culturing seedling breeding method of high-survival rate, step are as follows：
1）Ripe uncracked dendrobium candidum capsule is chosen, after being rinsed using clear water, is put into 0.1% mercuric chloride solution and soaks 4min,
After taking-up, using aseptic water washing 3 times, it is then placed in pretreatment fluid and soaks 8min, after taking-up, using aseptic water washing 5 times,
Surface moisture is blotted, obtains pretreatment dendrobium candidum capsule, the pretreatment fluid is made up of citric acid and alcohol, the pretreatment
The preparation method of liquid is：10g citric acids are taken, are put into 100mL 85% alcohol, after being uniformly mixed, you can；
2）Pretreatment dendrobium candidum capsule is cut, by seed uniformly dispersing on germination medium, trained in tissue culture room
Support, cultivated 22 days under conditions of temperature is 23 DEG C, light application time 8h/d, intensity of illumination are 1200lux, obtain protocorm,
The germination medium promotes liquid to form by 1/2MS culture mediums and sprouting, and the preparation method of the germination medium is as follows：Take open country
Horse chases after, cleaning, after drying, crushes, puts into percolator, carries out diacolation processing using 70% alcohol of 14 times of weight, is oozed
Filter extract solution and diacolation residue, diacolation residue is added to 70% alcohol of 4 times of weight, then ultrasonication 48min, filtering, obtain
Ultrasonic extract and ultrasonic wave extraction residue are obtained, percolate and ultrasonic extract are merged, adds gross weight 3%
Chitosan oligosaccharide, after being uniformly mixed, be concentrated by evaporation as the 20% of original volume, produce sprouting and promote liquid, will sprout promote liquid according to
30g/L amount is added into 1/2MS culture mediums, you can；
3）Protocorm is forwarded in proliferated culture medium, temperature be 25 DEG C, light application time 8h/d, intensity of illumination be
Cultivated 25 days under conditions of 2400lux, obtain protocorms, the proliferated culture medium is using MS culture mediums and adds 10g/L perfume
Any of several broadleaf plants juice and 2g/L disodium ethylene diamine tetraacetates composition；
4）Protocorms are forwarded in differential medium, temperature be 23 DEG C, light application time 12h/d, intensity of illumination be
Cultivated 27 days under conditions of 2300lux, obtain seedling, the differential medium is using MS culture mediums and adds 15g/L Succus Rhizoma Dioscoreaes
Formed with 0.4g/L methyl α-naphthyl acetates；
5）Seedling is forwarded in the tissue culture bottle containing seedling culture medium, temperature be 25 DEG C, light application time 15h/d, illumination
Intensity is cultivated 86 days under conditions of being 3000lux, obtains strong sprout, and the seedling culture medium promotes liquid group by MS culture mediums and nursery
Into the preparation method of the seedling culture medium is as follows：Japanese Cayratia Herb hay is taken, is cleaned, after drying, crushes, puts into percolator
In, diacolation processing is carried out using 70% alcohol of 11 times of weight, obtains percolate and diacolation residue, diacolation residue is added 8
70% alcohol of times weight, then ultrasonication 45min, filtering, obtains ultrasonic extract and ultrasonic wave extraction residue, will
Percolate and ultrasonic extract merge, and add the chitosan oligosaccharide of gross weight 5%, after being uniformly mixed, are concentrated by evaporation as original
The 15% of volume, produce nursery and promote liquid, nursery promotion liquid is added into MS culture mediums according to 20g/L amount, you can；
6）Tissue culture bottle containing strong sprout is sent into hardening room, carries out the hardening of 14 days, seedling after being refined；
7）Seedling after refining is taken out from tissue culture bottle, the culture medium of root carrying is removed using clear water, you can obtains tissue-cultured seedling.
Embodiment 5
A kind of candidum tissue culturing seedling breeding method of high-survival rate, step are as follows：
1）Ripe uncracked dendrobium candidum capsule is chosen, after being rinsed using clear water, is put into 0.1% mercuric chloride solution and soaks 4min,
After taking-up, using aseptic water washing 3 times, it is then placed in pretreatment fluid and soaks 10min, after taking-up, using aseptic water washing 5
It is secondary, surface moisture is blotted, obtains pretreatment dendrobium candidum capsule, the pretreatment fluid is made up of citric acid and alcohol, described pre-
The preparation method for the treatment of fluid is：10g citric acids are taken, are put into 100mL 85% alcohol, after being uniformly mixed, you can；
2）Pretreatment dendrobium candidum capsule is cut, by seed uniformly dispersing on germination medium, trained in tissue culture room
Support, cultivated 22 days under conditions of temperature is 24 DEG C, light application time 8h/d, intensity of illumination are 1200lux, obtain protocorm,
The germination medium promotes liquid to form by 1/2MS culture mediums and sprouting, and the preparation method of the germination medium is as follows：Take open country
Horse chases after, cleaning, after drying, crushes, puts into percolator, carries out diacolation processing using 70% alcohol of 15 times of weight, is oozed
Filter extract solution and diacolation residue, diacolation residue is added to 70% alcohol of 5 times of weight, then ultrasonication 50min, filtering, obtain
Ultrasonic extract and ultrasonic wave extraction residue are obtained, percolate and ultrasonic extract are merged, adds gross weight 3%
Chitosan oligosaccharide, after being uniformly mixed, be concentrated by evaporation as the 20% of original volume, produce sprouting and promote liquid, will sprout promote liquid according to
30g/L amount is added into 1/2MS culture mediums, you can；
3）Protocorm is forwarded in proliferated culture medium, temperature be 25 DEG C, light application time 8h/d, intensity of illumination be
Cultivated 25 days under conditions of 2500lux, obtain protocorms, the proliferated culture medium is using MS culture mediums and adds 10g/L perfume
Any of several broadleaf plants juice and 2g/L disodium ethylene diamine tetraacetates composition；
4）Protocorms are forwarded in differential medium, temperature be 25 DEG C, light application time 12h/d, intensity of illumination be
Cultivated 28 days under conditions of 2500lux, obtain seedling, the differential medium is using MS culture mediums and adds 15g/L Succus Rhizoma Dioscoreaes
Formed with 0.4g/L methyl α-naphthyl acetates；
5）Seedling is forwarded in the tissue culture bottle containing seedling culture medium, temperature be 25 DEG C, light application time 15h/d, illumination
Intensity is cultivated 90 days under conditions of being 3000lux, obtains strong sprout, and the seedling culture medium promotes liquid group by MS culture mediums and nursery
Into the preparation method of the seedling culture medium is as follows：Japanese Cayratia Herb hay is taken, is cleaned, after drying, crushes, puts into percolator
In, diacolation processing is carried out using 70% alcohol of 12 times of weight, obtains percolate and diacolation residue, diacolation residue is added 8
70% alcohol of times weight, then ultrasonication 50min, filtering, obtains ultrasonic extract and ultrasonic wave extraction residue, will
Percolate and ultrasonic extract merge, and add the chitosan oligosaccharide of gross weight 5%, after being uniformly mixed, are concentrated by evaporation as original
The 15% of volume, produce nursery and promote liquid, nursery promotion liquid is added into MS culture mediums according to 20g/L amount, you can；
6）Tissue culture bottle containing strong sprout is sent into hardening room, carries out the hardening of 15 days, seedling after being refined；
7）Seedling after refining is taken out from tissue culture bottle, the culture medium of root carrying is removed using clear water, you can obtains tissue-cultured seedling.
Comparative example 1
Compared with Example 3, pretreatment fluid is not used to be handled.Other are same as Example 3.
Comparative example 2
Compared with Example 3, using standard 1/2MS culture mediums as germination medium.Other are same as Example 3.
Comparative example 3
Compared with Example 3, using standard MS medium as seedling culture medium.Other are same as Example 3.
Survival test
Example 3 and 500 plants of tissue-cultured seedling prepared by comparative example 1-3 respectively, it is 1 to transplant to charcoal soil and liver moss volume ratio:1
In mixed-matrix, survival rate is counted.
Result of the test：The survival rate of embodiment 3 is 99%, and the survival rate of comparative example 1 is 96.2%, the survival rate of comparative example 2
For 95%, the survival rate of comparative example 3 is 90.6%.
As can be seen from the above results, the present invention is handled by using pretreatment fluid, using special sprouting culture
Base and the special seedling culture medium of use, be advantageous to improve the survival rate of candidum tissue culturing seedling.
Candidum tissue culturing seedling breeding method disclosed by the invention, the tissue-cultured seedling cultivated is healthy and strong, and transplanting survival rate is high, matter
Amount is stable, may advantageously facilitate the production-scale expansion of dendrobium candidum, has wide market prospects.Iron sheet stone disclosed by the invention
Dry measure used in former times tissue culture seeding cultivating method, is bred using tissue culture technology, and propagation efficiency is very high, can realize the big of selenium-enriched officinal dendrobium stem
Large-scale production, it is simple to operate, it is easy to spread.
The better embodiment of the present invention is explained in detail above, but the present invention is not limited to above-mentioned embodiment party
Formula, can also be on the premise of present inventive concept not be departed from one skilled in the relevant art's possessed knowledge
Various changes can be made.
Claims (8)
1. the candidum tissue culturing seedling breeding method of a kind of high-survival rate, it is characterised in that step is as follows：
1）Ripe uncracked dendrobium candidum capsule is chosen, after being rinsed using clear water, is put into 0.1% mercuric chloride solution and soaks 3-
4min, after taking-up, using aseptic water washing 2-3 times, it is then placed in pretreatment fluid and soaks 7-10min, after taking-up, use is sterile
Water rinses 4-5 times, blots surface moisture, obtains pretreatment dendrobium candidum capsule, the pretreatment fluid is by citric acid and alcohol group
Into；
2）Pretreatment dendrobium candidum capsule is cut, by seed uniformly dispersing on germination medium, trained in tissue culture room
Support, cultivated 19-22 days under conditions of temperature is 22-24 DEG C, light application time 8h/d, intensity of illumination are 1000-1200lux,
Obtain protocorm；
3）Protocorm is forwarded in proliferated culture medium, temperature be 23-25 DEG C, light application time 8h/d, intensity of illumination be
Cultivated 24-25 days under conditions of 2000-2500lux, obtain protocorms；
4）Protocorms are forwarded in differential medium, temperature be 23-25 DEG C, light application time 12h/d, intensity of illumination be
Cultivated 26-28 days under conditions of 2000-2500lux, obtain seedling；
5）Seedling is forwarded in the tissue culture bottle containing seedling culture medium, temperature be 23-25 DEG C, light application time 15h/d, light
Cultivated 80-90 days under conditions of being 2800-3000lux according to intensity, obtain strong sprout；
6）Tissue culture bottle containing strong sprout is sent into hardening room, carries out the hardening of 12-15 days, seedling after being refined；
7）Seedling after refining is taken out from tissue culture bottle, the culture medium of root carrying is removed using clear water, you can obtains tissue-cultured seedling.
2. the candidum tissue culturing seedling breeding method of high-survival rate according to claim 1, it is characterised in that the pre- place
Reason liquid preparation method be：10g citric acids are taken, are put into 100mL 85% alcohol, after being uniformly mixed, you can.
3. the candidum tissue culturing seedling breeding method of high-survival rate according to claim 1, it is characterised in that the sprouting
Culture medium promotes liquid to form by 1/2MS culture mediums and sprouting.
4. the candidum tissue culturing seedling breeding method of high-survival rate according to claim 3, it is characterised in that the sprouting
The preparation method of culture medium is as follows：Eupatorium lindleynun var. trifoliolatum is taken, is cleaned, after drying, crushes, puts into percolator, using 12-15 times of weight
70% alcohol carry out diacolation processing, obtain percolate and diacolation residue, diacolation residue added to 70% wine of 4-5 times of weight
Essence, then ultrasonication 40-50min, filtering, obtains ultrasonic extract and ultrasonic wave extraction residue, by percolate
Merge with ultrasonic extract, add the chitosan oligosaccharide of gross weight 3%, after being uniformly mixed, be concentrated by evaporation as the 20% of original volume,
Produce sprouting and promote liquid, promote liquid to be added according to 30g/L amount into 1/2MS culture mediums by sprouting, you can.
5. according to the candidum tissue culturing seedling breeding method of any described high-survival rates of claim 1-4, it is characterised in that institute
Proliferated culture medium is stated using MS culture mediums and adds 10g/L bananas juices and 2g/L disodium ethylene diamine tetraacetates composition.
6. the candidum tissue culturing seedling breeding method of high-survival rate according to claim 5, it is characterised in that the differentiation
Culture medium is using MS culture mediums and adds 15g/L Succus Rhizoma Dioscoreaes and 0.4g/L methyl α-naphthyl acetates composition.
7. the candidum tissue culturing seedling breeding method of high-survival rate according to claim 5, it is characterised in that the nursery
Culture medium promotes liquid to form by MS culture mediums and nursery.
8. the candidum tissue culturing seedling breeding method of high-survival rate according to claim 7, it is characterised in that the nursery
The preparation method of culture medium is as follows：Japanese Cayratia Herb hay is taken, is cleaned, after drying, crushes, puts into percolator, using 10-12
70% alcohol of times weight carries out diacolation processing, obtains percolate and diacolation residue, and diacolation residue is added into 6-8 times of weight
70% alcohol, then ultrasonication 40-50min, filtering, ultrasonic extract and ultrasonic wave extraction residue are obtained, by diacolation
Extract solution and ultrasonic extract merge, and add the chitosan oligosaccharide of gross weight 5%, after being uniformly mixed, are concentrated by evaporation as original volume
15%, produce nursery promote liquid, by nursery promotion liquid added according to 20g/L amount into MS culture mediums, you can.
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| CN103098713A (en) * | 2013-02-25 | 2013-05-15 | 富阳市文曲生态农业开发有限公司 | Dendrobium officinale domesticated breeding cultivation method |
| CN106359095A (en) * | 2016-08-29 | 2017-02-01 | 浙江金石生物科技有限公司 | Transplanting method of dendrobium candidum |
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| CN103098713A (en) * | 2013-02-25 | 2013-05-15 | 富阳市文曲生态农业开发有限公司 | Dendrobium officinale domesticated breeding cultivation method |
| CN106359095A (en) * | 2016-08-29 | 2017-02-01 | 浙江金石生物科技有限公司 | Transplanting method of dendrobium candidum |
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