CN106472319A - A kind of iris detoxification and fast breeding technique - Google Patents
A kind of iris detoxification and fast breeding technique Download PDFInfo
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- CN106472319A CN106472319A CN201610915724.8A CN201610915724A CN106472319A CN 106472319 A CN106472319 A CN 106472319A CN 201610915724 A CN201610915724 A CN 201610915724A CN 106472319 A CN106472319 A CN 106472319A
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- 238000001784 detoxification Methods 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000009395 breeding Methods 0.000 title claims abstract description 20
- 230000001488 breeding effect Effects 0.000 title claims abstract description 18
- 241000196324 Embryophyta Species 0.000 claims description 29
- 229920001817 Agar Polymers 0.000 claims description 26
- 229930006000 Sucrose Natural products 0.000 claims description 26
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 26
- 239000008272 agar Substances 0.000 claims description 26
- 239000005720 sucrose Substances 0.000 claims description 26
- 235000020415 coconut juice Nutrition 0.000 claims description 23
- 230000001954 sterilising effect Effects 0.000 claims description 20
- 238000004659 sterilization and disinfection Methods 0.000 claims description 20
- 238000005286 illumination Methods 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000012877 elongation medium Substances 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 7
- 230000004069 differentiation Effects 0.000 claims description 7
- 230000001939 inductive effect Effects 0.000 claims description 7
- 229920001542 oligosaccharide Polymers 0.000 claims description 7
- 230000003287 optical effect Effects 0.000 claims description 7
- 240000002514 Cymbidium ensifolium Species 0.000 claims description 6
- 229930024421 Adenine Natural products 0.000 claims description 5
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 5
- 241000218922 Magnoliophyta Species 0.000 claims description 5
- 241000921313 Phyllopodium Species 0.000 claims description 5
- 229960000643 adenine Drugs 0.000 claims description 5
- 239000000523 sample Substances 0.000 claims description 5
- 238000012549 training Methods 0.000 claims description 5
- 235000003140 Panax quinquefolius Nutrition 0.000 claims description 3
- 240000005373 Panax quinquefolius Species 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 241000233855 Orchidaceae Species 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 238000012546 transfer Methods 0.000 claims description 2
- 230000004083 survival effect Effects 0.000 abstract description 19
- 241000700605 Viruses Species 0.000 abstract description 12
- 230000008030 elimination Effects 0.000 abstract description 11
- 238000003379 elimination reaction Methods 0.000 abstract description 11
- 238000013459 approach Methods 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- 238000000053 physical method Methods 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 description 6
- 230000003000 nontoxic effect Effects 0.000 description 6
- 230000035800 maturation Effects 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 241000723826 Odontoglossum ringspot virus Species 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
- 241001505935 Phalaenopsis Species 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000737241 Cocos Species 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 241000732800 Cymbidium Species 0.000 description 1
- 241000710019 Cymbidium mosaic virus Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
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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
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to iris seedling breeding technical field is and in particular to a kind of iris detoxifying fast breeding method.The present invention by groping initial culture, stem apex elongation culture, virus-free culture, strong sprout, root culture, obtain a set of method and numerous approach soon carrying out secondary iris detoxification using physical method with reference to chemical reagent.Innovative point is to find out the method for stem apex elongation culture, growing point cuts size and combines chemical reagent and improves the method that survival rate and virus elimination rate, secondary detoxification improve virus elimination rate, establishes iris detoxification and numerous approach soon.The method has the advantages that survival rate, virus elimination rate are high, survival rate 80.13%, and virus elimination rate reaches 92.67%, has good technique effect and promotion prospect.
Description
(1) technical field：
The present invention relates to iris seedling breeding technical field is and in particular to a kind of iris detoxifying fast breeding method.
(2) background technology：
Iris (Phalaenopsis spp) belongs to tropical or semi-tropical aerial orchid, also known as phalaenopsis, because its attitude is graceful,
The beautiful in colour, florescence is lasting, have the laudatory title of " Cymbidium ensifolium (L.) Sw. queen ", is the important ornamental flower of indoor greening beautification, domestic and international city
Field is big to its demand.The virosiss of iris are the current a great problems perplexing blue boundary.
At present, more than 25 kinds, wherein endanger that the heaviest, distribution is the widest is sword-leaved cymbidium floral leaf to the virus being separated on iris
Viral (Cymbidium mosaic virus, CyMV) and odontoglossum ring spot virus (Odontoglossum ringspot virus,
ORSV).Disease plant mainly causes the symptoms such as floral leaf, necrosis, deformity, petal deformation, leads to plant strain growth bad, Hua little Er
Few, the florescence shortens, and has a strong impact on its ornamental values and the economic values.And in the domestic research with regard to iris detoxification survival rate is not
Higher than 30%, virus elimination rate is not higher than 80%, and therefore, a kind of survival rate of research is high, detoxification efficiency is good, and method easy and simple to handle has
Important realistic meaning.
(3) content of the invention
It is an object of the invention to provide a kind of iris detoxification and fast breeding technique, it includes following step：
1) selection of material and initial culture：
From robust growth, bloom 3-5, take viruliferous iris Flowering Plants after testing afterwards, cut bennet, 3-5cm
One section, often section comprise 1-2 full axillary bud, 75% ethanol surface sterilization 30-40s is used on superclean bench, then with use
0.1% HgCl2Concussion sterilization 8-10min, aseptic water washing 3-5 time, peel off axillary bud bract with probe forcepses, then 0.1%
HgCl2Concussion sterilization 2-3min, with aseptic water washing 5-8 time, excision is by HgCl2The two ends poisoned, are inoculated in 50mL inducing culture
First light culture 7-10 days on base, after proceed to illumination cultivation 2-3 month, treat that two panels true leaf launches to form Multiple Buds；
Described inducing culture is 1/3MS+6-BA5.0mg/L+NAA0.5mg/L+ coconut juice 100mL/L+ sucrose 20g/L+
Agar 6.0-6.5g/L, pH5.4-5.8；
During illumination cultivation, condition of culture is：25 ± 2 DEG C of temperature, intensity of illumination is 1500～2000lx, light irradiation time 8-10
Hour/sky；
2) stem apex elongation culture：
By step 1) Multiple Buds of lamina that induced proceed to 50ml and promote stem apex elongation medium, cultivate 40-60 days, treat
3rd or the 4th leaf maturation, obtain seedling；
Promoting stem apex elongation medium is：1/3MS+GA31～2mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0～
6.5g/L, pH5.4～5.8；
Condition of culture is：25 ± 2 DEG C of temperature, intensity of illumination is 1500～2000lx, light irradiation time 8～10 hour/day；
3) first time virus-free culture：
By step 2) seedling that obtains, be placed under the cold light source anatomical lens of 10X with dissecting needle strip a dimpling, vaulted,
Shinny growing point, cuts rapidly and is placed in 30mL virus-free culture primary surface, carries out light culture and proceeds to optical culture 2-3 after 7-15 days
Month, centre can carry out 1-2 switching, and anti-browning is dead, and every bottle of culture medium can inoculate the 3-5 stem apex containing growing point, and stem apex is cut
Take size to be 1-2mm, leave and take 1 phyllopodium；
Virus-free culture base is：1/3MS+6-BA5.0mg/L+NAA0.5mg/L+5% amino-oligosaccharide 0.2-0.3mL/L+ coconut palm
Juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8；
Condition of culture is：25 ± 2 DEG C of temperature, intensity of illumination is 1000～1500lx, light irradiation time 8～10 hour/day；
4) second virus-free culture：
By step 3) the detoxification stem apex that obtains, grow after 1 or 2 ripe lobule after it, carry out secondary virus-free culture, 10X
After stripping 1-2mm size growing point under cold light source anatomical lens, it is connected to rapidly 30mL virus-free culture primary surface, every bottle of culture medium inoculated
3-5 contains the stem apex of growing point, carries out light culture and proceeds to illumination cultivation 2-3 month after 7-15 days, monthly once transferred, changes
Culture medium, prevents browning dead.
Virus-free culture base is：1/3MS+6-BA5.0mg/L+NAA0.5mg/L+5% amino-oligosaccharide 0.2mL/L+ coconut juice
100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8；
Condition of culture is：25 ± 2 DEG C of temperature, intensity of illumination is 1000～1500lx, light irradiation time 8～10 hour/day；
5) enrichment culture of detoxic seedling：
By step 4) detoxic seedling that obtains, carry out enrichment culture after detection, excision blade and browning part are connected to Sheng 100mL
The Cymbidium ensifolium (L.) Sw. bottle of culture medium, inoculates 5 plants (after quantity increases, can increase to 21-35 strain/bottle depending on the condition of production) for every bottle；Culture 2-3
After individual month, robust growth, growth coefficient reaches 2-4, obtains final product detoxification differentiation Seedling；
Proliferated culture medium is：1/3MS+6-BA5.0mg/L+NAA0.5mg/L+ adenine 3-5mg/L+ coconut juice 100mL/L+
Sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8；
Condition of culture same 1).
6) strong seedling culture of detoxic seedling：
Treat step 5) to plant height 2-3cm, two leaves wholeheartedly proceed to Sheng 120mL strong seedling culture base to the detoxification that obtains differentiation Seedling length afterwards
Cymbidium ensifolium (L.) Sw. bottle after carry out strong seedling culture, transfer 16 plants for every bottle；After culture 45-60 days, plant height reaches 3-5cm, and wholeheartedly strong sprout is complete for two leaves
Become.
Strong seedling culture base is：1/2MS+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8.
Condition of culture:25 ± 2 DEG C of temperature, intensity of illumination is 1500～2000lx, light irradiation time 8～10 hour/day；
7) root culture of detoxic seedling：
By step 6) plant after the strong sprout that obtains, it is connected in the Cymbidium ensifolium (L.) Sw. bottle of 150mL root media and carries out root culture, often
Bottle switching 11-13 strain；After culture 3-4 month, to 3-4 piece leaf, root system reaches more than 3 to plant to be planted length, healthy and strong vibrant, you can to move
Enter greenhouse rooting culture.
Root media：1/2MS+NAA1.0mg/L+ Fructus Musae 30g/L+ Rhizoma Solani tuber osi 40g/L+ sucrose 20g/L+ agar 6.0-
6.5g/L, pH5.4～5.8.
Condition of culture:25 ± 2 DEG C of temperature, intensity of illumination is 1500～2000lx, light irradiation time 8～10 hour/day.
The present invention, by groping initial culture, stem apex elongation culture, virus-free culture, strong sprout, root culture, obtains an arbitrage
Carry out method and numerous approach soon of secondary iris detoxification with physical method with reference to chemical reagent.Innovative point is to find out stem apex
The method of elongation culture, growing point cut size and combine chemical reagent raising survival rate and virus elimination rate, secondary detoxification raising detoxification
The method of rate, establishes iris detoxification and numerous approach soon.The method has the advantages that survival rate, virus elimination rate are high, survival rate
80.13%, virus elimination rate reaches 92.67%, has good technique effect and promotion prospect.
Specific embodiment
Embodiment 1
Described iris detoxification and fast breeding technique, comprise the following steps that：
Firstth, from robust growth, bloom 3-5, take viruliferous iris Flowering Plants after testing afterwards, cut bennet
Intercept 3cm/ saves, and often saves 1 full axillary bud.75% ethanol surface sterilization 30s, 0.1% HgCl2Concussion sterilization 8min is (outward
After the concussion of implant surface till bubble-free), aseptic water washing 3 times, peel off axillary bud bract with probe forcepses, then 0.1% HgCl2Shake
Swing sterilization 3min, with aseptic water washing 5 times, excision is subject to HgCl2The two ends poisoned, are inoculated on inducing culture and carry out induction training
Support.
Secondth, by the Multiple Buds of lamina, plant division is connected to rush stem apex elongation medium, after cultivating 45 days, the 3rd leaf maturation.
Promoting stem apex elongation medium is：1/3MS+GA31.0mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L,
pH5.4-5.8.Condition of culture same 1).
3rd, seedling is excised after blade, be placed in and strip stem apex with dissecting needle under the cold light source anatomical lens of 10X, quickly cut
Under be placed in virus-free culture primary surface, proceed to optical culture 2 months after carrying out light culture 7 days, centre carry out 1 time switching.Each culture
Base can inoculate 4 stem apexs, and it is 1.0mm that stem apex cuts size, leaves and takes 1 phyllopodium.Virus-free culture base is：1/3MS+6-
BA5.0mg/L+NAA0.5mg/L+5% amino-oligosaccharide 0.2mL/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/
L, pH5.4-5.8.After growing two panels blade, carry out secondary detoxification.
4th, after nontoxic detection, nontoxic Seedling is carried out enrichment culture.Proliferated culture medium is：
1/3MS+6-BA5.0mg/L+NAA0.5mg/L+ adenine 3mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar
6.0-6.5g/L, pH5.4～5.8.Condition of culture same 1).
5th, treat detoxification differentiation Seedling length to plant height 2-3cm, two leaves wholeheartedly carry out strong seedling culture afterwards, and strong seedling culture base is：1/
2MS+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8.Condition of culture same 1).
6th, treat strong sprout plant length to plant height 3-5cm, two leaves wholeheartedly carry out root culture afterwards, culture 3-4, after individual month, plants
Strain is long, and to 3-4 piece leaf, root system reaches more than 3, healthy and strong vibrant, you can to move into greenhouse rooting culture.
In iris Primary culture in embodiment 1, secondary sterilization survival rate is 89.7%, compared with disposable sterilization survival rate
81%, improve 10.7%.After stem apex detoxification 60 days, survival rate 64.74%, virus elimination rate reaches 94%.
Embodiment 2
Described iris detoxification and fast breeding technique, comprise the following steps that：
Firstth, from robust growth, bloom 3-5, detection after take viruliferous iris Flowering Plants, cut bennet and cut
Section 4cm/ section, often saves 1 full axillary bud.75% ethanol surface sterilization 30s, 0.1% HgCl2Concussion sterilization 9min (explant
After the concussion of body surface face till bubble-free), aseptic water washing 3 times, peel off axillary bud bract with probe forcepses, then 0.1% HgCl2Concussion
Sterilization 2min, with aseptic water washing 5 times, excision is subject to HgCl2The two ends poisoned, are inoculated on inducing culture and carry out Multiple Buds and lure
Lead culture.
Secondth, by the Multiple Buds of lamina, plant division is connected to rush stem apex elongation medium, after cultivating 50 days, the 3rd leaf maturation.
Promoting stem apex elongation medium is：1/3MS+GA31.5mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L,
pH5.4-5.8.Condition of culture same 1).
3rd, seedling is excised after blade, be placed in and strip stem apex with dissecting needle under the cold light source anatomical lens of 10X, quickly cut
Under be placed in virus-free culture primary surface, proceed to optical culture 2 months after carrying out light culture 7 days, centre carry out 1 time switching.Each culture
Base can inoculate 4 stem apexs, and it is 1.5mm that stem apex cuts size, leaves and takes 1 phyllopodium.Virus-free culture base is：1/3MS+6-
BA5.0mg/L+NAA0.5mg/L+5% amino-oligosaccharide 0.25mL/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-
6.5g/L, pH5.4-5.8.After growing two panels blade, carry out secondary detoxification.
4th, after nontoxic detection, nontoxic Seedling is carried out enrichment culture.Proliferated culture medium is：
1/3MS+6-BA5.0mg/L+NAA0.5mg/L+ adenine 3mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar
6.0-6.5g/L, pH5.4～5.8.Condition of culture same 1).
5th, treat detoxification differentiation Seedling length to plant height 2-3cm, two leaves wholeheartedly carry out strong seedling culture afterwards, and strong seedling culture base is：1/
2MS+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8.Condition of culture same 1).
6th, treat strong sprout plant length to plant height 3-5cm, two leaves wholeheartedly carry out root culture afterwards, culture 3-4, after individual month, plants
Strain is long, and to 3-4 piece leaf, root system reaches more than 3, healthy and strong vibrant, you can to move into greenhouse rooting culture.
In iris Primary culture in embodiment 2, secondary sterilization survival rate is 93%, compared with disposable sterilization survival rate 81%,
Improve 14.8%.After stem apex detoxification 60 days, survival rate 68.74%, virus elimination rate reaches 93.1%.
Embodiment 3
Described iris detoxification and fast breeding technique, comprise the following steps that：
Firstth, from robust growth, bloom 3-5, detection after take viruliferous iris Flowering Plants, cut bennet and cut
Section 5cm/ section, often saves 2 full axillary buds.75% ethanol surface sterilization 30s, 0.1% HgCl2Concussion sterilization 10min (explant
After the concussion of body surface face till bubble-free), aseptic water washing 3 times, peel off axillary bud bract with probe forcepses, then 0.1% HgCl2Concussion
Sterilization 3min, with aseptic water washing 5 times, excision is subject to HgCl2The two ends poisoned, are inoculated on inducing culture and carry out Multiple Buds and lure
Lead culture.
Secondth, by the Multiple Buds of lamina, plant division is connected to rush stem apex elongation medium, after cultivating 50 days, the 3rd leaf maturation.
Promoting stem apex elongation medium is：1/3MS+GA32.0mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L,
pH5.4-5.8.Condition of culture same 1).
3rd, seedling is excised after blade, be placed in and strip stem apex with dissecting needle under the cold light source anatomical lens of 10X, quickly cut
Under be placed in virus-free culture primary surface, proceed to optical culture 2 months after carrying out light culture 7 days, centre carry out 1 time switching.Each culture
Base can inoculate 4 stem apexs, and it is 2.0mm that stem apex cuts size, leaves and takes 1 phyllopodium.Virus-free culture base is：1/3MS+6-
BA5.0mg/L+NAA0.5mg/L+5% amino-oligosaccharide 0.3mL/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/
L, pH5.4-5.8.After growing two panels blade, carry out secondary detoxification.
4th, after nontoxic detection, nontoxic Seedling is carried out enrichment culture.Proliferated culture medium is：
1/3MS+6-BA5.0mg/L+NAA0.5mg/L+ adenine 3mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar
6.0-6.5g/L, pH5.4～5.8.Condition of culture same 1).
5th, treat detoxification differentiation Seedling length to plant height 2-3cm, two leaves wholeheartedly carry out strong seedling culture afterwards, and strong seedling culture base is：1/
2MS+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8.Condition of culture same 1).
6th, treat strong sprout plant length to plant height 3-5cm, two leaves wholeheartedly carry out root culture afterwards, culture 3-4, after individual month, plants
Strain is long, and to 3-4 piece leaf, root system reaches more than 3, healthy and strong vibrant, you can to move into greenhouse rooting culture.
In iris Primary culture in embodiment 3, secondary sterilization survival rate is 93.2%, compared with disposable sterilization survival rate
81%, improve 15.1%.After stem apex detoxification 60 days, survival rate 76.74%, virus elimination rate reaches 87.3%.
Conclusion：By using the present invention carry out iris detoxification and numerous soon when, survival rate, de- in iris Primary culture
Malicious survival rate, detoxification success ratio are all higher., during late maintaining, sickness rate is relatively low for iris after detoxification, and flower is larger,
Florescence is long, is difficult bud, and blade chlorisis, abnormal phenomenon are improved, and substantially increase its ornamental value and economic worth.
Claims (10)
1. a kind of iris detoxification and fast breeding technique are it is characterised in that it includes following step：
1) selection of material and initial culture：
From robust growth, bloom 3-5, take viruliferous iris Flowering Plants after testing afterwards, cut bennet, 3-5cm mono- saves,
Often section comprises 1-2 full axillary bud, on superclean bench with 75% ethanol surface sterilization 30-40s, then with 0.1%
HgCl2Concussion sterilization 8-10min, aseptic water washing 3-5 time, peel off axillary bud bract with probe forcepses, then 0.1% HgCl2Concussion disappears
Malicious 2-3min, with aseptic water washing 5-8 time, excision is subject to HgCl2The two ends poisoned, are inoculated on 50mL inducing culture first dark training
Foster 7-10 days, after proceed to illumination cultivation 2-3 month, treat that two panels true leaf launches to form Multiple Buds；
2) stem apex elongation culture：
By step 1) Multiple Buds of lamina that induced proceed to 50ml and promote stem apex elongation medium, cultivate 40-60 days, treat the 3rd
Piece or the 4th leaf are ripe, obtain seedling；
3) first time virus-free culture：
By step 2) seedling that obtains, it is placed in and strip a dimpling, vaulted, shinny with dissecting needle under the cold light source anatomical lens of 10X
Growing point, cut rapidly and be placed in 30mL virus-free culture primary surface, carry out light culture and after 7-15 days, proceed to optical culture 2-3 month,
Centre can carry out 1-2 switching, and anti-browning is dead, and every bottle of culture medium can inoculate the 3-5 stem apex containing growing point, and stem apex cuts greatly
Little for 1-2mm, leave and take 1 phyllopodium；
4) second virus-free culture：
By step 3) the detoxification stem apex that obtains, grow after 1 or 2 ripe lobule after it, carry out secondary virus-free culture, 10X cold light
After stripping 1-2mm size growing point under the anatomical lens of source, it is connected to rapidly 30mL virus-free culture primary surface, every bottle of culture medium inoculated 3-5
Stem apex containing growing point, carries out light culture and proceeds to illumination cultivation 2-3 month after 7-15 days, monthly once transferred, change training
Foster base, prevents browning dead；
5) enrichment culture of detoxic seedling：
By step 4) detoxic seedling that obtains, carry out enrichment culture after detection, excision blade and browning part are connected to Sheng 100mL culture
The Cymbidium ensifolium (L.) Sw. bottle of base, inoculates 5 plants for every bottle；After culture 2-3 month, robust growth, growth coefficient reaches 2-4, obtains final product detoxification differentiation Seedling；
6) strong seedling culture of detoxic seedling：
Treat step 5) to plant height 2-3cm, two leaves wholeheartedly proceed to the orchid containing 120mL strong seedling culture base to the detoxification that obtains differentiation Seedling length afterwards
Carry out strong seedling culture after vase, transfer 16 plants for every bottle；After culture 45-60 days, plant height reaches 3-5cm, and wholeheartedly strong sprout completes two leaves；
7) root culture of detoxic seedling：
By step 6) plant after the strong sprout that obtains, it is connected in the Cymbidium ensifolium (L.) Sw. bottle of 150mL root media and carries out root culture, every bottle turns
Connect 11-13 strain；After culture 3-4 month, to 3-4 piece leaf, root system reaches more than 3 to plant to be planted length, healthy and strong vibrant, you can to move into temperature
Room rooting culture.
2. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 1) described in inducing culture
Base is 1/3MS+6-BA5.0mg/L+NAA0.5mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4-
5.8.
3. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 1) in illumination cultivation when, training
Foster condition is：25 ± 2 DEG C of temperature, intensity of illumination is 1500～2000lx, light irradiation time 8-10 hour/sky.
4. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 2) described in rush stem apex stretch
Long culture medium is：1/3MS+GA31～2mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0～6.5g/L, pH5.4～
5.8.
5. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 2) in culture 40-60 days when
Condition of culture be：25 ± 2 DEG C of temperature, intensity of illumination is 1500～2000lx, light irradiation time 8～10 hour/day.
6. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 3) in virus-free culture base be：
1/3MS+6-BA5.0mg/L+NAA0.5mg/L+5% amino-oligosaccharide 0.2-0.3mL/L+ coconut juice 100mL/L+ sucrose 20g/L+
Agar 6.0-6.5g/L, pH5.4～5.8；During optical culture, condition of culture is：25 ± 2 DEG C of temperature, intensity of illumination be 1000～
1500lx, light irradiation time 8～10 hour/day.
7. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 5) described in enrichment culture
Base is：1/3MS+6-BA5.0mg/L+NAA0.5mg/L+ adenine 3-5mg/L+ coconut juice 100mL/L+ sucrose 20g/L+ agar
6.0-6.5g/L, pH5.4～5.8.
8. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 6) described in strong seedling culture
Base is：1/2MS+ coconut juice 100mL/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8；Condition of culture:Temperature 25 ± 2
DEG C, intensity of illumination is 1500～2000lx, light irradiation time 8～10 hour/day.
9. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 7) described in root culture
Base：1/2MS+NAA1.0mg/L+ Fructus Musae 30g/L+ Rhizoma Solani tuber osi 40g/L+ sucrose 20g/L+ agar 6.0-6.5g/L, pH5.4～5.8；
Condition of culture:25 ± 2 DEG C of temperature, intensity of illumination is 1500～2000lx, light irradiation time 8～10 hour/day.
10. iris detoxification as claimed in claim 1 and fast breeding technique are it is characterised in that step 4) described in detoxification training
Foster base is：1/3MS+6-BA5.0mg/L+NAA0.5mg/L+5% amino-oligosaccharide 0.2mL/L+ coconut juice 100mL/L+ sucrose 20g/
L+ agar 6.0-6.5g/L, pH5.4～5.8；Condition of culture during optical culture is：25 ± 2 DEG C of temperature, intensity of illumination be 1000～
1500lx, light irradiation time 8～10 hour/day.
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