CN107455258B - The method for tissue culture of mini iris detoxic seedling - Google Patents
The method for tissue culture of mini iris detoxic seedling Download PDFInfo
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- CN107455258B CN107455258B CN201710729775.6A CN201710729775A CN107455258B CN 107455258 B CN107455258 B CN 107455258B CN 201710729775 A CN201710729775 A CN 201710729775A CN 107455258 B CN107455258 B CN 107455258B
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- 238000000034 method Methods 0.000 title claims abstract description 17
- 230000003612 virological effect Effects 0.000 claims abstract description 8
- 238000005520 cutting process Methods 0.000 claims abstract description 6
- 238000000338 in vitro Methods 0.000 claims abstract description 6
- 238000010438 heat treatment Methods 0.000 claims abstract description 5
- 238000005286 illumination Methods 0.000 claims description 26
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 15
- 241000234295 Musa Species 0.000 claims description 15
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 15
- 229910052799 carbon Inorganic materials 0.000 claims description 15
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 7
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 150000003851 azoles Chemical class 0.000 claims description 2
- 239000002574 poison Substances 0.000 claims description 2
- 231100000614 poison Toxicity 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 24
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 238000012090 tissue culture technique Methods 0.000 abstract description 5
- 239000002023 wood Substances 0.000 abstract description 2
- 229920001817 Agar Polymers 0.000 description 9
- 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 description 9
- 229930006000 Sucrose Natural products 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- 239000005720 sucrose Substances 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 7
- 241000723826 Odontoglossum ringspot virus Species 0.000 description 6
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 6
- 229940100050 virazole Drugs 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000710019 Cymbidium mosaic virus Species 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000012879 subculture medium Substances 0.000 description 4
- 230000009182 swimming Effects 0.000 description 4
- 241000233855 Orchidaceae Species 0.000 description 3
- 241001505935 Phalaenopsis Species 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 230000002073 mitogenetic effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 241000193006 Aphrodita Species 0.000 description 1
- 101100532034 Drosophila melanogaster RTase gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 244000127818 Phalaenopsis amabilis Species 0.000 description 1
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- 238000001035 drying Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002161 passivation Methods 0.000 description 1
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Classifications
-
- 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
-
- 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
Landscapes
- 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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to field of biotechnology, and in particular to the method for tissue culture of mini iris detoxic seedling.The present invention adopts the following technical scheme that, the method for tissue culture of mini iris detoxic seedling includes the following steps: that choosing mini iris in vitro cuttings is heat-treated, stem apex is taken to carry out Initial culture Multiple Buds from the in vitro cuttings after heat treatment, squamous subculture is carried out on the basis of Initial culture, the Multiple Buds survived are subjected to viral diagnosis, it picks out viral negative Multiple Buds and carries out squamous subculture, finally carry out culture of rootage.The present invention carries out seedling-wood breeding using tissue culture technique, can greatly speed up the breeding speed of mini iris.Seedling quality is high, thus improves ornamental value: can remove the main virus of mini iris using tissue culture technique, eliminates the harm shape of virus, improves the ornamental value of mini iris.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to the method for tissue culture of mini iris detoxic seedling.
Background technique
Mini iris is also known as small gaily-colored butterfly orchid, is the change of iris Phalaenopsis aphrodita Rchb.F.
Kind, height is commonly referred to as in 20-25cm, the iris that can be placed in 2 cun of basin devices.It is instant packed since its is small,
Foreign countries are often used as exquisite gift.Since the cultivation period of mini iris is short, take out from tissue culture bottle to the stalk stage can be urged
It only needs 7-9 months, recovery of the capital is fast, therefore becomes the first choice of orchid industry catechumen.
Although hardly carrying virus by the seed seedling of sexual propagation, and there are the advantages such as plant strain growth speed is fast,
It is since the uneven and pattern of its plant height is different, commodity property is poor, and production quantity is greatly lowered year by year.And pass through
The mitogenetic seedling of tissue culture technique (vegetative propagation) production gradually substitutes seed seedling, at present because its plant is consistent with pattern height
90% or more of the market share is occupied.But long-term vegetative propagation inevitably results in the accumulation of virus, and kind is caused increasingly to move back
Change, quality decline.Orchid virus is always the important disease of one kind for seriously endangering iris quality, and host range is extensive, easily
It infects, prevention and treatment is difficult.At present in the world it has been reported that orchid virus have 25 kinds, wherein with cymbidium mosaic virus (Cymbidium
Mosaic virus, CymMV) and odontoglossum ring spot virus (Odontoglossum Ring Spot Virus, ORSV) generation is the most
Generally, also most serious is endangered.Both viruses can make Phalaenopsis leaves form chlorisis striped, be recessed grey hickie or downright bad ring spot,
Cause plant strain growth bad, spend less, the florescence shortens, and frequent compound infection, harm aggravation, make iris ornamental value and
Economic value sharp fall.To avoid iris virus is extensive from occurring, virus is had to pass through before the mitogenetic breeding of parent
Detection, iris virus are also that nursery stock export quarantine must examine object.In order to improve the competitiveness of iris bottle seedling, ORSV is removed
With CymMV and more viruses, the inexorable trend that high-quality detoxic seedling is iris production development is cultivated.
Summary of the invention
To make up the deficiencies in the prior art, the present invention provides a kind of method for tissue culture of mini iris detoxic seedling, should
Method obtains seedling quality height, deviates from the main virus of mini iris, eliminates the harm shape of virus, improves mini butterfly
Blue ornamental value.
The present invention adopts the following technical scheme that the method for tissue culture of mini iris detoxic seedling includes the following steps: to select
It takes mini iris in vitro cuttings to be heat-treated, stem apex is taken to carry out Initial culture clump from the in vitro cuttings after heat treatment
It sprouts, squamous subculture is carried out on the basis of Initial culture, the Multiple Buds survived are subjected to viral diagnosis, pick out viral feminine gender
Multiple Buds carry out squamous subculture, finally carry out culture of rootage.Wherein, in initial culture base 6-BA concentration be 3.0-4.0mg/L,
NAA concentration is 0.3-0.5mg/L, banana puree 25-35g/L, active carbon 2-4g/L；In subculture medium, 6-BA concentration is
7.0-9.0mg/L, NAA concentration is 0.2-0.4mg/L, banana puree 25-35g/L, active carbon 2-4g/L；In root media
6-BA concentration be 3.5-4.5mg/L, NAA concentration be 0.7-0.9mg/L, banana puree 25-35g/L, active carbon 2-4g/L.
Preferably, taking mini iris stem apex size is 0.5-1.0mm.
Preferably, the sodium bicarbonate that concentration is 1-5/L and the virus that concentration is 20-40mg/L are additionally added in initial culture base
Azoles.
Preferably, the condition of the Initial culture are as follows: carry out two weeks dark cultures first, then in 25 DEG C of temperature, illumination is strong
2000lx, periodicity of illumination 14h/d are spent, surrounding is cultivated.
Since plant virus is to be propagated in plant by conducting tissue, and the separate living tissue of plant is due to dividing
It splits and forms conducting tissue quickly and not yet, therefore there is no virus in separate living tissue, the present invention takes inhibition and passivation virus
Heat treatment and chemical reagent handle explant, by the culture of different phase, by the virus detection techniques on molecular level,
The mini iris nursery stock that the main virus of removing may finally be obtained carries out mini iris without main virus
Quickly breeding.
It compared with prior art, can the beneficial effects of the present invention are: due to carrying out seedling-wood breeding using tissue culture technique
To greatly speed up the breeding speed of mini iris.Seedling quality is high, thus improves ornamental value: using tissue culture technique can be with
The main virus for deviating from mini iris eliminates the harm shape of virus, improves the ornamental value of mini iris.
Detailed description of the invention
Fig. 1 is to be endangered blade figure by iris virus；Wherein, left figure is chlorisis striped, and right figure is downright bad ring spot；
Fig. 2 is mini iris CyMV RT-PCR product electrophoretogram；Wherein, swimming lane 1,2,3,4 represent testing result as sun
Property, swimming lane 5,6 represent testing result as feminine gender；431bp is CyMV specific band；
Fig. 3 is mini iris ORSV RT-PCR product electrophoretogram, wherein swimming lane 1,2,3,4 represents testing result as yin
Property, swimming lane 5,6,7,8 represent testing result as the positive；332bp is ORSV specific band.
Specific embodiment
The present invention is described in detail below by the drawings and specific embodiments, but is not limited the scope of the invention.Such as without special
Illustrate, experimental method of the present invention is conventional method, and experiment equipment used, material, reagent etc. can be chemically public
Department's purchase.
Embodiment 1
The mini iris in vitro cuttings with poison are taken, are placed it in alternating temperature illumination box.Temperature when starting to process
It is 30 DEG C, increases by 1 DEG C after duration every other day and continue the mini iris of heat treatment culture, utilize the side for being stepped up temperature
Method makes mini iris adapt to the temperature condition being heat-treated, until 38 DEG C of temperature upper limit.After reaching 38 DEG C, continue culture 4 weeks, it is wet
Degree keeps 80% or so, and illumination condition is daily 16h, intensity of illumination 3000Lux.Still healthy mini butterfly after being heat-treated 4 weeks
Phalaenopsis aseptic seedling, strips shoot tip meristem under anatomical lens, and stem apex size is 0.5mm.It is seeded in initial culture base 1/2MS
On+6-BA3.0mg/L+NAA0.3mg/L+ agar 8g/L+ sucrose 30g/L+ banana puree 30g/L+ active carbon 2g/L, it is commissioned to train just
It supports and virazole and sodium bicarbonate is added in base, the concentration that virazole uses is 20mg/L, and the use concentration of sodium bicarbonate is 1.0g/
L.After first carrying out two weeks dark cultures, continue by condition of culture: 25 DEG C of temperature, intensity of illumination 2000lx, periodicity of illumination 14h/d,
Culture 4 weeks, the stem apex Multiple Buds survived are continued to be seeded in subculture medium (1/2MS+6-BA8.0mg/L+NAA0.2mg/L+
Agar 8g/L+ sucrose 30g/L+ banana puree 30g/L+ active carbon 2g/L) on, the Multiple Buds survived are subjected to extension culture, are trained
The condition of supporting are as follows: 25 DEG C of temperature, then intensity of illumination 2000lx, periodicity of illumination 14h/d carry out viral diagnosis.To nontoxic unrooted
Seedling carries out culture of rootage, and root media used is 1/2MS+6-BA4.0mg/L+NAA0.8mg/L+ agar 8g/L+ sucrose 30g/
L+ banana puree 30g/L+ active carbon 2g/L, culture of rootage condition are as follows: 25 DEG C of temperature, intensity of illumination 2000lx, periodicity of illumination 14h/
d。
Embodiment 2
Mini iris processing is the same as embodiment 1.Shoot tip meristem is stripped under anatomical lens, stem apex size is 0.8mm.By its
It is seeded in initial culture base 1/2MS+6-BA 3.5mg/L+0.4mg/LNAA agar 8g/L+ sucrose 30g/L+ banana puree 35g/L+
On active carbon 3g/L, virazole and sodium bicarbonate are added in initial culture base, the concentration that virazole uses is 30mg/L, carbonic acid
The use concentration of hydrogen sodium is 3.0g/L.After first carrying out two weeks dark cultures, continue by condition of culture: 25 DEG C of temperature, intensity of illumination
2000lx, periodicity of illumination 14h/d are cultivated 4 weeks, the stem apex Multiple Buds survived are continued to be seeded in subculture medium (1/2MS+6-
BA7.0mg/L+NAA0.3mg/L+ agar 8g/L+ sucrose 30g/L+ banana puree 35g/L+ active carbon 3g/L) on, the clump that will survive
It sprouts and carries out extension culture, condition of culture are as follows: 25 DEG C of temperature, then intensity of illumination 2000lx, periodicity of illumination 14h/d are carried out
Viral diagnosis.Culture of rootage is carried out to nontoxic no offspring, root media used is 1/2MS+6-BA4.5mg/L+
NAA0.9mg/L+ agar 8g/L+ sucrose 30g/L+ banana puree 35g/L+ active carbon 3g/L, culture of rootage condition are as follows: 25 DEG C of temperature,
Intensity of illumination 2000lx, periodicity of illumination 14h/d.
Embodiment 3
Mini iris processing is the same as embodiment 1.Shoot tip meristem is stripped under anatomical lens, stem apex size is 1.0mm.By its
It is seeded in initial culture base 1/2MS+6-BA4.0mg/L+NAA0.5mg/L+ agar 8g/L+ sucrose 30g/L+ banana puree 25g/L+
On active carbon 4g/L, virazole and sodium bicarbonate are added in initial culture base, the concentration that virazole uses is 40mg/L, carbonic acid
The use concentration of hydrogen sodium is 5.0g/L.After first carrying out two weeks dark cultures, continue by condition of culture: 25 DEG C of temperature, intensity of illumination
2000lx, periodicity of illumination 14h/d are cultivated 4 weeks, the stem apex Multiple Buds survived are continued to be seeded in subculture medium (1/2MS+6-
BA9.0mg/L+NAA0.4mg/L+ agar 8g/L+ sucrose 30g/L+ banana puree 25g/L+ active carbon 4g/L) on, the clump that will survive
It sprouts and carries out extension culture, condition of culture are as follows: 25 DEG C of temperature, then intensity of illumination 2000lx, periodicity of illumination 14h/d are carried out
Viral diagnosis.Culture of rootage is carried out to nontoxic no offspring, root media used is 1/2MS+6-BA3.5mg/L+
NAA0.7mg/L+ agar 8g/L+ sucrose 30g/L+ banana puree 25g/L+ active carbon 4g/L, culture of rootage condition are as follows: 25 DEG C of temperature,
Intensity of illumination 2000lx, periodicity of illumination 14h/d.
Test example
The main virus RT-PCR testing result of iris:
The extraction of the mini iris total serum IgE of one:
(1) the mini iris tissue-cultured seedling young leaflet tablet of 500mg is taken, liquid feeding nitrogen is ground into powder, and moves into 1.5mL rapidly
In Eppendorf pipe, 600 μ LRNA Extraction buffers (pH8.0) are added, 1 μ LRNasin is mixed.4 DEG C, 12 000rpm, centrifugation
15min。
(2) supernatant is taken, isometric chloroform: isoamyl alcohol (24:1) solution is added, is mixed, 4 DEG C, 12000rpm, centrifugation
10min.It repeats 1~2 time, removes albumin layer.
(3) water intaking phase, adds the dehydrated alcohol and 1/10 volume 3mol/LNaAc (pH5.3) of 2.5 times of volumes, mixes.-20℃
Set 2h.After taking-up, 4 DEG C, 12000rpm, it is centrifuged 15min.
(4) supernatant is abandoned, washes precipitating with 200 μ L, 70% dehydrated alcohol, 4 DEG C, 12000rpm, 5min, cleaning is multiple, until heavy
Forming sediment is pure white.
(5) 15min or so, is dissolved in 30 μ L, 1 ‰ DEPC water after drying at room temperature, and -20 DEG C of refrigerators save.
Two .CyMV RT-PCR detection architectures:
1.RT optimization system and program:
(1) reaction system:
5×Buffer 2.0μl；dNTPs 0.25mmol/L；RNasin 8U；1 μm of ol/L of primer CyMV-RP；Total serum IgE
1.0μl；Mo-MLVRTase 50U.10 μ l are supplied with DEPC water.
(2) response procedures: 37 DEG C of 2h；95 DEG C of 5min inactivate MO-MLVRTase；4 DEG C of preservations after reaction.
2.PCR optimization system and program:
(1) reaction system:
10×Buffer 2.5μl；dNTPs 0.2mmol/L；MgCl21.5mmol/L；CyMV-FP 0.8μmol/L；
CyMV-RP 0.8μmol/L；TagE 1U；10 μ l of RT product.25 μ l are supplied with DEPC water.
(2) response procedures: 94 DEG C of 1min；55℃2min；72℃2min；Circulation 35 times, last, which is taken turns, extends 5min, and 4 DEG C
It saves.
Three .ORSVRT-PCR detection architectures:
1.RT system: 5 × Buffer, 1.0 μ L；dNTPs 0.25mmol/L；RNasin 8U；2 μ of primer ORSV-RP
mol/L；1.0 μ L of total serum IgE；Mo-MLVRTase 50U.10 μ L are supplied with 1 ‰ DEPC water.
RT:37 DEG C of 2h is carried out by following response procedures；95 DEG C of 5min inactivate MO-MLV RTase；4 DEG C of guarantors after reaction
It deposits.
2.PCR amplification system is as follows: 10 × Buffer, 2.0 μ L；dNTPs 0.2mmol/L；MgCl21.5mmol/L；
ORSV-FP 0.8μmol/L；ORSV-RP 0.8μmol/L；TagE 0.5U；10 μ L of RT product.25 μ L are supplied with 1 ‰ DEPC water.
Response procedures are as follows: 94 DEG C of 1min；55℃2min；72℃2min；Circulation 35 times, last wheel extend 5min, 4 DEG C of guarantors
It deposits.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art within the technical scope of the present disclosure, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (1)
1. the method for tissue culture of mini iris detoxic seedling, which comprises the steps of: choose mini iris without
Bacterium test tube seedling is heat-treated, and is taken stem apex to carry out Initial culture Multiple Buds from the in vitro cuttings after heat treatment, is taken mini
Iris stem apex size is 0.5-1.0mm；Squamous subculture is carried out on the basis of Initial culture, and the Multiple Buds survived are subjected to disease
Poison detection picks out viral negative Multiple Buds and carries out squamous subculture, finally carries out culture of rootage, wherein 6- in initial culture base
BA concentration be 3.0-4.0mg/L, NAA concentration be 0.3-0.5mg/L, banana puree 25-35g/L, active carbon 2-4g/L；Subculture
In culture medium, 6-BA concentration be 7.0-9.0mg/L, NAA concentration be 0.2-0.4mg/L, banana puree 25-35g/L, active carbon are
2-4g/L；In root media 6-BA concentration be 3.5-4.5mg/L, NAA concentration be 0.7-0.9mg/L, banana puree 25-35g/
L, active carbon is 2-4g/L；The sodium bicarbonate that concentration is 1-5/L and the disease that concentration is 20-40mg/L are additionally added in initial culture base
Malicious azoles；
The condition of the Initial culture are as follows: two weeks dark cultures are carried out first, then in 25 DEG C of temperature, intensity of illumination 2000lx, light
According to period 14h/d, surrounding is cultivated；
The condition of the squamous subculture and culture of rootage are as follows: 25 DEG C of temperature, intensity of illumination 2000lx, periodicity of illumination 14h/d.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711358071.9A CN107926706B (en) | 2017-08-23 | 2017-08-23 | Tissue culture method of mini phalaenopsis virus-free seedlings |
| CN201710729775.6A CN107455258B (en) | 2017-08-23 | 2017-08-23 | The method for tissue culture of mini iris detoxic seedling |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710729775.6A CN107455258B (en) | 2017-08-23 | 2017-08-23 | The method for tissue culture of mini iris detoxic seedling |
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| CN201711358071.9A Division CN107926706B (en) | 2017-08-23 | 2017-08-23 | Tissue culture method of mini phalaenopsis virus-free seedlings |
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| CN107455258A CN107455258A (en) | 2017-12-12 |
| CN107455258B true CN107455258B (en) | 2019-05-31 |
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| CN201711358071.9A Active CN107926706B (en) | 2017-08-23 | 2017-08-23 | Tissue culture method of mini phalaenopsis virus-free seedlings |
| CN201710729775.6A Active CN107455258B (en) | 2017-08-23 | 2017-08-23 | The method for tissue culture of mini iris detoxic seedling |
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