CN104429976B - Efficient detoxification method for sweet cherry rootstocks - Google Patents
Efficient detoxification method for sweet cherry rootstocks Download PDFInfo
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Abstract
The invention relates to an efficient detoxification method for sweet cherry rootstocks; the method comprises the steps: 1), utilizing sweet cherry rootstock leaf buds in a field as explants for tissue culture, sterilizing and culturing to obtain tissue culture seedlings; 2) subculture; 3) thermal treatment; 4) pre-culturing and loading; 5) vitrification; 6) freezing; 7) unfreezing in a water bath after freezing, performing recovery processing for once or twice and then inducing plantlets; 8) inducing plantlets and 9) virus detection. The viruses of virus-free seedlings Gisela VI obtained by detoxifying prunus necrotic ring-spot viruses of sweet cherry rootstocks in the combination of thermal treatment and super-freezing therapy can be removed completely; stem tips can be easily peeled off; and the efficient detoxification method has wide application values in promoting healthy and sustainable development of sweet cherry industries.
Description
Technical field:
The present invention relates to a kind of sweet cherry rootstock poison-removing method, specifically a kind of to be treated using combined with heat treatment cryotherapy
Method removes the technology of sweet cherry rootstock Prunus necrotic ring spot virus, belongs to fruit tree virus Prevention Technique field.
Background technology:
Prunus avium (Prunus avium L.) is that China currently adjusts planting fruit trees structure, realizes the first-selection of increasing peasant income
Seeds.Virosiss have a strong impact on the yield and quality of Prunus avium, it has also become limit the major obstacle of China's Prunus avium industry development
One of.Prunus necrotic ring spot virus (Prunus Necrotic Ringspot Virus, PNRSV) are that most universal, harm occurs
More serious stone fruits virus, can cause tree body decline, yellowing leaf or necrosis, florescence postponement, fructescence to postpone
Or the underproduction, have no harvest when serious.According to investigations, in China's Prunus avium cultural area, PNRSV generally occurs, Shandong, Liaoning, Beijing etc.
Ground detects PNRSV infection.To the production garden virosiss RT-PCR identification of Shandong Prunus avium, Zong Xiaojuan etc. shows that PNRSV bands are malicious
Rate is 46.8%.
Due to the seriousness that virus is endangered to Fructus Pruni pseudocerasi, research and preventing and treating of the countries in the world to virus disease are all extremely paid attention to,
Mainly adopt detoxification technology to obtain detoxic seedling at present.European and American countries on the basis of clear and definite viral species, have been made just
The virus disease control measure of step, and be proved to cultivate the harm that virus-free nursery stock both can effectively prevent and treat fruit tree virus, can also show
Land and improve the yield and quality of fruit, be an effective advanced technology.Show in the production of Fructus Pruni pseudocerasi detoxic seedling following
Feature：Seedling growth is healthy and strong neat, as a result early, and fructescence is compared 5～10 days in advance, and the increase of fruit head is nearly more than 1 times,
Fruit total output improves 20～60%, and fruit quality improves, and soluble solid content is slightly higher, and fruit color degree is more than control,
Economic benefit increases by more than 1 times.In view of propagation by grafiting is one of major transmission path of Prunus avium PNRSV, at present in production still
Without the effective chemical pesticide control for PNRSV, therefore, viral removing is carried out to sweet cherry rootstock, virus-free sweet cherry is set up
The virus-free nursery stock production system such as Peach rootstock original seed farm and Seedlings nursery, is the key for preventing and treating Prunus avium virosiss at this stage.
At present, the basic skills of conventional acquisition detoxic seedling has following several：Shoot Tip Culture, shoot-tip grafting, heat treatment
With reference to Shoot Tip Culture.For example, Su Jiaming etc. adopts alternating temperature heat treatment method (37 DEG C of 16h/ nights of daytime to sweet cherry rootstock tissue cultured seedling
32 DEG C of 8h of evening) continuous processing 18 days combines Shoot Tip Culture and obtains detoxic seedling.Peng Xiang Yong etc. is by Prunus avium strain 6-7,6-3 and stock
GiselaNo.5 is processed 3 weeks in 38 DEG C, strips 0.5～1.0mm Micro-stem tip cultures, and Jing Screening and Identification obtains the tissue culture of removing PNRSV
Seedling.Cryotherapy therapy is the novel plant tissue poison-removing method of rising in recent years.Fu Hongqi etc. is combined using encapsulation dehydration
Vitrification cryotherapy Prunus avium stem apex, stem apex survival rate is up to 28.7%.
Although the detoxification to Prunus avium and its stock has been carried out preliminary study, problems are still suffered from, predominantly：
(1) traditional Shoot Tip Culture and combined with heat treatment Shoot Tip Culture detoxification are extremely inefficient.Dai Hongyan etc. proves that Prunus avium test tube seedling is micro-
Shoot Tip Culture can not effectively remove PNRSV.(2) complex operation, inefficiency.Traditional Shoot Tip Culture requires that stripping stem apex is less than
0.5mm, operation difficulty are big, and seedling efficiency is low.(3) encapsulation dehydration that Fu Hongqi is adopted combines the sweet cherry of Vitrification cryotherapy
Stem of Radix Persicae point, the method are relatively complicated, and vegetable material cultivation cycle is long.Showing is needed domestication by low temperature 30 days, is tied using Embedding drying
Close Vitrification to preserve stem apex material, and the method does not set up freezing regenerating system for sweet cherry rootstock material.
Additionally, the method has only inquired into the Prunus avium detoxification efficiency of single ultralow temperature therapy, survival rate and virus elimination rate are general.
The content of the invention
For the deficiencies in the prior art, the present invention provides a kind of sweet cherry rootstock high-efficiency detoxicating method.The method strips stem
Point is easily operated, meets requirement of experiment by taking 1mm stem apexs.Sweet cherry rootstock disease can effectively be removed using the method for the present invention
Poison, cryotherapy and resuscitation process simplify, time saving and energy saving, efficiency high, detoxification thoroughly, low cost, instrument and equipment is required not
It is high.
Summary of the invention：
A kind of sweet cherry rootstock poison-removing method, with field band poison sweet cherry rootstock leaf bud as explant, it is sterile-processed after,
Proceed to and build Seedling culture medium and set up test tube seedling, successive transfer culture is after 30 days, 38 DEG C of alternating temperature heat treatments 30 days strip 1mm stem apexs, carry out glass
Glass cryotherapy therapy, after renewal cultivation January, carries out RT-PCR identification PNRSV removing situations, filters out to detoxic seedling
Detoxic seedling Jing after subculture 3 times, viruses indentification is carried out with RT-PCR method again.
Detailed description of the invention：
Technical scheme is as follows：
A kind of sweet cherry rootstock high-efficiency detoxicating method is as follows including step：
1) build Seedling：Field sweet cherry rootstock leaf bud is taken as tissue culture explant, it is sterile-processed after be seeded in addition and swash
The MS of element builds in Seedling culture medium and cultivates, and obtains tissue cultured seedling；
2) successive transfer culture：By step 1) tissue cultured seedling that obtains successive transfer culture 25～30 days in MS increases Seedling culture medium,
3) heat treatment：By step 2) tissue cultured seedling after successive transfer culture is in 26～42 DEG C of temperature, light intensity 1000-2000lx condition
Lower culture 25～40 days；
4) preculture, loading：By step 3) tissue cultured seedling after heat treatment strips the stem apex group of 1～2mm in superclean bench
Knit, by stem-tip tissue preculture 2～5 days, then Jing room temperatures loaded 20～30min；
5) vitrification：During laden stem-tip tissue goes to cryoprotection solution PVS2, ice bath processes 60～90min,
6) freeze：Cryovial is put into rapidly cold in liquid nitrogen into cryopreservation tube by the stem-tip tissue subpackage after vitrification process
Freeze 20～24h；
7) thawed after freezing, then carry out recovering to process 1-2 time, then carry out induction seedling；
8) induce seedling：Stem-tip tissue after recovery is processed goes to MS and increases first light culture 3～7 days in Seedling culture medium, then turns
Enter illumination cultivation, successive transfer culture after seedling,
9) Viral diagnosis：Seedling PNRSV removal effects to be measured are detected using RT-PCR, detoxic seedling is filtered out.
Currently preferred, step 1) in disinfect be that explant is sterilized using secondary repeated disinfection method, first use
Detergent cleans 8～10min, then rinses 20～35min with tap water, in superclean bench, successively with 75% alcohol disinfecting 3～
5min, 2%NaClO 4～6min of sterilization, 0.1% mercuric chloride solution, 6～8min of sterilization, cuts axillary bud after peeling off axillary bud scale and uses again
0.1% mercuric chloride solution is sterilized 3～5min, with aseptic water washing 3～5 times after sterilizing every time, is finally exhausted bud with sterilizing filter paper
Body surface moisture.
Currently preferred, step 1) in the MS of addition hormone to build Seedling culture medium be to add the 6- that concentration is 0.5～1mg/L
The MS solid mediums of BA (6-benzyl aminopurine) and concentration for 0.1～0.2mg/L IBA (indolebutyric acid), condition of culture be
Temperature is 26 DEG C, light intensity is 2000-3000lx, light dark cycles are culture 30 days under conditions of 16h/8h.
Currently preferred, step 2) in successive transfer culture used by MS increase Seedling culture medium for addition concentration be 0.5～
The MS solid mediums of 0.8mg/L6-BA (6-benzyl aminopurine).
Currently preferred, step 3) in heat treatment actual conditions be, in 38 DEG C of daytime, light intensity 1000-2000lx, light
Cycle 16h；Heat treatment 30 days under 26 DEG C of night, dark cycle 8h, the culture medium of heat treatment and the culture medium phase of successive transfer culture
Together.
Currently preferred, step 4) by stem-tip tissue in the MS liquid cultures of addition 0.2～0.4mol/L sucrose concentrations
Preculture 2 days in base, the stem-tip tissue after preculture is processed in loading liquid 28～30min, described loading at room temperature
Liquid is the MS fluid mediums of the glycerol of 1～3mol/L of addition and 0.3～0.5mol/L sucrose concentrations.
Currently preferred, step 5) in the formula of cryoprotection solution PVS2 be：Add 30% in MS fluid mediums
Glycerol (v/v), 15% ethylene glycol (v/v), 15% dimethyl sulfoxide (v/v) and 0.4mol/L sucrose, ice bath process time is
90min。
Currently preferred, step 6) in freezing processing be specially：Per 5～6 stem apex subpackages to 1 cryopreservation tube in, it is cold
Freezing and medium being preserved for cryoprotection solution PVS2, cryoprotection solution PVS2 submergence stem-tip tissues freeze 24h in liquid nitrogen.
Currently preferred, step 7) in defrosting thawed for being placed in water-bath or metal bath, water-bath thaw be in
Thawed in 90s rapidly in 42 DEG C of water-bath, recovery be processed as by the stem-tip tissue after defrosting be placed in addition sucrose concentration 1.0～
Process 2 times in the MS fluid mediums (DS solution) of 1.2mol/L, it is per treatment for standing 10min under room temperature.
It is currently preferred, step 8) in, it is to add 6-BA (the 6- benzyls of 0.5mg/L to induce seedling MS to increase Seedling culture medium
Amidopurin) MS solid mediums, detoxic seedling successive transfer culture using with the addition of 5mg/L virazoles (Tribavirin) and
The MS solid mediums of 0.5mg/L 6-BA (6-benzyl aminopurine).
Currently preferred, described sweet cherry rootstock is No. 6 lignifyings of Gisela or semi-lignified branch, Virusfree
For Prunus necrotic ring spot virus (Prunus Necrotic Ringspot Virus, PNRSV).
The superclean bench of the present invention, cryovial are common commercial products, strip technology for this area routine techniquess, MS
For existing basal medium, it is obtained by existing conventional method.
The technical characterstic of the present invention and advantage：
Compared with the conventional method, the invention has the advantages that and good effect：
1) conventional Shoot Tip Culture and stem apex explant is less needed for heat treatment poison-removing method, the explant of usual below 0.5mm
Body can just obtain more satisfactory detoxification efficiency, and the inventive method passes through liquid nitrogen freezing by addition to shoot apical meristem
Plant cell is freezed to death, therefore required stem apex explant material compares that common poison-removing method is larger, and 1mm stem apexs can reach detoxification mesh
's.Therefore, the inventive method is to strip stem apex step more easy to operate.
2) existing Prunus avium cryotherapy poison-removing method carries out ultralow temperature therapy using conventional successive transfer culture tissue cultured seedling,
And heat treatment is combined by the inventive method with ultralow temperature therapy, i.e., the band poison tissue cultured seedling of successive transfer culture is in high temperature (38 DEG C/night of daytime
26 DEG C of evening) under process 30 days after, then carry out ultralow temperature therapy, stem apex survival rate and detoxification efficiency are above the Prunus avium reported
Cryotherapy detoxicity method；
Existing ultralow temperature detoxicity method is 30 days in the domestication by low temperature stage, present invention omits in the domestication by low temperature stage, shorten
Operating time；Additionally, existing method utilizes encapsulation- vitrification method, aseptically need to first carry out sodium alginate Embedding drying and take off
Water, then glass freezing process is carried out, the present invention adopts direct Vitrification, need not embed step, simplify operating procedure, contracts
The short operating time.Existing document proves that the survival rate of Vitrification and regeneration rate are superior to encapsulation- vitrification method.
3) it is one of most important mode that the stone fruits virus such as PNRSV are propagated that grafting passes poison, therefore, detoxification stock
Seedling is the key of Prunus avium Non-toxic nursery stock production.Existing ultralow temperature poison-removing method does not set up detoxification technology for stock material
System, the present invention set up the poison-removing method of Cherry dwarf rootstock, can be in the whole year production for efficiently realizing detoxification rootstock seedling.
Description of the drawings
Fig. 1 builds Seedling schematic diagram for step (1) field band poison ' Gisela 6 ' in embodiment 1, and wherein, in Fig. 1, I is leaf bud
After surface sterilization, the culture figure in MS builds Seedling culture medium；II obtains ' Gisela 6 ' tissue cultured seedling after two months for culture；
Fig. 2 is that 1 combined with heat treatment ultralow temperature detoxification of embodiment obtains regrowth procedure chart；
Fig. 3 is RT-PCR qualification figures after 1 combined with heat treatment ultralow temperature detoxification of embodiment, and A is the RT- for obtaining 16 plants of detoxic seedlings
PCR identifies that B is the secondary RT-PCR identifications of 7 plants of primary election detoxic seedlings.
Specific embodiment
Below by embodiment, the invention will be further described, it is necessary to is pointed out that embodiment is served only for the present invention
It is further described, it is impossible to be interpreted as limiting the scope of the invention, person skilled in art can be according to above-mentioned
The content of the invention makes some immaterial modifications and adaptations to the present invention, unspecified existing by this area in embodiment
Technology.
Embodiment 1：
A kind of sweet cherry rootstock high-efficiency detoxicating method is as follows including step：
(1) build Seedling：No. 6 leaf buds of sweet cherry rootstock Gisela for selecting field band poison are explant, are cut into 1 cm stem
Section, each stem section raw 1 leaf bud, first clean 10min with detergent, then rinse 30min with tap water, in superclean bench,
Successively with 75% alcohol disinfecting 5min, 2%NaClO sterilization 6min, 0.1% mercuric chloride solution sterilization 8min, after peeling off axillary bud scale
Cut axillary bud to be sterilized 5min with 0.1% mercuric chloride solution again, after sterilizing every time, sterilizing filter paper is finally used with aseptic water washing 5 times
Exhaustion sprout surface moisture.Explant after sterilization is seeded in the MS of addition hormone and builds culture, culturing room's condition in Seedling culture medium
For 26 DEG C of temperature, light intensity 2000lx, light dark cycles 16h/8h, tissue cultured seedling is obtained, it is 1mg/ that MS builds Seedling culture medium for addition concentration
L6-BA (6-benzyl aminopurine) and the MS solid mediums that concentration is 0.2mg/L IBA (indolebutyric acid).
10 field band poison ' Gisela 6 ' axillary buds are taken Jing after above-mentioned steps (1) sterilization, is cultivated in MS builds Seedling culture medium
' Gisela 6 ' tissue cultured seedling is obtained after two months, and wherein 8 leaf buds are survived and build up tissue cultured seedling, and pollution rate is 20%, by this
The sterilization of invention, both ensure that Disinfection Effect, and had not killed leaf bud, it is ensured that the survival rate of leaf bud, be further to process to carry out base
Plinth, is shown in accompanying drawing 1.
(2) successive transfer culture：By tissue cultured seedling successive transfer culture 30 days, culturing room's condition is 26 DEG C, light intensity 2000lx, light dark week
Phase 16h/8h, successive transfer culture culture medium are to add the MS solid mediums of 0.5mg/L 6-BA (6-benzyl aminopurine).；
(3) heat treatment：By the tissue cultured seedling after successive transfer culture in 38 DEG C of daytime, light intensity 1500lx, photoperiod 16h；Night 26
DEG C, heat treatment 30 days under dark cycle 8h, the culture medium of heat treatment is identical with the culture medium of successive transfer culture, is in addition concentration
For cultivating in the MS solid mediums of 0.5mg/L 6-BA (6-benzyl aminopurine).
(4) preculture：Tissue cultured seedling after process strips 1mm stem-tip tissues using anatomical lens in superclean bench, by stem
Point is cultivated 2 days in being organized in the MS fluid mediums of addition 0.3mol/L sucrose concentrations.
(5) load：Stem-tip tissue after preculture is processed into 30min in loading liquid at room temperature, loading liquid is addition
The MS fluid mediums of the glycerol and 0.4mol/L sucrose concentrations of 2mol/L.
(6) vitrification：Under condition of ice bath, vitrification during laden stem-tip tissue goes to cryoprotection solution PVS2 is processed
90min, the formula of cryoprotection solution PVS2 is：Add 30% glycerol, 15% ethylene glycol, 15% dimethyl in MS culture medium sub-
Sulfone and 0.4mol/L sucrose.
(7) freeze：Per 5～6 stem apex subpackages to 1 cryopreservation tube in, freezen protective medium be cryoprotection solution PVS2,
Cryoprotection solution PVS2 submergence stem-tip tissues, seal cryopreservation tube up for safekeeping, freeze 24h in input liquid nitrogen,
(8) thaw：Heat 90s in 42 DEG C of water-bath to thaw,
(9) recover：Processed with DS solution (formula is the MS fluid mediums of addition sucrose concentration 1.2mol/L) after thawing
Stem-tip tissue 2 times, it is per treatment for standing 10min under room temperature,
(10) induce seedling：Stem-tip tissue after recovery is processed goes to MS and increases first light culture 7 days in Seedling culture medium, then proceeds to
Illumination cultivation, the same successive transfer culture of illumination cultivation condition, successive transfer culture after seedling, it is addition that induction seedling MS increases Seedling culture medium
The MS solid mediums of the 6-BA (6-benzyl aminopurine) of 0.5mg/L, detoxic seedling successive transfer culture are adopted and with the addition of 5mg/L virazoles
(Tribavirin) and 0.5mg/L 6-BA (6-benzyl aminopurine) MS solid mediums.
(11) viruses indentification：RT-PCR detects PNRSV removal effects, filters out detoxic seedling.
PNRSV detection methods adopt " improvement of two kinds of main Prunus avium virus RT-PCR detection methods ", and ancestor knows beautiful etc., plants
Thing protects journal, and volume 38, the 3rd phase, the 285-286 page, detection method disclosed in June, 2011 is carried out, RT-PCR detection steps
It is rapid as follows：Detoxic seedling total serum IgE is extracted, with first chains of cDNA of random hexamers reverse transcription, with PNRSV capsid protein genes
Special primer PNRSV-S：5’-TGGTCCCACTCAGGGCTCAACAAAG-3’、PNRSV-A：5’-
ACGCGCAAAAGTGTCGAAATCTAAA-3 ' enters performing PCR amplification, and amplification condition is as follows：95℃ 5min、95℃50s-55℃
50s-72 DEG C of 50s 35 circulations, 72 DEG C of 5min, after reaction terminates, 1% agarose gel electrophoresiies are detected.
The 1mm stem apexs of 24 ' Giselas 6 ' are stripped, is obtained according to the present embodiment combined with heat treatment ultralow temperature detoxification step altogether
16 regrowths are obtained, survival rate is 66.7%；Jing PCR identify that wherein 7 plants are feminine gender, and virus elimination rate is 43.75%.7 plants of detoxifications
Seedling () Jing after 3 successive transfer culture, carries out RT-PCR identifications per 20 days subcultures once again, and wherein 3 plants show weak sun, 4 plants of performances
Feminine gender, the detoxic seedling strain Jing expanding propagation are shown in accompanying drawing 2-3 in being stored in this laboratory.
Embodiment 2：
With embodiment 1, difference is a kind of sweet cherry rootstock high-efficiency detoxicating method：
Heat treatment actual conditions in step (3) is, in 40 DEG C of daytime, light intensity 2000lx, photoperiod 16h；26 DEG C of night,
Heat treatment 30 days under dark cycle 8h, the culture medium of heat treatment is identical with the culture medium of successive transfer culture.
Step (4) preculture 2 days in the MS fluid mediums of addition 0.4mol/L sucrose concentrations by stem-tip tissue.
Stem-tip tissue after preculture is processed in loading liquid step (5) 28min at room temperature, and described loading liquid is
The MS fluid mediums of the glycerol and 0.5mol/L sucrose concentrations of addition 3mol/L.
Defrosting in step (8) is thawed for being placed in metal bath
Step (9) recovers the MS liquid cultures for being processed as the stem-tip tissue after defrosting is placed in addition sucrose concentration 1mol/L
Process 2 times in base (DS solution), it is per treatment for standing 10min under room temperature.
Embodiment 3：
With embodiment 1, difference is a kind of sweet cherry rootstock high-efficiency detoxicating method：
Explant in step (1) after sterilization is seeded in the MS of addition hormone and builds culture, culturing room's condition in Seedling culture medium
For 26 DEG C of temperature, light intensity 2500lx, light dark cycles 16h/8h, tissue cultured seedling is obtained, MS builds Seedling culture medium and for addition concentration is
0.8mg/L6-BA (6-benzyl aminopurine) and the MS solid mediums that concentration is 0.1mg/L IBA (indolebutyric acid).Step (2)
Middle successive transfer culture：By tissue cultured seedling successive transfer culture 30 days, culturing room's condition was 26 DEG C, light intensity 2500lx, light dark cycles 16h/8h,
Successive transfer culture culture medium is to add the MS solid mediums of 0.6mg/L 6-BA (6-benzyl aminopurine).
Comparative example 1：
A kind of sweet cherry rootstock shoot tip culture, it is as follows including step：
(1) build Seedling：No. 6 leaf buds of sweet cherry rootstock Gisela for selecting field band poison are explant, are cut into 1 cm stem
Section, each stem section raw 1 leaf bud, first clean 10min with detergent, then rinse 30min with tap water, in superclean bench,
Successively with 75% alcohol disinfecting 5min, 2%NaClO sterilization 5min, 0.1% mercuric chloride solution sterilization 8min, after peeling off axillary bud scale
Cut axillary bud to be sterilized 5min with 0.1% mercuric chloride solution again, after sterilizing every time, sterilizing filter paper is finally used with aseptic water washing 5 times
Exhaustion sprout surface moisture.Explant after sterilization is seeded in the MS of addition hormone and builds culture, culturing room's condition in Seedling culture medium
For 26 DEG C of temperature, light intensity 2000lx, light dark cycles 16h/8h, tissue cultured seedling is obtained, it is 1mg/ that MS builds Seedling culture medium for addition concentration
L6-BA (6-benzyl aminopurine) and the MS solid mediums that concentration is 0.2mg/L IBA (indolebutyric acid).
(2) successive transfer culture：By tissue cultured seedling successive transfer culture 30 days, culturing room's condition is 26 DEG C, light intensity 2000lx, light dark week
Phase 16h/8h, successive transfer culture culture medium are to add the MS solid mediums of 0.5mg/L 6-BA (6-benzyl aminopurine).
(3) Shoot Tip Culture：The band poison tissue cultured seedling of successive transfer culture is placed on superclean bench, 1mm size stem apexs is stripped, is put
Cultivate in MS increases Seedling culture medium, until inducing into detoxic seedling, increasing Seedling culture medium used is 0.5mg/L 6-BA for addition concentration
The MS solid mediums of (6-benzyl aminopurine).The same successive transfer culture of condition of culture.
(4) viruses indentification：RT-PCR detects PNRSV removal effects, filters out detoxic seedling.
Comparative example 2：
A kind of sweet cherry rootstock combined with heat treatment shoot tip culture, it is as follows including step：
(1) build Seedling：No. 6 leaf buds of sweet cherry rootstock Gisela for selecting field band poison are explant, are cut into 1 cm stem
Section, each stem section raw 1 leaf bud, first clean 10min with detergent, then rinse 30min with tap water, in superclean bench,
Successively with 75% alcohol disinfecting 5min, 2%NaClO sterilization 5min, 0.1% mercuric chloride solution sterilization 8min, after peeling off axillary bud scale
Cut axillary bud to be sterilized 5min with 0.1% mercuric chloride solution again, after sterilizing every time, sterilizing filter paper is finally used with aseptic water washing 5 times
Exhaustion sprout surface moisture.Explant after sterilization is seeded in the MS of addition hormone and builds culture, culturing room's condition in Seedling culture medium
For 26 DEG C of temperature, light intensity 2000lx, light dark cycles 16h/8h, tissue cultured seedling is obtained, it is 1mg/ that MS builds Seedling culture medium for addition concentration
L6-BA (6-benzyl aminopurine) and the MS solid mediums that concentration is 0.2mg/L IBA (indolebutyric acid).
(2) successive transfer culture：By tissue cultured seedling successive transfer culture 30 days, culturing room's condition is 26 DEG C, light intensity 2000lx, light dark week
Phase 16h/8h, successive transfer culture culture medium are to add the MS solid mediums of 0.5mg/L 6-BA (6-benzyl aminopurine).
(3) heat treatment：By the tissue cultured seedling after successive transfer culture in 38 DEG C of daytime, light intensity 1500lx, photoperiod 16h；Night 26
DEG C, heat treatment 30 days under dark cycle 8h, the culture medium of heat treatment is identical with the culture medium of successive transfer culture, is in addition concentration
For cultivating in the MS solid mediums of 0.5mg/L 6-BA (6-benzyl aminopurine).
(4) Shoot Tip Culture：Band poison tissue cultured seedling after heat treatment is placed in superclean bench and strips 1mm stem apexs, stem apex group
Knit and go to illumination cultivation in MS increasing Seedling culture medium, the same successive transfer culture of illumination cultivation condition, induction seedling MS increase Seedling culture medium and be
The MS solid mediums of the 6-BA (6-benzyl aminopurine) of addition 0.5mg/L, until induction seedling.
(5) viruses indentification：RT-PCR detects PNRSV removal effects, screens detoxic seedling.
Comparative example 3：
A kind of sweet cherry rootstock cryotherapy therapy shoot tip culture, it is as follows including step：
(1) build Seedling：No. 6 leaf buds of sweet cherry rootstock Gisela for selecting field band poison are explant, are cut into 1 cm stem
Section, each stem section raw 1 leaf bud, first clean 10min with detergent, then rinse 30min with tap water, in superclean bench,
Successively with 75% alcohol disinfecting 5min, 2%NaClO sterilization 5min, 0.1% mercuric chloride solution sterilization 8min, after peeling off axillary bud scale
Cut axillary bud to be sterilized 5min with 0.1% mercuric chloride solution again, after sterilizing every time, sterilizing filter paper is finally used with aseptic water washing 5 times
Exhaustion sprout surface moisture.Explant after sterilization is seeded in the MS of addition hormone and builds culture, culturing room's condition in Seedling culture medium
For 26 DEG C of temperature, light intensity 2000lx, light dark cycles 16h/8h, tissue cultured seedling is obtained, it is 1mg/ that MS builds Seedling culture medium for addition concentration
L6-BA (6-benzyl aminopurine) and the MS solid mediums that concentration is 0.2mg/L IBA (indolebutyric acid).
(2) successive transfer culture：By tissue cultured seedling successive transfer culture 30 days, culturing room's condition is 26 DEG C, light intensity 2000lx, light dark week
Phase 16h/8h, successive transfer culture culture medium are to add the MS solid mediums of 0.5mg/L 6-BA (6-benzyl aminopurine).；
(3) preculture：The malicious tissue cultured seedling of band strips 1mm stem-tip tissues using anatomical lens in superclean bench, by stem apex group
Cultivate 2 days in being woven in the MS fluid mediums of addition 0.3mol/L sucrose concentrations.
(4) load：Stem-tip tissue after preculture is processed into 30min in loading liquid at room temperature, loading liquid is addition
The MS fluid mediums of the glycerol and 0.4mol/L sucrose concentrations of 2mol/L.
(5) vitrification：Under condition of ice bath, vitrification during laden stem-tip tissue goes to cryoprotection solution PVS2 is processed
90min, the formula of cryoprotection solution PVS2 is：Add 30% glycerol, 15% ethylene glycol, 15% dimethyl in MS culture medium sub-
Sulfone and 0.4mol/L sucrose.
(6) freeze：Per 5～6 stem apex subpackages to 1 cryopreservation tube in, freezen protective medium be cryoprotection solution PVS2,
Cryoprotection solution PVS2 submergence stem-tip tissues, seal cryopreservation tube up for safekeeping, freeze 24h in input liquid nitrogen,
(7) thaw：Heat 90s in 42 DEG C of water-bath to thaw,
(8) recover：Processed with DS solution (formula is the MS fluid mediums of addition sucrose concentration 1.2mol/L) after thawing
Stem-tip tissue 2 times, it is per treatment for standing 10min under room temperature,
(9) induce seedling：Stem-tip tissue after recovery is processed goes to MS and increases first light culture 7 days in Seedling culture medium, then proceeds to
Illumination cultivation, the same successive transfer culture of illumination cultivation condition, successive transfer culture after seedling, it is addition that induction seedling MS increases Seedling culture medium
The MS solid mediums of the 6-BA (6-benzyl aminopurine) of 0.5mg/L, detoxic seedling successive transfer culture are adopted and with the addition of 5mg/L virazoles
(Tribavirin) and 0.5mg/L 6-BA (6-benzyl aminopurine) MS solid mediums.
(10) viruses indentification：RT-PCR detects PNRSV removal effects, filters out detoxic seedling.
Experimental example
Using " improvement of two kinds of main Prunus avium virus RT-PCR detection methods ", ancestor knows beautiful etc., and plant protection journal is public
The detection method opened, detects embodiment 1～3 and the detoxification efficiency after the process of comparative example 1～3,
Detection method：
Embodiment, comparative example set 6 process altogether, and each process strips 24 ' Giselas 6 ' using identical method
1mm stem apexs, after embodiment 1～3 and comparative example 1～3 are processed, each is processed in triplicate, statistics of averaging survival rate
And virus elimination rate, it is as a result as shown in table 1 below,
The sum into number of live vaccine/strip stem apex after survival rate=induction seedling
After virus elimination rate=detoxic seedling number/induction seedling into number of live vaccine
Table 1：Detoxification efficiency is contrasted
| Project | Survival rate | Virus elimination rate |
| Embodiment 1 | 66.7% | 43.75% |
| Embodiment 2 | 67% | 45.2% |
| Embodiment 3 | 62.3% | 43.6% |
| Comparative example 1 | 81.8% | 0% |
| Comparative example 2 | 82.9% | 18.2% |
| Comparative example 3 | 28.7% | 40.25% |
As can be seen from Table 1, from the point of view of the survival rate contrast of induction seedling, the survival rate of comparative example 1 and comparative example 2 is most
Height, the survival rate of comparative example 3 are minimum, the survival rate of embodiment of the present invention 1-3 65% or so, but from the point of view of detoxification efficiency,
Embodiment 1, embodiment 2, embodiment 3, the virus elimination rate of comparative example 3 are more or less the same, and the detoxification efficiency of comparative example 3 is slightly weaker than embodiment
1st, embodiment 2, the detoxification efficiency of embodiment 3；Comparative example 1 adopts common Shoot Tip Culture method, although the method stem apex survival rate
It is higher, but PNRSV can not be effectively removed, regrowth is detected all into the positive；Comparative example 2 adopts combined with heat treatment Shoot Tip Culture side
Method, survival rate are higher, but PNRSV removal efficiencies are relatively low, and only 18.2%；Comparative example 3 adopts cryotherapy therapy, the method
It is significantly improved in virus elimination rate index, but survival rate is relatively low, comprehensive analysis, embodiment of the present invention 1-3 is using change warm
Process with reference to ultralow temperature therapy Shoot Tip Culture, the method is ideal in two indexs of stem apex survival rate and virus elimination rate, right
The viral removal efficiency of sweet cherry rootstock Gisela 6 is ideal, stability is higher.
Claims (7)
1. a kind of sweet cherry rootstock high-efficiency detoxicating method, as follows including step：
1）Build Seedling：Field sweet cherry rootstock leaf bud is taken as tissue culture explant, it is sterile-processed after be seeded in addition hormone
MS builds in Seedling culture medium and cultivates, and obtains tissue cultured seedling；
2）Successive transfer culture：By step 1）The tissue cultured seedling of acquisition successive transfer culture 25～30 days in MS increases Seedling culture medium,
3）Heat treatment：By step 2）Tissue cultured seedling after successive transfer culture is under the conditions of 26～42 DEG C of temperature, light intensity 1000-2000lx
Culture 25～40 days；
4）Preculture, loading：By step 3）Tissue cultured seedling after heat treatment strips the stem-tip tissue of 1～2mm in superclean bench,
By stem-tip tissue in the MS fluid mediums of addition 0.2 ~ 0.4mol/ L sucrose concentrations preculture 2 days, after preculture
Stem-tip tissue process 28～30min in loading liquid at room temperature, the glycerol of described loading liquid for addition 1 ~ 3mol/ L
With the MS fluid mediums of 0.3 ~ 0.5mol/ L sucrose concentrations；
5）Vitrification：During laden stem-tip tissue goes to cryoprotection solution PVS2, ice bath processes 60～90min,
6）Freezing：Vitrification process after stem-tip tissue subpackage into cryopreservation tube, per 5 ~ 6 stem apex subpackages to 1 cryopreservation tube
In, freezen protective medium be cryoprotection solution PVS2, cryoprotection solution PVS2 submergence stem-tip tissues, in liquid nitrogen freeze
24h；
7）Thawed after freezing, then carry out recovering to process 1-2 time, then carry out induction seedling；Described defrosting is to be placed in
Thawed in water-bath or metal bath, it is that in being thawed in 90s rapidly in 42 DEG C of water-bath, recovery is processed as solving that water-bath is thawed
Stem-tip tissue after jelly is placed in the MS fluid mediums of addition 1.0～1.2mol/l of sucrose concentration and processes 2 times, per treatment
For standing 10min under room temperature；
8）Induction seedling：Stem-tip tissue after recovery is processed goes to MS and increases first light culture 3～7 days in Seedling culture medium, then proceeds to
Illumination cultivation, successive transfer culture after seedling,
9）Viral diagnosis：Seedling PNRSV removal effects to be measured are detected using RT-PCR, detoxic seedling is filtered out.
2. the sweet cherry rootstock high-efficiency detoxicating method according to claim 1, it is characterised in that step 1）In sterilization at
Reason is that explant is sterilized using secondary repeated disinfection method, first clean 8～10min with detergent, then with tap water flushing 20～
35min, in superclean bench, successively with 75% 3～5min of alcohol disinfecting, 2%NaClO 4～6min of sterilization, 0.1% mercuric chloride solution
6～8min of sterilization, cuts axillary bud again with 0.1% mercuric chloride solution, 3～5min of sterilization, uses after sterilizing every time after peeling off axillary bud scale
Aseptic water washing 3～5 times, finally with sterilizing filter paper exhaustion sprout surface moisture, the MS for adding hormone builds Seedling culture medium for adding
Plus the 6-BA that concentration is 0.5～1mg/L（6-benzyl aminopurine）It is 0.1～0.2 mg/L IBA with concentration（Indolebutyric acid）
MS solid mediums, condition of culture be temperature be 26 DEG C, light intensity be 2000-3000lx, light dark cycles be 16h/8h
Under the conditions of cultivate 30 days.
3. the sweet cherry rootstock high-efficiency detoxicating method according to claim 1, it is characterised in that step（2）In subculture training
Support MS used to increase Seedling culture medium for addition concentration is 0.5～0.8mg/L 6-BA（6-benzyl aminopurine）MS solid culture
Base.
4. the sweet cherry rootstock high-efficiency detoxicating method according to claim 1, it is characterised in that step 3）In heat treatment
Actual conditions is, in 38 DEG C of daytime, light intensity 1000-2000lx, photoperiod 16h；Heat treatment 30 under 26 DEG C of night, dark cycle 8h
My god, the culture medium of heat treatment is identical with the culture medium of successive transfer culture.
5. the sweet cherry rootstock high-efficiency detoxicating method according to claim 1, it is characterised in that step 5）Middle cryoprotection
The formula of solution PVS2 is：Add 30% glycerol in MS fluid mediums（v/v）, 15% ethylene glycol（v/v）, 15% dimethyl it is sub-
Sulfone（v/v）With concentration 0.4mol/L sucrose, ice bath process time is 90min.
6. the sweet cherry rootstock high-efficiency detoxicating method according to claim 1, it is characterised in that step 8）In, induce seedling
It is to add the 6-BA of 0.5mg/L to increase Seedling culture medium with MS（6-benzyl aminopurine）MS solid mediums, detoxic seedling subculture training
Foster employing with the addition of 5 mg/L virazoles（Tribavirin）With 0.5 mg/L 6-BA（6-benzyl aminopurine）MS solids training
Foster base.
7. the sweet cherry rootstock high-efficiency detoxicating method according to claim 1, it is characterised in that described sweet cherry rootstock
For No. 6 lignifyings of Gisela or semi-lignified branch, Virusfree is Prunus necrotic ring spot virus（Prunus Necrotic RingspotVirus, PNRSV）.
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| CN107409669A (en) * | 2017-04-22 | 2017-12-01 | 蚌埠市乔峰农业蔬菜专业合作社 | A kind of sweet potato is heat-treated poison-removing method |
| CN110384041A (en) * | 2019-05-23 | 2019-10-29 | 南京千亩休闲农业发展有限公司 | A kind of cultural method of sweet cherry rootstock asexual clonal detoxic seedling |
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