CN102893869B - Root tip detoxification and rapid propagation technology of strawberries - Google Patents

Abstract

The invention discloses a root tip detoxification and rapid propagation technology of strawberries. The method for detoxification and tissue culture of strawberries comprises the steps of: pre-processing the strawberries, to obtain disinfected root tips; carrying out induction culture on the root tips on a root tip inducing medium to obtain regenerated plant sprouts; carrying out multiplication culture on the sprouts into seedlings on a multiplication medium; culturing the seedlings into subculture seedlings on a subculture growth medium; culturing the subculture seedlings into test-tube seedlings on a rooting medium; and optionally detecting whether the test-tube seedlings are affected by virus. In addition, the invention discloses a multiple reverse transcription-polymerase chain reaction (RT-PCR) method for synchronous and rapid detection of strawberry virus, wherein a hybrid primer pair is adopted.

Description

Tip of a root detoxification and the fast breeding technique of strawberry

Technical field

The invention belongs to field of plant breeding, particularly, the present invention relates to detoxification and the tissue culture method of the strawberry tip of a root and can be used for detecting simultaneously and rapidly detection method of multiple RT-PCR that whether strawberry that the method produces infect virus.

Background technology

China is the abundantest country of strawberry wild resource in the world, begins very early to utilize fraises des bois, and follows so far always.The large fruited strawberry cultivation of China starts from 1915, but does not come into one's own slower development in the past.Strawberry production has had rapidly development since the eighties in 20th century, and present strawberry production area is 70,000 hm approximately ², area ranks first in the world, and wherein the main place of production is distributed in the areas such as Anhui, Liaoning, Hebei, Shandong, Jiangsu, Shanghai, Zhejiang, Sichuan, Xinjiang, Beijing.

Because strawberry is herbaceous plant, by the stolon breeding, easily form the serious harm of virus disease, anthracnose, epidemic disease etc. for a long time.Of a great variety through preliminary investigation strawberry virus and viroids, mainly contain yellow edge poison (Strawberry mild yellow edge virus, SMYEV), strawberry veinbanding virus (strawberry vein banding virus, SVBV), the harm of strawberry crinkle virus (Strawberry crinkle virus, SCV or SCrV) and Strawberry mottle virus (Strawberry mottle virus, SMoV).

More seriously, the symptom behind many virus infection strawberries (especially early symptom) is not obvious, so that control is difficult to be implemented in planting season.For example, strawberry light yellow edge virus infects separately cultivated species strawberry non-evident sympton, and diseased plant is slightly downgraded, and the strawberry growing way is died down, output and fruit quality degradation, and the underproduction can be up to 75%; When infecting separately the cultivated species strawberry, Strawberry mottle virus often do not show manifest symptom, but the decline of diseased plant growing way, fruit quality descends, but the underproduction 20%～30% just can produce mottled symptom during with other viral Combined Infection; When strawberry veinbanding virus infects separately the cultivated species strawberry, non-evident sympton, but cause that plant strain growth is weak, stolon pumping amount reduces, yield and quality descends, and causes diseased plant leaf-shrinkage, distortion behind mottle virus, the light-duty yellow edge poison Combined Infection, and plant is extremely downgraded.Only have strawberry crinkle virus to infect symptom comparatively obvious, disease plant shows as: leaf malformation, produce the chlorisis spot on the leaf, little, irregular chlorisis spot and necrotic plaque, vein chlorisis, transparent appear along vein; The spire growth is asymmetric, the distortion shrinkage, and the leaflet yellow, petiole shortens, and leaf is little, and plant is downgraded; Strain strawberry stolon pumping amount reduces, and fertility descends, and fruit diminishes, and velogen strain infects separately and can cause the underproduction 35%～40%, with other viral Combined Infections, endanger even more seriously, and Yield of Strawberry can significantly descend, even has no harvest.

For this reason, people are studied, and the stem apex/stolon by strawberry/flower pesticide detoxification tissue is cultivated with fast breeding technique and produced the virus-free Strawberry Seedlings of breeder's stock.For example, Chinese patent application 200510030445 discloses a kind of method of cultivating and producing detoxic seedling on strawberry stemp apex, has wherein substantially adopted conventional medium, and needs microscope to carry out craft to strip stem apex; Chinese patent 200510062199 discloses a kind of strawberry virus elimination method, has wherein adopted conventional medium, and need to strip stem apex, needs in addition at high temperature to cultivate, and greatly reduces the survival rate of Strawberry Seedlings; Chinese patent 200710022803 discloses a kind of strawberry anther that utilizes and has cultivated the method for carrying out detoxifying fast breeding; Chinese patent 200510062199 discloses the method that removes strawberry SMYEV, has wherein substantially adopted conventional medium, and need to strip stem apex, only carries out detoxification for SMYEV, and other viral detoxification efficiencies are not quite clear.Other open source literature also comprises Chinese patent (application) 200910304829,201010133426,201010197820,201010252871,201010253333,20101058534,201110096851,201110139539,201110327423,201110375059 etc.

Yet, the strawberry stem tip detoxification, need to peel off continuously more than ten layers with operation tool and be wrapped in the outer stem sheet of stem (point), operation easier is large, inefficiency, more seriously, although in most cases can detoxification, even experienced technical staff also be difficult to thoroughly peel off with sterilize (through we with multiple RT-PCR simultaneously and rapidly detection method detect, (mixing) pollution rate of 60% is arranged usually, cause the unstable of the quality of growing seedlings; And, adopt said method also to have explant differentiation speed and the detoxification test tube plantlet reproduction speed slower, growing way is weak and transplant the problems such as Production of Large Fields is on the low side.

For this reason, the inventor is through long-term and arduous research, rely on some fortune, adopt in a creative way take the tip of a root of strawberry and train with group as explant carries out detoxification, save and used microscopical step, and greatly reduce pollution rate, and worked out concrete strawberry detoxification and tissue culture method, its step and condition and prior art widely different for this reason.In addition, in order to check the effect of strawberry detoxification, especially take off the effect of multiple virus, the inventor also studies and has obtained simultaneously and rapidly detection method of multiple RT-PCR, can directly implement highly sensitive virus to the strawberry tissue detects, need not to separate, purifying, and wherein each primer and proportioning thereof be through optimizing, elimination the phase mutual interference in detecting.

Summary of the invention

The method of the strawberry detoxification that the object of the invention is to provide new and group training and can be with it supporting multiple RT-PCR detect the method for strawberry virus simultaneously and rapidly.The method of strawberry detoxification of the present invention and group training is low with respect to complex operation, the efficient of prior art, the problems such as sterilization is difficult thoroughly, tissue culture seedling pollution rate height, more easy and simple to handle, efficient is high, sterilization easily thoroughly, the tissue culture seedling pollution rate is low, reproduction speed fast, Miao Zhihao.

Particularly, in first aspect, the invention provides strawberry detoxification and the method for organizing training, it comprises

1) strawberry is carried out preliminary treatment, obtain the tip of a root of sterilization;

2) on tip of a root inducing culture, the tip of a root induced and cultivate into the regeneration plant young shoot;

3) on proliferated culture medium, young shoot propagation is cultivated into seedling;

4) on the subculture growth medium so that seedling grows up to the subculture seedling;

5) on root media so that the subculture seedling grows up to test-tube plantlet; With

6) whether optional test tubes seedling infects virus.

Preferably in the method for first aspect present invention,

The prescription of tip of a root inducing culture is: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/L+BA(6-Bian aminopurine) 0.3-0.5mg/L+2,4-D (the stupid fluoroacetic acid of 2,4-dichloro) 0.1-0.3mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

The prescription of proliferated culture medium is: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+BA 0.2-0.5 mg/L+ NAA(methyl α-naphthyl acetate) 0.1-0.3mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

The prescription of subculture growth medium is: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/0.5L+BA 0.1-0.3mg/L+lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L; And/or,

The prescription of root media is: MS minimal medium+NH ₄NO ₃400mg/L+ KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+NAA0.1-0.5mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L.

More preferably in the method for first aspect present invention, each step is

1) strawberry is put into the sterilization fine sand and heat-treat under 3 ℃ in 37 soil, until the pumping root system; Get the long main root tip of a root of 1-2cm, after sterilization and washing are inferior, directly the front end 0.1-0.2mm tip of a root is downcut as explant;

2) explant is seeded on the tip of a root inducing culture, in 3 ℃ in 25 soil, light intensity 1000 native 200lux, illumination 16 native 2 Xiao Shi ∕ cultivate all over the world, and rear light intensity is strengthened to 2000 native 500 lux all around, be strengthened to 4000 native 500 lux after six weeks, cultivate until induce the regeneration plant young shoot from the tip of a root;

3) the regeneration plant young shoot is seeded on the proliferated culture medium, in 3 ℃ in 25 soil, light intensity 2500 native 500lux, illumination 16 native 2 Xiao Shi ∕ cultivate all over the world, until grow up to the long seedling of 8-10cm;

4) seedling is cut into the long segment of 1-3cm, is seeded on the subculture growth medium, in 3 ℃ in 25 soil, light intensity 2500 native 500lux, illumination 16 native 2 Xiao Shi ∕ cultivate all over the world, until grow up to the long subculture seedling of 8-10cm; And/or

5) the subculture seedling is inoculated on the root media, in 3 ℃ in 25 soil, ∕ cultivated all over the world when light intensity 2500 native 500lux, illumination 16 soil 2 were little, until grow root system.

Also preferred in the method for first aspect present invention, whether the test tubes seedling infects virus is that right RT-PCR detects by adopting following mix primer:

SMoV upstream primer P1:5 '-TAACGGTTCACGTCCTAGTCTGAC-3 '

SMoV downstream primer P2:5 '-TCTTGGGCTTGGATCGTCACCTG-3 '

SMYEV upstream primer P3:5 '-GTGTCCTCAATCCAGCCAG-3 '

SMYEV downstream primer P4:5 '-CATGGCACTCATTGGACCTGGG-3 '

SVBV upstream primer P5:5 '-GAATGGGACAATGAAATGAC-3 '

SVBV downstream primer P6:5 '-GTGAGGAGAACTTAGGACA-3 '

SCrV upstream primer P7:5 '-CCCTTCGCAGGCGCGCTAACA-3 '

SCrV downstream primer P8:5 '-GGGGTCCAACTCATAAGCGAT-3 '.

Further preferably wherein, the content ratio of mix primer centering P1:P2:P3:P4:P5:P6:P7:P8 is 0.3 ~ 0.5:0.3 ~ 0.5:0.4 ~ 0.6:0.4 ~ 0.6:0.8 ~ 1:0.8 ~ 1:0.9 ~ 1.1:0.9 ~ 1.1, is preferably 0.4:0.4:0.5:0.5:0.9:0.9:1:1.

In second aspect, the invention provides the application of the strawberry tip of a root in strawberry detoxification and group are trained.Wherein preferred strawberry detoxification is the method for first aspect present invention with the group training.

In the third aspect, the invention provides the method that multiple RT-PCR detects strawberry virus simultaneously and rapidly, wherein adopt following mix primer pair:

SMoV upstream primer P1:5 '-TAACGGTTCACGTCCTAGTCTGAC-3 '

SMoV downstream primer P2:5 '-TCTTGGGCTTGGATCGTCACCTG-3 '

SMYEV upstream primer P3:5 '-GTGTCCTCAATCCAGCCAG-3 '

SMYEV downstream primer P4:5 '-CATGGCACTCATTGGACCTGGG-3 '

SVBV upstream primer P5:5 '-GAATGGGACAATGAAATGAC-3 '

SVBV downstream primer P6:5 '-GTGAGGAGAACTTAGGACA-3 '

SCrV upstream primer P7:5 '-CCCTTCGCAGGCGCGCTAACA-3 '

SCrV downstream primer P8:5 '-GGGGTCCAACTCATAAGCGAT-3 '.

Except detecting required primer, the reagent that RT-PCR is used and method flow are well-known to those skilled in the art, and many commercializations have been arranged.And increasing of primer easily causes the phase mutual interference, and the inventor is optimized for this reason.Preferably wherein, the content ratio of mix primer centering P1:P2:P3:P4:P5:P6:P7:P8 is 0.3 ~ 0.5:0.3 ~ 0.5:0.4 ~ 0.6:0.4 ~ 0.6:0.8 ~ 1:0.8 ~ 1:0.9 ~ 1.1:0.9 ~ 1.1, is preferably 0.4:0.4:0.5:0.5:0.9:0.9:1:1.

Preferably in third aspect present invention, detection to as if strawberry test-tube plantlet, the preferably test-tube plantlet that obtains of the method for first aspect present invention.

The beneficial effect that the present invention obtains is:

1, processing ease, efficient significantly improve: because strawberry " stem apex " stem apex by peripheral more than ten layers stem sheet parcel center forms, in the past at microscopically, with operation tool divest continuously more than ten layers of stem sheet be a difficulty greatly and the very low work of efficient; And outside strawberry " tip of a root " be directly exposed to, the tip that cuts its tip of a root just seemed very easy, and efficient can improve tens times to hundreds of times;

2, sterilization is thorough, and pollution rate is low: stem apex detoxify is because its peripheral stem sheet is difficult for Ex-all, and sterilization also just is difficult to thoroughly cause the tissue culture seedling pollution rate often up to about 60%; And tip of a root detoxification is sterilized easily and thoroughly because it is peripheral without any parcel, therefore its tissue culture seedling pollution rate can greatly descend;

3, improved the quality of group training seedling: the present invention has not only successfully cultivated Plantlets of Strawberry take the tip of a root as explant, and by clear and definite after the test, tip of a root group training seedling breaks up speed, plant regeneration speed, test-tube plantlet reproduction speed and output than Shoot-tip Culture seedling at explant and is improved;

4, clear and definite after the field planting test, its rooting rate of tip of a root tissue cultural seedlings of free and transplanting survival rate are up to more than 98%;

5, whole method fully has been fit to the characteristics of the tip of a root, and medium at different levels have been done comprehensive improvement and adjustment, has guaranteed the success of tip of a root group training;

6, developed in addition the technology that multiple RT-PCR detects strawberry virus simultaneously and rapidly, unite use with aforementioned detoxification and tissue culture method, can guarantee zero band poison of Plantlets of Strawberry, thereby lay a good foundation for the high-quality of Plantlets of Strawberry, safety, high yield and spread.

Description of drawings

Fig. 1 is the growing way photo of the Strawberry Seedlings cultivated of strawberry detoxification of the present invention and tissue culture method, and wherein A, B and C have shown respectively the plantation situation of Strawberry Seedlings in experimental field, land for growing field crops and facility (booth).

Fig. 2 is the growing way photo of the Strawberry Seedlings cultivated of prior art.

For the ease of understanding, below will describe in detail the present invention by specific embodiment.It needs to be noted, instantiation only is in order to illustrate, not consist of limitation of the scope of the invention.Obviously those of ordinary skill in the art can illustrate according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.In addition, the present invention has quoted open source literature, and these documents also are in order more clearly to describe the present invention, and their full text content is all included the present invention in and carried out reference, just look like they full text in specification of the present invention repeated description excessively the same.

Embodiment

Below describe by specific embodiment, wherein special material, the reagent that describes in detail is well known to the skilled person, and can buy from market that (strawberry cultivars is red chivalrous and a chapter Ji; The MS minimal medium can be available from the bright inferior Bioisystech Co., Ltd in Wuhan, and model M524(has wherein added agar), also can be referring to books or the laboratory manual of plant tissue culture.

Embodiment 1 multiple RT-PCR detects the virus infection of strawberry tissue simultaneously and rapidly

Entrust the following strawberry virus-specific primer pair of synthesizing ribonucleotide sequence, and two kinds of primer content that are mixed into SMoV two kinds of primer content being respectively 4 μ M, SMYEV two kinds of primer content being respectively 5 μ M, SVBV two kinds of primer content being respectively 9 μ M, SCrV are respectively the mix primer solution of 10 μ M:

SMoV upstream primer P1:5 '-TAACGGTTCACGTCCTAGTCTGAC-3 '

SMoV downstream primer P2:5 '-TCTTGGGCTTGGATCGTCACCTG-3 '

SMYEV upstream primer P3:5 '-GTGTCCTCAATCCAGCCAG-3 '

SMYEV downstream primer P4:5 '-CATGGCACTCATTGGACCTGGG-3 '

SVBV upstream primer P5:5 '-GAATGGGACAATGAAATGAC-3 '

SVBV downstream primer P6:5 '-GTGAGGAGAACTTAGGACA-3 '

SCrV upstream primer P7:5 '-CCCTTCGCAGGCGCGCTAACA-3 '

SCrV downstream primer P8:5 '-GGGGTCCAACTCATAAGCGAT-3 '.

Get strawberry and organize 1g, put into 100mL and contain 0.5%Triton X-100 and 0.4 %Na ₂SO ₃Solution in 37 ℃ of lixiviate 20min, then in room temperature (25 ℃) lixiviate 1h, filter, get filtrate with the centrifugal 10min of 12,000r/ min, get sample solution.

Employing RT-PCR reverse transcription kit (can be available from the sea base bio tech ltd; goods number D0501); mix above-mentioned sample solution 2.5 μ L, 5 * RT MasterMix, 2 μ L and Oligo (dT) 20 Primer 0.5 μ L; complement to 10 μ L with deionized water; response procedures is 25 ℃ of incubation 10mim; 42 ℃ of reverse transcription 45mim, 95 ℃ of deactivation 2mim obtain reverse transcription cDNA solution.

Mix above-mentioned reverse transcription cDNA solution 4 μ L, above-mentioned mix primer solution 1 μ L and 2 * Taq PCR Mix, 12.5 μ L, complement to 25 μ L with deionized water, carry out PCR, condition is 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, 72 ℃ are extended 1 min, totally 30 circulations; Last 72 ℃ are extended 10 min.

Above primer and proportioning cause the phase mutual interference detecting strawberry tissue (especially strawberry test-tube plantlet tissue) Shi Buhui, and SMoV virus is positive, and will amplify the 15kb band, otherwise not amplify, and are negative; SMYEV virus is positive, and will amplify the 21kb band, otherwise not amplify, and is negative; SVBV virus is positive, and will amplify the 16kb band, otherwise not amplify, and is negative; Virus is positive, and will amplify the 23kb band, otherwise not amplify, and is negative.This is easy to bring resolution by the agarose electrophoresis bar.

Embodiment 2 strawberry detoxifications and tissue culture method 1

Be formulated as follows medium:

1) MS minimal medium;

2) tip of a root inducing culture: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/L+BA 0.5mg/L+2,4-D 0.1mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

3) proliferated culture medium: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+BA 0.5 mg/L+ NAA 0.2mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

4) subculture growth medium: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/0.5L+BA 0.2mg/L+lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

5) root media: MS minimal medium+NH ₄NO ₃400mg/L+ KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+NAA 0.5mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

Tip of a root detoxification is as follows with group training step:

1) pretreatment: strawberry is put into the sterilization fine sand heat-treat 6-12 week in 37 soil under 2 ℃, until the pumping root system; Get the long main root tip of a root of 1-2cm, successively through 70%(V/V) alcohol disinfecting 30 seconds, add that a tween shook sterilization 8 minutes and with after aseptic washing 3-5 time with 0.1% mercury chloride, directly with the cutting-out of the front end 0.1-0.2mm tip of a root as explant;

2) tip of a root regeneration induction plant is cultivated: explant is seeded on the tip of a root inducing culture, in 2 ℃ in 25 soil, light intensity 1000lux, illumination 16 Xiao Shi ∕ cultivate all over the world, light intensity is strengthened to 2000 lux after extremely, be strengthened to 4000 lux after six weeks, cultivated 3.5-4.5 month, until induce the regeneration plant young shoot from the tip of a root;

3) young shoot propagation is cultivated: the regeneration plant young shoot is seeded on the proliferated culture medium, in 2 ℃ in 25 soil, cultivated 1 month under light intensity 2500lux, the illumination 16 Xiao Shi ∕ d, until grow up to the long seedling of 8-10cm;

4) subculture grown cultures: seedling is cut into the long segment of 2-2.5cm, is seeded on the subculture growth medium, in 2 ℃ in 25 soil, cultivate under light intensity 2500lux, the illumination 16 Xiao Shi ∕ d, until grow up to the long subculture seedling of 8-10cm;

5) culture of rootage: the subculture seedling is inoculated on the root media, in 2 ℃ in 25 soil, cultivates under light intensity 2500lux, the illumination 16 Xiao Shi ∕ d, be test-tube plantlet until approximately send out roots to be tied to form after 1 week.Finish thus detoxification and group training process, can transplant land for growing field crops or greenhouse gardening.

Embodiment 3 strawberry detoxifications and tissue culture method 2

Be formulated as follows medium:

2) tip of a root inducing culture: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/L+BA 0.8mg/L+2,4-D 0.2mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

3) proliferated culture medium: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+BA 0.35 mg/L+ NAA 0.35mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

4) subculture growth medium: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/0.5L+BA 0.35mg/L+lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

5) root media: MS minimal medium+NH ₄NO ₃400mg/L+ KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+NAA 0.3Mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L.

Tip of a root detoxification and the step of group training step with embodiment 2.

Embodiment 4 strawberry detoxifications and tissue culture method 3

Be formulated as follows medium:

2) tip of a root inducing culture: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/L+BA 1.0mg/L+2,4-D 0.3mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

3) proliferated culture medium: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+BA 0.2mg/L+ NAA 0.5mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

4) subculture growth medium: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/0.5L+BA 0.5mg/L+lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

5) root media: MS minimal medium+NH ₄NO ₃400mg/L+ KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+NAA 0.1mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L.

Tip of a root detoxification and the step of group training step with embodiment 2.

The comparative result of embodiment 5 the present invention and prior art

Academy of agricultural sciences, Zhejiang Province virology and biotechnology research satellite car between in (academy of agricultural sciences, Ningbo City, Zhejiang Province organizes in the Culture Center) carry out detoxification and group training test, produce the strawberry test-tube plantlet.Experimental group is carried out with reference to the described method of embodiment 2-5, carries out altogether 96 groups (every kind of method is not less than and does 20 groups), and wherein the photo of growing way as shown in Figure 1; Control group carries out with reference to method and similar approach that Chinese patent application 200510030445 and Chinese patent 200510062199 are put down in writing, carries out altogether 96 groups, and wherein part growing way photo as shown in Figure 2.Get each group training and the test-tube plantlet that produces, after 96 tissues of every group of random sampling mix, detect according to embodiment 1 described method.Testing result is as shown in table 1, and the pollution that experimental group detects (even if any one sample detects any this group of virus pollution in the group) has 16, and pollution rate is 16.6%, and control group pollutes 64, and pollution rate is up to 66.7%; The average callus of experimental group is 32 days, is shorter than 38 days of control group; Experimental group regeneration plant average out to 126 days is shorter than 132 days of control group; The monthly average reproduction speed of the Strawberry Seedlings of experimental group is 5.1 times, is higher than 4.8 times of control group; The transplanting survival rate 98% of the test-tube plantlet of experimental group also is higher than 96% of control group; The every strain of initial seedling of experimental group can on average be bred and be produced seedling 113.1 strains, and only 102 strains of control group.This shows, the Strawberry Seedlings that method of the present invention is cultivated is improved at aspects such as explant differentiation speed, plant regeneration speed, test-tube plantlet reproduction speed group training seedling and land for growing field crops output than prior art.

The Contrast on effect table of the detoxification of table 1 the present invention and prior art and group training

Claims (6)

1. strawberry detoxification and the method for organizing training, it comprises

1) strawberry is put into the sterilization fine sand and under 34-40 ℃, heat-treat, until the pumping root system; Get the long main root tip of a root of 1-2cm, after this sterilization and washing, directly downcut the front end 0.1-0.2mm tip of a root as explant;

2) explant is seeded on the tip of a root inducing culture, at 22-28 ℃, light intensity 800-1200lux, illumination 14-18 Xiao Shi ∕ cultivate all over the world, light intensity is strengthened to 1500-2500 lux all around, be strengthened to 3500-4500 lux after six weeks, cultivate until induce the regeneration plant young shoot from the tip of a root, wherein, the prescription of tip of a root inducing culture is: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/L+6-BA 0.3-0.5mg/L+2,4-D 0.1-0.3mg/L+ lactoalbumin hydrolysate 1g/L+ sucrose 20 g/L;

3) the regeneration plant young shoot is seeded on the proliferated culture medium, at 22-28 ℃, light intensity 2000-3000lux, illumination 14-18 Xiao Shi ∕ cultivate all over the world, until grow up to the long seedling of 8-10cm, wherein, the prescription of proliferated culture medium is: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+6-BA 0.2-0.5 mg/L+ NAA 0.1-0.3mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

4) seedling is cut into the long segment of 1-3cm, is seeded on the subculture growth medium, at 22-28 ℃, light intensity 2000-3000lux, illumination 14-18 Xiao Shi ∕ cultivate all over the world, until grow up to the long subculture seedling of 8-10cm, wherein, the prescription of subculture growth medium is: MS minimal medium+NH ₄NO ₃400mg/L+KNO ₃500mg/L+KH ₂PO ₄50mg/0.5L+6-BA 0.1-0.3mg/L+lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L;

5) the subculture seedling is inoculated on the root media, at 22-28 ℃, light intensity 2000-3000lux, illumination 14-18 Xiao Shi ∕ cultivate all over the world, until grow root system, so that the subculture seedling grows up to test-tube plantlet, wherein, the prescription of root media is: MS minimal medium+NH ₄NO ₃400mg/L+ KNO ₃500 mg/L+KH ₂PO ₄50 mg/L+NAA 0.1-0.5mg/L+ lactoalbumin hydrolysate 1 g/L+ sucrose 20 g/L; With

6) whether optional test tubes seedling infects virus.

2. method claimed in claim 1, wherein whether to infect virus be that right RT-PCR detects by adopting following mix primer to the test tubes seedling:

SMoV upstream primer P1:5 '-TAACGGTTCACGTCCTAGTCTGAC-3 '

SMoV downstream primer P2:5 '-TCTTGGGCTTGGATCGTCACCTG-3 '

SMYEV upstream primer P3:5 '-GTGTCCTCAATCCAGCCAG-3 '

SMYEV downstream primer P4:5 '-CATGGCACTCATTGGACCTGGG-3 '

SVBV upstream primer P5:5 '-GAATGGGACAATGAAATGAC-3 '

SVBV downstream primer P6:5 '-GTGAGGAGAACTTAGGACA-3 '

SCrV upstream primer P7:5 '-CCCTTCGCAGGCGCGCTAACA-3 '

SCrV downstream primer P8:5 '-GGGGTCCAACTCATAAGCGAT-3 ',

Wherein the content of mix primer centering P1:P2:P3:P4:P5:P6:P7:P8 is than being 0.4:0.4:0.5:0.5:0.9:0.9:1:1.

3. the application of the strawberry tip of a root in strawberry detoxification and group are trained, wherein the strawberry detoxification is the arbitrary described method of claim 1-2 with the group training.

4. multiple RT-PCR detects the method for strawberry virus simultaneously and rapidly, wherein adopts following mix primer pair:

SMoV upstream primer P1:5 '-TAACGGTTCACGTCCTAGTCTGAC-3 '

SMoV downstream primer P2:5 '-TCTTGGGCTTGGATCGTCACCTG-3 '

SMYEV upstream primer P3:5 '-GTGTCCTCAATCCAGCCAG-3 '

SMYEV downstream primer P4:5 '-CATGGCACTCATTGGACCTGGG-3 '

SVBV upstream primer P5:5 '-GAATGGGACAATGAAATGAC-3 '

SVBV downstream primer P6:5 '-GTGAGGAGAACTTAGGACA-3 '

SCrV upstream primer P7:5 '-CCCTTCGCAGGCGCGCTAACA-3 '

SCrV downstream primer P8:5 '-GGGGTCCAACTCATAAGCGAT-3 ',

Wherein the content of mix primer centering P1:P2:P3:P4:P5:P6:P7:P8 is than being 0.4:0.4:0.5:0.5:0.9:0.9:1:1.

5. method claimed in claim 4, wherein detect to as if the strawberry test-tube plantlet.

6. method claimed in claim 5, wherein detect to as if the test-tube plantlet that obtains of the arbitrary described method of claim 1-2.

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