CN104429940A - Method for acquiring virus-free strawberry seedlings - Google Patents
Method for acquiring virus-free strawberry seedlings Download PDFInfo
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Abstract
The invention provides a rapid propagation method for acquiring virus-free strawberry seedlings. The method comprises the following steps: (1) selecting a raw material and sterilizing the raw material; (2) bud culture: removing sepals of a sterilized bud, then inoculating the bud into a prepared culture medium of MS+6-BA1.0mg/L+NAA0.1mg/l for culture till clumpy buds grow out; (3) propagation culture: transferring the clumpy buds onto a culture medium of MS+6-BA0.1mg/L for propagation culture; (4) induced rooting: cutting off strawberry seedlings from the clumpy buds, transferring the strawberry seedlings to the culture medium of 1/2MS+NAA0.1mg/L culture medium, inducing the rooting, and transferring the rest of callus to the culture medium of MS+6-BA0.1mg/L for culture; (5) seedling hardening: transplanting the rooted strawberry seedlings into a culture plate from a culture bottle, and carrying out the seedling hardening growth in a greenhouse. By utilizing the method, the method for acquiring the virus-free seedlings is simple, the acquired seedlings are strong, stolon reproduce quickly, and the quality is good.
Description
Technical field
This patent is a kind of method utilizing strawberry bud to carry out strawberry detoxic seedling factorial praluction, relates to the method such as Strawberry tissue culture, detoxic seedling factorial praluction, belongs to modern agriculture seedling growing process field.
Background technology
Rapid propagation in vitro (
in vitropropagation) and plant toxic (virus-free) be current Plant Tissue Breeding application at most, the most effective aspect.In production, many asexually propagated crops are all subject to infecting of virus, thus cause the serious degradation of kind, the underproduction and reduction quality.Utilizing plant meristematic tissue to carry out cultivation can make the plant newly grown up to slough virus, and it is numerous to carry out expansion to virus free plants.Profit is produced virus-free crops in this way and is had potato, sweet potato, garlic, strawberry, apple, banana etc., and large-scale application in production.
Strawberry as the rose family (Rosaceae) Fragaria (
fragaria) herbaceos perennial, demand is increasing in the market.According to the breeding of this traditional stolon and propagation by division modes of reproduction, after cultivation for many years, be easy to infect multiple virus, cause the bad and resistance reduction of the reduction of kind of a property serious degradation, output, product qualitative change etc.Therefore, strawberry quality how is improved and improves its disease resistance that oneself more and more receives the concern of people.Strawberry cultivars improvement adopts conventional cross-breeding technology, but breeding cycle is long, and efficiency of selection is low.The modern biotechnology of development in recent years is that Strwberry Breeding provides new way, and it can obtain the newtype that conventional breeding is difficult to maybe cannot obtain, thus creates the kind matter made new advances.The application of modern biotechnology in plant is risen along with the development of plant tissue culture technique.The tissue cultures of strawberry is carried out comparatively early, since the eighties, the cultivation of the various organs of strawberry, tissue and cell develops rapidly, has carried out the research of the aspects such as anther culture, leaf culture, Shoot Tip Culture, Protoplast cuhnre and cell suspension cultures, and has made some progress.
Since the careless malicious stem apex of utilization carried out obtaining regeneration plant from in-vitro culture from Miller reported first in 1963, many scholars have in succession been had to carry out the research of this respect both at home and abroad.Mainly utilize Shoot Tip Culture to reach Fast-propagation and remove the object of careless viral disease poison.Qin Lanying etc. establish the in vitro amount reproduction system of careless malicious stem apex, and 14 d can obtain 10 young plants from a young plant, and rooting rate and transplanting survival rate all very high.Li Qing etc. think, root media can with the medium of 1/2 MS macroelement, and the consumption of sugar can reduce to 15 g/L, and available granulated sugar replaces sucrose, also can save agar and carry out liquid culture, all not affect growth of seedling, thus can reduce seedling cost.
Guo Chunyuan etc. propose strawberry anther tissue culture rapid propagation technique, and condition of culture is 22-25
oc, illumination every day 18 hours 3000 LX, can form a large amount of callus in about 15-25 days.Callus inducing medium consists of: MS+6-BA 2.0 mg/L+NAA 0.2 mg/L.Liu Yadong, Wu Tao, Zhu Yuling etc. have carried out introduction and the research of group training detoxification technology of new varieties " terrible anger is sweet ".Shoot-tip Culture detoxification result of the test shows: the best of this kind lures bud medium to be MS+6-BA 2.0 mg/L+NAA 0.1 mg/L, and best root media is MS+NAA 0.05 mg/L; And have studied hardening and the transplantation technique of nontoxic seedling.
Numerous experimental studies have found that, in tissue culture procedures, the kind of hormone and proportioning are important outside regulating measures.In the anther culture process of grass poison, the subject matter of existence is that healing rate is low and formation indefinite bud is difficult, about Hormone Conditions on the malicious anther cultural impact of grass, all has report both at home and abroad.The different culture media such as Ma Hongmin cultivates flower pesticide, find that only additional NAA flower pesticide agensis becomes callus: if cultivate with F+IAA 0.2 mg/L+BA 2.0 mg/L, then flower pesticide can form embryo callus regeneration plant or bud, also can be formed directly in plant; Hang Ling etc. take MS as minimal medium, and add BA O.5 mg/L+2,4-D O.25 mg/L, induction of anther callus rate reaches 97.7%; The discoveries such as Wu Weimin, as the exogenous hormone BA that differential medium uses, when ZT, CPPU, KT concentration is 2 mg/L, BA is better than ZT, CPPU and KT, and is conducive to using 1 mg/LCPPU as the exogenous hormone of squamous subculture the embryo keeping callus; The reports such as Hou Xilin, MS ten NAA O.l mg/L+BA 5.O mg/L is conducive to indefinite bud most; Faedi etc. find that the KT of additional 0.Ol mg/L on MS medium can induce embryo callus subculture high-frequency to occur.Some researchers find, except hormone, when differentiation-inducing, add 5-15 mg/L AgNO in differential medium
3all can improve the differentiation rate of indefinite bud, with 8 mg/L AgNO
3best results; Han Xuemei proposes total 15 mol/L of medium (wherein nitrate nitrogen 1O mol/L, by state nitrogen 5 mol/L), and other compositions are significantly improved than MS with the anther plant differentiation rate of MS.Separately some researchers have reported that flower pesticide 4 DEG C of Cold pretreatment 48 h have obvious effect for the ability improving flower pesticide generation callus.But current strawberry seedling is cultivated still exists seedling quality difference, virus-free culture cycle long two problems.
Summary of the invention
The present invention be directed to current Problems existing, the method for a set of new strawberry detoxic seedling Fast-propagation is provided, to reach the object of efficient factorial praluction, form the technology of a set of strawberry detoxic seedling Fast-propagation.
The present invention utilizes plant tissue culture technique, and the bud for strawberry carries out Fiber differentiation as explant, selects maximum hormone ratio, and make it develop into tissue cultures that complete plant is strawberry provides a new technology.Fast-propagation virus-free Strawberry seedlings, the production system of setting up a factory Plantlets of Strawberry, for China's Strawberry industry provides a large amount of high-quality seedling, makes up the deficiency in strawberry market.
For achieving the above object, the present invention realizes by the following technical solutions:
Utilize the method for plant tissue rapid propagation in vitro, comprise the following steps:
1) to draw materials sterilizing: utilize the unopened bud of strawberry, sterilization treatment;
2) flower bud culture: by the bud removing sepal after sterilizing, be inoculated in the medium configured and cultivate, described medium is MS medium, adds the 6-BA that concentration is 0.4-2.5mg/L, concentration is that the NAA of 0.02-0.2 mg/L cultivates, and grows clump bud;
3) expand numerous cultivation: by the clump bud grown up to directly or the cutting MS medium that is transferred to the 6-BA adding concentration 0.05-2.0mg/L carry out expanding numerous cultivation;
4) root induction: after expanding numerous cultivation, cuts strawberry seedlings from clump bud, is transferred to add on 1/2 MS medium that concentration is 0.1 mg/L NAA to induce it to take root, and residue callus is cultivated on the medium being forwarded to MS+6-BA 0.1 mg/L;
5) strong sprout hardening: before transplanting, seedling is transferred to and is not added on the MS medium of any hormone; Strawberry Seedlings after being processed strong sprout is transplanted to culture plate from blake bottle, carries out growth in strong sprout, as the seedling that next step stolon is bred in greenhouse.
Preferably, described step 2) in the concentration of 6-BA be 1.0-1.5mg/L; Preferred, the concentration of 6-BA is 1.0mg/L.
Preferably, described step 2) in the concentration of NAA be 0.05-0.15 mg/L; Preferred, the concentration of NAA is 0.1 mg/L
Preferably, in described step 3), the concentration of 6-BA is 0.1 mg/L.
The plant tissue rapid propagation in vitro that the present invention adopts obtains the method for detoxic seedling, has following advantage and good effect than existing Virus-elimination Techniques For Strawberry:
Why bud becomes the best explant being superior to other Strawberry tissue culture: on the one hand, and from the viewpoint of drawing materials and explant sterilization, cauline leaf handle is relatively close to soil, be bacterial contamination comparatively serious, once sterilization is unclean, just easily cause medium to pollute, explant is dead.And bud is just different, wherein, will open because bud is in and not have open state, bud is surrounded by petal, and inside is almost in aseptic state, very easily surface sterilization; Meanwhile, overcome and choose stem or leaf as the situation often producing brown stain during explant; Again, the bud of this time period neither be too young tender, is convenient to aseptically peel calyx off, and avoid destroying explant during cutting, thus avoid sterilization difficulty and operating difficulties etc.
On the other hand, the easy degree of inductivity, survival rate and differentiation is analyzed.Comparatively speaking, the very easily differentiation-inducing seedling of bud, on the medium of hormon proportioning easier evoking adventive bud generation and differentiation-inducingly to take root.And easy brownization of blade, petiole, vitrifying, the callus of generation not easily root induction germinates, finally dead.
In addition, what is more important, bud induces one-step-seedling formation in MS+6-BA 1.0 mg/L+NAA 0.1 mg/L medium, and Callus formation is little, in inducing culture, can be divided into stem, leaf after squamous subculture.This, for keeping the excellent moral character of original kind, identifies that its genetic stability is significant.
Further, before transplanting, seedling is transferred to and does not add on the MS medium of any hormone, the survival rate of transplantation of seedlings can be improved.
Through field experiment, send out seedling rate detoxic seedling year more than 100/, be better than malicious Strawberry Seedlings in spite of illness far away, actual effect is fine.
Embodiment
Describe the present invention below in conjunction with embodiment.
The technical method utilizing strawberry bud to carry out strawberry detoxic seedling Fast-propagation provided by the present invention, its embodiment is as follows:
1) acquisition of explant and sterilization
Get strawberry bud, rinse 1-2 hour under flowing tap water.Have perianth to be wrapped in outside bud, make it be isolated from the outside, its inside is in germ-free condition, very easily surface sterilization, and can carry out disinfection after current through 1-2 hour operation.
With 70% alcohol surface sterilization 30 seconds on superclean bench, then sterilize 15 minutes with the hydrogen peroxide of about 14%, then use aseptic water washing 3 times, be inoculated in after then bud being removed outer bract on preprepared inducing culture.
2) the choosing of hormone
After the disinfecting of bud routine, on superclean bench, bud removes outer sepal, and the explant handled well to be seeded in NAA fixed concentration be 0.1 mg/L, 6-BA is on the medium of 0.4-3.5 mg/L; And 6-BA fixed concentration is on the medium of 1.0 mg/L, different N AA concentration.Experimental result is in table 1
The developmental state of bud on hormon concentration cultures: table 1
(when 6-BA concentration is 2.0-2.5 mg/L, though inductivity is higher, there is browning)
Can be obtained by table 1: draw the screening of hormone concentration, the Fiber differentiation of bud combines best with MS+6-BA 1.0 mg/L+NAA 0.1 mg/L.
3) induced bud differentiation and expand numerous
After bud cultivates several generations on inducing culture, callus constantly expands, and has the differentiation of immature sprout, and sprout grows up to plantlet gradually.These sprouts that grow thickly are cut, then the MS being inoculated into hormon proportioning cultivates the differentiation carrying out induced bud.In this test, we divide four process, and result is in table 2.
Table 2
Can be obtained by table 2: best expansion breeding culture medium is MS+6-BA0.1 mg/L
4) root induction
After expanding numerous cultivation, cut strawberry seedlings from clump bud, residue callus is cultivated on the medium being forwarded to MS+6-BA 0.1 mg/L.When Regenerated plant grows to about 4 ~ 5cm, these seedlings under cutting, separately proceed to sprout in 1/2 MS+NAA 0.1 mg/L medium to cultivate and take root.About about 10 days, new root occurred, then every about 5 days, average every strain took root 2 ~ 3, in continuous generation.
5) strong sprout hardening
Before transplanting, in order to improve the survival rate of transplantation of seedlings, seedling being transferred to and not adding on the MS medium of any hormone.Strawberry Seedlings after being processed strong sprout is transplanted to culture plate from blake bottle, carries out growth in strong sprout, as the seedling that next step stolon is bred in greenhouse.
Embodiment 1
1) acquisition of explant and sterilization
Get strawberry bud, rinse 1-2 hour under flowing tap water.With 70% alcohol surface sterilization 30 seconds on superclean bench, then sterilize 15 minutes with the hydrogen peroxide of about 14%, then use aseptic water washing 3 times, be inoculated in after then bud being removed outer bract on preprepared inducing culture.
2) the choosing of hormone
After the disinfecting of bud routine, on superclean bench, bud removes outer sepal, and the explant handled well to be seeded in NAA concentration be 0.1 mg/L, 6-BA is on the medium of 1.0 mg/L.
3) induced bud differentiation and expand numerous
After bud cultivates several generations on inducing culture, callus constantly expands, and has the differentiation of immature sprout, and sprout grows up to plantlet gradually.These sprouts that grow thickly are cut, then is inoculated that to add concentration be that the MS of 0.1 mg/L 6-BA cultivates and carries out the differentiation of induced bud.In this test, we divide four process, and result is in Table
4) root induction
After expanding numerous cultivation, cut strawberry seedlings from clump bud, residue callus is cultivated on the medium being forwarded to MS+6-BA 0.1 mg/L.When Regenerated plant grows to about 4 ~ 5cm, these seedlings under cutting, separately proceed to sprout in 1/2 MS+NAA 0.1 mg/L medium to cultivate and take root.About about 10 days, new root occurred, then every about 5 days, average every strain took root 2 ~ 3, in continuous generation.
5) strong sprout hardening
Before transplanting, seedling is transferred to and does not add on the MS medium of any hormone.Strawberry Seedlings after being processed strong sprout is transplanted to culture plate from blake bottle, carries out growth in strong sprout, as the seedling that next step stolon is bred in greenhouse.
Embodiment 2
In the present embodiment, other steps are identical with embodiment 1, and unique difference is step 2) in the choosing of hormone, NAA concentration is 0.1 mg/L, 6-BA concentration is on the medium of 1.2 mg/L.
Embodiment 3
In the present embodiment, other steps are identical with embodiment 1, and unique difference is step 2) in the choosing of hormone, NAA concentration is 0.1 mg/L, 6-BA concentration is on the medium of 1.5 mg/L.
Embodiment 4
In the present embodiment, other steps are identical with embodiment 1, and unique difference is step 2) in the choosing of hormone, NAA concentration is 0.05mg/L, 6-BA concentration is on the medium of 1.0 mg/L.
Embodiment 5
In the present embodiment, other steps are identical with embodiment 1, and unique difference is step 2) in the choosing of hormone, NAA concentration is 0.15mg/L, 6-BA concentration is on the medium of 1.0 mg/L.
Claims (4)
1. obtain a method for strawberry detoxic seedling, it is characterized in that, comprise the following steps:
1) to draw materials sterilizing: utilize the unopened bud of strawberry, sterilization treatment;
2) flower bud culture: by the bud removing sepal after sterilizing, be inoculated in the medium configured and cultivate, described medium is MS medium, adds the 6-BA that concentration is 0.4-2.5mg/L, concentration is that the NAA of 0.02-0.2 mg/L cultivates, and grows clump bud;
3) expand numerous cultivation: by the clump bud grown up to directly or the cutting MS medium that is transferred to the 6-BA adding concentration 0.05-2.0mg/L carry out expanding numerous cultivation;
4) root induction: after expanding numerous cultivation, cuts strawberry seedlings from clump bud, is transferred to add on 1/2 MS medium that concentration is 0.1 mg/L NAA to induce it to take root, and residue callus is cultivated on the medium being forwarded to MS+6-BA 0.1 mg/L;
5) strong sprout hardening: before transplanting, seedling is transferred to and is not added on the MS medium of any hormone; Strawberry Seedlings after being processed strong sprout is transplanted to culture plate from blake bottle, carries out growth in strong sprout, as the seedling that next step stolon is bred in greenhouse.
2. the method for acquisition strawberry detoxic seedling according to claim 1, is characterized in that, described step 2) in the concentration of 6-BA be 1.0-1.5mg/L.
3. the method for acquisition strawberry detoxic seedling according to claim 1, is characterized in that, described step 2) in the concentration of NAA be 0.05-0.15 mg/L.
4. the method for acquisition strawberry detoxic seedling according to claim 1, it is characterized in that, in described step 3), the concentration of 6-BA is 0.1 mg/L.
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Cited By (3)
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| CN107333659A (en) * | 2017-09-15 | 2017-11-10 | 江苏省农业科学院 | A kind of day neutrality Strawberry Plantlets fast breeding culture medium and tissue culture and rapid propagation method |
| CN109076960A (en) * | 2018-09-29 | 2018-12-25 | 河南云帮农业科技有限公司 | A kind of Plantlets of Strawberry detoxication and tissue culture strong sprout method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106069757A (en) * | 2016-06-20 | 2016-11-09 | 热作两院种苗组培中心 | A kind of Daphniphyllum calycinum method for tissue culture |
| CN106069757B (en) * | 2016-06-20 | 2018-04-10 | 热作两院种苗组培中心 | A kind of daphniphyllum calycinum method for tissue culture |
| CN107333659A (en) * | 2017-09-15 | 2017-11-10 | 江苏省农业科学院 | A kind of day neutrality Strawberry Plantlets fast breeding culture medium and tissue culture and rapid propagation method |
| CN107333659B (en) * | 2017-09-15 | 2019-01-22 | 江苏省农业科学院 | A kind of day neutral Strawberry Plantlets fast breeding culture medium and tissue culture and rapid propagation method |
| CN109076960A (en) * | 2018-09-29 | 2018-12-25 | 河南云帮农业科技有限公司 | A kind of Plantlets of Strawberry detoxication and tissue culture strong sprout method |
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