CN103947554B - Lily is by the method for somatic embryo development ways detoxification - Google Patents
Lily is by the method for somatic embryo development ways detoxification Download PDFInfo
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- CN103947554B CN103947554B CN201410183779.5A CN201410183779A CN103947554B CN 103947554 B CN103947554 B CN 103947554B CN 201410183779 A CN201410183779 A CN 201410183779A CN 103947554 B CN103947554 B CN 103947554B
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Abstract
The invention discloses the method for a kind of lily by the detoxification of somatic embryo development ways, comprise the steps: 1) embryonic callus induction: the lily bud scale cultivated under aseptic condition is as explant, and Fiber differentiation goes out embryo callus.2) the selectivity squamous subculture of embryo callus: select the newborn spherical cell stage produced by embryo callus as subculture material.3) squamous subculture of spherical cell stage: spherical cell stage is inoculated in solid multiplication medium or liquid proliferated culture medium and carries out squamous subculture, until do not detect virus in the somatic embryo of squamous subculture.Method of operating of the present invention is simple, carries out repeatedly subculture by routine experimentation person to plan detoxification material, less demanding to experimental condition and operating personnel.By the Fast-propagation of somatic embryo, thoroughly can slough virus, detoxification efficiency is better.
Description
Technical field
The present invention relates to lily detoxification breeding technical field, be specifically related to the method for a kind of lily by the detoxification of somatic embryo development ways.
Background technology
Lily, as the flower of luckiness, have the meaning of life-long happiness and perfect harmony and is loved by the people.Fragrant Lily is the queen in lily, and because of its pleasant aroma, the fine work more in fresh cut-flowers, first choice of making a gift to someone.In recent years, a rising star after the large cut-flower (Chinese rose, carnation, chrysanthemum, sword lily, flameray gerbera) in the lily cut flowers Shi Ji world five is also one of current cut-flower most popular in the world.But problem maximum in lily planting process is exactly that the deterioration of variety problem of lily---virus is one of its main cause.In lily, common virus has cucumber mosaic virus (Cucumber Mosaic Virus, CMV), the broken brocade virus of tulip (Tulip Breaking Virus, TBV), lily asymptomatic virus (Lily Symptomless Virus, etc. LSV) more than ten plant, these viruses by aphid bite or soil nematodes is propagated.The lily be infected by the virus shows growth potential weakening, plant is downgraded, early flowering, flower deformity, diminish, pattern goes down, early spring there is the phenomenons such as floral leaf, shrinkage, distortion, top spot, necrosis in blade, having had a strong impact on the yield and quality of fresh cut lily flowers, is the producing and selling of fresh cut lily flowers, and especially outlet brings very large threat.The diffusion velocity of lily virus in its organizer quickly, once infect virus, be then with poison all the life, be injured for a long time by lily; Add the insect Vector transmission such as aphid, virus spreads rapidly and infects between each plant, expands damaging range.For lily virus, be still difficult at present adopt chemical agent or biologic product to carry out direct effectively preventing.Therefore, the countermeasure preventing lily from infecting virus disease adopts virus-free culture technology acquisition detoxic seedling, and isolation is bred, produce healthy kind ball.
At present, the poison-removing method of lily mainly contains: Shoot Tip Culture, thermal treatment and antiviral agent method etc. three kinds.Shoot Tip Culture detoxification principle is that the cell that plant meristematic tissue newly produces does not have virus, plant corpus inner virus can only be moved by fibrovascular system, vascular bundle is not had in meristematic tissue, virus can only move by protoplasmic connection, speed is very slow, be difficult to the meristematic tissue catching up with active growth, so the tip of a root of vigorous growth, stem apex are generally all virus-free or seldom have viral distribu-tion.When utilizing Shoot Tip Culture to carry out lily detoxification, only when stripping the stem apex or shoot apical meristem that size is 0.3 ~ 0.5mm, just likely obtain detoxic seedling.Because the stem apex peeled off is little, need to operate under anatomical lens, operation easier is large, and after inoculation, small stem apex is owing to being subject to physical injury when peeling off, and be difficult to survive, virus elimination rate is also lower.Thermal treatment occurs passivation according at high temperature virus, and it copies and obviously weakens or no longer breed.In general, temperature is higher, the duration is longer, and detoxification efficiency is better, but the survival rate of plant is also lower simultaneously.The action principle of antiviral agent method is the formation that antiviral agent can stop viral RNA cap sequence under three squama acid conditions.Antivirus inhibitor destroys the formation of RNA polymerase in early days, in the formation of later stage break virus coat protein, stops the breeding of virus, reaches detoxification object.Thermal treatment and antiviral agent method generally all need to be combined with Shoot Tip Culture.After Shoot Tip Culture complicated operation, inoculation easily there is brownization in stem apex, and survival rate is low.Therefore, traditional poison-removing method needs inhibition process test material being carried out to physics or chemistry, and detoxification success or not is relevant with the meristematic tissue size manually cut off, and the impact by environment and human factor is comparatively large, there is the halfway problem of detoxification.
Summary of the invention
For above-mentioned deficiency of the prior art, the invention provides the method for a kind of lily by the detoxification of somatic embryo development ways, simplify detoxification flow process, reduction operating personnel and culture environment are on the impact of detoxification result.
To achieve these goals, the technical solution used in the present invention is as follows:
Lily, by the method for somatic embryo development ways detoxification, comprises the steps:
1) embryonic callus induction: the lily bud scale cultivated under aseptic condition is as explant, and be inoculated on embryonic callus induction medium, half-light cultivates 28 days, and Fiber differentiation goes out embryo callus.
2) screening of embryo callus: screen the embryo callus obtained in step 1), the spherical cell stage that selecting makes new advances produces is as subculture material.
3) squamous subculture of spherical cell stage: by step 2) in the spherical cell stage that screens be inoculated in solid multiplication medium or liquid proliferated culture medium and carry out first generation squamous subculture, temperature remains on 24 ± 2 DEG C, half-light cultivates 14 days, obtains first generation squamous subculture somatic embryo.
The spherical cell stage making new advances and produce is screened in first generation squamous subculture somatic embryo, be inoculated in solid multiplication medium or liquid proliferated culture medium and carried out second generation squamous subculture, temperature remains on 24 ± 2 DEG C, and half-light cultivates 14 days, obtains second generation squamous subculture somatic embryo; Circulation like this, until do not detect virus in the somatic embryo of squamous subculture.
Wherein, the consisting of of described embryonic callus induction medium: MS medium+PIC 1 ~ 8mgL
-1+ agar 7gL
-1+ sucrose 30gL
-1, pH value is 5.8.
Consisting of of described solid multiplication medium: MS medium+PIC 1 ~ 4mgL
-1+ agar 7gL
-1+ sucrose 30gL
-1, pH value is 5.8.
Consisting of of described liquid proliferated culture medium: MS medium+PIC 1 ~ 4mgL
-1+ sucrose 30gL
-1, pH value is 5.8.
Further, described liquid proliferated culture medium is added in bio-reactor, and the throughput of bio-reactor remains on 20mlmin
-1.
Further, the screening of described spherical cell stage adopts aperture to be that the sieve of 0.5-1.0 mm screens.
Relative to prior art, the present invention has following beneficial effect:
1, method of operating of the present invention is simple.In During Detoxification, without the need to carrying out the loaded down with trivial details pre-treatment of high temperature or virazole to test material, being the specific operations such as the small meristematic tissue of 0.3 ~ 0.5 mm without the need to cutting volume, by routine experimentation person, repeatedly subculture being carried out to test material, less demanding to experimental condition and operating personnel.By the fast breeding of somatic embryo, thoroughly can slough virus, detoxification efficiency is better.
2, incubation time is shortened.In conventional method, the plan detoxification material of 0.3 ~ 0.5 mm has to pass through long-term cultivation and can reach the minimum sample size of Viral diagnosis, and somatic embryo development ways poison-removing method of the present invention can meet the requirement of Viral diagnosis minimum amount of sample amount by the fast breeding of somatic embryo within the relatively short time.
3, detoxification efficiency is improved.The detoxification result of tradition poison-removing method without guarantee, by artificial and environmental influence is larger.Varying in size of each meristematic tissue cutting causes its detoxification efficiency different, therefore needs to carry out Viral diagnosis to each meristematic tissue, as Viral diagnosis finds that then this meristematic tissue is useless, and long-term cultivation work is invalid still containing virus.In somatic embryo poison-removing method, can detecting the somatic embryo of different subculture number, as do not sloughed virus, can Multiplying culture be proceeded until slough virus.As sloughed virus, also can carry out Multiplying culture, realizing the amount reproduction of detoxification material in a short time, do not waste material.
4, detoxification efficiency is good.By artificial and environmental influence comparatively greatly, this depends on and cuts the size that shoot apical meristem cuts more greatly tradition poison-removing method, and virus can not thoroughly remove; Cut less, stem apex cannot survive.Often exist and slough a kind of virus, the phenomenon that other kind of virus is not sloughed.Somatic embryo detoxification is subject to artificial and environmental influence is less, and detoxification efficiency is better, can slough multiple virus simultaneously.
Accompanying drawing explanation
Fig. 1 is embryo callus containing PIC2 mgL
-1liquid culture condi under the growth perfonnance of embryo callus.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described.
Embodiment one:
Lily, by the method for somatic embryo development ways detoxification, comprises the steps:
1) embryonic callus induction: the lily bud scale cultivated under aseptic condition, as explant, is inoculated on embryonic callus induction solid culture medium, cultivate 28 days, Fiber differentiation goes out embryo callus.
Wherein, the consisting of of embryonic callus induction medium: MS medium+PIC 1 ~ 3mgL
-1+ agar 7gL
-1+ sucrose 30gL
-1, pH value is 5.8.PIC is the one in plant growth regulator, and its English full name is Picloram, and Chinese is picloram.
Embryonic callus induction medium adds 7gL in MS minimal medium
-1agar and 30gL
-1sucrose, keeps pH value to be 5.8, and adds the material of following formula:
| PIC（mg·L -1） | BA（mg·L -1） | NAA（mg·L -1） | Effect | |
| Formula 1 | 1 | 0 | 0 | Scale base portion forms less callus, and with a small amount of little young ball differentiation, embryo callus yield is lower |
| Formula 2 | 2 | 0 | 0 | Scale base portion forms the callus of more short texture, and without little young ball differentiation, embryo callus yield is higher |
| Formula 3 | 3 | 0 | 0 | Scale forms a large amount of hard-packed callus, and without little young ball differentiation, embryo callus yield is higher |
| Comparative formula 1 | 1 | 2 | 1 | Scale base portion forms a large amount of callus, and with a large amount of little young ball differentiation, embryo callus yield is lower, and mixed raw with non embryogenic callus |
| Comparative formula 2 | 2 | 2 | 1 | Scale base portion forms a large amount of callus, and with a small amount of little young ball differentiation, embryo callus yield is general, and mixed raw with non embryogenic callus |
| Comparative formula 3 | 3 | 2 | 1 | Scale base portion forms small amount callus, and without little young ball differentiation, embryo callus yield is higher, presents graininess and is distributed on scale |
From above-mentioned formula, BA(6-benzyl aminoadenine) and NAA(methyl α-naphthyl acetate) less on the impact of lily embryonic callus induction.In comparative formula 1 ~ comparative formula 3, BA, NAA and PIC are used in combination, easily cause the mixobiosis of embryo callus and non embryogenic callus, cause difficulty to the abstraction and purification of lily embryo callus.When PIC concentration is lower than BA and NAA, particularly when PIC concentration is 1mgL
-1time, lily bud scale more easily differentiates little young ball, and non-callus, useless to the virus-free culture of follow-up lily.Therefore, the present invention only adds PIC in minimal medium, does not add BA and NAA, Fiber differentiation embryo callus.
2) screening of embryo callus: screen the embryo callus obtained in step 1), selects immature spherical cell stage as subculture material.Embryo callus shows as the nutty structure of loose distribution, and can be separated easily with tweezers, it is translucent that color is bright yellow or milky, and spherical cell stage can be grown to whole plant in MS minimal medium.
3) squamous subculture of spherical cell stage in solid culture medium: by step 2) in the immature spherical cell stage that screens be inoculated in solid multiplication medium and carry out first generation squamous subculture, temperature remains on 24 ± 2 DEG C, half-light cultivates 14 days, obtains first generation squamous subculture somatic embryo.
The spherical cell stage making new advances and produce is screened in first generation squamous subculture somatic embryo, be inoculated in solid multiplication medium and carried out second generation squamous subculture, temperature remains on 24 ± 2 DEG C, and half-light cultivates 14 days, obtains second generation squamous subculture somatic embryo; Circulation like this, until the somatic embryo of squamous subculture does not detect virus.
Wherein, the consisting of of solid multiplication medium: MS medium+PIC 1 ~ 2mgL
-1+ agar 7gL
-1+ sucrose 30gL
-1, pH value is 5.8.
Embodiment two:
In the present embodiment, step 1) and step 2) identical with embodiment one, step 3) is the squamous subculture of spherical cell stage in bio-reactor: by step 2 in embodiment one) the spherical cell stage that obtains is inoculated in the bio-reactor being added with liquid proliferated culture medium and carries out first generation squamous subculture, the mass volume ratio concentration of the spherical cell stage inoculated in each bio-reactor is 0.5-5%(w/v), namely add the liquid proliferated culture medium of 100ml in each bio-reactor, the spherical cell stage of 0.5 ~ 5g can be inoculated.In the present embodiment, inoculate the spherical cell stage of 1g in each bio-reactor, after inoculation, the throughput of bio-reactor is adjusted to 20mlmin
-1, temperature remains on 24 ± 2 DEG C, cultivates 14d under being placed in half-light condition of culture, obtains first generation squamous subculture somatic embryo.
In first generation squamous subculture somatic embryo, screen the spherical cell stage making new advances and produce, be inoculated in the bio-reactor being added with liquid proliferated culture medium and carry out second generation squamous subculture, after inoculation, the throughput of bio-reactor is adjusted to 20mlmin
-1, temperature remains on 24 ± 2 DEG C, and half-light cultivates 14 days, obtains second generation squamous subculture somatic embryo; Circulation like this, until the somatic embryo of squamous subculture does not detect virus.
When screening spherical cell stage, because the diameter of spherical cell stage is less than or equal to 1mm, therefore, aperture can be adopted to be that the sieve of 0.5 ~ 1.0 mm screens, to be immature spherical cell stage by the material of sieve.
Wherein, the consisting of of liquid proliferated culture medium: MS medium+PIC 1 ~ 2mgL
-1+ sucrose 30gL
-1, pH value is 5.8.
In the present invention, embryonic callus induction medium, solid multiplication medium and liquid proliferated culture medium are all at 1.05kgcm
-2with under the condition of 121 DEG C, sterilizing 20min.
For the squamous subculture of spherical cell stage, solid multiplication medium can be only adopted to carry out cultivating (as embodiment one), also the liquid proliferated culture medium of bio-reactor only can be adopted to carry out cultivating (as embodiment two), the liquid proliferated culture medium of solid multiplication medium and bio-reactor can also be adopted to hocket cultivation; As first generation squamous subculture carries out in solid multiplication medium, second generation squamous subculture carries out in the liquid proliferated culture medium of bio-reactor, and third generation squamous subculture carries out in solid multiplication medium, alternate cycles like this; Also can carry out in solid multiplication medium by 1st ~ 4 squamous subculture, 5th ~ 10 subcultures such as to carry out at the training method in the liquid proliferated culture medium of bio-reactor.
The principle of detoxification of the present invention is: somatic embryo originates from single or minority is not broken up or the somatic cell of dedifferentiation, and the initial stage shows as spherical cell stage, and each somatic embryo can develop into a complete plant individual.In the new spherical cell stage formed, rarely have virus to exist.Owing to not having skeleton to be communicated with its maternal tissue, therefore virus is difficult to diffuse to the new spherical cell stage formed from maternal tissue.Spherical cell stage is easy to be separated from parent.The spherical cell stage be separated is injured few, can continue under suitable condition to form how new spherical cell stage or become mature somatic embryo tire through torpedo embryo growth period, and then develop into complete plant.
The present invention can guaranteeing that vegetable material efficiently breaks up, under the prerequisite that grows fast, according to the difference of cell and virus multiplication speed, by the Fast-propagation of somatic embryo, reduces until slough intracellular virus gradually.The present invention has efficiently, simple to operate, and plant tissue hyperplasia is fast, the advantage that detoxification efficiency is good.
In Subculture, the somatic embryo that subculture produces each time all directly can carry out Viral diagnosis, namely after squamous subculture each time, all the somatic embryo that squamous subculture produces is screened, therefrom filter out the material that immature spherical cell stage is used as squamous subculture next time, and remaining somatic embryo is used for Viral diagnosis, as do not detected virus in remaining somatic embryo, then detoxification success.Certainly, in actual mechanical process, not the somatic embryo that subculture produces each time all being carried out Viral diagnosis, generally needing the kind according to sloughing virus different, after squamous subculture 4 times, the somatic embryo of squamous subculture each time backward is all carried out Viral diagnosis.Also the somatic embryo produced in Subculture can be inoculated in MS solid culture medium and differentiate little young ball and be used for Viral diagnosis.For the present invention, generally after 8-10 subculture, the virus in lily explant can remove completely.
For the somatic embryo sloughing virus, Multiplying culture can be proceeded, to turn out more detoxification somatic embryo.Also can be placed in MS solid culture medium and carry out differentiation cultivation, with the little young ball of the detoxification differentiating robust growth, carry out acclimatization and transplants.
Finally it should be noted that, above embodiment is only in order to illustrate technical scheme of the present invention but not restriction technologies scheme, although applicant's reference preferred embodiment is to invention has been detailed description, those of ordinary skill in the art is to be understood that, those are modified to technical scheme of the present invention or equivalent replacement, and do not depart from aim and the scope of the technical program, all should be encompassed in the middle of right of the present invention.
Claims (3)
1. lily is by the method for somatic embryo development ways detoxification, it is characterized in that, comprises the steps:
1) embryonic callus induction: the lily bud scale cultivated under aseptic condition is as explant, and be inoculated on embryonic callus induction medium, half-light cultivates 28 days, and Fiber differentiation goes out embryo callus;
2) screening of embryo callus: screen the embryo callus obtained in step 1), selects spherical cell stage as subculture material;
3) squamous subculture of spherical cell stage: by step 2) in the spherical cell stage that screens be inoculated in solid multiplication medium or liquid proliferated culture medium and carry out first generation squamous subculture, temperature remains on 24 ± 2 DEG C, half-light cultivates 14 days, obtains first generation squamous subculture somatic embryo;
The spherical cell stage making new advances and produce is screened in first generation squamous subculture somatic embryo, be inoculated in solid multiplication medium or liquid proliferated culture medium and carried out second generation squamous subculture, temperature remains on 24 ± 2 DEG C, and half-light cultivates 14 days, obtains second generation squamous subculture somatic embryo; Circulation like this, until do not detect virus in the somatic embryo of squamous subculture;
Wherein, the consisting of of described embryonic callus induction medium: MS medium+PIC 1 ~ 3mgL
-1+ agar 7gL
-1+ sucrose 30gL
-1, pH value is 5.8;
Consisting of of described solid multiplication medium: MS medium+PIC 1 ~ 2mgL
-1+ agar 7gL
-1+ sucrose 30gL
-1, pH value is 5.8;
Consisting of of described liquid proliferated culture medium: MS medium+PIC 1 ~ 2mgL
-1+ sucrose 30gL
-1, pH value is 5.8.
2. lily according to claim 1 is by the method for somatic embryo development ways detoxification, and it is characterized in that, described liquid proliferated culture medium is added in bio-reactor, and the throughput of bio-reactor remains on 20mlmin
-1.
3. lily according to claim 2 is by the method for somatic embryo development ways detoxification, it is characterized in that, the screening of described spherical cell stage adopts aperture to be that the sieve of 0.5-1.0 mm screens.
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| CN104186319B (en) * | 2014-08-19 | 2016-05-11 | 上海市农业科学院 | Tip of a root detoxification and the quick-breeding method of a kind of lily |
| CN104303997B (en) * | 2014-08-25 | 2017-02-01 | 连云港市农业科学院 | Method for detoxifying lily based on embryogenic callus specific induction |
| CN106718931B (en) * | 2017-01-19 | 2019-07-23 | 重庆市风景园林科学研究院 | The method for carrying out succulent breeding using bioreactor |
| CN108464242B (en) * | 2018-06-19 | 2021-07-13 | 沈阳农业大学 | Lily tenuifolius somatic embryo direct generation method capable of remarkably shortening induction time |
| CN110268979B (en) * | 2019-06-03 | 2022-03-11 | 南京农业大学 | Method for establishing efficient regeneration system of pollen-free lily and application |
| CN110972955A (en) * | 2020-01-02 | 2020-04-10 | 齐齐哈尔大学 | Method for inducing somatic embryogenesis and plant regeneration of lily leaves |
| CN111280059A (en) * | 2020-03-26 | 2020-06-16 | 南京农业大学 | Efficient lily detoxification method |
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| CN101658135B (en) * | 2009-09-02 | 2011-08-03 | 北京林业大学 | Method for desorbing lily bulb virus |
| CN103314864B (en) * | 2013-07-16 | 2015-05-06 | 李锋 | Method for obtaining detoxified seedling from bulb scales of tulip |
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