CN105557512A - Lily devirusing culture method by using compound amantadine hydrochloride - Google Patents
Lily devirusing culture method by using compound amantadine hydrochloride Download PDFInfo
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Abstract
The invention discloses a lily devirusing culture method by using compound amantadine hydrochloride, and belongs to the field of plant tissue culture for rapid propagation and the biotechnological field. The lily devirusing culture method comprises the following steps: detecting lily bulb viruses, preparing a culture medium for use, sterilizing and inoculating explants, culturing the explants, detecting viruses of tissue culture seedlings and the like, so as to quickly obtain a large number of devirused tissue culture seedlings. The best formula of an induction medium for directly differentiating lily into buds and rootless tissue culture seedlings is obtained according to an orthogonal method; by preparing antiviral compound amantadine hydrochloride reagents with a series of concentration gradients, the optimal devirusing concentration of virus-free is selected; the devirusing efficiency can reach 87%; by the lily devirusing culture method, not only is the cycle of induction culture shortened, but also the devirusing efficiency and the propagating factor are high; the lily devirusing culture method provides a technical basis for culturing virus-free lily plants and promoting a research on virus-free planting.
Description
Technical field
A kind of method using compound hydrochloric acid amantadine medicament to cultivate lily detoxification of the present invention, belongs to plant tissue culture fast breeding technique and biological technical field.
Background technology
Lily is the general name of Monocotyledonae Liliaceae (Liliaceae) lilium (Lilium), is perennial root herbage flower.Lilium has more than 90 to plant in the world, and what originate in China about has 47 kinds, accounts for 51% of the world total.Lily culture is various in style, and its flower is very large, rich color, and flower appearance is graceful, and can do cut-flower, potted flower, can apply again in Garden Greenland, and most of lily being edible, is first-class excellent tonic product.The meaning that lily have " life-long happiness and perfect harmony ", very popular.Lily is mainly distributed in China, Japan, North America and European zone of constant temperature area, and China's lily cut flowers production development is in recent years very fast, and in Kunming, Shanghai, Shenzhen formed three large production bases.But its breeding mainly still adopts conventional bulb separation, point bulbil scale cuttage, the mode of the breeding such as scale embedding.Long-term vegetative propagation makes to accumulate a large amount of virus in lily body gradually and causes the ill viral disease of lily, affects the seed output and quality of lily.Therefore setting up simple and easy to do, that virus elimination rate is high detoxification system is reduce virus to one of the harm of lily, key issue of recovery kind of property.
Since Stewart (1896) describes the downright bad striped of lily, in succession report the virus causing disease 14 kinds of lily.Wherein occur general, endanger serious virus and have 5 kinds of i.e. lily cryptovirus (Lilysymptomlessvirus, LSV), cucumber mosaic virus (Cucumbermosaicvirus, CMV), tulip design of scattered small flowers and plants virus (Tulipbreakingvirus, TBV), different name lily mottle virus (Lilymottlevirus, and lily rosette virus (Lilyrosettevirus, LRV) LMoV); Other 9 kinds of viruses are all in the few generation in cultivation area, lily is produced and does not constitute widespread harm, as Lily virus X (LilyvirusX, LVX), lily ring spot virus (Lilyringspotvirus, LRSV), daffodil virus (Narcissusmosaicvirus, and nepovirus (Tobaccoringspotvirus, TRSV) NMV).The present invention is mainly to lily asymptomatic virus (Lilysymptomlessvirus, LSV), cucumber mosaic virus (Cucumbermosaicvirus, CMV), lily mottle virus (Lilymottlevirus, LMoV) virus, lily rosette virus (Lilyrosettlevirus, LRV) detect.
Carrying out lily detoxification research is Phillips (1962) the earliest, after he utilizes lily Shoot Tip Culture detoxification success, successively has again many foreign scholars to utilize stem apex detoxify success.Have several national profit to obtain the virus-free seedling of lily in this way in the world at present, and production on large-scale application.Two key techniques that stem apex detoxify is cultivated are survival rate and the virus elimination rate of stem apex.Shoot Tip Culture survival rate is low.Brownization reduces the primary factor of Shoot Tip Culture survival rate.The generation of brownization is relevant with many factors, and stem apex size, season of drawing materials, training method, hormone concentration, minimal medium, medium additives all have impact to stem apex brownization, and such as stem apex is less, and brownization is more serious, and survival rate is lower.Be the generation of Browning control when Shoot Tip Culture, need to optimize above-mentioned each factor.Application antioxidant AC and VC also effectively can suppress the browning of Shoot Tip Culture.Given this, the present invention adopts lily bulb to carry out the cultivation of detoxic seedling as material.
The method that conventional detoxification technology has the cultivation of stem-apex Meristem culture, bulbil, heat treating process, chemotherapy and is combined with each other.In addition, other organ culture detoxifications of floral organ official rank are also a kind of effective poison-removing methods.Present in-vitro breeding virus-free plant has become the important step of the production of flowers and plants.Just find that the virus near growing tips of the plant is seldom even virus-free as far back as nineteen forty-three White.Within 1962, Phillips carries out lily detoxification research the earliest; Within 1966, Mori and Hamaya obtains lily virus-free plant by Shoot Tip Culture; Within 1993, Zhao Xiangyun utilizes 0.3 ~ 0.8mm bulbil growing point to carry out cultured in vitro to Lilium sulphureum Baker, and the seedling cultivated successfully can slough nepovirus; Calendar year 2001 Xi Mengli cultivates Yixing lily detoxification and draws, the stem apex detoxify effect of inoculation 0.3 ~ 0.5mm is best, reaches 40% to the virus elimination rate of CMV, and the stem apex being less than 0.3mm can not be cultivated and survive; The third-class discovery detoxification efficiency of Xu Pin in 2003 because of lily cultivar and viral species and to some extent difference, in " Casablanca " lily, LSV easily sloughs, and virus elimination rate reaches 76%, and in " evil spirit is beautiful " lily, CMV can all slough; The direct stem apexs such as Yun Hui in the wrong in 2003 slough Elle lily CMV virus, and inoculation 0.2 ~ 0.3mm stem apex detoxify rate reaches 43%.Kim research in 1994 reports virazole has stronger inhibitory action effect to CMV.Xu in 1999 etc. have studied by chemical treatment (virazole or dihydrouracil) and 35 DEG C of thermal treatment produce slough in bulb with the virus such as LSV, and obtain detoxification bulb by induction oriental hybrid lily Georgia kind callus.After Yun Hui in the wrong in 2003 etc. process 5d to the Lilium Pollyanna Elle with CMV at 40 DEG C, strip virus elimination rate when 0.2 ~ 0.3mm stem-tip tissue is cultivated and can reach 67%, and planting percent also reaches 48%.Calendar year 2001 Xi Mengli etc. with Yixing lily for examination material, 3 kinds of poison-removing methods (Shoot Tip Culture, combined with heat treatment Shoot Tip Culture, anther culture) are studied, result shows, bulbil is through 50 ± 1 DEG C of hot water treatment 40min, after cultivating 30d, the detoxification efficiency cutting 0.8 ~ 1.0mm Shoot Tip Culture is best, and virus elimination rate reaches 100%, cuts Shoot Tip Culture and also can improve detoxification efficiency after 38 ± 1 DEG C of hot air treatment.
Therefore, carry out virus to remove process, promote Non-toxic culture technique and have important realistic meaning to the ornamental value and economic worth that improve lily.
Leading reference:
[1] Zhou Xiaoyan. lily Major Diseases and control [J] thereof. plant protection, 2002,28 (1): 57-58.
[2] Zhong Jinghui, Cai Qiujin. Researches of Lilium Diseases and sustainable harnessing [J] thereof. forest transition communication, 2000,19 (2): 23,28-31.
[3] Fu Yulan. Floriculture Course [M]. Beijing: Chinese agriculture publishing house, 2001.216-217.
[4] Shen Shulin. lily viral diseases and inspection [J] .1996 thereof, 10 (1): 253-256
[5] Wang little Jing, Li Ling. plant growth regulator is in the application [M] of Plant Tissue Breeding. Beijing: Chemical Industry Press .2002,9:11-12.
[6] Zhao Xiangyun, Cheng Qian, Xing Youmei, etc. the training of lily bulbil group and detoxification research [J]. gardening journal, 1993,20 (3): 284-288.
[7] Xi Mengli, Wang Jieping, Zhang Jingjuan, etc. Virus-elimination Techniques for Lilium lancifalium Thunb [J]. Jiangsu's agriculture journal, 2001,17 (1): 492511.
[8] Xu Pinsan, Luan Yushi, Liu Jiwen etc. the research [J] that lily Adventitious bud culture detoxification kind ball is produced. Botany Gazette, 2003,20 (3): 3012318.
[9] Hong Yanhua, Yin Guangfeng, Zhang Lijun. lily detoxification and virus detection techniques progress [J]. Agricultural University Of Shenyang's journal, 2003,34 (3): 2552527.
[10] noble scholar. the quarantine of flower virus disease and removing method [J] thereof. plant quarantine, 1994, (6): 3402341.
[11]KimJY，LeeSY，ChoiJK，etal.Effectofvirazoleoneliminationofviru2ses(LSVandCMV)frominfectedlilyPlants[J].RDAJournalofAgriculturalScience(KoreaRepublic)，1994，36：3702374.
[12]XuPS，NiimiY.Evaluationofvirus2freebulbetProductionbyantiviraland/orheattreatmentinvitroscaleculturesofLiliumlongiflorum‘Georgia’andL.‘Casabl2anca’[J].JournaloftheJapanesesocietyforHorticulturalScience，1999，68：6402647.
[13] Wang little Jing, Li Ling. plant growth regulator is in the application [M] of Plant Tissue Breeding. Beijing: Chemical Industry Press .2002,9:11-12.
[14] Liu Yongsheng, Li Youyong. the use [J] of active carbon in Plant Tissue Breeding. Plant Physiology Communications .1994,30 (3): 214-217.
[15] Yan Xianwei. sweet cherry Shoot Tip Culture and Fast-propagation research [J]. gardening journal, 1990, 17 (4): 275-279.ASJESCJ, BLOM-BARNHOORNGJ.Air-bornefieldspreadoftulipbreakingviru s, lilysymptomlessvirusandlilyvirusXinlilyaffectedbyseasona lincidenceofflyingaphidsandcontrolbyspraysofmineraloil, vegetableoil, insecticideandpheromoneintheNetherlands [J] .ActaHorticulture, 1994, 377:301-324.ASJESCJ.Controlofaphid-borneLilysymptomlessv irusandLilymottlevirusinLiliumintheNetherlands [J] .VirusResearch, 2000, 71 (1-2): 23-32.
Summary of the invention
The object of the invention is to find the new method being better applicable to tissue-cultured derived plant lily detoxification, for cultivating lily virus-free plant later, promote Non-toxic cultivation and technical foundation is provided.
The object of the invention is achieved through the following technical solutions:
The method using compound hydrochloric acid amantadine medicament to cultivate lily detoxification, carry out as follows:
(1) material selection: " Siberia " (Siberia), " Suo Bang " (Sorbonne) plant ball;
(2) detection of lily bulb virus: choose the bulb scale for planting experimentally ball, extract total serum IgE, as template after reverse transcription, adopt reverse transcription PCR (reversetranscriptionPCR, RT-PCR) detect, determine whether kind of a ball scale carries virus.
(3) preparation, for subsequent use of medium: minimal medium is MS medium; The impact adopting Orthogonal Method research 6-BA and NAA concentration to train lily group, determining can the suitableeest 6-BA and the NAA concentration of directly differentiation and bud formation and unrooted plantlet in vitro, prepares best inducing culture; Adopt the medium of concentration preparation containing antiviral drugs of 1,3,5,6,7,8,9,10,12,14,16,18mg/L.Antiviral drugs adopts 0.2u membrane filtration degerming.
(4) explant sterilization, inoculation: detection spent the night by the lily bulb tap water of virus infections, 0.1% mercuric chloride sterilizing 10 minutes, then at 75% alcohol-pickled 20 seconds.Take out material, with aseptic water washing 5-6 time.Material is cut into 2.0mm × 2.0mm blockage, be inoculated on best inducing culture and cultivate.
(5) explant is cultivated: temperature 25 ± 1 DEG C, cultivate 45-50 days under the environmental condition of illumination 12h/d, intensity of illumination 1500Lx, lily bulb explant is directly divided into bulb bud and unrooted plantlet in vitro.Further blade is cut, group training clove rip cutting 2-4 block is inoculated in control group (inducing culture) and experimental group (inducing culture+certain density antiviral drugs) respectively, continues to cultivate; Continuous cutting, after subculture three times, RT-PCR detects viral seedling and takes viruliferous situation.
(6) plantlet in vitro Viral diagnosis: the kind according to detecting virus: lily asymptomatic virus (Lilysymptomlessvirus, LSV), cucumber mosaic virus (CMV), lily mottle virus (Lilymottlevirus, LMoV) virus, lily rosette virus (Lilyrosettlevirus, LRV) is according to known sequences Design Auele Specific Primer, and detecting without amplification through RT-PCR is negative detoxic seedling.
Virus elimination rate=
rT-PCR detects without amplified band sample seedling number
RT-PCR always detects seedling number
Positive rate=
rT-PCR detects amplified band sample seedling number
RT-PCR always detects seedling number
A kind of described method using compound hydrochloric acid amantadine medicament to cultivate lily detoxification, is characterized in that, best Fiber differentiation based formulas: MS+6-BA2.0mg/L+NAA0.35mg/L+4.0% sucrose+agar powder 6.0g/L (PH5.8).
A kind of described method using compound hydrochloric acid amantadine medicament to cultivate lily detoxification, it is characterized in that, the optimum concentration 14mg/L of the medicament compound hydrochloric acid amantadine (AmantadiniHydrochloridum) that experimental group uses, virus elimination rate can reach 87%.
Research finds, the present invention determines by Orthogonal Method the suitableeest 6-BA and the NAA concentration making the direct differentiation and bud formation of lily and unrooted plantlet in vitro, and lily is carried out to the optimum concentration of the compound hydrochloric acid amantadine medicament that detoxification uses, reach detoxification efficiency high, the advantages such as the time is short.
Compared with prior art, its remarkable advantage is the detoxification efficiency greatly improving plantlet in vitro in the present invention, and for cultivating lily virus-free plant, the research promoting Non-toxic cultivation provides technical foundation.
Embodiment
By the following examples to further instruction of the present invention.
Embodiment: (Viral diagnosis of tissue cultural seedlings of free)
(1) vegetable material
Respectively with the plantlet in vitro of control group and experimental group for material.
(2) extraction of viral RNA
Adopt TRIzol reagent, respectively each test sample is carried out to the extraction of total serum IgE, the compound method of the RNase-free water used in leaching process: 0.1% (v/v) DEPC ultra-pure water will be filled it up with in clean glass bottle, glass bottle after process of spending the night
Autoclaving; The plastic and glass vessel processing method of RNase-free: glassware can 150 DEG C of bakings 6 hours; Plastic ware soaks 10 minutes in 0.5MNaOH, then thoroughly cleans with DEPC water, then autoclaving.Concrete operation step is as follows:
1) test sample directly puts into mortar, adds a small amount of liquid nitrogen, rapid grind into powder, and every 50-100mg plant sample adds 1mlTRIzol.After sample adds TRIzol, room temperature places 5min, makes the abundant cracking of sample;
2) 4 DEG C, centrifugal 10 minutes of 12,000rpm, gets supernatant;
3) every 1mlTRIzol adds 200 μ l chloroforms, and after thermal agitation mixing, room temperature is placed 3-5min and made its natural phase-splitting;
4) 4 DEG C, the centrifugal 10-15min of 12,000rpm.The superiors' aqueous phase is transferred in new pipe;
5) in supernatant, add the ice-cold isopropyl alcohol of equal-volume, room temperature places 10-20min.4 DEG C, the centrifugal 10min of 12,000rpm, abandons supernatant, and RNA is deposited at the bottom of pipe;
6) add 1ml75% ethanol (with the preparation of RNase-free water) in RNA precipitation, gentle vibration centrifuge tube, suspend precipitation;
7) 4 DEG C, the centrifugal 1-2min of 5,000-8,000rpm, abandons supernatant; Of short duration centrifugal fast, carefully inhale with pipettor and abandon supernatant, room temperature is placed and is dried precipitation in 1-2 minute;
8) add 50-100 μ lRNase-free water in precipitation, flick tube wall, fully to dissolve RNA ,-70 DEG C of preservations.
(3) amplification of viral each gene order
A) design of virus sequence pcr amplification primer
According to the lily asymptomatic virus (Lilysymptomlessvirus reported, LSV), cucumber mosaic virus (CMV), lily mottle virus (Lilymottlevirus, LMoV) virus, the PCR atopic primer of sequences Design Tm=55 DEG C of the coat protein gene of lily rosette virus (Lilyrosettlevirus, LRV).
B) synthesis of cDNA first chain
Adopt virus coat protein gene PCR downstream primer as the initial primers of reverse transcription first chain, reverse transcription synthetic reaction system is as follows: 1 μ g total serum IgE is placed in centrifuge tube, is placed in 70 DEG C of water-bath 10min, and of short duration centrifugal rear horse back is transferred in ice.Mixed with total serum IgE after sex change by reactant liquor, reaction system is as follows:
Reversetranscription10×buffer2.0μl
dNTPmixture，10mM2.0μl
MgCl2，25mM4.0μl
RecombinantRNasin?Ribonucleaseinhibitor0.5μl
AMVreversetranscriptase(HighConc.)1.5u
ViruscoatproteingenePCRreverseprimers，10mM1.0μl
TotalRNA1.0μg
Nuclease-Freewatertoafinalvolumeof20μl
Reverse transcription reaction carries out 15min at 42 DEG C, has reacted latter 95 DEG C, 5min inactivation reverse transcriptase, proceeds to 4 DEG C of insulation 5min.-20 DEG C save backup.
C) reverse transcriptase chain reaction (RT-PCR)
With reverse transcription cDNA product for template, arranging the PCR reaction not adding template cDNA is negative control, is undertaken by following system:
10×PCRreactionbuffer(Mg2+plus)2μl
dNTPmixture，10mM1μl
ViruscoatproteingenePCRforwardprimer，10mM1μl
ViruscoatproteingenePCRreverseprimer，10mM1μl
ReversetranscriptioncDNAproducts1μg
Taq，5U/μl0.2μl
Nuclease-Freewatertoafinalvolumeof20μl
PCR program:
94 DEG C, 5min; 94 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 1min, 30 circulations; 72 DEG C, 10min.PCR primer 1% agarose gel electrophoresis, SYBRGreen dyes, and observes, detect with or without target amplification fragment, film recording under uviol lamp.
(4) RT-PCR product detects
When the negative control PCR not adding template cDNA reacts driftlessness amplified production, have the individuality of target amplification band to be virus infections plant, the individuality of driftlessness amplified band is virus-free infection plant, i.e. detoxification plant.
Claims (3)
1. the method using compound hydrochloric acid amantadine medicament to cultivate lily detoxification, is characterized in that carrying out as follows:
(1) material selection: " Siberia " (Siberia), " Suo Bang " (Sorbonne) plant ball;
(2) detection of lily bulb virus: choose the bulb scale for planting experimentally ball, extract total serum IgE, as template after reverse transcription, adopt reverse transcription PCR (reversetranscriptionPCR, RT-PCR) detect, determine whether kind of a ball scale carries virus;
(3) preparation, for subsequent use of medium: minimal medium is MS medium; The impact adopting Orthogonal Method research 6-BA and NAA concentration to train lily group, determining can the suitableeest 6-BA and the NAA concentration of directly differentiation and bud formation and unrooted plantlet in vitro, prepares best inducing culture; Adopt the medium of concentration preparation containing antiviral drugs of 1,3,5,6,7,8,9,10,12,14,16,18mg/L; Antiviral drugs adopts 0.2u membrane filtration degerming;
(4) explant sterilization, inoculation: detection is spent the night by the lily bulb tap water of virus infections, 0.1% mercuric chloride sterilizing 10 minutes, then at 75% alcohol-pickled 20 seconds; Take out material, with aseptic water washing 5-6 time; Material is cut into 2.0mm × 2.0mm blockage, be inoculated on best inducing culture and cultivate;
(5) explant is cultivated: temperature 25 ± 1 DEG C, cultivate 45-50 days under the environmental condition of illumination 12h/d, intensity of illumination 1500Lx, lily bulb explant is directly divided into bulb bud and unrooted plantlet in vitro; Further blade is cut, group training clove rip cutting 2-4 block is inoculated in control group (inducing culture) and experimental group (inducing culture+certain density antiviral drugs) respectively, continues to cultivate; Continuous cutting, after subculture three times, RT-PCR detects viral seedling and takes viruliferous situation;
(6) plantlet in vitro Viral diagnosis: the kind according to detecting virus: lily asymptomatic virus (Lilysymptomlessvirus, LSV), cucumber mosaic virus (CMV), lily mottle virus (Lilymottlevirus, LMoV) virus, lily rosette virus (Lilyrosettlevirus, LRV) is according to known sequences Design Auele Specific Primer, and detecting without amplification through RT-PCR is negative detoxic seedling.
2. a kind of method using compound hydrochloric acid amantadine medicament to cultivate lily detoxification according to claims 1, is characterized in that best Fiber differentiation based formulas: MS+6-BA2.0mg/L+NAA0.35mg/L+4.0% sucrose+agar powder 6.0g/L (PH5.8).
3. a kind of method using compound hydrochloric acid amantadine medicament to cultivate lily detoxification according to claims 1, it is characterized in that the optimum concentration 14mg/L of the medicament compound hydrochloric acid amantadine (AmantadiniHydrochloridum) that experimental group uses, virus elimination rate can reach 87%.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101061790A (en) * | 2007-04-13 | 2007-10-31 | 浙江省农业科学院 | Quick virus-free group-cultivating and propagating method of OT type lily of hybrid group |
| CN102440183A (en) * | 2010-10-14 | 2012-05-09 | 安利清 | Method for using rimantadine hydrochloride drug to culture lily under virus-free state |
| CN102440189A (en) * | 2010-10-14 | 2012-05-09 | 杨凯 | Method for lily virus-free cultivation through using moroxydine drug |
| CN102440188A (en) * | 2010-10-14 | 2012-05-09 | 杨凯 | Method for using amantadine hydrochloride drug to culture lily under virus-free state |
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- 2014-10-28 CN CN201410582897.3A patent/CN105557512A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101061790A (en) * | 2007-04-13 | 2007-10-31 | 浙江省农业科学院 | Quick virus-free group-cultivating and propagating method of OT type lily of hybrid group |
| CN102440183A (en) * | 2010-10-14 | 2012-05-09 | 安利清 | Method for using rimantadine hydrochloride drug to culture lily under virus-free state |
| CN102440189A (en) * | 2010-10-14 | 2012-05-09 | 杨凯 | Method for lily virus-free cultivation through using moroxydine drug |
| CN102440188A (en) * | 2010-10-14 | 2012-05-09 | 杨凯 | Method for using amantadine hydrochloride drug to culture lily under virus-free state |
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Application publication date: 20160511 |