CN102440188A - Method for using amantadine hydrochloride drug to culture lily under virus-free state - Google Patents
Method for using amantadine hydrochloride drug to culture lily under virus-free state Download PDFInfo
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Abstract
The invention discloses a method for using an amantadine hydrochloride drug to culture lily under a virus-free state and belongs to the fields of plant tissue culturing fast propagation technique and biologic technique. The method comprises the following steps of: inspecting lily mother bulb virus, preparing a culture medium and reserving, sterilizing and inoculating explants, culturing the explants, inspecting virus of tissue culturing seedlings, and the like, thereby quickly obtaining lots of tissue culturing seedlings. According to an orthogonal method, a formula of the best induced culture medium of buds and root-less tissue culturing seedlings directly differentiated from lily is obtained; an amantadine hydrochloride antiviral drug with a series of concentration gradients is prepared and an optimum detoxifying concentration is selected, so that the detoxifying efficiency can reach 87%; a period of induced culturing is shortened, and meanwhile, the detoxifying efficiency is high and the enhancement factor is high; and a technical basis for researching on the culturing for lily virus-free plants and the popularization for virus-free culturing is provided.
Description
One, technical field
The present invention uses the amantadine hydrochloride medicament to the lily detoxification cultured method, belongs to plant tissue culture fast breeding technique and biological technical field.
Two, background technology
Lily is the general name of Monocotyledonae Liliaceae (Liliaceae) lilium (Lilium), is perennial perennial root herbage flower.Kind surplus lilium has 90 in the world, what originate in China has 47 kinds approximately, accounts for 51% of the world total.Lily culture is various in style, and its flower is very large, rich color, and the flower appearance is graceful, can do cut-flower, potted flower, can in the greenery patches, gardens, use again, and most of lily is edible, be first-class excellent tonic product.Lily have the meaning of " life-long happiness and perfect harmony ", and is very popular.Lily mainly is distributed in China, Japan, North America and European zone of constant temperature area, and the lily cut flowers production development of China recent years is very fast, in Kunming, Shanghai, Shenzhen formed three big production bases.But conventional bulb separation, the cuttage of branch bulbil scale are mainly still adopted in its breeding, the mode of breeding such as scale embedding.Long-term vegetative propagation makes and accumulates a large amount of viruses in the lily body gradually and cause the ill viral disease of lily, influences output and the quality of lily.Therefore setting up the detoxification system simple and easy to do, that virus elimination rate is high is to 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, 14 kinds of the virus causing diseases of lily have been reported in succession.Wherein take place general; Endangering serious virus has 5 kinds, promptly the lily cryptovirus (Lily symptomless virus, LSV), cucumber mosaic virus (Cucumber mosaicvirus; CMV), tulip design of scattered small flowers and plants virus (Tulip breaking virus; TBV), the different name lily mottle virus (Lily mottle virus, LMoV) and lily rosette virus (Lily rosette virus, LRV); Other 9 kinds of viruses are few generation in the cultivation area all; Lily production is not constituted generally harm; As lily X virus (Lily virus X, LVX), lily ring spot virus (Lily ring spot virus, LRSV), daffodil virus (Narcissus mosaicvirus; NMV) and nepovirus (Tobacco ring spot virus, TRSV).The present invention is mainly to lily asymptomatic virus (Lily symptomlessvirus; LSV), cucumber mosaic virus (Cucumber mosaic virus; CMV), lily mottle virus (Lily mottle virus; LMoV) virus, (Lily rosettle virus LRV) detects lily rosette virus.
Carrying out lily detoxification research is Phillips (1962) the earliest, and he utilizes after the lily Shoot Tip Culture detoxification success, successively has many foreign scholars to utilize the stem apex detoxify success again.Have several countries to utilize this method to obtain the virus-free seedling of lily at present in the world, and on producing large-scale application.Survival rate and virus elimination rate that two key techniques that stem apex detoxify is cultivated are stem apexs.The Shoot Tip Culture survival rate is low.Brownization is to reduce the primary factor of Shoot Tip Culture survival rate.The generation of brownization is relevant with many factors, and stem apex size, the season of drawing materials, training method, hormone concentration, minimal medium, medium additives are all influential to brownization of stem apex, and for example stem apex is more little, and brownization is serious more, and survival rate is low more.When Shoot Tip Culture,, need to optimize above-mentioned each factor for suppressing the generation of brownization.Use the browning that antioxidant AC and VC also can effectively suppress Shoot Tip Culture.Given this, the present invention adopts lily bulb to carry out the cultivation of detoxic seedling as material.
The method that detoxification technology commonly used has shoot apical meristem cultivation, bulbil cultivation, heat treating process, chemotherapy and interosculates.In addition, other organ culture detoxifications of floral organ official rank also are a kind of effective poison-removing methods.In-vitro breeding virus-free plant has become the important step of the production of flowers and plants now.Near nineteen forty-three White just finds growing tips of the plant virus seldom even virus-free.Phillips carried out lily detoxification research the earliest in 1962; Mori in 1966 and Hamaya have obtained lily virus-free plant through Shoot Tip Culture; Zhao Xiangyun utilized 0.3～0.8mm bulbil growing point that yellowish colored lily is carried out cultured in vitro in 1993, and the seedling of cultivating can successfully slough nepovirus; Calendar year 2001 Xi Mengli cultivates Yixing lily detoxification and draws, and the stem apex detoxify effect of inoculation 0.3～0.5mm is best, and the virus elimination rate of CMV is reached 40%, can not cultivate into alive less than the stem apex of 0.3mm; The third-class discovery detoxification efficiency of Xu Pin in 2003 is because of lily kind and viral species difference to some extent, and LSV sloughs easily in " Casablanca " lily, and virus elimination rate reaches 76%, and CMV can all slough in " evil spirit is beautiful " lily; Yun Hui in the wrong in 2003 etc. directly slough Elle lily CMV virus with stem apex, and inoculation 0.2～0.3mm stem apex detoxify rate reaches 43%.Kim research in 1994 has reported that virazole has stronger inhibitory action effect to CMV.Xu in 1999 etc. have studied through chemical treatment (virazole or dihydrouracil) and 35 ℃ of treatment production and have sloughed the viruses such as LSV that have in the bulb, and through inducing oriental hybrid lily Georgia kind callus to obtain the detoxification bulb.Yun Hui in the wrong in 2003 etc. to the Asia lily Elle that has CMV 40 ℃ handle 5d down after, strip 0.2～0.3mm stem-tip tissue when cultivating virus elimination rate can reach 67%, and planting percent also reaches 48%.Calendar year 2001 Xi Mengli etc. serve as the examination material with Yixing lily; 3 kinds of poison-removing methods (Shoot Tip Culture, combined with heat treatment Shoot Tip Culture, anther culture) are studied, and the result shows that bulbil is through 50 ± 1 ℃ of hot water treatment 40min; After cultivating 30d; The detoxification efficiency that cuts 0.8～1.0mm Shoot Tip Culture is best, and virus elimination rate reaches and cuts Shoot Tip Culture after 100%, 38 ± 1 ℃ of hot air treatment and also can improve detoxification efficiency.
Therefore, carrying out virus removes processing, promote not to have and to poison culture technique ornamental value and the economic worth that improves lily had important practical sense.
The main reference document:
[1] Zhou Xiaoyan. main disease of lily and control [J] thereof. plant protection, 2002,28 (1): 57-58.
[2] Zhong Jinghui, Cai Qiujin. lily disease and lasting administer [J] thereof. the sick worm communication of forest, 2000,19 (2): 23,28-31.
[3] Fu Yulan. flowers are learned [M]. Beijing: Chinese agriculture publishing house, 2001.216-217.
[4] Shen Shulin. lily disease viral disease and check thereof [J] .1996,10 (1): 223-226
[5] Wang Xiaojing, Li Ling. plant growth regulator is in the application [M] of Plant Tissue Breeding. Beijing: the .2002 of Chemical Industry Press, 9:11-12.
[6] Zhao Xiangyun, Cheng Qian, Xing Youmei, etc. training of lily bulbil group and detoxification research [J]. gardening journal, 1993,20 (3): 284-288.
[7] Xi Mengli, Wang Jieping, Zhang Jingjuan, etc. Yixing lily detoxification technology [J]. Jiangsu agricultural journal, 2001,17 (1): 492511.
[8] Xu Pinsan, Luan Yushi, Liu Jiwen etc. the lily indefinite bud is cultivated the research [J] that 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): 2252227.
[10] noble scholar. the quarantine of flower virus disease and removing method thereof [J]. plant quarantine, 1994, (6): 3402341.
[11]Kim?J?Y，Lee?SY，Choi?J?K，et?al.Effect?of?virazole?on?elimination?of?viru2?ses(LSV?and?CMV)from?infectedlily?Plants[J].RDA?Journal?of?Agricultural?Science(Korea?Republic)，1994，36：3702374.
[12]Xu?PS，Niimi?Y.Evaluation?of?virus2?free?bulbet?Production?by?antiviral?and/or?heat?treatment?in?vitro?scalecultures?of?Lilium?longiflorum‘Georgia’and?L.‘Casabl2anca’[J].Journal?ofthe?Japanese?society?for?HorticulturalScience，1999，68：6402647.
[13] Wang Xiaojing, Li Ling. plant growth regulator is in the application [M] of Plant Tissue Breeding. Beijing: the .2002 of Chemical Industry Press, 9:11-12.
[14] Liu Yongsheng, Li Youyong. the use of active carbon [J] in the Plant Tissue Breeding. Plant Physiology Communications .1994,30 (3): 214-217.
[15] Yan Xianwei. sweet cherry Shoot Tip Culture and breeding research [J] fast. gardening journal, 1990,17 (4): 275-279.
[16]ASJES?C?J，BLOM-BARNHOORN?G?J.Air-borne?field?spread?of?tulip?breaking?virus，lily?symptomlessvirus?and?lily?virus?X?in?lily?affected?by?seasonal?incidence?of?flying?aphids?and?control?by?sprays?of?mineral?oil，vegetable?oil，insecticide?and?pheromone?in?the?Netherlands[J].Acta?Horticulture，1994，377：301-324.
[17]ASJ?ES?C?J.Control?of?aphid-borne?Lily?symptomless?virus?and?Lily?mottle?virus?in?Lilium?in?theNetherlands[J].Virus?Research，2000，71(1-2)：23-32.
Three, summary of the invention
The objective of the invention is to seek the new method of better suitable tissue-cultured derived plant lily detoxification, is to cultivate lily virus-free plant later on, and promoting not have to poison to cultivate provides technical foundation.
The object of the invention realizes through following technical scheme:
Use the amantadine hydrochloride medicament to the lily detoxification cultured method, carry out as follows:
(1) material selection: " Siberia " (Siberia), " Suo Bang " (Sorbonne) plant ball;
(2) detection of lily bulb virus: choose the bulb scale that supplies to plant experimentally ball, extract total RNA, as template, (reverse transcription PCR RT-PCR) detects, and confirms whether kind of a ball scale carries virus to adopt reverse transcription PCR after the reverse transcription.
(3) culture medium preparation, subsequent use: minimal medium is the MS medium;
Adopt the influence to the training of lily group of Orthogonal Method research 6-BA and NAA concentration, the righttest 6-BA and the NAA concentration of definite directly differentiation and bud formation and unrooted tissue cultivating seedling are prepared best inducing culture; Adopt 1,3,5,6,7,8,9,10,12,14,16, the concentration preparation of 18mg/L contains the medium of antiviral drugs.Antiviral drugs adopts the degerming of 0.2u membrane filtration.
(4) explant sterilization, inoculation: will detect by the lily bulb of virus infections and spend the night with running water flushing, 0.1% mercuric chloride sterilization 10 minutes is then at 70% alcohol-pickled 20 seconds.Take out material, with aseptic water washing 5-6 time.Material cut is become 2.0mm * 2.0mm blockage, be inoculated on the best inducing culture and cultivate.
(5) explant is cultivated: 22 ± 1 ℃ of temperature, illumination 12h/d cultivated 45-50 days under the environmental condition of intensity of illumination 1500Lx, and the lily bulb explant directly is divided into bulb bud and unrooted tissue cultivating seedling.Further blade is downcut, will organize training clove rip cutting 2-4 piece and be inoculated in respectively on control group (inducing culture) and the experimental group (inducing culture+certain density antiviral drugs), continue to cultivate; After the continuously cutting, subculture three times, RT-PCR detects the situation that viral seedling carries virus.
(6) tissue cultivating seedling virus detects: according to detecting viral kind: lily asymptomatic virus (Lily symptomless virus; LSV), cucumber mosaic virus (CMV), lily mottle virus (Lily mottle virus; LMoV) virus; (Lily rosettle virus LRV) according to known sequences Design Auele Specific Primer, detects the negative detoxic seedling of nothing amplification through RT-PCR to lily rosette virus.
Described use amantadine hydrochloride medicament is characterized in that best inducing culture based formulas to the lily detoxification cultured method: MS+6-BA2.0mg/L+NAA0.35mg/L+4.0% sucrose+agar powder 6.0g/L (PH 5.8).
Described use amantadine hydrochloride medicament is characterized in that the optimum concentration 14mg/L of the employed medicament amantadine hydrochloride of experimental group (Amantadini Hydrochloridum) to the lily detoxification cultured method, and virus elimination rate can reach 87%.
Discover; The present invention confirms to make the righttest 6-BA and the NAA concentration of direct differentiation and bud formation of lily and unrooted tissue cultivating seedling through Orthogonal Method; And lily carried out the optimum concentration of the employed amantadine hydrochloride medicament of detoxification, advantage such as it is high to reach detoxification efficient, and the time is short.
The present invention compared with prior art, its remarkable advantage is the detoxification efficient that has improved tissue cultivating seedling greatly, be to cultivate lily virus-free plant, promoting does not have the research that poisons cultivation technical foundation is provided.
Four, embodiment
Embodiment through following further explains the present invention.
Embodiment: (virus of tissue cultural seedlings of free detects)
(1) vegetable material
Tissue cultivating seedling with control group and experimental group is a material respectively.
(2) extraction of viral RNA
Adopt TRIzol reagent, supply test agent to carry out the extraction of total RNA, the compound method of the RNase-free water that uses in the leaching process to each respectively: will clean and fill it up with 0.1% (v/v) DEPC ultra-pure water, glass bottle autoclaving after the overnight treatment in the glass bottle; The plastic and glass vessel processing method of RNase-free: glassware can be 150 ℃ of bakings 6 hours; Plastic ware soaked 10 minutes in 0.5M NaOH, thoroughly cleaned with DEPC water then, autoclaving again.The concrete operations step is following:
1) supply test agent directly to put into mortar, add a small amount of liquid nitrogen, rapid grind into powder, every 50-100mg plant sample adds 1ml TRIzol.After sample added TRIzol, room temperature was placed 5min, makes the abundant cracking of sample;
2) 4 ℃, 12, centrifugal 10 minutes of 000rpm gets supernatant;
3) every 1ml TRIzol adds 200 μ l chloroforms, and room temperature is placed 3-5min and made its natural phase-splitting behind the thermal agitation mixing;
4) 4 ℃, 12, the centrifugal 10-15min of 000rpm.The superiors' water is transferred in the new pipe;
5) in supernatant, add the ice-cold isopropyl alcohol of equal-volume, room temperature is placed 10-20min.4 ℃, 12, the centrifugal 10min of 000rpm abandons supernatant, and RNA is deposited in the pipe end;
6) add 1ml 75% ethanol (with the preparation of RNase-free water) in the RNA deposition, gentle vibration centrifuge tube, deposition suspends;
7) 4 ℃, 5,000-8, the centrifugal 1-2min of 000rpm abandons supernatant; Of short duration centrifugal fast, abandon supernatant with careful suction of pipettor, room temperature is placed and was dried deposition in 1-2 minute;
8) add 50-100 μ l RNase-free water in the deposition, flick tube wall, with abundant dissolving RNA ,-70 ℃ of preservations.
(3) amplification of each gene order of virus
A) design of virus sequence pcr amplification primer
According to the lily asymptomatic virus of having reported (Lily symptomless virus; LSV); Cucumber mosaic virus (CMV), lily mottle virus (Lily mottle virus, LMoV) virus; Lily rosette virus (Lily rosettle virus, the sequences Design Tm=55 of coat protein gene LRV) ℃ PCR atopic primer.
B) cDNA first chain is synthetic
Adopt the initial primers of virus coat protein gene PCR downstream primer as reverse transcription first chain, reverse transcription synthetic reaction system is following:
The total RNA of 1 μ g is placed centrifuge tube, place 70 ℃ of water-bath 10min, of short duration centrifugal back is transferred in the ice at once.Total RNA after reactant liquor and the sex change is mixed, and reaction system is following:
Reverse?transcription?10×buffer 2.0μl
dNTP?mixture，10mM 2.0μl
MgCl2，25mM 4.0μl
Recombinant
Ribonuclease?inhibitor 0.5μl
AMV?reverse?transcriptase(High?Conc.) 1.5u
Virus?coat?protein?gene?PCR?reverse?primers，10mM 1.0μl
Total?RNA 1.0μg
Nuclease-Free?water?to?a?final?volume?of 20μl
Reverse transcription reaction carries out 15min under 42 ℃, after reaction is accomplished 95 ℃, 5min inactivation reverse transcriptase changes 4 ℃ of insulation 5min over to.-20 ℃ of preservations are subsequent use.
C) reverse transcription-PCR (RT-PCR)
With reverse transcription cDNA product is template, the PCR that does not add template cDNA is set reacts negative contrast, is undertaken by following system:
10×PCR?reaction?buffer(Mg2+plus) 2μl
dNTP?mixture，10mM 1μl
Virus?coat?protein?gene?PCR?forward?primer，10mM 1μl
Virus?coat?protein?gene?PCR?reverse?primer，10mM 1μl
Reverse?transcription?cDNA?products 1μg
Taq，5U/μl 0.2μl
Nuclease-Free?water?to?a?final?volume?of 20μl
The PCR program:
94 ℃, 5min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1min, 30 circulations; 72 ℃, 10min.PCR product 1% agarose gel electrophoresis, SYBR Green dyeing, uviol lamp is observed down, detects to have or not target amplification fragment, film recording.
(4) the RT-PCR product detects
When not adding the negative control PCR reaction driftlessness amplified production of template cDNA, the individuality that the target amplification band is arranged is the virus infections plant, and the individuality of driftlessness amplified band is the virus-free infection plant, i.e. the detoxification plant.
Claims (3)
1. use the amantadine hydrochloride medicament to the lily detoxification cultured method, it 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 that supplies to plant experimentally ball, extract total RNA, as template, (reverse transcription PCR RT-PCR) detects, and confirms whether kind of a ball scale carries virus to adopt reverse transcription PCR after the reverse transcription;
(3) culture medium preparation, subsequent use: minimal medium is the MS medium;
Adopt the influence to the training of lily group of Orthogonal Method research 6-BA and NAA concentration, the righttest 6-BA and the NAA concentration of definite directly differentiation and bud formation and unrooted tissue cultivating seedling are prepared best inducing culture; Adopt 1,3,5,6,7,8,9,10,12,14,16, the concentration preparation of 18mg/L contains the medium of antiviral drugs.Antiviral drugs adopts the degerming of 0.2u membrane filtration.
(4) explant sterilization, inoculation: will detect by the lily bulb of virus infections and spend the night with running water flushing, 0.1% mercuric chloride sterilization 10 minutes is then at 70% alcohol-pickled 20 seconds.Take out material, with aseptic water washing 5-6 time.Material cut is become 2.0mm * 2.0mm blockage, be inoculated on the best inducing culture and cultivate.
(5) explant is cultivated: 22 ± 1 ℃ of temperature, illumination 12h/d cultivated 45-50 days under the environmental condition of intensity of illumination 1500Lx, and the lily bulb explant directly is divided into bulb bud and unrooted tissue cultivating seedling.Further blade is downcut, will organize training clove rip cutting 2-4 piece and be inoculated in respectively on control group (inducing culture) and the experimental group (inducing culture+certain density antiviral drugs), continue to cultivate; After the continuously cutting, subculture three times, RT-PCR detects the situation that viral seedling carries virus.
(6) tissue cultivating seedling virus detects: according to detecting viral kind: lily asymptomatic virus (Lily symptomless virus; LSV), cucumber mosaic virus (CMV), lily mottle virus (Lily mottle virus; LMoV) virus; (Lily rosettle virus LRV) according to known sequences Design Auele Specific Primer, detects the negative detoxic seedling of nothing amplification through RT-PCR to lily rosette virus.
According to claims 1 described use amantadine hydrochloride medicament to the lily detoxification cultured method, it is characterized in that best inducing culture based formulas: MS+6-BA2.0mg/L+NAA0.35mg/L+4.0% sucrose+agar powder 6.0g/L (PH 5.8).
According to claims 1 described use amantadine hydrochloride medicament to the lily detoxification cultured method; It is characterized in that the optimum concentration 14mg/L of the employed medicament amantadine hydrochloride of experimental group (Amantadini Hydrochloridum), virus elimination rate can reach 87%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104273171A (en) * | 2014-09-22 | 2015-01-14 | 陈本建 | Soluble hydrofluoride pesticide comprehensive formula |
| CN105532447A (en) * | 2014-10-28 | 2016-05-04 | 松桃宏发肉食品有限责任公司 | Method for detoxification culture of lily by using acyclovir agent |
| CN105557512A (en) * | 2014-10-28 | 2016-05-11 | 松桃宏发肉食品有限责任公司 | Lily devirusing culture method by using compound amantadine hydrochloride |
| CN105613284A (en) * | 2014-10-28 | 2016-06-01 | 松桃宏发肉食品有限责任公司 | Method for accelerating expanding culture of lily virus-free cormels |
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| CN101173251A (en) * | 2007-12-04 | 2008-05-07 | 中国农业科学院蔬菜花卉研究所 | Method for rapidly removing three viruses of lily |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104273171A (en) * | 2014-09-22 | 2015-01-14 | 陈本建 | Soluble hydrofluoride pesticide comprehensive formula |
| CN105532447A (en) * | 2014-10-28 | 2016-05-04 | 松桃宏发肉食品有限责任公司 | Method for detoxification culture of lily by using acyclovir agent |
| CN105557512A (en) * | 2014-10-28 | 2016-05-11 | 松桃宏发肉食品有限责任公司 | Lily devirusing culture method by using compound amantadine hydrochloride |
| CN105613284A (en) * | 2014-10-28 | 2016-06-01 | 松桃宏发肉食品有限责任公司 | Method for accelerating expanding culture of lily virus-free cormels |
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Application publication date: 20120509 |