CN1926964A - Process for expelling Danshen virus by using finely cellular agglomerate regeneration method - Google Patents
Process for expelling Danshen virus by using finely cellular agglomerate regeneration method Download PDFInfo
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- CN1926964A CN1926964A CN 200610048342 CN200610048342A CN1926964A CN 1926964 A CN1926964 A CN 1926964A CN 200610048342 CN200610048342 CN 200610048342 CN 200610048342 A CN200610048342 A CN 200610048342A CN 1926964 A CN1926964 A CN 1926964A
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Abstract
The invention relates to a method for using finely cellular regeneration method to remove Danshen virus. Wherein, it comprises callus induction, callus adjustment, cell suspension cultivation, callus induction plant regeneration, and the virus check of regenerated plant (enzyme immunity adsorption method) to obtain the Danshen plant without CMV virus. The invention can avoid general thermal treatment, but directly use somatic cell induction to differentiate it into cleavage cell (callus); the conidium cell via state adjustment is directly inducted and regenerated to form plant, to remove the virus.
Description
Technical field
The invention belongs to the purification and rejuvenation of plant and breed, be meant a kind of method that removes red sage root virus with the microcell agglomerate regeneration method especially.
Background technology
Conventional " thermal treatment+Shoot Tip Culture " exists thermal treatment passivation virus though remove the plant virus method, and continuous high temperature is handled and also plant damaged, even dead, easily causes plant withered, dead.The detoxification treatment process time is long, the problem of weak effect.
Summary of the invention
The objective of the invention is to is not to be subjected to virus infection substantially in the cell according to plant division, do not contain virus in mitogenetic summit (stem apex) or the somatoblast, the plant that is obtained by summit (stem apex) or noble cells does not contain virus or contains the very low such basic principle of viral rate.Avoid the heat treatment process of conventional method virus-free, provide a kind of method of red sage root virus that removes with the microcell agglomerate regeneration method to remove the cucumber mosaic virus (CMV) in the red sage root.
Overall technology design of the present invention is:
Remove the method for red sage root virus with the microcell agglomerate regeneration method, comprise following processing step:
(1) callus induction: after red sage root explant washing and sterilizing, be seeded in the callus that under dark condition, carries out on the callus of induce medium after callus induction obtains inducing;
(2) callus is regulated and control: after the callus that induces is separated from parent, carry out successive transfer culture, make callus be loose, graininess;
(3) cell suspension cultures: loose, graininess callus 1～2 gram with obtaining in the step (2), be inoculated on the medium after the crushing and cultivate, filter and obtain small cell mass;
(4) callus induction plant regeneration:, transfer on the medium and cultivate, the regeneration induction plant with the small cell mass that obtains in the step (3);
(5) virus of regeneration plant detects: detect with enzyme chain immunoabsorption, obtain to have removed viral red sage root plant and carried out tissue culture expanding propagation.
Concrete processing step of the present invention and technological parameter are:
Red sage root explant in the step (1) comprises its rhizome, blade, young stem.
Step (1) is that red sage root explant is sterilized, wash, scrub with suds again, on superclean bench, adopted for 70% alcohol surface sterilizing 10-20 second, with 0.1% mercuric chloride solution sterilization 1～3 minute, be seeded in the callus after callus induction obtains inducing on the callus of induce medium then then;
Wherein callus of induce medium proportioning is: MS+6-BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L, sucrose 20～30g/L, agar 5g/L, pH5.6-5.8; Condition of culture is: temperature is 25 ± 2 ℃, cultivates under dark condition.
Step (2) is after the callus that induces that will obtain in the step (1) is separated from parent, be inoculated into successive transfer culture on the medium, the medium proportioning is: N6+BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L+ sucrose 3%+ agar 5g/L, pH5.6-5.8, and interpolation potassium chloride 100-500mg/L makes callus be loose, graininess in medium.
Step (3) is loose, graininess callus 1～2 gram that will obtain in the step (2), is inoculated in after the crushing in the 250ml triangular flask that the 50ml liquid nutrient medium is housed, and is shaken cultivation on the constant temperature rotary shaker of 110rpm in rotating speed, and temperature is 27～28 ℃; The medium proportioning is: N6+BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L+ sucrose 3%, pH5.6-5.8; With fresh liquid nutrient medium, change the culture fluid of 1/3 volume in the triangular flask after one week, change culture fluid later on weekly one time, cultivate after 1 month, filter and obtain small cell mass.
Step (4) be with step (3) obtain with small cell mass, transfer on the medium and cultivated 20-40 days, the regeneration of callus induction plant, callus of induce plant regeneration medium is: MS+BA1-4mg/L+NAA0.1mg/L+ sugar 30g/l+ agar 5g/l; Through 30 days, the regeneration bud point appearred in the microcell agglomerate, and regeneration bud behind 2-4cm, carries out culture of rootage through differentiation culture length, obtains plant to be checked; Differential medium is: MS+BA0.5mg/L+NAA0.1mg/L+ sugar 30g/l+ agar 5g/l, root media is a 1/2MS+NAA0.2mg/L+ sucrose 2%, pH5.6-5.8.
BA:6-benzylaminopurine in more than describing, NAA: methyl, 2,4-D:2,4-dichlorphenoxyacetic acid (down together).
The obtained technological progress of the present invention is:
The present invention has avoided heat treated process, directly adopts the somatic induction dedifferentiation to become somatoblast (callus), and meristematic cell forms plant through the direct regeneration induction of condition regulation, removes virus.Therefore, this method has overcome the thermal treatment that causes because of conventional " thermal treatment+Shoot Tip Culture " poison-removing method and has made withered, the dead problem of plant.Be specially adapted to red sage root virus-free, also be applicable to most plant virus-frees.
Embodiment
Below in conjunction with embodiment the present invention is further described, but not as a limitation of the invention.
Remove the method for red sage root virus with the microcell agglomerate regeneration method, comprise following processing step:
(1) callus induction: with red sage root explant (rhizome, blade, young stem etc.) sterilization, wash, scrub with suds again, on superclean bench, adopted for 70% alcohol surface sterilizing 10-20 second, sterilized 1～3 minute with 0.1% mercuric chloride solution then; Be seeded in the callus after callus induction obtains inducing on the callus of induce medium then.Wherein callus of induce medium proportioning is: MS+6-BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L, sucrose 20～30g/L, agar 5g/L, pH5.6-5.8; Condition of culture is: temperature is 25 ± 1 ℃, carries out under dark condition.
(2) callus regulation and control: be after the callus that induces that will obtain in the step (1) is separated from parent, be inoculated into successive transfer culture on the medium, the medium proportioning is: N6+BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L+ sucrose 3%+ agar 5g/L, pH5.6-5.8, and interpolation potassium chloride 100-500mg/L makes callus be loose, graininess in medium;
(3) cell suspension cultures: step (2) is restrained loose, the graininess callus 1～2 that obtains in the step (2), be inoculated in after the crushing in the 250ml triangular flask that the 50ml liquid nutrient medium is housed, be shaken cultivation on the constant temperature rotary shaker of 110rpm in rotating speed, temperature is 27～28 ℃; The medium proportioning is: N6+BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L+ sucrose 3%, pH5.6-5.8; The culture fluid of 1/3 volume in the triangular flask is changed with fresh liquid nutrient medium in one week back, changes culture fluid later on weekly one time, cultivate January after, filter the small cell mass of acquisition;
(4) callus induction plant regeneration: with step (3) obtain with small cell mass, transfer on the medium and cultivated 20-40 days, the regeneration of callus induction plant, callus of induce plant regeneration medium is: MS+BA1-4mg/L+NAA0.1mg/L+ sugar 30g/l+ agar 5g/l.Through 30 days, the regeneration bud point appearred in the microcell agglomerate, and regeneration bud behind 2-4cm, carries out culture of rootage through differentiation culture length, obtains plant to be checked; Differential medium is: MS+BA0.5mg/L+NAA0.1mg/L+ sugar 30g/l+ agar 5g/l, root media is a 1/2MS+NAA0.2mg/L+ sucrose 2%, pH5.6-5.8.
(5) virus of regeneration plant detects: detect with enzyme chain immunoabsorption, obtain to have removed viral red sage root plant and carried out tissue culture expanding propagation.
Claims (6)
1, remove the method for red sage root virus with the microcell agglomerate regeneration method, it is characterized in that comprising following processing step:
(1) callus induction: after red sage root explant washing and sterilizing, be seeded in and under dark condition, carry out callus induction, the callus after obtaining inducing on the callus of induce medium;
(2) callus is regulated and control: after the callus that induces is separated from parent, carry out successive transfer culture, make callus be loose, graininess;
(3) cell suspension cultures: loose, graininess callus 1～2 gram with obtaining in the step (2), be inoculated on the medium after the crushing and cultivate, filter and obtain small cell mass;
(4) callus induction plant regeneration:, transfer on the medium and cultivate, the regeneration induction plant with the small cell mass that obtains in the step (3);
(5) virus of regeneration plant detects: detect with enzyme chain immunoabsorption, obtain to have removed viral red sage root plant.
2, according to claim 1ly remove the method for red sage root virus, it is characterized in that the red sage root explant in the described step (1) comprises its rhizome, blade, young stem with the microcell agglomerate regeneration method.
3, the method that removes red sage root virus with the microcell agglomerate regeneration method according to claim 1, it is characterized in that described step (1) is that red sage root explant is sterilized, wash, scrub with suds again, on superclean bench, adopted for 70% alcohol surface sterilizing 10-20 second, sterilized 1～3 minute with 0.1% mercuric chloride solution then; Be seeded in callus induction on the callus of induce medium then, the callus after obtaining inducing;
Wherein callus of induce medium proportioning is: MS+6-BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L, sucrose 20～30g/L, agar 5g/L, PH5.6-5.8; Condition of culture is: temperature is 25 ± 2 ℃, cultivates under dark condition.
4, the method that removes red sage root virus with the microcell agglomerate regeneration method according to claim 1, it is characterized in that described step (2) is after the callus that induces that will obtain in the step (1) is separated from parent, be inoculated into successive transfer culture on the medium, the medium proportioning is: N6+BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L+ sucrose 3%+ agar 5g/L, PH5.6-5.8, and interpolation potassium chloride 100-500mg/L makes callus be loose, graininess in medium.
5, the method that removes red sage root virus with the microcell agglomerate regeneration method according to claim 1, it is characterized in that described step (3) is loose, graininess callus 1～2 gram that will obtain in the step (2), be inoculated in after the crushing in the 250ml triangular flask that the 50ml liquid nutrient medium is housed, be shaken cultivation on the constant temperature rotary shaker of 110rpm in rotating speed, temperature is 27～28 ℃; The medium proportioning is: N6+BA1.0mg/L+NAA1.0mg/L+2,4-D1.0mg/L+ sucrose 3%, pH5.6-5.8; The culture fluid of 1/3 volume in the triangular flask is changed with fresh liquid nutrient medium in one week back, changes culture fluid later on weekly one time, cultivate January after, filter the small cell mass of acquisition.
6, the method that removes red sage root virus with the microcell agglomerate regeneration method according to claim 1, it is characterized in that described step (4) be with step (3) obtain with small cell mass, transfer on the medium and cultivated 20-40 days, the regeneration of callus induction plant, callus of induce plant regeneration medium is: MS+BA1-4mg/L+NAA0.1mg/L+ sugar 30g/l+ agar 5g/l; Through about 30 days, the regeneration bud point appears in the microcell agglomerate, and regeneration bud behind 2-4cm, carries out culture of rootage through differentiation culture length, obtains plant to be checked; Differential medium is: MS+BA0.5mg/L+NAA0.1mg/L+ sugar 30g/l+ agar 5g/l, root media is a 1/2MS+NAA0.2mg/L+ sucrose 2%, pH5.6-5.8.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428875A (en) * | 2011-12-29 | 2012-05-02 | 四川农业大学 | Tissue culture and rapid propagation method of Szechuan salvia miltiorrhiza bunge |
| CN104542276A (en) * | 2014-11-10 | 2015-04-29 | 浙江省萧山棉麻研究所 | Method for detoxifying culture of anthurium andraeanum |
| CN105613285A (en) * | 2014-10-31 | 2016-06-01 | 中国人民解放军第二军医大学 | Method for quickly increasing content of rosmarinic acid in salvia miltiorrhiza bunge |
| CN106665352A (en) * | 2016-12-01 | 2017-05-17 | 大连大学 | Virus-elimination method of plant tissue cultured virus-free explant |
-
2006
- 2006-09-26 CN CNB2006100483426A patent/CN100444720C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428875A (en) * | 2011-12-29 | 2012-05-02 | 四川农业大学 | Tissue culture and rapid propagation method of Szechuan salvia miltiorrhiza bunge |
| CN105613285A (en) * | 2014-10-31 | 2016-06-01 | 中国人民解放军第二军医大学 | Method for quickly increasing content of rosmarinic acid in salvia miltiorrhiza bunge |
| CN104542276A (en) * | 2014-11-10 | 2015-04-29 | 浙江省萧山棉麻研究所 | Method for detoxifying culture of anthurium andraeanum |
| CN106665352A (en) * | 2016-12-01 | 2017-05-17 | 大连大学 | Virus-elimination method of plant tissue cultured virus-free explant |
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| CN100444720C (en) | 2008-12-24 |
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