CN1907013A - Method for saussurea involucrate callus induction and tissue culture sprout quick reproduction - Google Patents
Method for saussurea involucrate callus induction and tissue culture sprout quick reproduction Download PDFInfo
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Abstract
The invention discloses a saussurea involucrate callus inducing and tissue-cultivating plant rapid-growing method, which comprises the following steps: preparing aseptic explant material; inducing callus at different parts; adopting solid cultivation, suspension cultivation or biological reactor to breed root.
Description
Technical field
The invention belongs to the Biochemical Engineering field, relate to a kind of saussurea involucrata callus induction and tissue culture sprout quick breeding method.
Background technology
Saussurea involucrata belongs to the composite family hieracioides and belongs to (Saussurea DC.) plant, and several kinds are arranged, and can be used as medicine on an equal basis, and quality also has the branch of quality.Be mainly used in dispelling cold and removing dampness, activating blood to promote menstruation, anti-inflammatory and antalgic etc.The rheumatic arthritis that is used for the treatment of among the people, gynaecological disease etc.Pharmacological evaluation proves that lanatechead saussurea herb with flower has anticancer, and hemangiectasis is hypotensive, fatigue-resisting function.Particularly the flavones that contains in the saussurea involucrata (jaceosidin) has significant curative effect to treatment ascitic type tumour, and its preparation is well received at home and abroad.Owing to can not meet the need of market far away, the coyoting of wild saussurea involucrata is dug excessively not only make natural resources endangered, also ecotope has been caused heavy damage simultaneously.Though realized artificial planting on a small scale by seed, exist wild seed rare, be difficult to gather, problems such as germplasm degeneration easily take place in the seed of artificial planting continuous plantation of many generations back.Because the saussurea involucrata growing environment is special, provenance is rare, the artificial cultivation difficulty, growth cycle is long, many generations plantation back germplasm is easily degenerated, and therefore, utilizing plant tissue culture, fast breeding technique to carry out saussurea involucrata seedling production, to go forward side by side that pedestrian's work post plants be the effective way that solves present saussurea involucrata shortage of resources.
Watt Cuba promise baby of China once reported the stem, blade, holder and the little capitulum that utilize saussurea involucrata nineteen ninety and added 2 milligrams every liter methyl and 0.2 milligram every liter N for explant utilizes MS
6The medium callus induction of-benzyladenine, wherein little capitate inductivity is 85%, and the inductivity of other explants only is 20%, and the callus poor growth, and browning is aging easily.With little capitulum is that the callus of induce of explant becomes bud, utilizes low temperature treatment just can take root in tens days when taking root simultaneously.Because saussurea involucrata only blooms in the 6～August in every year, take the time of explant to be restricted, and owing to when taking root, utilize low temperature treatment just can take root in tens days, so the breeding cycle of the method is longer relatively.It is 100% that Institute of Botany, Chinese Academy of Sciences once utilizes stem of saussurea involucrata and inductivity that leaf induces the callus stem at report in 1997, the inductivity of leaf is about 50%, but for the callus of being induced is embryo callus subculture, whether has the germination potentiality not have follow-up report.
Summary of the invention
The purpose of this invention is to provide a kind of saussurea involucrata callus induction and tissue culture sprout quick breeding method, it is indoor numerous soon on a large scale to utilize method of the present invention that saussurea involucrata is carried out, to solve saussurea involucrata breeds difficulty under native state problem, it is deficient day by day to solve the saussurea involucrata resource, so that problems such as species are endangered finally realize the extensive artificial planting of wild saussurea involucrata; And breed saussurea involucrata fast by extensive tissue culture technique such as bioreactor culture, and enlarge the saussurea involucrata cultivated area, improve the reserves of saussurea involucrata.
Saussurea involucrata callus induction of the present invention and tissue culture sprout quick breeding method may further comprise the steps:
(1). directly gather fresh explants such as wild saussurea involucrata leaf, root from the field and be used for callus induction; Or MS (referring to Xue Qingshan, Xiao Yuping, Lin Juan.Philosophy and technique Beijing Science Press of culture in vitro, 2001) on the minimal medium with the saussurea involucrata seed germination, cultivate aseptic seedling, be the explant induction callus with its leaf, root or plumular axis then.The method that saussurea involucrata sprouts aseptic seedling is to select full saussurea involucrata seed, with mass concentration is that 70% ethanol and mass concentration are 2% clorox sterilization, be inoculated on the MS minimal medium, in temperature is 15～35 degrees centigrade, illumination 1000～5000 luxs were cultivated 10～30 days down, can grow the saussurea involucrata aseptic seedling.
(2). be explant (leaf of wild saussurea involucrata seedling, root need be that 70% ethanol or mass concentration are 2% clorox sterilization through mass concentration) with leaf, root or the plumular axis of the saussurea involucrata seedling of step (1) respectively, aseptic explant directly inserts MS interpolation N respectively
6In the medium of-benzyladenine, methyl, 2,4 dichlorophenoxyacetic acid and sucrose, the N in the MS medium wherein
6-benzyladenine concentration is 0.01～3.0 milligram every liter, the concentration of methyl is 0.01～5.0 milligram every liter, 2, the concentration of 4-dichlorphenoxyacetic acid is 0～2.5 milligram every liter, concentration of sucrose is every liter of 5～80 gram, the pH value 5.7～6.0 of medium, add every liter of agar of 1～9 gram of cultivating base unit weight, sterilized 15～20 minutes down for 115～125 ℃ in temperature, the cooling medium, 15～35 degrees centigrade of temperature, illumination 1000～5000 luxs are cultivated down, induce the callus of leaf, root or the plumular axis explant of saussurea involucrata seedling, various callus of induce rates reach more than 85%.
(3). adopt solid culture, suspension culture or bio-reactor large-scale culture to expand the embryo callus subculture or the body embryo of callus of leaf, root or the plumular axis explant of numerous step (2) saussurea involucrata seedling, go into MS and add N expanding embryo callus subculture after numerous or body embryonic breeding
6On the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea, wherein, N in the MS medium
6The concentration of-benzyladenine is 0.01～2.5 milligram every liter, and the concentration of methyl is 0.05～2.0 milligram every liter, and the concentration of 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea is 0～5 milligram every liter; In temperature is 15～35 degrees centigrade, and illumination 1000～5000 luxs were cultivated 10～30 days down, can sprout and sprout or plant; Cut out greater than 1 centimetre of simple bud sprouting the bud of growing thickly that, add N at MS less than 3 centimetres
6Expand in the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea numerous, N in the MS medium wherein
6The concentration of-benzyladenine is 0.01～2.5 milligram every liter, the concentration of methyl is 0.01～5.0 milligram every liter, 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) concentration of urea is 0～5 milligram every liter, treat simple bud grow into 〉=change in the root media 4 centimetres the time and take root; Maybe with sprout 〉=4 centimetres simple bud changes in the root media and takes root; Maybe the plant that sprouts is changed on the MS minimal medium and grow.
(4) simple bud of step (3) is changed in the root media taking root, is to adopt interior sterile rootage of container (as bottle) or open taking root; When in container, taking root, simple bud is changed in the incubator of MS interpolation heteroauxin, indolebutyric acid, methyl and active carbon, wherein, the concentration of heteroauxin is 0.01～2.0 milligram every liter in the MS medium, the concentration of indolebutyric acid is 0.01～2.0 milligram every liter, the concentration of methyl is 0.01～2.0 milligram every liter, and the mass concentration of active carbon is 0～2.0%; Perhaps simple bud is changed in the open container that the vermiculite after the sterilization is housed, it is 0.01～2.0 milligram every liter heteroauxin that described vermiculite is added with concentration with the MS of 1/2 concentration, concentration is 0.01～2.0 milligram every liter indolebutyric acid, concentration is that the culture fluid of 0.01～2.0 milligram every liter methyl soaks into, in temperature is 15～35 degrees centigrade, illumination 1000～5000 luxs are cultivated down, and rooting rate can reach more than 85% after 10～30 days; The plant that sprouts changed on the MS minimal medium grow;
(5). the saussurea involucrate tissue culture sprout that the saussurea involucrate tissue culture sprout of taking root in step (4) container or container are taken root outward, or change the plant of MS minimal medium growth in the step (3) over to; Be transferred to and carry out hardening in loam+vermiculite, wherein, the volume ratio of loam and vermiculite is 10: 1～1: 10.
Described step (2) is to adjust pH with inorganic acid or inorganic base.Example hydrochloric acid, sulfuric acid etc.; Sodium hydroxide, potassium hydroxide etc.
The present invention utilizes extensive tissue culture technique to breed the saussurea involucrata seedling fast, and key step comprises: the preparation of aseptic explant material, the explant induction callus with different parts, employing solid culture, suspension culture or bio-reactor large-scale culture expand numerous embryo callus subculture and body embryo, sprout and sprout or plant, simple bud growth and take root, the grow thickly breeding fast of bud subculture, hardening, transplanting.The bud rooting rate can reach more than 85%, and the open-air survival rate of transplantation of seedlings can reach more than 80%.Plant by the extensive tissue-culturing rapid propagation saussurea involucrata seedling pedestrian's work post of going forward side by side; can overcome the many generation plantations of seed seedling-raising easily degenerates; easily produce damage by disease and insect; shortcomings such as the growth and the synchronism of blooming are relatively poor; help the optimization of saussurea involucrata germ plasm resource, improve the saussurea involucrata resource reserve; preserve the ecological environment, promote the sound development of saussurea involucrata industrial chain.
Embodiment
Embodiment 1:
(1). with saussurea involucrata seed germination, cultivation aseptic seedling, be the explant induction callus with its leaf then.Saussurea involucrata sprouts the aseptic seedling method: select full saussurea involucrata seed mass concentration and be 70% ethanol and mass concentration and be 2% clorox and sterilize, be inoculated on the MS minimal medium, in temperature is 18 degrees centigrade, and illumination 2000 luxs were cultivated 25 days down, can grow the saussurea involucrata aseptic seedling.
(2). with the leaf of the saussurea involucrata seedling of step (1) is that explant directly inserts MS and adds N
6In the medium of-benzyladenine, methyl, 2,4 dichlorophenoxyacetic acid and sucrose, the N in the MS medium wherein
6-benzyladenine concentration is 0.5 milligram every liter, the concentration of methyl is 2.0 milligrams every liter, 2, the concentration of 4-dichlorphenoxyacetic acid is 1.0 milligrams every liter, concentration of sucrose is every liter of 30 gram, utilize hydrochloric acid and sodium hydroxide solution to adjust the pH value 5.85 of medium, add every liter of agar of 5.5 grams of cultivating base unit weight, sterilized 20 minutes down for 121 degrees centigrade in temperature, the cooling medium, 25 degrees centigrade of temperature, illumination 2000 luxs are cultivated down, induce the callus of the leaf explant of saussurea involucrata seedling, the callus of induce rate reaches 88%.
(3). adopt suspension culture to cultivate the embryo callus subculture of the callus of the leaf explant that expands numerous step (2) saussurea involucrata seedling, insert MS and add N expanding embryo callus subculture after numerous
6On the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea, wherein, N in the MS medium
6The concentration of-benzyladenine is 0.2 milligram every liter, and the concentration of methyl is 0.05 milligram every liter, and the concentration of 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea is 3 milligrams every liter; In temperature is 25 degrees centigrade, and illumination 2000 luxs were cultivated 25 days down, can sprout and sprout; With sprout 〉=4 centimetres simple bud directly changes in the root media and takes root; Cut out greater than 1 centimetre of simple bud sprouting the bud of growing thickly that, add N at MS less than 3 centimetres
6Expand in the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea numerous, N in the MS medium wherein
6The concentration of-benzyladenine is 0.2 milligram every liter, and the concentration of methyl is 0.05 milligram every liter, and the concentration of 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea is 3 milligrams every liter, treat simple bud grow into 〉=change in the root media 4 centimetres the time and take root.
(4) simple bud of step (3) is changed in the root media taking root, is to adopt sterile rootage in the container; When in container, taking root, simple bud is changed in the incubator of MS interpolation heteroauxin, indolebutyric acid, methyl and active carbon, wherein, the concentration of heteroauxin is 0.5 milligram every liter in the MS medium, and the concentration of indolebutyric acid is 0.2 milligram every liter, and the concentration of methyl is 0.2 milligram every liter, the mass concentration of active carbon is 1%, in temperature is 25 degrees centigrade, and under illumination 2000 luxs, rooting rate can reach 95% after 20 days.
(5). with the saussurea involucrate tissue culture sprout of taking root in step (4) container, be transferred to and carry out hardening in loam+vermiculite, the volume ratio of its medium loam and vermiculite is 1: 5.
Embodiment 2:
(1). with saussurea involucrata seed germination, cultivation aseptic seedling, be the explant induction callus with its root then.Saussurea involucrata sprouts the aseptic seedling method: select full saussurea involucrata seed mass concentration and be 70% ethanol and mass concentration and be 2% clorox and sterilize, be inoculated on the MS minimal medium, in temperature is 25 degrees centigrade, and illumination 2000 luxs were cultivated 25 days down, can grow the saussurea involucrata aseptic seedling.
(2). with the root of the saussurea involucrata seedling of step (1) is that explant directly inserts MS and adds N
6In the medium of-benzyladenine, methyl, 2,4 dichlorophenoxyacetic acid and sucrose, the N in the MS medium wherein
6-benzyladenine concentration is 0.5 milligram every liter, the concentration of methyl is 2.0 milligrams every liter, 2, the concentration of 4-dichlorphenoxyacetic acid is 1.0 milligrams every liter, concentration of sucrose is every liter of 30 gram, utilize hydrochloric acid and sodium hydroxide solution to adjust the pH value 5.85 of medium, add every liter of agar of 5.5 grams of cultivating base unit weight, sterilized 15 minutes down for 125 degrees centigrade in temperature, the cooling medium, 25 degrees centigrade of temperature, illumination 2000 luxs are cultivated down, induce the callus of the root explant of saussurea involucrata seedling, the callus of induce rate reaches 98%.
(3). adopt bioreactor culture to expand the embryo callus subculture of callus of the root explant of numerous step (2) saussurea involucrata seedling, insert MS and add N expanding embryo callus subculture after numerous
6On the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea, wherein, N in the MS medium
6The concentration of-benzyladenine is 0.5 milligram every liter, and the concentration of methyl is 0.05 milligram every liter, and the concentration of 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea is 1 milligram every liter; In temperature is 25 degrees centigrade, and illumination 2000 luxs were cultivated 25 days down, can sprout and sprout; The simple bud that sprouts 3～5 centimetres directly changed in the root media take root; Cut out greater than 1 centimetre of simple bud sprouting the bud of growing thickly that, add N at MS less than 3 centimetres
6Expand in the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea numerous, N in the MS medium wherein
6The concentration of-benzyladenine is 0.5 milligram every liter, and the concentration of methyl is 0.05 milligram every liter, and the concentration of 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea is 1 milligram every liter, treat simple bud grow into 〉=change in the root media 4 centimetres the time and take root.
(4) simple bud of step (3) is changed in the root media taking root, is to adopt sterile rootage in the container; When in container, taking root, simple bud is changed in the incubator of MS interpolation heteroauxin, indolebutyric acid, methyl and active carbon, wherein, the concentration of heteroauxin is 0.2 milligram every liter in the MS medium, and the concentration of indolebutyric acid is 0.2 milligram every liter, and the concentration of methyl is 0.2 milligram every liter, the mass concentration of active carbon is 1.5%, in temperature is 25 degrees centigrade, and under illumination 2000 luxs, rooting rate can reach 96% after 15 days.
(5). with the saussurea involucrate tissue culture sprout of taking root in step (4) container, be transferred to and carry out hardening in loam+vermiculite, the volume ratio of its medium loam and vermiculite is 1: 5.
Embodiment 3:
(1). adopting saussurea involucrata wild[l blade as explant, is that 70% ethanol and mass concentration are that 2% clorox is sterilized with mass concentration.
(2). with the leaf of the saussurea involucrata seedling of step (1) is that explant directly inserts MS and adds N
6In the medium of-benzyladenine, methyl, 2,4 dichlorophenoxyacetic acid and sucrose, the N in the MS medium wherein
6-benzyladenine concentration is 0.02 milligram every liter, the concentration of methyl is 4.0 milligrams every liter, 2, the concentration of 4-dichlorphenoxyacetic acid is 2.0 milligrams every liter, concentration of sucrose is every liter of 20 gram, utilize hydrochloric acid and sodium hydroxide solution to adjust the pH value 5.85 of medium, add every liter of agar of 5.5 grams of cultivating base unit weight, sterilized 25 minutes down for 115 degrees centigrade in temperature, the cooling medium, 25 degrees centigrade of temperature, illumination 2000 luxs are cultivated down, induce the callus of the leaf explant of saussurea involucrata seedling, the callus of induce rate reaches 88%.
(3). adopt suspension culture to cultivate the embryo callus subculture of the callus of the leaf explant that expands numerous step (2) saussurea involucrata seedling, insert MS and add N expanding embryo callus subculture after numerous
6On the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea, wherein, N in the MS medium
6The concentration of-benzyladenine is 2 milligrams every liter, and the concentration of methyl is 1.5 milligrams every liter, and the concentration of 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea is 4 milligrams every liter; In temperature is 25 degrees centigrade, and illumination 2000 luxs were cultivated 25 days down, can sprout and sprout; The simple bud that sprouts 3～5 centimetres directly changed in the root media take root; Cut out greater than 1 centimetre of simple bud sprouting the bud of growing thickly that, add N at MS less than 3 centimetres
6Expand in the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea numerous, N in the MS medium wherein
6The concentration of-benzyladenine is 0.2 milligram every liter, and the concentration of methyl is 2 milligrams every liter, and the concentration of 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea is 2 milligrams every liter, treat simple bud grow into 〉=change in the root media 4 centimetres the time and take root.
(4) simple bud of step (3) is changed in the root media taking root, is to adopt sterile rootage in the container; When in container, taking root, simple bud is changed in the incubator of MS interpolation heteroauxin, indolebutyric acid, methyl and active carbon, wherein, the concentration of heteroauxin is 0.05 milligram every liter in the MS medium, and the concentration of indolebutyric acid is 0.15 milligram every liter, and the concentration of methyl is 1.5 milligrams every liter, the mass concentration of active carbon is 1%, in temperature is 25 degrees centigrade, and under illumination 2000 luxs, rooting rate can reach 90% after 30 days.
(5). with the saussurea involucrate tissue culture sprout of taking root in step (4) container, be transferred to and carry out hardening in loam+vermiculite, the volume ratio of its medium loam and vermiculite is 1: 5.
Claims (5)
1. saussurea involucrata callus induction and tissue culture sprout quick breeding method is characterized in that this method may further comprise the steps:
(1). directly gather wild saussurea involucrata leaf from the field, the fresh explant of root is used for callus induction; Or on the MS minimal medium, use saussurea involucrata seed germination, cultivation aseptic seedling, be the explant induction callus with its leaf, root or plumular axis then;
(2). leaf, root or the plumular axis with the saussurea involucrata seedling of step (1) is explant respectively, and aseptic explant directly inserts MS interpolation N respectively
6In the medium of-benzyladenine, methyl, 2,4 dichlorophenoxyacetic acid and sucrose, the N in the MS medium wherein
6-benzyladenine concentration is 0.01～3.0 milligram every liter, the concentration of methyl is 0.01～5.0 milligram every liter, 2, the concentration of 4-dichlorphenoxyacetic acid is 0～2.5 milligram every liter, concentration of sucrose is every liter of 5～80 gram, the pH value 5.7～6.0 of medium, add every liter of agar of 1～9 gram of cultivating base unit weight, in 115～125 ℃ of sterilizations down of temperature, the cooling medium, 15～35 degrees centigrade of temperature, illumination 1000～5000 luxs are cultivated down, induce the callus of leaf, root or the plumular axis explant of saussurea involucrata seedling;
(3). adopt solid culture, suspension culture or bio-reactor large-scale culture to expand the embryo callus subculture or the body embryo of callus of leaf, root or the plumular axis explant of numerous step (2) saussurea involucrata seedling, go into MS and add N expanding embryo callus subculture after numerous or body embryonic breeding
6On the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea, wherein, N in the MS medium
6The concentration of-benzyladenine is 0.01～2.5 milligram every liter, and the concentration of methyl is 0.05～2.0 milligram every liter, and the concentration of 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea is 0～5 milligram every liter; In temperature is 15～35 degrees centigrade, and illumination 1000～5000 luxs are cultivated down, can sprout and sprout or plant; Cut out greater than 1 centimetre of simple bud sprouting the bud of growing thickly that, add N at MS less than 3 centimetres
6Expand in the medium of-benzyladenine, methyl and 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) urea numerous, N in the MS medium wherein
6The concentration of-benzyladenine is 0.01～2.5 milligram every liter, the concentration of methyl is 0.01～5.0 milligram every liter, 1-phenyl-3-(1,2,3-thiadiazoles-5-yl) concentration of urea is 0～5 milligram every liter, treat simple bud grow into 〉=change in the root media 4 centimetres the time and take root; Maybe with sprout 〉=4 centimetres simple bud changes in the root media and takes root; Maybe the plant that sprouts is changed on the MS minimal medium and grow;
(4) simple bud of step (3) is changed in the root media taking root, is to adopt sterile rootage or open taking root in the container; When in container, taking root, simple bud is changed in the incubator of MS interpolation heteroauxin, indolebutyric acid, methyl and active carbon medium, wherein, the concentration of heteroauxin is 0.01～2.0 milligram every liter in the MS medium, the concentration of indolebutyric acid is 0.01～2.0 milligram every liter, the concentration of methyl is 0.01～2.0 milligram every liter, and the mass concentration of active carbon is 0～2.0%; Perhaps simple bud is changed in the open container that the vermiculite after the sterilization is housed, it is 0.01～2.0 milligram every liter heteroauxin that described vermiculite is added with concentration with the MS of 1/2 concentration, concentration is 0.01～2.0 milligram every liter indolebutyric acid, concentration is that the culture fluid of 0.01～2.0 milligram every liter methyl soaks into, in temperature is 15～35 degrees centigrade, and illumination 1000～5000 luxs are cultivated down;
(5). the saussurea involucrate tissue culture sprout that the saussurea involucrate tissue culture sprout of taking root in step (4) container or container are taken root outward, or change the plant of MS minimal medium growth in the step (3) over to; Be transferred to and carry out hardening in loam+vermiculite, wherein, the volume ratio of loam and vermiculite is 10: 1～1: 10.
2. method according to claim 1, it is characterized in that: the method that described step (1) saussurea involucrata sprouts aseptic seedling is to select full saussurea involucrata seed, with mass concentration is that 70% ethanol and mass concentration are 2% clorox sterilization, be inoculated on the MS minimal medium, in temperature is 15～35 degrees centigrade, illumination 1000～5000 luxs were cultivated 10～30 days down, can grow the saussurea involucrata aseptic seedling.
3. method according to claim 1 is characterized in that: the leaf of the wild saussurea involucrata seedling of described step (1), root need be that 70% ethanol or mass concentration are 2% clorox sterilization through mass concentration.
4. method according to claim 1 is characterized in that: described step (2) is to adjust pH with inorganic acid or inorganic base.
5. method according to claim 1 is characterized in that: described step (2) was sterilized 15～20 minutes down for 115～125 ℃ in temperature.
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| CN106069766A (en) * | 2016-06-29 | 2016-11-09 | 无锡南理工科技发展有限公司 | A kind of Herba Saussureae Involueratae in-vitro breeding method |
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| CN115786232A (en) * | 2022-12-19 | 2023-03-14 | 中国科学院青岛生物能源与过程研究所 | Rapid suspension culture and genetic transformation method of saussurea involucrate cells |
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| CN1606905A (en) * | 2003-10-14 | 2005-04-20 | 刘杰龙 | Xinjiang snow lotus tube seedling large scale cloning and breeding production technology and its uses |
| CN1257667C (en) * | 2004-10-27 | 2006-05-31 | 中国科学院新疆理化技术研究所 | Quick method for inducing and clustering bud in tissue culture for herba saussureae involucratae |
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2005
- 2005-08-01 CN CNB2005100122832A patent/CN100459850C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103518622A (en) * | 2013-10-21 | 2014-01-22 | 西北大学 | Method for prolonging life time of Sinkiang saussurea involucrata test-tube plantlet at low latitude under low atmospheric pressure |
| CN103518622B (en) * | 2013-10-21 | 2016-01-20 | 西北大学 | A kind of low atmospheric pressure extends the method for time of Sinkiang saussurea involucrata test-tube plantlet in low altitude area life span |
| CN106069766A (en) * | 2016-06-29 | 2016-11-09 | 无锡南理工科技发展有限公司 | A kind of Herba Saussureae Involueratae in-vitro breeding method |
| CN107318649A (en) * | 2017-06-29 | 2017-11-07 | 河北农业大学 | It is a kind of to reduce the culture medium and cultural method of walnut Explant browning rate |
| CN109220806A (en) * | 2018-11-06 | 2019-01-18 | 云南省红河热带农业科学研究所 | A kind of papaya seedling rooting and the method for transplanting |
| CN115786232A (en) * | 2022-12-19 | 2023-03-14 | 中国科学院青岛生物能源与过程研究所 | Rapid suspension culture and genetic transformation method of saussurea involucrate cells |
| CN115786232B (en) * | 2022-12-19 | 2023-10-24 | 中国科学院青岛生物能源与过程研究所 | Quick suspension culture and genetic transformation method for saussurea involucrata cells |
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