CN101671648B - Separation and culturing method of saussurea involucrate protoplast - Google Patents
Separation and culturing method of saussurea involucrate protoplast Download PDFInfo
- Publication number
- CN101671648B CN101671648B CN2009101134708A CN200910113470A CN101671648B CN 101671648 B CN101671648 B CN 101671648B CN 2009101134708 A CN2009101134708 A CN 2009101134708A CN 200910113470 A CN200910113470 A CN 200910113470A CN 101671648 B CN101671648 B CN 101671648B
- Authority
- CN
- China
- Prior art keywords
- protoplastis
- protoplast
- cpw
- callus
- condition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a separation and culturing method of Sinkiang saussurea protoplast, comprising four steps of culturing of sterile saussurea involucrate seedlings, inducement and differentiation of embryonic callus, separation and purification of plasmogen and culturing of the protoplast which are all applicable to the separation and culturing of the Sinkiang saussurea involucrate protoplast. The invention has simple and easily-operated method and lower requirements on the equipment and culturing conditions; in addition, the enzymolysis method is practical, the conditions for cell wall removal are mild, and the separated protoplast has high yield and strong activity, and is easy for the cultuing of the saussurea involucrate protoplast and a plant regeneration, thus laying the foundations for the culturing and fusing of the protoplast as well as the germplasm resource innovation of the Sinkiang saussurea involucrate and the research of transgenic saussurea involucrate.
Description
Technical field
The present invention relates to the foundation of Herba Saussureae Involueratae suspension cell line, relate in particular to the separation and the cultural method of Herba Saussureae Involueratae protoplastis.
Background technology
Saussurea involucrata, (Saussurea involucrata Kar.et Kir.et Maxim) belongs to composite family, is national secondary endangered plant, is per nnial herb, high 15-25 (35) centimetre; Root stock is thick, and chocolate, base portion are brown; Stem Dan Sheng, upright, hollow, diameter 2-4 centimetre, do not have hair, leaf is intensive, nearly keratin, green, blade Long Circle or ovum shape Long Circle are about 14 centimetres, and wide 2-4 centimetre, the blunt or little point of tip, base portion is downward, and there is sparse little sawtooth at the edge, tool corpora mammillaria glandular hairs, topmost bud 13-17, the thin tooth membranous transparent, faint yellow, that the edge tool is neat is slightly by glandular hairs, the blunt point of tip, base portion shrinks, and often exceeds 2 times of inflorescences.Head inflorescence 10-30 is gathered in the stem end and is spherical; The involucre semisphere, phyllary 3-4 layer, closely membranous, lanceolar, anxious point, edge black, those hairs; The pistil purple is about 14 millimeters.The achene Long Circle, tool is indulged rib; 2 layers of pappus, outer short, rough hair shape, internal layer is long, featheriness.
Saussurea involucrata is grown in below the high mountain snow line usually, between 4000 meters of the height above sea level upper limits, is distributed in the high and cold mountain region of the northwestward 2400 meters of height above sea level lower limits in China.The highest monthly mean temperature 3-5 ℃, the subzero 21-19 of minimum monthly mean temperature ℃, about 800 millimeters of annual precipitation, frostless season only had about 50 days.Soil is based on alpine meadow soil, and organic content is 8.5--11%, nitrogen content 4.5--10%.Because envrionment conditions is abominable, general plant is difficult to growth, has only cold-resistant, the low temperature resistant sedge of minority to belong to Carex spp., and high grass belongs to Kobresia spp. and the association with it of various high mountain per nnial herbs.The saussurea involucrata seed is 0 ℃ of germination, 3-5 ℃ of growth.Seedling can be stood-21 ℃ severe cold.Vegetative period less than bimestrial environment in, highly can surpass five to seven times of other plant, just can bloom though it takes 5 years, but actual growth fate has only eight months, this also is fairly individual biologically.
Saussurea involucrata also is the rare famous and precious medicinal plant of a kind of high mountain, owing to excessively excavate, rate of emergence is low, the breeding difficulty, and the characteristics of poor growth etc., as not adopting an effective measure, strictly protection will face the danger of extinction.Therefore protect saussurea involucrata germ plasm resource, on science or pharmaceutically all significant.So the nature conservation point is set up in Yi Li or Altay mountain area that suggestion is concentrated in the saussurea involucrata distribution, is laid special stress on protecting; Also available seed is bred fast.But during the saussurea involucrata seed maturity, high and cold mountain area has been the heavy snow time, and the difficult seed of gathering causes the difficulty on the introducing culture, and biological characteristics solid and that germinate is studied in suggestion in the wilderness area, and carries out saussurea involucrata introducing culture and the test of being transplanted to former habitat.
By the extensive artificial breeding saussurea involucrata of method for plant tissue culture, can not be subjected to the restriction of season and weather at present, accelerate reproduction speed, enlarge germ plasm resource fast.It is the aseptic technique method of using, and the isolated organ of culturing plants, tissue or cell make it on the substratum of synthetic, by division, propagation, differentiation, the growth of cell, finally grows up to the process of complete regenerated plant.Therefore Herba Saussureae Involueratae not only can be protected the wild germplasm resource by the measure of tissue culture regeneration plant, and provides new way for the Herba Saussureae Involueratae large-scale production.
On this basis, the present invention carries out separation and Extraction to the Herba Saussureae Involueratae protoplastis, because the protoplastis of saussurea involucrata cell all contains whole genetic information of this individuality, the totipotency that under suitable culture condition, has regeneration and the similar individuality of its parent, a large amount of protoplastiss that obtain at one time are homogeneity in heredity; The protoplasma physical efficiency overcomes the not affine obstacle of sexual cell, is convenient to carry out edge somatic hybridization far away, thereby obtains remote hybrid; Protoplastis can directly absorb DNA, organoid, virus, plasmid of external source etc., is the desirable acceptor that carries out Study on Genetic Transformation, can obtain the transgenosis saussurea involucrata; Simultaneously in the process that protoplastis is cultivated, this gives birth to meta-bolitess can to produce some, can separation and Extraction under the condition of technology maturation; This external protoplastis is cultivated in the regeneration plant process, tends to produce somaclonal variation, also can obtain mutant etc. by chemical factors mutagenesis.Therefore, the germ plasm resource of utilizing protoplastis cultivation approach to carry out Herba Saussureae Involueratae is innovated a kind of feasible and valid approach of can yet be regarded as.
Summary of the invention
The present invention relates to the separation and the cultural method of Herba Saussureae Involueratae protoplastis, this method by the cultivation of aseptic saussurea involucrata seedling, embryo callus induce and differentiation, plasmic isolation and purification and protoplastis are cultivated four steps and are finished.The culture medium prescription of the disinfection treatment method of acquisition saussurea involucrata aseptic seedling, best induced embryonic callus, best saussurea involucrate protoplast enzymolysis and separation condition, and the culture medium prescription and the culture condition of protoplastis after the separation, purifying, all be applicable to the separation and the cultivation of the protoplastis of Herba Saussureae Involueratae, also lay a good foundation for the research of innovation of Herba Saussureae Involueratae germ plasm resource and transgenosis saussurea involucrata.
Invent the separation and the cultural method of described Herba Saussureae Involueratae protoplastis, finished by the steps such as enzymolysis, separation and cultivation that obtain aseptic seedling, embryo callus, protoplastis, concrete operations follow these steps to carry out:
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2-3 hour with the tap water flushing, first with 75% alcohol-pickled 10-60s on Bechtop then, 0.1% mercuric chloride sterilization 1-10min, 15% hydrogen peroxide, handle 20-40min, aseptic water washing 3-4 time soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated on the MS substratum, 23 ± 1 ℃ of temperature, illumination 2500-3000lx, time 16h/d, 10-20d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 1-2cm, insert substratum MS+NAA 1.0-1.5mg/L+6-BA 0.1-1.0mg/L+2,4-D0.1-0.5mg/L in, place 23 ± 1 ℃ of temperature, illumination 3000-4000lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 1-3 time, take by weighing the CPW+13% mannitol solution that the chopping of 1g embryo callus places 10ml, carry out plasmolysis, under 25 ℃ of dark conditions of temperature, handled 3-5 hour;
E, the plasmolysis liquid of steps d is removed at leisure, keep the embryo callus after the plasmolysis, place 10ml enzyme liquid to carry out enzymolysis, place rotating speed 80rpm, 26 ± 2 ℃ of temperature, under the dark condition, enzymolysis 12-16h;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant liquor, the CPW+10% mannitol solution that adds 3-4ml, carrying out protoplastis cleans, place centrifugal 2-5min under the rotating speed 1000rpm condition again, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on 20% sucrose solution (5ml centrifuge tube dress 3ml 20% sucrose solution) gently, centrifugal 5-10min under rotating speed 800rpm condition then is because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, puts into another clean centrifuge tube, the CPW+10% mannitol solution that adds 2-5ml, carry out protoplastis and clean once more, the centrifugal 2-5min of rotating speed 1000rpm abandons supernatant liquor;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by Safranin B Extra dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis, and the culture density of protoplastis of the present invention is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, through 1-2 macroscopic cell mass appearred on protoplast culture medium after the month roughly, promptly form little callus, described callus inducing medium is the additional hormone 1.0mg/LNAA+0.3mg/L 6-BA+0.5mg/L 2 of NT substratum or DPD substratum, 4-D.
Hormone concentration is preferably among the step c: NAA 1.0mg/L+6-BA 0.3mg/L+2,4-D0.1mg/L.
Enzymolysis solution is among the step e: cellulase 1.0%-2.0%, and macerozyme 0.1%-0.5%, polygalacturonase 0.1%-0.5%, 5mmol/L MES is dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulates pH5.6-5.8.
Herba Saussureae Involueratae protoplastis of the present invention separates and its advantage of cultural method is:
At first, protoplastis is the same with cell, has totipotency, and contains whole genetic information of this individuality, under the suitable culture condition, and the similar individuality of renewable Cheng Yuqi parent; The a large amount of protoplastiss that obtain at one time are homogeneity in heredity, can be used as the ideal material of plant physiology, cytobiology, the research of somatic cell genetics scheduling theory; Protoplastis merges without fertilization process, can overcome the not affine obstacle of sexual cell, creates between the kind that sexual hybridization can not obtain or bigeners, thereby enlarge physical basis of heredity; Protoplastis can directly be taken in various macromole such as foreign DNA, organoid, bacterium or virion, can be used as the desirable acceptor material of fundamental research and crop improvement; Protoplastis might produce somaclonal variation in culturing process, this may become genetic resources important in the crop improvement, and can therefrom choose good varient and be applied to produce; The monoploid protoplastis has very important value to the screening of varient with bringing out.
Secondly, the simple easy handling of this method, the culture medium prescription inductivity height of the induced embryonic callus that adopts, the method practicality, lower to the requirement of equipment and condition, the embryo callus vitality that obtains simultaneously is vigorous, regenerative power is strong; In addition, the method for enzymolysis is simple, removes the cell walls mild condition, and isolating protoplastis yield height, active cultivation and the plant regeneration that is easy to saussurea involucrate protoplast are by force laid a good foundation for protoplastis merges.At last, the preservation of the very low temperature of protoplastis also has extremely important value for the preservation of the germ plasm resource with good character.
Description of drawings
The aseptic saussurea involucrata seedling that Fig. 1 obtains for high-effective disinfecting method of the present invention
The embryo callus that Fig. 2 induces the saussurea involucrata blade to obtain for the present invention
Fig. 3 is the design sketch behind the embryo callus suspension culture 20-30d of the present invention
Fig. 4 is the saussurea involucrate protoplast figure behind enzymolysis of the present invention and the purifying
Fig. 5 is used to separate the saussurea involucrata cytological map of protoplastis for the present invention
Fig. 6 cultivates the small callus figure that obtains the 2-3 month for protoplastis of the present invention
Embodiment
The Herba Saussureae Involueratae seed that the present invention adopts is from the Hejing County, Xinjiang.
Embodiment 1
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2 hours with the tap water flushing, then on Bechtop with 75% alcohol-pickled 10s, 0.1% mercuric chloride sterilization 1min, 15% hydrogen peroxide is handled 20min, aseptic water washing 3 times soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated on the MS substratum, 23 ± 1 ℃ of temperature, illumination 2500lx, time 16h/d, 10d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 1cm, insert substratum MS+NAA 1.0mg/L+6-BA 0.1mg/L+2, among the 4-D 0.1mg/L, place 23 ± 1 ℃ of temperature, illumination 30001x, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 1 time, take by weighing the chopping of 1g embryo callus and place 10ml CPW+13% mannitol solution, carry out plasmolysis, under 25 ℃ of dark conditions, handled 3 hours;
E, the plasmolysis liquid of steps d is removed at leisure, keep the embryo callus after the plasmolysis, place 10ml enzyme liquid, be positioned over rotating speed 80rpm, 26 ℃ of temperature are carried out enzymolysis under the dark condition, enzymolysis 12h, enzyme liquid is formed: cellulase 1.0%, macerozyme 0.1%, polygalacturonase 0.1%, 5mmol/L MES (2, (N-morphine quinoline)-ethylsulfonic acid), is dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulates pH 5.6;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant liquor, add 3ml CPW+10% mannitol solution, carrying out protoplastis cleans, place centrifugal 2min under the rotating speed 1000rpm condition, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently (5ml centrifuge tube dress 3ml 20% sucrose solution) on 20% sucrose solution, centrifugal 5min under rotating speed 800rpm condition is because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, puts into another clean centrifuge tube, add 2ml CPW+10% mannitol solution, carry out protoplastis and clean once more, and then the centrifugal 2min of rotating speed 1000rpm, supernatant liquor abandoned;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by the Safranin B Extra dyestuff dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis, and the culture density of this primary plastid is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, substratum is NT+NAA 1.0mg/L+6-BA 0.1mg/L+2, the hormone of 4-D 0.1mg/L, place the artificial climate incubator, 26 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, after 1 month, macroscopic cell mass on protoplast culture medium, occurs, promptly form little callus.
Embodiment 2
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2 hours with the tap water flushing, then on Bechtop with 75% alcohol-pickled 20s, 0.1% mercuric chloride sterilization 3min, 15% hydrogen peroxide is handled 25min, aseptic water washing 4 times soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated on the MS substratum, 23 ± 1 ℃ of temperature, illumination 2800lx, time 16h/d, 12d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 1.5cm, insert substratum MS+NAA 1.2mg/L+6-BA 0.3mg/L+2, among the 4-D 0.2mg/L, place 23 ± 1 ℃ of temperature, illumination 3200lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 1 time, take by weighing the CPW+13% mannitol solution that the chopping of 1g embryo callus places 10ml, carry out plasmolysis, under 25 ℃ of dark conditions of temperature, handled 3.5 hours;
E, the plasmolysis liquid of steps d is removed at leisure, keep the embryo callus after the plasmolysis, place 10ml enzyme liquid, be positioned over rotating speed 80rpm, 26 ± 2 ℃ of temperature are carried out enzymolysis under the dark condition, time 14h, enzyme liquid is formed: cellulase 1.2%, macerozyme 0.2%, polygalacturonase 0.2%, 5mmol/L MES, be dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulate pH5.7;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant liquor, add 4ml CPW+10% mannitol solution, carrying out protoplastis cleans, place centrifugal 2min under the rotating speed 1000rpm condition, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently (5ml centrifuge tube dress 3ml 20% sucrose solution) on 20% sucrose solution, centrifugal 5min under rotating speed 800rpm condition is because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, puts into another clean centrifuge tube, add 2ml CPW+10% mannitol solution, carry out protoplastis and clean once more,, abandon supernatant liquor at the centrifugal 2min of rotating speed 1000rpm;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by phenol safron dyestuff redly dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis, and the culture density of this primary plastid is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, substratum is DPD+NAA 1.2mg/L+6-BA0.2mg/L+2,4-D 0.2mg/L, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, after 35 days, macroscopic cell mass on protoplast culture medium, occurs, promptly form little callus.
Embodiment 3
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 3 hours with the tap water flushing, then on Bechtop with 75% alcohol-pickled 60s, 0.1% mercuric chloride sterilization 10min, 15% hydrogen peroxide treatment 30min, aseptic water washing 4 times soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated into the MS substratum of no hormone, 23 ± 1 ℃ of temperature, illumination 2700lx, time 16h/d, 15d begin sprouting;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 2cm, insert substratum MS+NAA 1.5mg/L+6-BA 1.0mg/L+2, among the 4-D 0.5mg/L, place 23 ± 1 ℃ of temperature, illumination 3800lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 3 times, take by weighing the CPW+13% mannitol solution that the chopping of 1g embryo callus places 10ml, carry out plasmolysis, under 25 ℃ of dark conditions, handled 5 hours;
E, the plasmolysis liquid of steps d is removed at leisure, keep the embryo callus after the plasmolysis, place 10ml enzyme liquid, be positioned over rotating speed 80rpm, 26 ± 2 ℃ of temperature are carried out enzymolysis, time 16h under the dark condition, enzyme liquid is formed: cellulase 2.0%, macerozyme 0.5%, polygalacturonase 0.5%, 5mmol/L MES, be dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulate pH5.8.
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant, the protoplastis scavenging solution that adds 4ml CPW+10% N.F,USP MANNITOL, place centrifugal 5min under the rotating speed 1000rpm condition, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently (5ml centrifuge tube dress 3ml 20% sucrose solution) on 20% sucrose solution, centrifugal 10min under rotating speed 800rpm condition, because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, and broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, put into another clean centrifuge tube, add the 5mlCPW+10% mannitol solution, carry out protoplastis and clean, at the centrifugal 5min of rotating speed 1000rpm, abandon supernatant liquor;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by Safranin B Extra dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis, and the culture density of this primary plastid is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, substratum is NT+NAA 1.3mg/L+6-BA 0.4mg/L+2, the hormone of 4-D 0.5mg/L, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, after 40 days, macroscopic cell mass on protoplast culture medium, occurs, promptly form little callus.
Embodiment 4
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2.5 hours with the tap water flushing, then on Bechtop with 75% alcohol-pickled 30s, 0.1% mercuric chloride sterilization 5min, 15% hydrogen peroxide is handled 28min, aseptic water washing 4 times soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated on the MS substratum, 23 ± 1 ℃ of temperature, illumination 2600lx, time 16h/d, 13d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 2cm, insert substratum MS+NAA 1.3mg/L+6-BA 0.6mg/L+2, among the 4-D 0.4mg/L, place 23 ± 1 ℃ of temperature, illumination 3600lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 2 times, take by weighing the chopping of 1g embryo callus and place 10ml CPW+13% mannitol solution, carry out plasmolysis, under 25 ℃ of dark conditions, handled 3 hours;
E, the plasmolysis liquid of steps d is removed at leisure, keep the embryo callus after the plasmolysis, place 10ml enzyme liquid, be positioned over rotating speed 80rpm, 26 ± 2 ℃ of temperature are carried out enzymolysis, time 16h under the dark condition, enzyme liquid is formed: cellulase 2.0%, macerozyme 0.5%, polygalacturonase 0.5%, 5mmol/L MES, be dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulate pH5.8;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant liquor, the CPW+10% mannitol solution that adds 3.5ml, carrying out protoplastis cleans, place centrifugal 3min under the rotating speed 1000rpm condition, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently (5ml centrifuge tube dress 3ml 20% sucrose solution) on 20% sucrose solution, centrifugal 6min under rotating speed 800rpm condition, because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, put into another clean centrifuge tube, the CPW+10% mannitol solution that adds 3ml carries out protoplastis and cleans once more, at the centrifugal 3min of rotating speed 1000rpm, abandon supernatant liquor;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by Safranin B Extra dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis.The culture density of this primary plastid is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, through on protoplast culture medium, occurring macroscopic cell mass after February, promptly form little callus, described callus inducing medium is DPD+NAA 1.3mg/L+6-BA 0.6mg/L+2,4-D 0.3mg/L.
Embodiment 5
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2 hours with the tap water flushing, then on Bechtop earlier with 75% alcohol-pickled 45s, 0.1% mercuric chloride sterilization 7min, 15% hydrogen peroxide is handled 30min, aseptic water washing 4 times soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated on the MS substratum, 23 ± 1 ℃ of temperature, illumination 2700lx, time 16h/d, 14d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 1.5cm, insert substratum MS+NAA 1.25mg/L+6-BA 0.5mg/L+2, among the 4-D 0.2mg/L, place 23 ± 1 ℃ of temperature, illumination 3300lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 1 time, take by weighing the chopping of 1g embryo callus and place 10ml CPW+13% mannitol solution, carry out plasmolysis, under 25 ℃ of dark conditions, handled 4.5 hours;
E, the plasmolysis liquid of steps d is removed at leisure, keep the embryo callus after the plasmolysis, place 10ml enzyme liquid, be positioned over rotating speed 80rpm, 26 ± 2 ℃ of temperature are carried out enzymolysis, time 13h under the dark condition, enzyme liquid is formed: cellulase 1.25%, macerozyme 0.2%, polygalacturonase 0.3%, 5mmol/L MES, be dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulate PH5.7;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant liquor, add 3.5ml CPW+10% mannitol solution and carry out the protoplastis cleaning, place centrifugal 2.5min under the rotating speed 1000rpm condition, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently (5ml centrifuge tube dress 3ml 20% sucrose solution) on 20% sucrose solution, centrifugal 5.5min under rotating speed 800rpm condition, because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, and broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, put into another clean centrifuge tube, the CPW+10% mannitol solution that adds 2.5ml carries out protoplastis and cleans once more, at the centrifugal 2.5min of rotating speed 1000rpm, abandons supernatant liquor;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by Safranin B Extra dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis.The culture density of this primary plastid is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, macroscopic cell mass appears on protoplast culture medium after 50 days, promptly form little callus, callus inducing medium is NT+NAA 1.25mg/L+6-BA 0.5mg/L+2,4-D 0.2mg/L.
Embodiment 6
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2.5 hours with the tap water flushing, then on Bechtop with 75% alcohol-pickled 55s, 0.1% mercuric chloride sterilization 9min, 15% hydrogen peroxide is handled 38min, aseptic water washing 3 times soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated on the MS substratum, 23 ± 1 ℃ of temperature, illumination 2900lx, time 16h/d, 18d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 2cm, insert substratum MS+NAA 1.0mg/L+6-BA 0.3mg/L+2, among the 4-D 0.1mg/L, place 23 ± 1 ℃ of temperature, illumination 3900lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 2 times, take by weighing the chopping of 1g embryo callus and place 10ml CPW+13% mannitol solution, carry out plasmolysis, under 25 ℃ of dark conditions, handled 4 hours;
E, the plasmolysis liquid of steps d is removed at leisure, keep the embryo callus after the plasmolysis, place 10ml enzyme liquid, rotating speed 80rpm, 26 ± 2 ℃ of temperature are carried out enzymolysis, time 14h under the dark condition, enzyme liquid is formed: cellulase 1.65%, macerozyme 0.4%, polygalacturonase 0.25%, 5mmol/L MES, be dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulate pH5.6;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant liquor, add 3ml CPW+10% mannitol solution, carrying out protoplastis cleans, place centrifugal 4.5min under the rotating speed 1000rpm condition, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently (5ml centrifuge tube dress 3ml 20% sucrose solution) on 20% sucrose solution, centrifugal 7min under rotating speed 800rpm condition is because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, puts into another clean centrifuge tube, the CPW+10% mannitol solution that adds 4.5ml, carry out protoplastis and clean once more,, abandon supernatant liquor at the centrifugal 4.5min of rotating speed 1000rpm;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by Safranin B Extra dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis, and the culture density of this primary plastid is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, macroscopic cell mass appears on protoplast culture medium after 55 days, promptly form little callus, callus inducing medium is DPD+NAA 1.35mg/L+6-BA 0.8mg/L+2,4-D 0.15mg/L.
Embodiment 7
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2 hours with the tap water flushing, then on Bechtop with 75% alcohol-pickled 18s, 0.1% mercuric chloride sterilization 7min, 15% hydrogen peroxide is handled 30min, aseptic water washing 4 times soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated on the MS substratum, 23 ± 1 ℃ of temperature, illumination 3000lx, time 16h/d, 17d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 2cm, insert substratum MS+NAA 1.4mg/L+6-BA 0.7mg/L+2, among the 4-D 0.3mg/L, place 23 ± 1 ℃ of temperature, illumination 4000lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 3 times, take by weighing the chopping of 1g embryo callus and place 10ml CPW+13% mannitol solution, carry out plasmolysis, under 25 ℃ of dark conditions, handled 3.5 hours;
E, the plasmolysis liquid of steps d is removed at leisure, embryo callus after the reservation plasmolysis places 10ml enzyme liquid medium speed 80rpm, 26 ± 2 ℃ of temperature, carry out enzymolysis under the dark condition, time 15h, enzyme liquid is formed: cellulase 1.4%, macerozyme 0.35%, polygalacturonase 0.35%, 5mmol/L MES is dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulates pH5.75;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant liquor, add 4ml CPW+10% mannitol solution and carry out the protoplastis cleaning, place centrifugal 3.5min under the rotating speed 1000rpm condition, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently (5ml centrifuge tube dress 3ml 20% sucrose solution) on 20% sucrose solution, centrifugal 10min under rotating speed 800rpm condition, because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, and broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, put into another clean centrifuge tube, the protoplastis that adding 4.5ml CPW+10% mannitol solution carries out cleans once more, at the centrifugal 5min of rotating speed 1000rpm, abandons supernatant liquor;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by Safranin B Extra dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis, and the culture density of this primary plastid is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, macroscopic cell mass appears on protoplast culture medium after 40 days, promptly form little callus, callus inducing medium is DPD+NAA 1.4mg/L+6-BA 0.7mg/L+2,4-D 0.3mg/L.
Embodiment 8
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2 hours with the tap water flushing, then on Bechtop with 75% alcohol-pickled 60s, 0.1% mercuric chloride sterilization 3min, 15% hydrogen peroxide is handled 28min, aseptic water washing 4 times soaks 10min with sterilized water at last;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated on the MS substratum, 23 ± 1 ℃ of temperature, illumination 2650lx, time 16h/d, 17d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 2cm, insert substratum MS+NAA 1.45mg/L+6-BA 0.65mg/L+2, among the 4-D 0.35mg/L, place 23 ± 1 ℃ of temperature, illumination 4000lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with growth among the step c rapidly, color is bud green, compact structure, in conjunction with loose embryo callus subculture 2 times, take by weighing the chopping of 1g embryo callus and place 10ml CPW+13% mannitol solution, carry out plasmolysis, under 25 ℃ of dark conditions, handled 3 hours;
E, the plasmolysis liquid of steps d is removed at leisure, embryo callus after the reservation plasmolysis places 10ml enzyme liquid medium speed 80rpm, 26 ± 2 ℃ of temperature, carry out enzymolysis under the dark condition, time 16h, enzyme liquid is formed: cellulase 2.0%, macerozyme 0.1%, polygalacturonase 0.1%, 5mmol/L MES is dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulates pH5.8;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under rotating speed 1000rpm condition, abandon supernatant liquor, add 3ml CPW+10% mannitol solution and carry out the protoplastis cleaning, place centrifugal 5min under the rotating speed 1000rpm condition, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently (5ml centrifuge tube dress 3ml 20% sucrose solution) on 20% sucrose solution, centrifugal 10min under rotating speed 800rpm condition, because density gradient centrifugation, vitality is strong, the protoplastis that state is good swims between 20% the sucrose solution and CPW+10% mannitol solution, and broken cell residue sinks to the pipe end, with gently that state is the good protoplastis sucking-off of 200 μ l liquid-transfering guns, put into another clean centrifuge tube, adding 5ml CPW+10% mannitol solution carries out protoplastis and cleans once more, at the centrifugal 5min of rotating speed 1000rpm, abandons supernatant liquor;
G, from the saussurea involucrate protoplast of step f separation and purification, get the centrifuge tube that 100 μ l are put in 1ml and be used for microscopy, at first use the Olympus microscope, under 40 times of object lens, observe the yield of protoplastis; Again by Safranin B Extra dye, film-making, the situation of under same multiple, observing viable cell; Protoplastis strong to vitality with blood counting chamber at last, that state is good is counted, and is used to adjust the culture density of protoplastis, and the culture density of this primary plastid is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, observe the living condition and the cell walls regenerated process of protoplastis, macroscopic cell mass appears on protoplast culture medium after 2 months, promptly form little callus, callus inducing medium is DPD+NAA 1.45mg/L+6-BA 0.65mg/L+2,4-D 0.35mg/L.
Claims (1)
1. the separation of a saussurea involucrate protoplast and cultural method is characterized in that following these steps to carrying out:
A, filter out the saussurea involucrata seeds of full grains, carried out surface cleaning in 2-3 hour with the tap water flushing, then on Bechtop with 75% alcohol-pickled 10-60s, 0.1% mercuric chloride sterilization 1-10min, 15% hydrogen peroxide treatment 20-40min, aseptic water washing 3-4 time soaks 10min with sterilized water then;
B, the aseptic saussurea involucrata seed after handling among the step a is inoculated into the MS substratum, 23 ± 1 ℃ of temperature, illumination 2500-3000lx, time 16h/d, 10-20d begin to sprout;
C, with the cotyledon of the aseptic seedling sprouted among the step b and the fragment that true leaf is cut into 1-2cm, insert substratum MS+NAA 1.0-1.5mg/L+6-BA 0.1-1.0mg/L+2,4-D0.1-0.5mg/L in, place 23 ± 1 ℃ of temperature, illumination 3000-4000lx, time 16h/d carries out inducing and breaking up of embryo callus;
D, with step c embryo callus subculture 1-3 time, take by weighing the chopping of 1g embryo callus and place 10ml CPW+13% mannitol solution, carry out plasmolysis, under 25 ℃ of dark conditions, handled 3-5 hour;
E, the plasmolysis liquid of steps d is poured out at leisure, embryo callus is placed 10ml enzyme liquid, be positioned over rotating speed 80rpm, 26 ± 2 ℃ of temperature, carry out enzymolysis under the dark condition, enzymolysis time 12-16h, wherein enzymolysis solution is: cellulase 1.0%-2.0%, macerozyme 0.1%-0.5%, polygalacturonase 0.1%-0.5%, 5mmol/L MES is dissolved in the solution of CPW+10% N.F,USP MANNITOL, regulates pH5.6-5.8;
F, enzymolysis tissue among the step e is removed by filter the saussurea involucrata fragment of tissue of not complete digestion with 500 purpose sieves, then with filtrate centrifugal 5min under the 1000rpm condition, abandon supernatant liquor, the CPW+10% mannitol solution that adds 3-4ml carries out protoplastis and cleans, place centrifugal 2-5min under the rotating speed 1000rpm condition then, abandon supernatant liquor, keep the 1ml washing lotion, to be mixed with the 1ml washing lotion sucking-off of protoplastis with liquid-transfering gun, be laid on gently on 20% sucrose solution, centrifugal 5-10min under rotating speed 800rpm condition, use 200 μ l liquid-transfering guns gently with the protoplastis sucking-off again, the CPW+10% mannitol solution that adds 2-5ml carries out protoplastis and cleans once more, at the centrifugal 2-5min of rotating speed 1000rpm, abandons supernatant liquor;
G, the centrifuge tube that the 100 μ l of the saussurea involucrate protoplast after the step f separation and purification are put in 1ml are used for microscopy, and the culture density of adjusting protoplastis with blood counting chamber is 1 * 10
5Individual/ml;
H, the protoplastis suspension of adjusting culture density in the step g is laid on carries out shallow-layer on the callus inducing medium and cultivate, place the artificial climate incubator, 26 ± 2 ℃ of temperature, dark condition is cultivated down, added a subculture took a sample simultaneously and carries out microscopy every 7 days, macroscopic cell mass appearred on protoplast culture medium after the month through 1-2, promptly form little callus, described callus inducing medium is additional hormone NAA1.0-1.5mg/L+6-BA 0.1-1.0mg/L+2 among NT or the DPD, 4-D 0.1-0.5mg/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101134708A CN101671648B (en) | 2009-09-30 | 2009-09-30 | Separation and culturing method of saussurea involucrate protoplast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101134708A CN101671648B (en) | 2009-09-30 | 2009-09-30 | Separation and culturing method of saussurea involucrate protoplast |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101671648A CN101671648A (en) | 2010-03-17 |
| CN101671648B true CN101671648B (en) | 2011-01-05 |
Family
ID=42019071
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101134708A Expired - Fee Related CN101671648B (en) | 2009-09-30 | 2009-09-30 | Separation and culturing method of saussurea involucrate protoplast |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101671648B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433294A (en) * | 2011-11-23 | 2012-05-02 | 中国科学院新疆理化技术研究所 | Method for extracting lettuce protoplast |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013904A (en) * | 2013-01-19 | 2013-04-03 | 安徽科技学院 | Enzymatic hydrolysis preparation method for protoplasts of sorghum |
| CN103013905B (en) * | 2013-01-19 | 2014-03-26 | 安徽科技学院 | Technique for preparing Sudan grass protoplast by using vacuum enzymolysis method |
| CN103834611A (en) * | 2014-02-11 | 2014-06-04 | 西北农林科技大学 | Separation and purification method for Salvia Miltiorrhiza protoplast |
| CN105219693B (en) * | 2015-09-19 | 2019-03-29 | 安徽科技学院 | A kind of soybean leaves protoplast electrofusion method for subcellular localization |
| CN105385649A (en) * | 2015-12-03 | 2016-03-09 | 安徽科技学院 | Method for rapid preparation of leaf protoplasts of Stevia rebaudiana |
| CN109554327B (en) * | 2018-11-14 | 2022-07-01 | 广州中医药大学(广州中医药研究院) | Method for separating and purifying amomum villosum mesophyll protoplast |
| CN109370976A (en) * | 2018-12-20 | 2019-02-22 | 江苏省农业科学院 | Chinese small iris mesophyll protoplast and preparation method thereof |
| CN112980764B (en) * | 2021-02-24 | 2022-11-01 | 上海辰山植物园 | Rapid and efficient preparation method of citronella protoplast |
| CN113862212A (en) * | 2021-11-15 | 2021-12-31 | 安赛搏(重庆)生物技术有限公司 | Process for large-scale subculture and large-scale culture of saussurea involucrate cells |
| CN114836365A (en) * | 2022-04-26 | 2022-08-02 | 成都阿斯它文化传媒有限公司 | Application of pluripotent nuclear organic cells in delaying senescence |
| CN115607492A (en) * | 2022-10-18 | 2023-01-17 | 安赛搏(重庆)生物技术有限公司 | Radix ceratostigmae callus extract and preparation method and application thereof |
| 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 |
-
2009
- 2009-09-30 CN CN2009101134708A patent/CN101671648B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433294A (en) * | 2011-11-23 | 2012-05-02 | 中国科学院新疆理化技术研究所 | Method for extracting lettuce protoplast |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101671648A (en) | 2010-03-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101671648B (en) | Separation and culturing method of saussurea involucrate protoplast | |
| CN100553445C (en) | New lilium longiflorum production of hybrid seeds parent's tissue-culturing rapid propagation and cross-breeding method | |
| CN102144553B (en) | Method for rapidly propagating Paris polyphylla Smith | |
| CN102301952B (en) | Method for breeding chamomile | |
| CN104255489B (en) | The asexual quick breeding technology method of the tender stem of tuniclike psammosilene root band axillalry bud | |
| CN105309311A (en) | Method for breeding improved variety of scrophularia ningpoensis Hemsl. | |
| CN109757373A (en) | A kind of Jing Banxia quick breeding method for tissue culture | |
| CN106342679A (en) | Breeding method of perennial rice and application | |
| CN104429952A (en) | Method for efficiently obtaining regeneration plant by cultivating isolated microspores of brassica oleracea L.var.capitata L. | |
| CN102388803A (en) | Chilli cytoplasm male sterile line protoplast separation purification and callus forming method | |
| CN107173016A (en) | Narrow leaf Herba Stachydis introduce a fine variety artificial purification and rejuvenation implantation methods | |
| CN105265320B (en) | A kind of tissue culture propagation of aristolochia mollissima | |
| CN112335549A (en) | Method for obtaining larch regeneration plant through tissue in-vitro culture | |
| CN101422128B (en) | Separation and regeneration method of gulfweed protoplast | |
| CN101156550A (en) | Method for breeding new flower-shaped chrysanthemum | |
| CN102217532B (en) | Simplified efficient culture method for brassica napus microspores | |
| CN106538382A (en) | A kind of method that eremochloa ophiuroides high-efficiency regeneration system is set up as explant with young fringe | |
| CN101983555A (en) | Method for inducing indirect somatic embryogenesis of chrysanthemum | |
| CN101637130B (en) | Cephalotaxus hainanensis embryo culturing and seedling breeding method | |
| CN101766123B (en) | Method for rapid propagation of zephyr lily | |
| CN109479718A (en) | A kind of rooting method of tissue culture of shinyleaf yellowhorn seedling | |
| CN101564009B (en) | Propagation method for keeping cabbage RGMS male sterile line | |
| CN102599065A (en) | Quick propagation method for humulus scandens | |
| CN102232359B (en) | In-vitro rapid propagation method of double-petal Jasminum sambac | |
| CN106613970B (en) | The quick breeding by group culture method of sealwort leaf elegant jessamine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110105 Termination date: 20150930 |
|
| EXPY | Termination of patent right or utility model |