CN101946699A - Method for breeding autotetraploid saussurea involucrata plant - Google Patents
Method for breeding autotetraploid saussurea involucrata plant Download PDFInfo
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- CN101946699A CN101946699A CN 201010235095 CN201010235095A CN101946699A CN 101946699 A CN101946699 A CN 101946699A CN 201010235095 CN201010235095 CN 201010235095 CN 201010235095 A CN201010235095 A CN 201010235095A CN 101946699 A CN101946699 A CN 101946699A
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Abstract
The invention relates to a method for breeding a saussurea involucrata plant. In the method, the lower hypocotyl stem section of the saussureae involucratae aseptic seedling after cutting off root and leaves is used as the explant, the explant is induced by using solid culture media with different concentrations of colchicine to generate a tetraploid saussureae involucratae plant, and the tetraploid saussureae involucratae is identified by using the tetraploid screening identification technology. The method provides an improvement method for optimal colchicine concentration, processing time and processing temperature for inducing generation of tetraploid, and morphology, chromosome counting and flow cytometry for identifying tetraploid. The method can be used for quickly breeding the autotetraploid saussureae involucratae plants; and the method shortens the breeding cycle, thereby effectively increasing the content of active ingredients, and overcomes the defect of variety degeneration of the saussureae involucratae in natural propagation and artificial planting.
Description
Technical field
The present invention relates to medicinal plant breeding field, specifically, relate to the breeding method of a kind of rare ethnic drug Herba Saussureae Involueratae autotetraploid plant.
Background technology
Herba Saussureae Involueratae (Saussurea involucrata Kar et Kir.) is the perennial disposable large-scale herbaceous plant that blossoms and bears fruit of composite family phoenix hair Chrysanthemum, has another name called lanatechead saussurea herb with flower or He Lian, is three grades of protection kinds in imminent danger of country, the peculiar distribution kind in Xinjiang.Grow on the inferior high mountain and high mountain steep cliff cliff at height above sea level 2400～4100m place, among the glacial drift rock seam.For distinctive rare rare Chinese herbal medicine in Xinjiang and high mountain flowers, has important medicinal and ornamental value.Saussurea involucrata is low temperature resistant, wind resistance is cold, and growing environment is very harsh, natural propagation rate low (under the wild environment, seed germination rate is less than 5%), growth cycle is long and artificial cultivation is difficult.At present, the population distribution of suitable saussurea involucrata existence is also dwindled day by day along with global warming, and artificial digging excessively excessively disorderly adopted, and causes wild saussurea involucrata resource deficient day by day, and species are endangered.
Herba Saussureae Involueratae reaches 5～8 years vegetative period, root, stem, leaf are rich in secondary metabolite with medical active such as alkaloid, flavonoids, volatile oil, lactones, steroidal class, polysaccharide etc., its bud more is rich in trace element and amino acid etc., has effects such as promoting blood circulation and removing obstruction in channels, dispelling cold and removing dampness, anti-inflammatory and antalgic, antifatigue, terminal pregnancy and nourishing and strengthening vital.Polyploid plant can be satisfied the requirement that medicinal material is produced preferably because chromosome doubling often has the huge property of organ, and promptly the output of root, stem and leaf is higher; On the other hand, the variation of plant chromosome ploidy also tends to cause the variation of secondary metabolite content, might obtain the high medicinal plant of active constituent content.The polyploid plant annidation reaches the also enhancing to some extent of resistance to adverse circumstance simultaneously.In recent years, the research of Herba Saussureae Involueratae is concentrated on physiology, effective aspects such as medicinal ingredient, pharmacology and gene engineering mostly, but study very few about its polyploid breeding.Therefore, fundamentally solve the breed breeding of medicinal plant Herba Saussureae Involueratae, all have very important significance not only for the popularization and the development that promote artificial genuineness plantation industry, and for protection, the development and utilization of high mountain medicinal plant in imminent danger.
Monoploid is meant the bion that contains these species gametic chromosome number in the somatic cell.And monoploid is meant that somatic cell contains a genomic individuality.Most biologies are diplont, contain a chromosome set in its haploid somatic cell, if former species itself are polyploid, the chromosome set number that contains in its haploid somatic cell is necessarily more than one so.Monoploid as the tetraploid paddy rice contains two chromosome sets, and the monoploid of hexaploid wheat contains three chromosome sets.Polyploid just is meant and contains genomic bion more than three in the somatic cell.Autopolyploid is the polyploid that is doubled to form by original chromosome set.Given this, in the cultivating process that carries out autotetraploid Herba Saussureae Involueratae plant, will guarantee really that intracellular chromosome number is integer and doubles, rather than only refer to simply increasing of chromosome number, otherwise the polyploid that obtains is heterozygote (the irregular increase of chromosome number) or chimera (the various cells that contain Different Ploidy), and this type of polyploid tends to take place back mutation in the shoot proliferation process, and the proterties instability.In order to obtain the autotetraploid Herba Saussureae Involueratae plant of isozygotying, the present invention has carried out improving significantly on the 2007100089255.X basis.Optimize the surface of the seed sterilization method, tetraploid plant inductive condition particularly treatment temperature and later stage propagation temperature strictness control, to use shoot tip meristem instead be that material carries out the chromosome compressing tablet, utilizes the advanced person to do refinement and improvement in aspect such as dna content flow cytometer detection technique accurately, and the greenhouse transplanting of successfully realization group training tetraploid plant, found out one by a large amount of experiments and overlapped the experimental technique of cultivating the autotetraploid Herba Saussureae Involueratae plant of isozygotying.
In addition, the present invention is also in conjunction with saussurea involucrata aseptic seedling rapid induction grow thickly bud and multiplication technique, utilize colchicine to handle hypocotyl stem section and make the regeneration plant chromosome doubling, induce to produce tetraploid Herba Saussureae Involueratae plant, combining form, anatomy, chromosome compressing tablet and low cytometric analysis Screening and Identification have the tetraploid plant that merit and biomass and medicinal ingredient all increase.Can overcome the deterioration of variety problem that Herba Saussureae Involueratae occurs in natural propagation and artificial planting, for enlarging the peculiar rare medicinal plant cultivated area in Xinjiang, improve Herba Saussureae Involueratae plantation income, meeting the need of market and promoting conventional Chinese medicine develops significant.
Summary of the invention
The object of the present invention is to provide the breeding method of a kind of autotetraploid Herba Saussureae Involueratae plant.The hypocotyl stem section of the Herba Saussureae Involueratae aseptic seedling behind this method utilization excision root, the leaf is explant, induce processing by the solid culture medium that contains the variable concentrations colchicine, induce and produce tetraploid Herba Saussureae Involueratae plant, make plant produce merit by changing ploidy, obtain the tetraploid Herba Saussureae Involueratae by tetraploid Screening and Identification to improve the content of its biomass and medicinal ingredient.This method provides induces effective colchicine concentration, processing time, the treatment temperature that tetraploid produces and is used for improving one's methods of morphology, chromosome counting and flow cytometer that tetraploid identifies.Can realize the cultivation of autotetraploid Herba Saussureae Involueratae plant fast by the method, and then effectively improve the content of its active component, overcome the deterioration of variety problem that Herba Saussureae Involueratae occurs in natural propagation and artificial planting.
Invent the breeding method of described a kind of autotetraploid Herba Saussureae Involueratae plant, follow these steps to carry out:
A, with Herba Saussureae Involueratae seed emerge in worm water 20min, use 75% ethanol surface sterilization 20-50sec again, use 15%H then
2O
2Solution disinfection 15-50min with rinsed with sterile water 2-5 time of the seed after the sterilization, reseeds on the MS medium, every bottle of MS medium sowing 10-30 grain seed, and the saussurea involucrata seed can be sprouted the generation aseptic seedling after 7-15 days;
B, the aseptic seedling of germination and growth among the step a is excised its root and blade, retain plumular axis stem section part;
C, hypocotyl stem section among the step b is inoculated in to be added with on the inducing culture that sprouts that 2% dimethyl sulfoxide (DMSO) and concentration are the 0-0.20% colchicine handles 1-3d, cultivation temperature 25-30 ℃, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1, the inducing culture that sprouts is MS+BA0.3-0.5mg/L+TDZ0.3-0.5mg/L+NAA0.05-0.1mg/L;
D, the hypocotyl stem section of handling among the step c changed in the MS+BA0.5mg/L+TDZ0.5mg/L+NAA0.1mg/L proliferated culture medium cultivate 25-30d, 20 ± 1 ℃ of cultivation temperature, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1
E, the seedling of breeding in the steps d is cut into to be forwarded in the 1/2MS+NAA0.05-0.2mg/L root media behind the simple bud takes root;
The screening of f, Herba Saussureae Involueratae autotetraploid: plant among the step e and dliploid adjoining tree are done comparative analysis, according to the size of the two Stomacal guard cell on mode of appearance such as width of blade, leaf area and anatomical structure and the difference between the density, tentatively be defined as the autotetraploid plant;
One of evaluation of g, Herba Saussureae Involueratae autotetraploid: the young tender tip of a root of plant and young tender indefinite bud stem-tip tissue among the clip step e at random, at temperature 0-4 ℃, through 0.002mol/L oxine preliminary treatment or the preliminary treatment of the saturated paracide aqueous solution, the Kano fixer is fixed, 60 ℃ of temperature, 1-2mol/L salt acid dissociation 6-15min, the pinkish red dyeing liquor dyeing of improvement phenol 10-15min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud stem tip chromosome, tentatively is defined as the autotetraploid plant;
Two of the evaluation of h, Herba Saussureae Involueratae autotetraploid: the blade of part plant among the clip step e at random shreds it with scissors 200 order nylon net filters, 500-1000rmin in being equipped with under the normal temperature in the culture dish that the 3-5ml cell nucleus extracts buffer solution
～1Centrifugal rinsing 3 times, flow cytometer detect and are defined as the autotetraploid plant;
The transplanting of i, Herba Saussureae Involueratae autotetraploid: the residual medium that the plant flush away root of cultivating among the step e is adhered to, be colonizated in turfy soil: perlite: on the seedbed, greenhouse of vermiculite=3: 1: 1, build shed, enclose film, ventilate once every day, each 30min watered once, after the week in per two days, remove film and shed, regularly water, hot-house culture, Herba Saussureae Involueratae autotetraploid plant acclimatization and transplants survival rate reaches 82%.
Medium among step c, d and the e is solid culture medium.
The oxine pretreatment time is 2-6h in the step g, and saturated paracide aqueous solution pretreatment time is 5-10h, and the Kano fixer set time is 12-36h.
Seedbed, step I greenhouse humidity is 60%, and the hot-house culture temperature is controlled at 20-22 ℃.
The breeding method advantage of autotetraploid Herba Saussureae Involueratae plant of the present invention is:
The improvement of sterilization method.Laboratory the surface of the seed sterilization is in order to guarantee that material does not pollute in incubation.And former sterilization method has adopted HgCl mostly
2, this tends to cause the murder by poisoning of Hg ion and residual for medicinal plant, and waste liquid also can cause environment heavy metal pollution etc., uses HgCl
2Sterilization also can make seed germination rate reduce.And the discoloration of hydrogen peroxide has remarkable Disinfection Effect for the distinctive fold epidermal structure of saussurea involucrata seed, and the step of two after improvement sterilization has been saved the time, has reduced the pollution of Hg, has protected environment.In addition, will use the flowing water flushing to use the purpose that emerge in worm water can also reach cost such as conserve water resource instead before the seed disinfection.
The selection of abductive approach.Herba Saussureae Involueratae is a perennial herb, grain weight few and obtain very difficult.Handle saussurea involucrata seed and seed seedling in the experimental field with approach such as method for soaking seed, semar techniques, effect is all undesirable, does not also obtain to isozygoty tetraploid, but also is subjected to the restriction in season.In the laboratory, sprout the Herba Saussureae Involueratae seed with the solid culture medium of additional colchicine, obtain the seedling that makes a variation easily, but mostly be lopsided seedling, the later stage survival rate is low.The seedling of this new sprouting simultaneously is comparatively fragile, to the toxic action sensitivity of colchicine.And adopt the present invention will handle aseptic seedling after growth a period of time through inducing the tetraploid better effects if of acquisition, suitably rising temperature (25～30 ℃) can make inductivity significantly improve during handling, then need suitable low temperature (20 ± 1 ℃) in the multiplicative stage, regulation and control by temperature can make tetraploid regeneration plant morphological differences remarkable, the induced mutation rate height.Method is feasible, the screening operation amount is little.
The selection of methods for ploidy determination.Phytomorph changes identifies that tetraploid is the easiest, also the most rough, can be used for the primary dcreening operation of variant; The big property that pore showed can be used as identifies tetraploid major criterion; Yet the variation of mode of appearance can only explain plant variation may take place, wanted to judge more like clockwork that its ploidy also needs the evidence of chromosome and dna content molecular level aspect.On the present invention basis that form and pore are observed in early days, comprehensively identify inducing the tetraploid plant that obtains in conjunction with chromosome counting and flow cytometer again, easy operating, the result is accurately and reliably.Four kinds of different methods for ploidy determination can also expand each tissue culture stage numerous, that induce screening in propagation and carry out simultaneously, guaranteed the accurately qualitative of gained tetraploid material, particularly low cytometric analysis is introduced in the ploidy breeding and evaluation of rare medicinal plant, can realize the in good time tracking of tetraploid material is detected, with strong points, efficient is high.
The selection of Methods of Ploidy Identification method.Adopt chromosome counting to carry out in the ploidy detection to mutagenesis gained plant, how to identify around materials such as the tip of a root, group training bud, callus, pollen, endosperm.But Herba Saussureae Involueratae is the perennial disposable herbaceous plant that blossoms and bears fruit, and realize the in good time tracking of inducing seedling is detected, and the group training bud and the tip of a root are undoubtedly optimal material.Saussurea involucrata seedling leaves base is given birth to, and has only a growing point position, with carrying out compressing tablet after the excision of group training bud, waste is caused to the precious material that screening gets in the capital, carry out compressing tablet again and it is cultivated the stage of taking root, then the cycle longer, this is very disadvantageous for the medicinal plant breeding.The present invention utilizes hypocotyl stem section growing point successfully to observe chromosome, has realized effective, fast detecting to polyploid Herba Saussureae Involueratae seedling process of growth chromosome number.
Description of drawings
Fig. 1 sprouts the seed mode of appearance figure of Herba Saussureae Involueratae aseptic seedling for the present invention.
Fig. 2 excises the hypocotyl stem section figure that root, leaf stay for the present invention.
Fig. 3 is dliploid of the present invention and tetraploid phenotype comparison diagram, and wherein a1, b1, c1 are dliploid; A2, b2, c2 are tetraploid, and a is that blade, b are that root, c are whole strain.
Fig. 4 is dliploid a of the present invention and tetraploid b pore comparison diagram.
Fig. 5 is dliploid chromosome a of the present invention and tetraploid chromosomes b figure.
Fig. 6 is dliploid a of the present invention and tetraploid b～e DNA relative amount curve comparison diagram.
Embodiment
Embodiment 1:
With Herba Saussureae Involueratae seed emerge in worm water 20min, use concentration 75% ethanol surface sterilization 20sec again, use 15%H then
2O
2Solution disinfection 15min with rinsed with sterile water 3 times of the seed after the sterilization, reseeds on the MS medium, 10 seeds of every bottle of MS medium sowing, and the saussurea involucrata seed can be sprouted the generation aseptic seedling after 7-15 days; Seed germination rate is 73.3% (the normal chitting piece number/seed sum of participating in the experiment).
Aseptic seedling excision root and blade with germination and growth retain plumular axis stem section part;
Hypocotyl stem section is inoculated into is added with inducing of 2% dimethyl sulfoxide (DMSO) and sprouts and handle 3d (contrast) among the medium MS+BA0.3mg/L+TDZ0.3mg/L+NAA0.05mg/L, cultivation temperature 25-30 ℃, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1
The hypocotyl stem section of handling changed in the MS+BA0.5mg/L+TDZ0.5mg/L+NAA0.1mg/L proliferated culture medium cultivate 25d, 20 ± 1 ℃ of cultivation temperature, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1
The seedling of propagation is cut into to be forwarded in the 1/2MS+NAA0.1mg/L root media behind the simple bud takes root;
The screening of Herba Saussureae Involueratae autotetraploid: comparative analysis is done in doubtful strain and dliploid adjoining tree, according to the size of the two Stomacal guard cell on mode of appearance such as width of blade, leaf area and anatomical structure and the difference between the density, tentatively determine that it is dliploid;
One of evaluation of Herba Saussureae Involueratae autotetraploid: tissues such as the tender tip of a root of clip control group children and young tender indefinite bud stem apex at random, under 4 ℃, through 0.002mol/L oxine preliminary treatment 4h, the Kano fixer is 24h fixedly, 60 ℃ of temperature, 1mol/L salt acid dissociation 10min, the pinkish red dyeing liquor dyeing of improvement phenol 15min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud stem tip chromosome, determines that tentatively chromosome is 2n=2x=32, is liploid plant;
Two of the evaluation of Herba Saussureae Involueratae autotetraploid: clip control group young leaflet tablet at random shreds it with scissors 200 order nylon net filters, 1000rmin in being equipped with under the normal temperature in the culture dish that the 3ml cell nucleus extracts buffer solution
-1Centrifugal rinsing 3 times, flow cytometer detect determines that the diplontic fluorescence channel value of plant PI is 300;
The transplanting of Herba Saussureae Involueratae autotetraploid: the residual medium with the plant flush away root of cultivating adheres to is colonizated in turfy soil: perlite: on the seedbed, greenhouse of vermiculite=3: 1: 1, build shed, enclose film, ventilate once every day, and each 30min watered once in per two days, humidity 60%, after one week, remove film and shed, regularly water, the hot-house culture temperature is controlled at 22 ℃, and Herba Saussureae Involueratae plant acclimatization and transplants survival rate reaches 90%;
Indefinite bud survival rate 100%, POLYPLOID INDUCEMENT rate 0% (the bud number of inductivity=polyploid number/propagation).
Embodiment 2:
With Herba Saussureae Involueratae seed emerge in worm water 20min, use concentration 75% ethanol surface sterilization 20sec again, use 15%H then
2O
2Solution disinfection 15min with rinsed with sterile water 2 times of the seed after the sterilization, reseeds on the MS medium, 15 seeds of every bottle of MS medium sowing, and the saussurea involucrata seed can be sprouted the generation aseptic seedling after 7-15 days; Seed germination rate is 73.3% (the normal chitting piece number/seed sum of participating in the experiment).
The aseptic seedling of germination and growth is excised its root and blade, retain plumular axis stem section part;
Hypocotyl stem section is inoculated into all is added with inducing of 2% dimethyl sulfoxide (DMSO) and 0.05% colchicine and sprouts and handle 3d, 25 ℃ of cultivation temperature, intensity of illumination 50 μ molm among the medium MS+BA0.3mg/L+TDZ0.3mg/L+NAA0.05mg/L
-2S
-1, light application time 16hd
-1
The hypocotyl stem section of handling changed in the MS+BA0.5mg/L+TDZ0.5mg/L+NAA0.1mg/L proliferated culture medium cultivate 25d, 20 ± 1 ℃ of cultivation temperature, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1
The seedling of propagation is cut into to be forwarded in the 1/2MS+NAA0.05mg/L root media behind the simple bud takes root;
The screening of Herba Saussureae Involueratae autotetraploid: comparative analysis is done in doubtful strain and dliploid adjoining tree, according to the size of the two Stomacal guard cell on mode of appearance such as width of blade, leaf area and anatomical structure and the difference between the density, tentatively determine that it is tetraploid;
One of evaluation of Herba Saussureae Involueratae autotetraploid: tissues such as the doubtful strain of the clip tender tip of a root of children and young tender indefinite bud stem apex at random, under 4 ℃, through 0.002mol/L oxine preliminary treatment 2h, the Kano fixer is 12h fixedly, 60 ℃ of temperature, 1mol/L salt acid dissociation 6min, the pinkish red dyeing liquor dyeing of improvement phenol 10min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud stem tip chromosome, and it is tetraploid plant that chromosome is defined as 2n=4x=64;
Two of the evaluation of Herba Saussureae Involueratae autotetraploid: clip control group young leaflet tablet at random shreds it with scissors 200 order nylon net filters, 500rmin in being equipped with under the normal temperature in the culture dish that the 4ml cell nucleus extracts buffer solution
～1Centrifugal rinsing 3 times, flow cytometer detect determines that the tetraploid fluorescence channel value of plant PI is 600;
The transplanting of Herba Saussureae Involueratae autotetraploid: the residual medium that flush away tetraploid plant root adheres to is colonizated in turfy soil: perlite: on the seedbed, greenhouse of vermiculite=3: 1: 1, build shed, enclose film, ventilate once every day, and each 30min watered once in per two days, humidity 60%, after one week, remove film and shed, regularly water, the hot-house culture temperature is controlled at 20 ℃, and Herba Saussureae Involueratae autotetraploid plant acclimatization and transplants survival rate reaches 78%.
The indefinite bud survival rate reaches 56%, POLYPLOID INDUCEMENT rate 27% (the bud number of inductivity=polyploid number/propagation).
Embodiment 3:
With Herba Saussureae Involueratae seed emerge in worm water 20min, use 75% ethanol surface sterilization 30sec again, use 15%H then
2O
2Solution disinfection 30min with rinsed with sterile water 4 times of the seed after the sterilization, reseeds on the MS medium, 20 seeds of every bottle of MS medium sowing, and the saussurea involucrata seed can be sprouted the generation aseptic seedling after 7-15 days; Seed germination rate is 75% (the normal chitting piece number/seed sum of participating in the experiment).
The aseptic seedling of germination and growth is excised its root and blade, retain plumular axis stem section part;
Hypocotyl stem section is inoculated into is added with inducing of 2% dimethyl sulfoxide (DMSO) and 0.10% colchicine and sprouts and handle 2d, 28 ℃ of cultivation temperature, intensity of illumination 50 μ molm among the medium MS+BA0.4mg/L+TDZ0.4mg/L+NAA0.08mg/L
-2S
-1, light application time 16hd
-1
The hypocotyl stem section of handling changed in the MS+BA0.5mg/L+TDZ0.5mg/L+NAA0.1mg/L proliferated culture medium cultivate 28d, 20 ± 1 ℃ of cultivation temperature, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1
The seedling of propagation is cut into to be forwarded in the 1/2MS+NAA0.1mg/L root media behind the simple bud takes root;
The screening of Herba Saussureae Involueratae autotetraploid: comparative analysis is done in doubtful strain and dliploid adjoining tree, according to the size of the two Stomacal guard cell on mode of appearance such as width of blade, leaf area and anatomical structure and the difference between the density, tentatively determine that it is tetraploid;
One of evaluation of Herba Saussureae Involueratae autotetraploid: tissues such as the doubtful strain of the clip tender tip of a root of children and young tender indefinite bud stem apex at random, under 4 ℃, through supersaturation paracide aqueous solution preliminary treatment 5h, the Kano fixer is 24h fixedly, 60 ℃ of temperature, 2mol/L salt acid dissociation 10min, the pinkish red dyeing liquor dyeing of improvement phenol 13min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud stem tip chromosome, and it is tetraploid plant that chromosome is defined as 2n=4x=64;
Two of the evaluation of Herba Saussureae Involueratae autotetraploid: the doubtful strain young leaflet tablet of clip at random shreds it with scissors 200 order nylon net filters, 1000rmin in being equipped with under the normal temperature in the culture dish that the 3ml cell nucleus extracts buffer solution
～1Centrifugal rinsing 3 times, flow cytometer detect determines that the tetraploid fluorescence channel value of plant PI is 600;
The transplanting of Herba Saussureae Involueratae autotetraploid: the residual medium that flush away tetraploid plant root adheres to is colonizated in turfy soil: perlite: on the seedbed, greenhouse of vermiculite=3: 1: 1, build shed, enclose film, ventilate once every day, and each 30min watered once in per two days, humidity 60%, after one week, remove film and shed, regularly water, the hot-house culture temperature is controlled at 21.5 ℃, and Herba Saussureae Involueratae autotetraploid plant acclimatization and transplants survival rate reaches 85%.
Indefinite bud survival rate 43%, POLYPLOID INDUCEMENT rate reach 61% (the bud number of inductivity=polyploid number/propagation).
Embodiment 4:
With Herba Saussureae Involueratae seed emerge in worm water 20min, use concentration 75% ethanol surface sterilization 40sec again, use 15%H then
2O
2Solution disinfection 40min with rinsed with sterile water 5 times of the seed after the sterilization, reseeds on the MS medium, 30 seeds of every bottle of MS medium sowing, and the saussurea involucrata seed can be sprouted the generation aseptic seedling after 7-15 days; Seed germination rate is 86.4% (the normal chitting piece number/seed sum of participating in the experiment);
The aseptic seedling of germination and growth is excised its root and blade, retain plumular axis stem section part;
Hypocotyl stem section is inoculated into is added with inducing of 2% dimethyl sulfoxide (DMSO) and 0.15% colchicine and sprouts and handle 1d, 30 ℃ of cultivation temperature, intensity of illumination 50 μ molm among the medium MS+BA0.5mg/L+TDZ0.5mg/L+NAA0.1mg/L
-2S
-1, light application time 16hd
-1
The hypocotyl stem section of handling changed in the MS+BA0.5mg/L+TDZ0.5mg/L+NAA0.1mg/L proliferated culture medium cultivate 30d, 20 ± 1 ℃ of cultivation temperature, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1
The seedling of propagation is cut into to be forwarded in the 1/2MS+NAA0.15mg/L root media behind the simple bud takes root;
The screening of Herba Saussureae Involueratae autotetraploid: comparative analysis is done in doubtful strain and dliploid adjoining tree, according to the size of the two Stomacal guard cell on mode of appearance such as width of blade, leaf area and anatomical structure and the difference between the density, tentatively determine that it is tetraploid;
One of evaluation of Herba Saussureae Involueratae autotetraploid: tissues such as the doubtful strain of the clip tender tip of a root of children and young tender indefinite bud stem apex at random, under 4 ℃, through 0.002mol/L oxine preliminary treatment 6h, the Kano fixer is 36h fixedly, 60 ℃ of temperature, 1.5mol/L salt acid dissociation 12min, the pinkish red dyeing liquor dyeing of improvement phenol 15min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud stem tip chromosome, and it is tetraploid plant that chromosome is defined as 2n=4x=64;
Two of the evaluation of Herba Saussureae Involueratae autotetraploid: the doubtful strain young leaflet tablet of clip at random shreds it with scissors 200 order nylon net filters, 1000rmin in being equipped with under the normal temperature in the culture dish that the 5ml cell nucleus extracts buffer solution
～1Centrifugal rinsing 3 times, flow cytometer detect determines that the tetraploid fluorescence channel value of plant PI is 600;
The transplanting of Herba Saussureae Involueratae autotetraploid: the residual medium that flush away tetraploid plant root adheres to is colonizated in turfy soil: perlite: on the seedbed, greenhouse of vermiculite=3: 1: 1, build shed, enclose film, ventilate once every day, and each 30min watered once in per two days, humidity 60%, after one week, remove film and shed, regularly water, the hot-house culture temperature is controlled at 21 ℃, and Herba Saussureae Involueratae autotetraploid plant acclimatization and transplants survival rate reaches 74%.
Indefinite bud survival rate 35%, POLYPLOID INDUCEMENT rate 49% (the bud number of inductivity=polyploid number/propagation).
Embodiment 5:
With Herba Saussureae Involueratae seed emerge in worm water 20min, use 75% ethanol surface sterilization 50sec again, use 15%H then
2O
2Solution disinfection 50min with rinsed with sterile water 2 times of the seed after the sterilization, reseeds on the MS medium, 25 seeds of every bottle of MS medium sowing, and the saussurea involucrata seed can be sprouted the generation aseptic seedling after 7-15 days; Seed germination rate is 55.1% (the normal chitting piece number/seed sum of participating in the experiment).
The aseptic seedling of germination and growth is excised its root and blade, retain plumular axis stem section part;
Hypocotyl stem section is inoculated into is added with inducing of 2% dimethyl sulfoxide (DMSO) and 0.20% colchicine and sprouts and handle 1d, 29 ℃ of cultivation temperature, intensity of illumination 50 μ molm among the medium MS+BA0.3mg/L+TDZ0.4mg/L+NAA0.08mg/L
-2S
-1, light application time 16hd
-1
The hypocotyl stem section of handling changed in the MS+BA0.5mg/L+TDZ0.5mg/L+NAA0.1mg/L proliferated culture medium cultivate 30d, 20 ± 1 ℃ of cultivation temperature, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1
The seedling of propagation is cut into to be forwarded in the 1/2MS+NAA0.2mg/L root media behind the simple bud takes root;
The screening of Herba Saussureae Involueratae autotetraploid: comparative analysis is done in doubtful strain and dliploid adjoining tree, according to the size of the two Stomacal guard cell on mode of appearance such as width of blade, leaf area and anatomical structure and the difference between the density, tentatively determine that it is tetraploid;
One of evaluation of Herba Saussureae Involueratae autotetraploid: tissues such as the doubtful strain of the clip tender tip of a root of children and young tender indefinite bud stem apex at random, under 4 ℃, through supersaturation paracide aqueous solution preliminary treatment 10h, the Kano fixer is 20h fixedly, 60 ℃ of temperature, 2mol/L salt acid dissociation 8min, the pinkish red dyeing liquor dyeing of improvement phenol 13min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud stem tip chromosome, and it is tetraploid plant that chromosome is defined as 2n=4x=64;
Two of the evaluation of Herba Saussureae Involueratae autotetraploid: the doubtful strain young leaflet tablet of clip at random shreds it with scissors 200 order nylon net filters, 900rmin in being equipped with under the normal temperature in the culture dish that the 4ml cell nucleus extracts buffer solution
～1Centrifugal rinsing 3 times, flow cytometer detect determines that the tetraploid fluorescence channel value of plant PI is 600;
The transplanting of Herba Saussureae Involueratae autotetraploid: the residual medium that flush away tetraploid plant root adheres to is colonizated in turfy soil: perlite: on the seedbed, greenhouse of vermiculite=3: 1: 1, build shed, enclose film, ventilate once every day, and each 30min watered once in per two days, humidity 60%, after one week, remove film and shed, regularly water, the hot-house culture temperature is controlled at 20.5 ℃, and Herba Saussureae Involueratae autotetraploid plant acclimatization and transplants survival rate reaches 70%.
Indefinite bud survival rate 40%, POLYPLOID INDUCEMENT rate 33% (the bud number of inductivity=polyploid number/propagation).
Claims (4)
1. the breeding method of an autotetraploid Herba Saussureae Involueratae plant is characterized in that following these steps to carrying out:
A, with Herba Saussureae Involueratae seed emerge in worm water 20min, use 75% ethanol surface sterilization 20-50sec again, use 15%H then
2O
2Solution disinfection 15-50min with rinsed with sterile water 2-5 time of the seed after the sterilization, reseeds on the MS medium, every bottle of MS medium sowing 10-30 grain seed, and the saussurea involucrata seed can be sprouted the generation aseptic seedling after 7-15 days;
B, the aseptic seedling of germination and growth among the step a is excised its root and blade, retain plumular axis stem section part;
C, hypocotyl stem section among the step b is inoculated in to be added with on the inducing culture that sprouts that 2% dimethyl sulfoxide (DMSO) and concentration are the 0-0.20% colchicine handles 1-3d, cultivation temperature 25-30 ℃, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1, the inducing culture that sprouts is MS+BA0.3-0.5mg/L+TDZ0.3-0.5mg/L+NAA0.05-0.1mg/L;
D, the hypocotyl stem section of handling among the step c changed in the MS+BA0.5mg/L+TDZ0.5mg/L+NAA0.1mg/L proliferated culture medium cultivate 25-30d, 20 ± 1 ℃ of cultivation temperature, intensity of illumination 50 μ molm
-2S
-1, light application time 16hd
-1
E, the seedling of breeding in the steps d is cut into to be forwarded in the 1/2MS+NAA0.05-0.2mg/L root media behind the simple bud takes root;
The screening of f, Herba Saussureae Involueratae autotetraploid: plant among the step e and dliploid adjoining tree are done comparative analysis, according to the size of the two Stomacal guard cell on mode of appearance such as width of blade, leaf area and anatomical structure and the difference between the density, tentatively be defined as the autotetraploid plant;
One of evaluation of g, Herba Saussureae Involueratae autotetraploid: the young tender tip of a root of plant and young tender indefinite bud stem-tip tissue among the clip step e at random, at temperature 0-4 ℃, through 0.002mol/L oxine preliminary treatment or the preliminary treatment of the saturated paracide aqueous solution, the Kano fixer is fixed, 60 ℃ of temperature, 1-2mol/L salt acid dissociation 6-15min, the pinkish red dyeing liquor dyeing of improvement phenol 10-15min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud stem tip chromosome, tentatively is defined as the autotetraploid plant;
Two of the evaluation of h, Herba Saussureae Involueratae autotetraploid: the blade of part plant among the clip step e at random shreds it with scissors 200 order nylon net filters, 500-1000rmin in being equipped with under the normal temperature in the culture dish that the 3-5ml cell nucleus extracts buffer solution
～1Centrifugal rinsing 3 times, flow cytometer detect and are defined as the autotetraploid plant;
The transplanting of i, Herba Saussureae Involueratae autotetraploid: the residual medium that the plant flush away root of cultivating among the step e is adhered to, be colonizated in turfy soil: perlite: on the seedbed, greenhouse of vermiculite=3: 1: 1, build shed, enclose film, ventilate once every day, each 30min watered once, after the week in per two days, remove film and shed, regularly water, hot-house culture, Herba Saussureae Involueratae autotetraploid plant acclimatization and transplants survival rate reaches 82%.
2. method according to claim 1 is characterized in that the medium among step c, d and the e is solid culture medium.
3. method according to claim 1 is characterized in that the oxine pretreatment time is 2-6h in the step g, and saturated paracide aqueous solution pretreatment time is 5-10h, and the Kano fixer set time is 12-36h.
4. method according to claim 1 is characterized in that seedbed, step I greenhouse humidity is 60%, and the hot-house culture temperature is controlled at 20-22 ℃.
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