CN104170729B - A kind of method of utilizing thin slice culture technique to obtain Bai Jianghua tetraploid plant - Google Patents
A kind of method of utilizing thin slice culture technique to obtain Bai Jianghua tetraploid plant Download PDFInfo
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Abstract
The present invention discloses a kind of method of utilizing thin slice culture technique to obtain Bai Jianghua tetraploid plant. The method cuts thin slice from test-tube plantlet stem section, and thin slice is processed with colchicine, then adopts thin slice culture technique to carry out adventitious bud inducing to thin slice after treatment, obtains regeneration plant; By regeneration plant is detected, screening obtains tetraploid plant. While carrying out induced mutation in the present invention, explant used is thin slice, thickness only has 0.3～0.5mm, cell number is few, in the time processing with colchicine, can make the cell of explant fully be exposed in liquid, the plant overwhelming majority who obtains is homozygote, has simplified the screening process of polyploid plant; Chromosome Analysis technology used, have simple to operate, required time is short, the advantage such as the chromosome image of acquisition is clear. That method of the present invention has is simple to operate, plant regeneration frequency is high, the regeneration period is short, the easy advantage of drawing materials, and strong operability is easy to universal and promotes.
Description
Technical field
The invention belongs to Zingiber (Zingiberaceae) Hedychium Koenig (Hedychium) Bai Jianghua (HedychiumCoronarium) breeding technical field, particularly one utilizes thin slice culture technique to obtain Bai Jianghua tetraploidThe method of plant. Set up fast numerous cultivating system of Bai Jianghua by thin slice culture technique, and utilize and set upFast numerous cultivating system, in conjunction with colchicine processing, can obtain Bai Jianghua polyploid plant fast, thereby realThe object of new varieties is improved, obtained to existing dialogue ginger flower variety.
Background technology
Bai Jianghua is Zingiber Hedychium Koenig, perennial rhizome shape herbage flower plant, because it is beautiful in colour and smellFragrance, as odor type Fresh Cutting flower, courtyard greening and garden landscape plant, is mainly the flower garden virtue being extensively popularFragrant flower grass, market demand is huge. In addition, Bai Jianghua or a kind of good fragrant seasoning plant, it is plantedIn strain, contain linalool, the multiple aromatic such as farnesene, can extract aromatic oil, as high-grade fragrantEssence.
But at present in the large-scale application of Bai Jianghua, also there are many problems, for example cut flower vase phase short (2～3 days), extract effective medicinal ingredient inefficiency, plant tillering coefficient is low, reproduction speed is slow etc. In vainGinger gives fewer birth in Guangzhou or sterile restriction by the method for artificial sexual hybridization obtains new varieties, by biologyTechnology is expected to the solution of problem of implementation.
Polyploid breeding (polyploidbreeding) technology is to utilize the way such as induced mutations or natural variation screeningFootpath obtains polyploid material, in order to the technology of seed selection New MS Polyploid Variety. Polyploid plant has corolla conventionallyLarge and abundant, fragrance is denseer, and the florescence is elongated, stem chap strong, resistance to lodging, and blade relents, photosynthetic workBy features such as enhancings, be bred as a collection of flowers with higher ornamental value by artificial induction's polyploid meansKind.
Carrying out in plant polyploid breeding process, conventional colchicine carries out mutagenesis processing. Its inductionChromosome doubling mainly contains live body and processes double method and ex vivo treatment double method. Live body is processed the plant obtainingMostly be chimera, be subject to environmental disturbances large, often can not genetic stability, very easily reply for original chromosome doublyNumber. Double often to adopt stem apex, terminal bud, axillalry bud, Multiple Buds, excised leaf etc. to lure as explant in vitroLead material, the variation plant obtaining is also much chimera, also exists that aberration rate is low, ploidy is unstableThe phenomenon such as fixed, thus must carry out a large amount of screening operations. These become restricted passage induced mutation and carry out manyThe principal element of times physical culture kind. Therefore, setting up suitable plant regeneration system reduces before chimera generationPutting forward condition, is an important bottleneck problem that need to solve while adopting polyploid mutagenesis breeding.
The in vitro plant regeneration system of Bai Jianghua of report is mainly the lower embryo that adopts the aseptic seedling of mature seed at presentAxle is as explant, or the bud breeding of employing adventitious bud inducing clump regeneration of plantlet, also there is no Bai JianghuaThin slice culture technique and successfully report of polyploid plant induction.
Summary of the invention
In order to overcome the shortcoming and deficiency of prior art, the object of the present invention is to provide one to utilize thin slice trainingThe technology of supporting obtains the method for Bai Jianghua tetraploid plant. The method cuts thin slice from test-tube plantlet stem section, to thin sliceProcess with colchicine, then adopt thin slice culture technique to carry out adventitious bud inducing to thin slice after treatment,Obtain regeneration plant. By regeneration plant is detected, screening obtains tetraploid plant.
Object of the present invention is achieved through the following technical solutions: one utilizes thin slice culture technique to obtain Bai JianghuaThe method of tetraploid plant, comprises the steps:
(1) sterilization of explant and the induction of adventitious bud: the bud of sprouting taking the root-like stock of Bai Jianghua is as explant,Be inoculated in bud inducing culture A and cultivate, induce adventitious bud;
(2) adventitious bud seedling: the adventitious bud of step (1) induction is proceeded in seedling culture medium and cultivated,Obtain healthy and strong test-tube plantlet;
(3) expansion of test-tube plantlet is numerous: get the test-tube plantlet that step (2) obtains, cut thin slice from its base portion, connectPlant and cultivate in bud inducing culture B, obtain healthy and strong adventitious bud; Adventitious bud is proceeded to seedling to be cultivatedIn base, cultivate, obtain test-tube plantlet;
(4) repeating step (3) as required, until obtain enough test-tube plantlets;
(5) induction of tetraploid plant: get the test-tube plantlet that step (3) or (4) obtain, cut from its base portionGet thin slice, carry out immersion treatment with colchicine solution; Thin slice after treatment is seeded in to bud inducing culture BIn cultivate, obtain adventitious bud; Adventitious bud is transferred on seedling culture medium and cultivated, obtain mutagenic treatmentAfter test-tube plantlet;
(6) test-tube plantlet obtaining in step (5) is tamed, is transplanted to the flowerpot that Nutrition Soil is housed,Obtain the Bai Jianghua regeneration plant of mutagenesis;
(7) the Bai Jianghua regeneration plant of mutagenesis step (6) being obtained and original seed liploid plant seedling stageContrast, blade is broadened greatly, color is dark green, and vein highlights, the micro-sagging growth of blade, and it is large that pore becomes,The plant that stomatal frequency diminishes is elected candidate plant as;
(8) get the tip of a root of the candidate plant obtaining in step (7), carry out Chromosome Analysis, chooseChromosome number is the seedling of 2n=4x=68, obtains Bai Jianghua tetraploid plant;
(9) tetraploid plant step (8) being obtained carries out field observation, chooses floral organ and obviously becomesGreatly, plant strain growth has the plant of clear superiority, is defined as having the Bai Jianghua tetraploid of good agronomic traitsPlant.
In step (1):
The bud that the root-like stock of described Bai Jianghua is sprouted preferably adopts following methods to carry out pre-treatment: get Bai JianghuaThe bud sprouted of root-like stock, water is rinsed well rear with liquid detergent bubble 25～30 minutes, then water rinse 25～30 minutes, carry out surface sterilization 60～90 seconds with 70～75wt% alcohol, finally disappear with 0.1wt% mercuric chloridePoison 15～20 minutes, cuts the bud of long 0.5～2.0cm as explant;
Described bud inducing culture A is preferably MS culture medium+(3～6) mgL-16-BA+(0.1～0.2)mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
Described more preferably MS culture medium+5mgL of bud inducing culture A-16-BA+0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
The time of described cultivation is preferably 20～40 days;
In step (2):
Described seedling medium optimization is 1/2MS culture medium+(0.5～1) gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
More preferably 1/2MS culture medium+1gL of described seedling culture medium-1Active carbon+15gL-1Sucrose+7g·L-1Agar;
The time of described cultivation is preferably 15～45 days;
In step (3):
Described bud inducing culture B is preferably MS culture medium+(1～5) mgL-16-BA or (0.05～0.2)mg·L-1TDZ+(0.1～0.2)mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
Described more preferably MS culture medium+3mgL of bud inducing culture B-16-BA+0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
In step (3), described adventitious bud length is 1～2cm; The described same step of seedling culture medium (2)In seedling culture medium;
Described test-tube plantlet is that adventitious bud is transferred to the plant more than high 6cm obtaining on seedling culture medium; InstituteThe thickness of the thin slice of stating is preferably 0.3～0.5mm;
In step (5), described colchicine solution is preferably MS culture medium+(0.2～0.8) wt% autumnNarcissus element+1% (v/v) DMSO (dimethyl sulfoxide (DMSO)); The time of described immersion treatment is preferably 12～36h；
More preferably, described colchicine solution is MS culture medium+0.6wt% colchicine+1% (v/v)DMSO (dimethyl sulfoxide (DMSO)); The time of described immersion treatment is 24h;
The described time of cultivating in bud inducing culture B is preferably 20～40 days;
The described time of cultivating on seedling culture medium of transferring to is preferably 15～45 days;
The pH of the described culture medium of step (1), (2), (3), (5) is 5.6～5.9, except TDZ adoptsWith joining after filtration sterilization outside the culture medium of autoclave sterilization, remaining culture medium all adopts HTHPSterilizing (121 DEG C, 1.06kg/cm2Lower sterilizing 15min);
The condition of the described cultivation of step (1), (2), (3), (5) is all at 26～28 DEG C, 30 μ molm-2s-1Under light intensity (white fluorescent lamp), 16L/8D illumination condition, carry out;
In step (6), the time of described domestication is preferably 3～7 days;
Described Nutrition Soil is preferably and is rich in organic soil, as planting for flowers of buying from flowers marketOrganoclay, the beautiful soil of training;
It is that the inorganic salt solution of 1/20MS culture medium once irrigates that described soil preferably adopts concentration, whole mistakeJourney does not refertilize, and suitably keeps the skin wet depending on the dry wet situation of soil;
In step (6), the test-tube seedling transplanting obtaining in step (5), after flowerpot, should suitably be sheltered from heat or light,And keep ground moistening, cultivate after a month plant to be planted survives and can proceed to normal cultivation management;
In step (7):
Described pore opening with density inspect method is: choose the consistent liploid plant of development degree and autumnThe plant that narcissus element obtains after processing, gets the consistent blade in joint position, and from blade same area, tear and take off epidermis,Be placed on slide, make Temporary slide, under microscope, measure length and the density of pore;
The assay method of described blade size: choose the consistent liploid plant of development degree and colchicineThe plant obtaining after processing, gets the identical blade in blade joint position, measure leaf long (leaf base is long to blade tip) andLeaf wide (position that blade is the widest);
Chromosome Analysis method described in step (8): choose liploid plant and process colchicineProcess the candidate plant obtaining, experiment 17:00 hole irrigation the previous day foot water, 9:00 o'clock morning next day is to 10:00 point, gets the delicate sturdy tip of a root of root, and " dissociate-hypotonic-dye-compressing tablet-observation " method of employing is dyedColour solid number is analyzed; Concrete steps are as follows: scrape off tip of a root epidermis with scalpel, be soaked in dissociation solution (37%HCl:95％C2H5OH=1:3～1:5, volume ratio) in, 5～8min dissociates under normal temperature; Take out the tip of a root, with steamingHeat up in a steamer water and rinse 3～4 times, then be put into hypotonic 25～30min in distilled water; Take a morsel meristematic zone material with dissectingPin is dialled loose, drips the pinkish red dye liquor dyeing of phenol 3～5min; Covered, sops up unnecessary dye liquor, thenCover on cover glass with filter paper, knock gently cover plate across filter paper along diagonal with pencil eraser head, make materialComposition is uniformly dispersed; The slice, thin piece pressing is placed in to micro-Microscopic observation, qualification chromosome number; That observes is thinBorn of the same parents' number is no less than 30, in somatoblast, more than 85% is dying of this plant for constant consistent chromosome numberColour solid number;
Described dissociation solution is preferably volume ratio 37%HCl:95%C2H5OH=1:3; Described dissociate timeBetween be preferably 6min; Wherein, 37%HCl refers to that mass fraction is 37% HCl; 95%C2H5OH isRefer to the C that percentage by volume is 95%2H5OH；
The big or small mensuration of floral organ described in step (9), the length of employing lip, ala and width (Wide), the filigree of stamen, the length of flower pesticide measure;
The present invention has following advantage and effect with respect to prior art:
(1) the present invention utilize thin slice culture technique set up Bai Jianghua plant regeneration system, have simple to operate,Plant regeneration frequency is high, the regeneration period is short, the easy advantage of drawing materials, and adopts this technology, carries out Bai Jianghua manyThe induction of times body, strong operability, is easy to universal and promotes.
(2) while carrying out induced mutation, explant used is thin slice, and thickness only has 0.3～0.5mm, thinBorn of the same parents' number is few, in the time processing with colchicine, can make the cell of explant fully be exposed in liquid,The plant overwhelming majority who obtains is homozygote, has simplified the screening process of polyploid plant.
(3) the Chromosome Analysis technology adopting in the present invention, have simple to operate, required time is short,The advantages such as chromosome image is clear that obtain, have solved the consumption of utilizing chromosome number to carry out the detection of plant ploidyTime consume the difficult problem of power.
Brief description of the drawings
Fig. 1 is that embodiment 1 utilizes thin slice culture technique to set up the process of Bai Jianghua plant regeneration system.
Fig. 2 is the tetraploid plant that obtains of embodiment 1 and plant profile, pore and the dyeing of liploid plantThe comparative result figure of body;
Fig. 3 is the comparative result of the floral organ size of the tetraploid plant that obtains of embodiment 1 and liploid plantFigure.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present inventionBe not limited to this.
Various culture mediums required in embodiment are as follows:
Bud inducing culture A is MS culture medium+(3～6) mgL-16-BA+(0.1～0.2)mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
Seedling culture medium is 1/2MS culture medium+(0.5～1) gL-1Active carbon+15gL-1Sucrose+7 g·L-1Agar;
Bud inducing culture B is MS culture medium+(1～5) mgL-16-BA or (0.05～0.2) mgL-1TDZ+(0.1～0.2)mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
Above-mentioned medium pH is 5.6～5.9, goes out except joining HTHP after TDZ employing filtration sterilizationOutside the culture medium of bacterium, remaining culture medium all adopts autoclave sterilization (sterilizing under 121 DEG C, 1.06kg/cm215min); All incubation are all at 26～28 DEG C, 30 μ molm-2s-1Light intensity (white fluorescent lamp), 16L/8DUnder illumination condition, carry out;
In the composition of the above culture medium, 6-BA refers to 6-benzyladenine; NAA refers to a-methyl α-naphthyl acetate; TDZRefer to thiadiazole phenylurea, trade name Thidiazuron (Thidiazuron, N-Phenyl-N '-1,2,3-thiadiazol-5-Ylurea, N-phenyl-N-1,2,3-thiadiazoles-5-urea, TDZ); 1/2MS culture medium refers to MS culture mediumA great number of elements, trace element, amino acid and vitamin content all reduce by half.
Embodiment 1
(1) sterilization of explant and the induction of adventitious bud: get Bai Jianghua (Hedychiumcoronarium) (citySell routine. Root-like stock down together) is sprouted the sprouting, and rinses well rear with 30 points of liquid detergent bubbles with running waterClock, then after 30 minutes, carry out surface sterilization 60 second with 70wt% alcohol by running water flow wash, finally use0.1wt% mercuric chloride sterilization 18 minutes cuts the bud of 0.5cm as explant on superclean bench, inoculationIn bud inducing culture A, cultivate, induce adventitious bud through the cultivation of 20 days; Described bud inductionCulture medium A is MS culture medium+3mgL-16-BA+0.2mg·L-1NAA+30g·L-1Sucrose+7g·L-1Agar;
(2) adventitious bud seedling: the adventitious bud of step (1) induction is proceeded in seedling culture medium, promote seedling rawRoot maturation, cultivates and can obtain healthy and strong test-tube plantlet more than high 6cm after 15 days; Described seedling culture medium is1/2MS culture medium+1gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(3) induction of adventitious bud and the acquisition of test-tube plantlet: the test-tube plantlet that step (2) is obtained cuts thin slice(the thick 0.3～0.5mm of thin slice), is seeded in upper cultivation of bud inducing culture B, and cultivating can after 20 daysObtain healthy and strong adventitious bud; Described bud inducing culture B is MS culture medium+2mgL-16-BA+0.1mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
The adventitious bud that grows to 1cm is transferred on seedling culture medium and carried out seedling cultivation, cultivate and can obtain for 15 daysObtain healthy and strong test-tube plantlet; Described seedling culture medium is 1/2MS culture medium+1gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(4) repeating step (3) as required, until obtain enough test-tube plantlets;
(5) the induction processing of thin slice: get the test-tube plantlet that step (3) or (4) obtain, cut from its base portionThin slice (the thick 0.3～0.5mm of thin slice), use MS culture medium+0.2wt% colchicine+1% (v/v)DMSO fluid nutrient medium carries out immersion treatment 36 hours; Thin slice after treatment distilled water flushing 1～2 time,Be seeded in bud inducing culture B (MS culture medium+5mgL-16-BA+0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar) in cultivate, cultivate and within 20 days, obtain afterwards healthy and strong adventitious bud; Adventitious bud is turnedMove on to seedling culture medium (1/2MS culture medium+0.5gL-1Active carbon+15gL-1Sucrose+7gL-1Fine jadeFat) upper cultivation 15 days, obtain the test-tube plantlet of mutagenic treatment;
(6) test-tube plantlet obtaining in step (5) is carried out to uncork domestication, under room scattering illumination, place 7My god, keep moistening in bottle. Then seedling is cleaned up, be transplanted to the flowerpot that Nutrition Soil is housed, Nutrition Soil isThe organoclay for flower culture of buying from flowers market is that 1/20MS cultivates by concentration before plantlet of transplantThe inorganic salt solution of base once irrigates, and or else whole process applies fertilizer, depending on the dry wet suitable supplementing water of situation of soilPoint, keep ground moistening and suitably shelter from heat or light, after 1 month, obtain the Bai Jianghua regeneration plant of mutagenesis, canTo proceed to normal cultivation management.
(7) Bai Jianghua regeneration plant and the original seed liploid plant of mutagenesis step (6) being obtained carry outContrast, broadens blade greatly, and color is dark green, and vein highlights, the micro-sagging growth of blade, and it is large that pore becomes, gasThe plant that hole density diminishes is elected candidate plant as; Choose the consistent liploid plant of development degree and colchicineThe plant obtaining after processing, gets the consistent blade in joint position, tears and takes off epidermis from blade same area, is placed in and carriesOn slide, make Temporary slide, under microscope, measure length and the density of pore; Choose development degree oneThe plant that the liploid plant causing and colchicine obtain after processing, gets the identical blade in blade joint position, measuresLeaf long (leaf base is long to blade tip) and leaf wide (position that blade is the widest); Result is respectively as in table 1 and Fig. 2Shown in A, B, E, F.
(8) the candidate plant obtaining in selecting step (7), contrasts with liploid plant. Test the previous day17:00 hole irrigation foot water, morning next day 9:00 point to 10:00 point, get the delicate sturdy tip of a root of root, adoptCarry out Chromosome Analysis by " dissociate-hypotonic-dye-compressing tablet-observation " method; Concrete steps are as follows: useScalpel scrapes off tip of a root epidermis, is soaked in dissociation solution (37%HCl:95%C2H5OH=1:3, volume ratio)In, 6min dissociates under normal temperature; Take out the tip of a root, with distilled water flushing 3 times, then be put in distilled water hypotonic 30Min; The meristematic zone material dissecting needle that takes a morsel is dialled loose, drips the pinkish red dye liquor dyeing of phenol 3min; Close the lidSlide, sops up unnecessary dye liquor, then covers on cover glass with filter paper, with pencil eraser head across filter paper along rightLinea angulata knocks cover plate gently, and material composition is uniformly dispersed; The slice, thin piece pressing is placed in to micro-Microscopic observation,Qualification chromosome number; The cell number of observing is no less than 30, more than 85% is 2n=4x=68 in somatoblastThe plant of bar chromosome number, is defined as Bai Jianghua tetraploid plant (as shown in Fig. 2 C, D).
(9) tetraploid plant step (8) being obtained carries out field observation, measure floral organ lip,The length of ala and width (the widest), and measure the filigree of stamen, the length of flower pesticide; Floral organ is obviously becomeLarge plant is defined as having the Bai Jianghua tetraploid plant of good agronomic traits. Table 2 and Figure 3 shows that twoThe comparison of times body plant and tetraploid plant floral organ.
The blade of table 1 liploid plant and tetraploid plant and stomata characteristics comparison
| Leaf long (cm) | Leaf wide (cm) | (μ m) for pore length | Stomatal frequency (individual/mm2) | |
| Dliploid | 21.55±0.20A | 4.83±0.05A | 33.23±0.28A | 130.28±3.36A |
| Tetraploid | 25.00±0.51B | 5.36±0.09B | 44.76±0.99B | 86.17±3.90B |
Table 2 Bai Jianghua liploid plant and relatively (cm) of tetraploid plant floral organ size
| Lip is long | Lip is wide | Ala is long | Ala is wide | Filigree is long | Flower pesticide is long | |
| Dliploid | 4.32±0.09A | 4.24±0.09A | 4.21±0.03A | 1.93±0.03A | 2.89±0.07A | 1.19±0.01A |
| Tetraploid | 5.33±0.14B | 5.13±0.27B | 5.47±0.08B | 2.50±0.09B | 4.04±0.10B | 1.55±0.04B |
Fig. 1 is that embodiment 1 utilizes thin slice culture technique to set up the process of Bai Jianghua plant regeneration system, wherein:A is for cutting the ripe plant (bar=1cm) that thin slice is used; The part that B cuts from the ripe plant of a strainThin slice (bar=5mm); The adventitious bud (bar=1cm) that C grows from a thin slice; The regeneration that D obtains is plantedStrain.
Fig. 2 is the tetraploid plant that obtains of embodiment 1 and plant profile, pore and the chromosome of liploid plantComparative result figure; A is liploid plant, and B is tetraploid plant; C is the chromosome of liploid plant, DThe chromosome of tetraploid plant; E is the pore of liploid plant, and F is the pore of tetraploid plant.
Fig. 3 is the comparative result of the floral organ size of the tetraploid plant that obtains of embodiment 1 and liploid plantFigure; A is the diplontic open bud that is about to, and B is the tetraploid open bud that is about to; C is tetraploidFlower pesticide and filigree, D is diplontic flower pesticide and filigree; E is the flower of tetraploid plant, and F is that dliploid is plantedThe flower of strain.
Embodiment 2
(1) sterilization of explant and the induction of adventitious bud: get Bai Jianghua's (Hedychiumcoronarium)Root-like stock is sprouted the sprouting, and rinses well afterwards with liquid detergent bubble 28 minutes with running water, then uses current from the beginningMoving flushing after 28 minutes carried out surface sterilization 75 seconds with 75wt% alcohol, finally with the sterilization of 0.1wt% mercuric chloride15 minutes, on superclean bench, cut the bud of 1.0cm as explant, be inoculated into bud inducing culture AIn cultivate, can induce adventitious bud through the cultivations of 30 days; Described bud inducing culture A is MSCulture medium+4mgL-16-BA+0.1mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
(2) adventitious bud seedling: the adventitious bud of step (1) induction is proceeded in seedling culture medium, promote seedling rawRoot maturation, cultivates the healthy and strong test-tube plantlet that can obtain for 30 days more than high 6cm; Described seedling culture medium is 1/2MS culture medium+0.5gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(3) induction of adventitious bud and the acquisition of test-tube plantlet: the test-tube plantlet that step (2) is obtained cuts thin slice(the thick 0.3～0.5mm of thin slice), is seeded in upper cultivation of bud inducing culture B, cultivates acquisition in 20 daysHealthy and strong adventitious bud; Described bud inducing culture B is MS culture medium+5mgL-16-BA+0.15mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
The adventitious bud that grows to 1.5cm is transferred on seedling culture medium and carried out seedling cultivation, cultivate and obtain after 15 daysObtain healthy and strong test-tube plantlet; Described seedling culture medium is 1/2MS culture medium+0.5gL-1Active carbon+15g·L-1Sucrose+7gL-1Agar;
(4) repeating step (3) as required, until obtain enough test-tube plantlets;
(5) the induction processing of thin slice: get the test-tube plantlet that step (3) or (4) obtain, cut from its base portionThin slice (the thick 0.3～0.5mm of thin slice), use MS culture medium+0.4wt% colchicine+1% (v/v)DMSO fluid nutrient medium carries out immersion treatment 24 hours; Thin slice after treatment distilled water flushing 1～2 time,Be seeded in bud inducing culture B (MS culture medium+3mgL-16-BA+0.1mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar) in, cultivate and within 30 days, obtain healthy and strong adventitious bud; Adventitious bud is transferred to intoSeedling culture medium (1/2MS culture medium+1gL-1Active carbon+15gL-1Sucrose+7gL-1Agar) upper trainingSupport 20 days, obtain the test-tube plantlet of mutagenic treatment;
(6) test-tube plantlet obtaining in step (5) is carried out to uncork domestication, under room scattering illumination, place 3My god, keep moistening in bottle. Then seedling is cleaned up, be transplanted to the flowerpot that Nutrition Soil is housed, Nutrition Soil isThe organoclay for flower culture of buying from flowers market is that 1/20MS cultivates by concentration before plantlet of transplantThe inorganic salt solution of base once irrigates, and whole process does not refertilize, depending on the dry wet suitable supplementing water of situation of soilPoint, keep ground moistening and suitably shelter from heat or light, after 1 month, obtain the Bai Jianghua regeneration plant of mutagenesis, canTo proceed to normal cultivation management.
(7) Bai Jianghua regeneration plant and the original seed liploid plant of mutagenesis step (6) being obtained carry outContrast, broadens blade greatly, and color is dark green, and vein highlights, the micro-sagging growth of blade, and it is large that pore becomes, gasThe plant that hole density diminishes is elected candidate plant as; Choose the consistent liploid plant of development degree and colchicineThe plant obtaining after processing, gets the consistent blade in joint position, tears and takes off epidermis from blade same area, is placed in and carriesOn slide, make Temporary slide, under microscope, measure length and the density of pore; Choose development degree oneThe plant that the liploid plant causing and colchicine obtain after processing, gets the identical blade in blade joint position, measuresLeaf long (leaf base is long to blade tip) and leaf wide (position that blade is the widest).
(8) the candidate plant obtaining in selecting step (7), contrasts with liploid plant. Test the previous day17:00 hole irrigation foot water, morning next day 9:00 point to 10:00 point, get the delicate sturdy tip of a root of root, adoptCarry out Chromosome Analysis by " dissociate-hypotonic-dye-compressing tablet-observation " method; Concrete steps are as follows: useScalpel scrapes off tip of a root epidermis, is soaked in dissociation solution (37%HCl:95%C2H5OH=1:4, volume ratio)In, 6min dissociates under normal temperature; Take out the tip of a root, with distilled water flushing 3 times, then be put in distilled water hypotonic 30Min; The meristematic zone material dissecting needle that takes a morsel is dialled loose, drips the pinkish red dye liquor dyeing of phenol 3min; Close the lidSlide, sops up unnecessary dye liquor, then covers on cover glass with filter paper, with pencil eraser head across filter paper along rightLinea angulata knocks cover plate gently, and material composition is uniformly dispersed; The slice, thin piece pressing is placed in to micro-Microscopic observation,Qualification chromosome number; The cell number of observing is no less than 30, more than 85% is 2n=4x=68 in somatoblastThe plant of bar chromosome number, is defined as Bai Jianghua tetraploid plant.
(9) tetraploid plant step (8) being obtained carries out field observation, measure floral organ lip,The length of ala and width (the widest), and measure the filigree of stamen, the length of flower pesticide; Floral organ is obviousBecome the Bai Jianghua tetraploid plant that large plant is defined as having good agronomic traits.
Embodiment 3
(1) sterilization of explant and the induction of adventitious bud: get Bai Jianghua's (Hedychiumcoronarium)Root-like stock is sprouted the sprouting, and rinses well afterwards with liquid detergent bubble 25 minutes with running water, then uses current from the beginningMoving flushing after 25 minutes carried out surface sterilization 90 seconds with 72wt% alcohol, finally with the sterilization of 0.1wt% mercuric chloride20 minutes, on superclean bench, cut the bud of 1.5cm as explant, be inoculated into bud inducing culture AIn cultivate, induce adventitious bud through the cultivation of 40 days; Described bud inducing culture A is MS trainingSupport base+5mgL-16-BA+0.15mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
(2) adventitious bud seedling: the adventitious bud of step (1) induction is proceeded in seedling culture medium, promote seedling rawRoot maturation, cultivates the healthy and strong test-tube plantlet that can obtain for 45 days more than high 6cm; Described seedling culture medium is 1/2MS culture medium+0.75gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(3) induction of adventitious bud and the acquisition of test-tube plantlet: the test-tube plantlet that step (2) is obtained cuts thin slice(the thick 0.3～0.5mm of thin slice), is seeded in upper cultivation of bud inducing culture B, cultivates acquisition in 20 daysHealthy and strong adventitious bud; Described bud inducing culture B is MS culture medium+1mgL-16-BA+0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
The adventitious bud that grows to 2cm is transferred to and on seedling culture medium, carried out seedling cultivation; Cultivate and obtain afterwards for 15 daysHealthy and strong test-tube plantlet; Described seedling culture medium is 1/2MS culture medium+0.75gL-1Active carbon+15g·L-1Sucrose+7gL-1Agar;
(4) repeating step (3) as required, until obtain enough test-tube plantlets;
(5) the induction processing of thin slice: get the test-tube plantlet that step (3) or (4) obtain, cut from its base portionThin slice (the thick 0.3～0.5mm of thin slice), use MS culture medium+0.6wt% colchicine+1% (v/v)DMSO fluid nutrient medium carries out immersion treatment 24 hours; Thin slice after treatment distilled water flushing 1～2 time,Be seeded in bud inducing culture B (MS culture medium+4mgL-16-BA+0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar) in, cultivate and within 40 days, obtain healthy and strong adventitious bud; Adventitious bud is transferred to intoSeedling culture medium (1/2MS culture medium+0.75gL-1Active carbon+15gL-1Sucrose+7gL-1Agar) onCultivate 30 days, obtain the test-tube plantlet of mutagenic treatment;
(6) test-tube plantlet obtaining in step (5) is carried out to uncork domestication, under room scattering illumination, place 5My god, keep moistening in bottle. Then seedling is cleaned up, be transplanted to the flowerpot that Nutrition Soil is housed, Nutrition Soil isThe organoclay for flower culture of buying from flowers market is that 1/20MS cultivates by concentration before plantlet of transplantThe inorganic salt solution of base once irrigates, and or else whole process applies fertilizer, depending on the dry wet suitable supplementing water of situation of soilPoint, keep ground moistening and suitably shelter from heat or light, after 1 month, obtain the Bai Jianghua regeneration plant of mutagenesis, canTo proceed to normal cultivation management.
(7) Bai Jianghua regeneration plant and the original seed liploid plant of mutagenesis step (6) being obtained carry outContrast, broadens blade greatly, and color is dark green, and vein highlights, the micro-sagging growth of blade, and it is large that pore becomes, gasThe plant that hole density diminishes is elected candidate plant as; Choose the consistent liploid plant of development degree and colchicineThe plant obtaining after processing, gets the consistent blade in joint position, tears and takes off epidermis from blade same area, is placed in and carriesOn slide, make Temporary slide, under microscope, measure length and the density of pore; Choose development degree oneThe plant that the liploid plant causing and colchicine obtain after processing, gets the identical blade in blade joint position, measuresLeaf long (leaf base is long to blade tip) and leaf wide (position that blade is the widest).
(8) the candidate plant obtaining in selecting step (7), contrasts with liploid plant. Test lastIts 17:00 hole irrigation foot water, morning next day 9:00 point to 10:00 point, get the delicate sturdy tip of a root of root," dissociate-hypotonic-dye-compressing tablet-observation " method of employing is carried out Chromosome Analysis; Concrete steps are as follows:Scrape off tip of a root epidermis with scalpel, be soaked in dissociation solution (37%HCl:95%C2H5OH=1:3, volume ratio)In, 6min dissociates under normal temperature; Take out the tip of a root, with distilled water flushing 3 times, then be put in distilled water hypotonic 30Min; The meristematic zone material dissecting needle that takes a morsel is dialled loose, drips the pinkish red dye liquor dyeing of phenol 3min; Close the lidSlide, sops up unnecessary dye liquor, then covers on cover glass with filter paper, with pencil eraser head across filter paper along rightLinea angulata knocks cover plate gently, and material composition is uniformly dispersed; The slice, thin piece pressing is placed in to micro-Microscopic observation,Qualification chromosome number; The cell number of observing is no less than 30, more than 85% is 2n=4x=68 in somatoblastThe plant of bar chromosome number, is defined as Bai Jianghua tetraploid plant.
(9) tetraploid plant step (8) being obtained carries out field observation, measure floral organ lip,The length of ala and width (the widest), and measure the filigree of stamen, the length of flower pesticide; Floral organ is obviousBecome the Bai Jianghua tetraploid plant that large plant is defined as having good agronomic traits.
Embodiment 4
(1) sterilization of explant and the induction of adventitious bud: get Bai Jianghua's (Hedychiumcoronarium)Root-like stock is sprouted the sprouting, and rinses well afterwards with liquid detergent bubble 25 minutes with running water, then uses current from the beginningMoving flushing after 25 minutes carried out surface sterilization 90 seconds with 70wt% alcohol, finally with the sterilization of 0.1wt% mercuric chloride20 minutes, on superclean bench, cut the bud of 2.0cm as explant, be inoculated into bud inducing culture AIn cultivate, induce adventitious bud through the cultivation of 20 days; Described bud inducing culture A is MS trainingSupport base+5mgL-16-BA+0.15mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
(2) adventitious bud seedling: the adventitious bud of step (1) induction is proceeded in seedling culture medium, promote seedling rawRoot maturation, cultivates the healthy and strong test-tube plantlet that can obtain for 20 days more than high 6cm; Described seedling culture medium is 1/2MS culture medium+0.75gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(3) induction of adventitious bud and the acquisition of test-tube plantlet: the test-tube plantlet that step (2) is obtained cuts thin slice(the thick 0.3～0.5mm of thin slice), is seeded in upper cultivation of bud inducing culture B, cultivates acquisition in 20 daysHealthy and strong adventitious bud; Described bud inducing culture B is MS culture medium+0.05mgL-1TDZ+0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
The adventitious bud that grows to 2cm is transferred on seedling culture medium and carried out seedling cultivation, cultivate to obtain for 15 days and be good forStrong test-tube plantlet; Described seedling culture medium is 1/2MS culture medium+0.75gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(4) repeating step (3) as required, until obtain enough test-tube plantlets;
(5) the induction processing of thin slice: get the test-tube plantlet that step (3) or (4) obtain, cut from its base portionThin slice (the thick 0.3～0.5mm of thin slice), use MS culture medium+0.8wt% colchicine+1% (v/v)DMSO fluid nutrient medium carries out immersion treatment 12 hours; Thin slice after treatment distilled water flushing 1～2 time,Be seeded in bud inducing culture B (MS culture medium+0.05mgL-1TDZ+0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar) in, cultivate and within 20 days, obtain healthy and strong adventitious bud; Adventitious bud is shiftedTo seedling culture medium (1/2MS culture medium+0.75gL-1Active carbon+15gL-1Sucrose+7gL-1Agar)Upper cultivation 35 days, obtains the test-tube plantlet of mutagenic treatment;
(6) test-tube plantlet obtaining in step (5) is carried out to uncork domestication, under room scattering illumination, place 5My god, keep moistening in bottle. Then seedling is cleaned up, be transplanted to the flowerpot that Nutrition Soil is housed, Nutrition Soil isThe organoclay for flower culture of buying from flowers market is that 1/20MS cultivates by concentration before plantlet of transplantThe inorganic salt solution of base once irrigates, and or else whole process applies fertilizer, depending on the dry wet suitable supplementing water of situation of soilPoint, keep ground moistening and suitably shelter from heat or light, after 1 month, obtain the Bai Jianghua regeneration plant of mutagenesis, canTo proceed to normal cultivation management.
(7) Bai Jianghua regeneration plant and the original seed liploid plant of mutagenesis step (6) being obtained carry outContrast, broadens blade greatly, and color is dark green, and vein highlights, the micro-sagging growth of blade, and it is large that pore becomes, gasThe plant that hole density diminishes is elected candidate plant as; Choose the consistent liploid plant of development degree and colchicineThe plant obtaining after processing, gets the consistent blade in joint position, tears and takes off epidermis from blade same area, is placed in and carriesOn slide, make Temporary slide, under microscope, measure length and the density of pore; Choose development degree oneThe plant that the liploid plant causing and colchicine obtain after processing, gets the identical blade in blade joint position, measuresLeaf long (leaf base is long to blade tip) and leaf wide (position that blade is the widest).
(8) the candidate plant obtaining in selecting step (7), contrasts with liploid plant. Test lastIts 17:00 hole irrigation foot water, morning next day 9:00 point to 10:00 point, get the delicate sturdy tip of a root of root," dissociate-hypotonic-dye-compressing tablet-observation " method of employing is carried out Chromosome Analysis; Concrete steps are as follows:Scrape off tip of a root epidermis with scalpel, be soaked in dissociation solution (37%HCl:95%C2H5OH=1:5, volume ratio)In, 6min dissociates under normal temperature; Take out the tip of a root, with distilled water flushing 3 times, then be put in distilled water hypotonic 30Min; The meristematic zone material dissecting needle that takes a morsel is dialled loose, drips the pinkish red dye liquor dyeing of phenol 3min; Close the lidSlide, sops up unnecessary dye liquor, then covers on cover glass with filter paper, with pencil eraser head across filter paper along rightLinea angulata knocks cover plate gently, and material composition is uniformly dispersed; The slice, thin piece pressing is placed in to micro-Microscopic observation,Qualification chromosome number; The cell number of observing is no less than 30, more than 85% is 2n=4x=68 in somatoblastThe plant of bar chromosome number, is defined as Bai Jianghua tetraploid plant.
(9) tetraploid plant step (8) being obtained carries out field observation, measure floral organ lip,The length of ala and width (the widest), and measure the filigree of stamen, the length of flower pesticide; Floral organ is obviousBecome the Bai Jianghua tetraploid plant that large plant is defined as having good agronomic traits.
Embodiment 5
(1) sterilization of explant and the induction of adventitious bud: get Bai Jianghua's (Hedychiumcoronarium)Root-like stock is sprouted the sprouting, and rinses well afterwards with liquid detergent bubble 28 minutes with running water, then uses current from the beginningMoving flushing after 28 minutes carried out surface sterilization 75 seconds with 75wt% alcohol, finally with the sterilization of 0.1wt% mercuric chloride15 minutes, on superclean bench, cut the bud of 1.0cm as explant, be inoculated into bud inducing culture AIn cultivate, induce adventitious bud through the cultivation of 30 days; Described bud inducing culture A is MS trainingSupport base+4mgL-16-BA+0.1mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
(2) adventitious bud seedling: the adventitious bud of step (1) induction is proceeded in seedling culture medium, promote seedling rawRoot maturation, cultivates the healthy and strong test-tube plantlet that can obtain for 30 days more than high 6cm; Described seedling culture medium is 1/2MS culture medium+0.5gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(3) induction of adventitious bud and the acquisition of test-tube plantlet: the test-tube plantlet that step (2) is obtained cuts thin slice(the thick 0.3～0.5mm of thin slice), is seeded in upper cultivation of bud inducing culture B, cultivates acquisition in 20 daysHealthy and strong adventitious bud; Described bud inducing culture B is MS culture medium+0.2mgL-1TDZ+0.15mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
The adventitious bud that grows to 1.5cm is transferred on seedling culture medium and carried out seedling cultivation, cultivate acquisition in 15 daysHealthy and strong test-tube plantlet; Described seedling culture medium is 1/2MS culture medium+0.5gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(4) repeating step (3) as required, until obtain enough test-tube plantlets;
(5) the induction processing of thin slice: get the test-tube plantlet that step (3) or (4) obtain, cut from its base portionThin slice (the thick 0.3～0.5mm of thin slice), use MS culture medium+0.4wt% colchicine+1% (v/v)DMSO fluid nutrient medium carries out immersion treatment 24 hours; Thin slice after treatment distilled water flushing 1～2 time,Be seeded in bud inducing culture B (MS culture medium+0.2mgL-1TDZ+0.1mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar) in, cultivate and within 30 days, obtain healthy and strong adventitious bud; Adventitious bud is shiftedTo seedling culture medium (1/2MS culture medium+1gL-1Active carbon+15gL-1Sucrose+7gL-1Agar)Upper cultivation 40 days, obtains the test-tube plantlet of mutagenic treatment;
(6) test-tube plantlet obtaining in step (5) is carried out to uncork domestication, under room scattering illumination, place 3My god, keep moistening in bottle. Then seedling is cleaned up, be transplanted to the flowerpot that Nutrition Soil is housed, Nutrition Soil isThe organoclay for flower culture of buying from flowers market is that 1/20MS cultivates by concentration before plantlet of transplantThe inorganic salt solution of base once irrigates, and or else whole process applies fertilizer, depending on the dry wet suitable supplementing water of situation of soilPoint, keep ground moistening and suitably shelter from heat or light, after 1 month, obtain the Bai Jianghua regeneration plant of mutagenesis, canTo proceed to normal cultivation management.
(7) Bai Jianghua regeneration plant and the original seed liploid plant of mutagenesis step (6) being obtained carry outContrast, broadens blade greatly, and color is dark green, and vein highlights, the micro-sagging growth of blade, and it is large that pore becomes, gasThe plant that hole density diminishes is elected candidate plant as; Choose the consistent liploid plant of development degree and colchicineThe plant obtaining after processing, gets the consistent blade in joint position, tears and takes off epidermis from blade same area, is placed in and carriesOn slide, make Temporary slide, under microscope, measure length and the density of pore; Choose development degree oneThe plant that the liploid plant causing and colchicine obtain after processing, gets the identical blade in blade joint position, measuresLeaf long (leaf base is long to blade tip) and leaf wide (position that blade is the widest).
(8) the candidate plant obtaining in selecting step (7), contrasts with liploid plant. Test the previous day17:00 hole irrigation foot water, morning next day 9:00 point to 10:00 point, get the delicate sturdy tip of a root of root, adoptCarry out Chromosome Analysis by " dissociate-hypotonic-dye-compressing tablet-observation " method; Concrete steps are as follows: useScalpel scrapes off tip of a root epidermis, is soaked in dissociation solution (37%HCl:95%C2H5OH=1:3, volume ratio)In, 6min dissociates under normal temperature; Take out the tip of a root, with distilled water flushing 3 times, then be put in distilled water hypotonic 30Min; The meristematic zone material dissecting needle that takes a morsel is dialled loose, drips the pinkish red dye liquor dyeing of phenol 3min; Close the lidSlide, sops up unnecessary dye liquor, then covers on cover glass with filter paper, with pencil eraser head across filter paper along rightLinea angulata knocks cover plate gently, and material composition is uniformly dispersed; The slice, thin piece pressing is placed in to micro-Microscopic observation,Qualification chromosome number; The cell number of observing is no less than 30, more than 85% is 2n=4x=68 in somatoblastThe plant of bar chromosome number, is defined as Bai Jianghua tetraploid plant.
(9) tetraploid plant step (8) being obtained carries out field observation, measure floral organ lip,The length of ala and width (the widest), and measure the filigree of stamen, the length of flower pesticide; Floral organ is obviousBecome the Bai Jianghua tetraploid plant that large plant is defined as having good agronomic traits.
Embodiment 6
(1) sterilization of explant and the induction of adventitious bud: get Bai Jianghua's (Hedychiumcoronarium)Root-like stock is sprouted the sprouting, and rinses well afterwards with liquid detergent bubble 28 minutes with running water, then uses current from the beginningMoving flushing after 28 minutes carried out surface sterilization 75 seconds with 73wt% alcohol, finally with the sterilization of 0.1wt% mercuric chloride15 minutes, on superclean bench, cut the bud of 1.5cm as explant, be inoculated into bud inducing culture AIn cultivate, induce adventitious bud through the cultivation of 40 days; Described bud inducing culture A is MS trainingSupport base+4mgL-16-BA+0.1mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
(2) adventitious bud seedling: the adventitious bud of step (1) induction is proceeded in seedling culture medium, promote seedling rawRoot maturation, cultivates the healthy and strong test-tube plantlet that can obtain for 40 days more than high 6cm; Described seedling culture medium is 1/2MS culture medium+0.5gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(3) induction of adventitious bud and the acquisition of test-tube plantlet: the test-tube plantlet that step (2) is obtained cuts thin slice(the thick 0.3～0.5mm of thin slice), is seeded in upper cultivation of bud inducing culture B, cultivates acquisition in 20 daysHealthy and strong adventitious bud; Described bud inducing culture B is MS culture medium+0.1mgL-1TDZ+0.15mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
The adventitious bud that grows to 1.5cm is transferred on seedling culture medium and carried out seedling cultivation, cultivate acquisition in 15 daysHealthy and strong test-tube plantlet; Described seedling culture medium is 1/2MS culture medium+0.5gL-1Active carbon+15gL-1Sucrose+7gL-1Agar;
(4) repeating step (3) as required, until obtain enough test-tube plantlets;
(5) the induction processing of thin slice: get the test-tube plantlet that step (3) or (4) obtain, cut from its base portionThin slice (the thick 0.3～0.5mm of thin slice), use MS culture medium+0.6wt% colchicine+1% (v/v)DMSO fluid nutrient medium carries out immersion treatment 24 hours; Thin slice after treatment distilled water flushing 1～2 time,Be seeded in bud inducing culture B (MS culture medium+0.1mgL-1TDZ+0.1mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar) in, cultivate and within 40 days, obtain healthy and strong adventitious bud; Adventitious bud is shiftedTo seedling culture medium (1/2MS culture medium+1gL-1Active carbon+15gL-1Sucrose+7gL-1Agar)Upper cultivation 45 days, obtains the test-tube plantlet of mutagenic treatment;
(6) test-tube plantlet obtaining in step (5) is carried out to uncork domestication, under room scattering illumination, place 3My god, keep moistening in bottle. Then seedling is cleaned up, be transplanted to the flowerpot that Nutrition Soil is housed, Nutrition Soil isThe organoclay for flower culture of buying from flowers market is that 1/20MS cultivates by concentration before plantlet of transplantThe inorganic salt solution of base once irrigates, and or else whole process applies fertilizer, depending on the dry wet suitable supplementing water of situation of soilPoint, keep ground moistening and suitably shelter from heat or light, after 1 month, obtain the Bai Jianghua regeneration plant of mutagenesis, canTo proceed to normal cultivation management.
(7) Bai Jianghua regeneration plant and the original seed liploid plant of mutagenesis step (6) being obtained carry outContrast, broadens blade greatly, and color is dark green, and vein highlights, the micro-sagging growth of blade, and it is large that pore becomes, gasThe plant that hole density diminishes is elected candidate plant as; Choose the consistent liploid plant of development degree and colchicineThe plant obtaining after processing, gets the consistent blade in joint position, tears and takes off epidermis from blade same area, is placed in and carriesOn slide, make Temporary slide, under microscope, measure length and the density of pore; Choose development degree oneThe plant that the liploid plant causing and colchicine obtain after processing, gets the identical blade in blade joint position, measuresLeaf long (leaf base is long to blade tip) and leaf wide (position that blade is the widest).
(8) the candidate plant obtaining in selecting step (7), contrasts with liploid plant. Test the previous day17:00 hole irrigation foot water, morning next day 9:00 point to 10:00 point, get the delicate sturdy tip of a root of root, adoptCarry out Chromosome Analysis by " dissociate-hypotonic-dye-compressing tablet-observation " method; Concrete steps are as follows: useScalpel scrapes off tip of a root epidermis, is soaked in dissociation solution (37%HCl:95%C2H5OH=1:3, volume ratio)In, 6min dissociates under normal temperature; Take out the tip of a root, with distilled water flushing 3 times, then be put in distilled water hypotonic 30Min; The meristematic zone material dissecting needle that takes a morsel is dialled loose, drips the pinkish red dye liquor dyeing of phenol 3min; Close the lidSlide, sops up unnecessary dye liquor, then covers on cover glass with filter paper, with pencil eraser head across filter paper along rightLinea angulata knocks cover plate gently, and material composition is uniformly dispersed; The slice, thin piece pressing is placed in to micro-Microscopic observation,Qualification chromosome number; The cell number of observing is no less than 30, more than 85% is 2n=4x=68 in somatoblastThe plant of bar chromosome number, is defined as Bai Jianghua tetraploid plant.
(9) tetraploid plant step (8) being obtained carries out field observation, measure floral organ lip,The length of ala and width (the widest), and measure the filigree of stamen, the length of flower pesticide; Floral organ is obviousBecome the Bai Jianghua tetraploid plant that large plant is defined as having good agronomic traits.
Above-described embodiment is preferably embodiment of the present invention, but embodiments of the present invention are not subject to above-mentioned enforcementThe restriction of example, other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, replacesGeneration, combination, simplification, all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (8)
1. utilize thin slice culture technique to obtain a method for Bai Jianghua tetraploid plant, it is characterized in that comprisingFollowing steps:
(1) sterilization of explant and the induction of adventitious bud: the bud of sprouting taking the root-like stock of Bai Jianghua is as explant,Be inoculated in bud inducing culture A and cultivate, induce adventitious bud;
(2) adventitious bud seedling: the adventitious bud of step (1) induction is proceeded in seedling culture medium and cultivated,Obtain healthy and strong test-tube plantlet;
(3) expansion of test-tube plantlet is numerous: get the test-tube plantlet that step (2) obtains, cut thin slice from its base portion, connectPlant and cultivate in bud inducing culture B, obtain healthy and strong adventitious bud; Adventitious bud is proceeded to seedling to be cultivatedIn base, cultivate, obtain test-tube plantlet;
(4) repeating step (3) as required, until obtain enough test-tube plantlets;
(5) induction of tetraploid plant: get the test-tube plantlet that step (3) or (4) obtain, cut from its base portionGet thin slice, carry out immersion treatment with colchicine solution; Thin slice after treatment is seeded in to bud inducing culture BIn cultivate, obtain adventitious bud; Adventitious bud is transferred on seedling culture medium and cultivated, obtain mutagenic treatmentAfter test-tube plantlet;
(6) test-tube plantlet obtaining in step (5) is tamed, is transplanted to the flowerpot that Nutrition Soil is housed,Obtain the Bai Jianghua regeneration plant of mutagenesis;
(7) the Bai Jianghua regeneration plant of mutagenesis step (6) being obtained and original seed liploid plant seedling stageContrast, blade is broadened greatly, color is dark green, and vein highlights, the micro-sagging growth of blade, and it is large that pore becomes,The plant that stomatal frequency diminishes is elected candidate plant as;
(8) get the tip of a root of the candidate plant obtaining in step (7), carry out Chromosome Analysis, chooseChromosome number is the seedling of 2n=4x=68, obtains Bai Jianghua tetraploid plant;
The thickness of described thin slice is 0.3～0.5mm;
Described bud inducing culture B is MS culture medium+1～5mgL-16-BA or 0.05～0.2mg·L-1TDZ+0.1～0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar;
In step (5), described colchicine solution is MS culture medium+0.2～0.8wt% colchicine+bodyLong-pending percentage 1%DMSO;
The time of described immersion treatment is 12～36h;
In step (1):
The bud that the root-like stock of described Bai Jianghua is sprouted adopts following methods to carry out pre-treatment: the root of getting Bai JianghuaThe bud that shape stem is sprouted, water is rinsed well rear with liquid detergent bubble 25～30 minutes, then water rinses 25～30Minute, carry out surface sterilization 60～90 seconds with 70～75wt% alcohol, finally with the sterilization of 0.1wt% mercuric chloride15～20 minutes, cut the bud of long 0.5～2.0cm as explant.
2. method according to claim 1, is characterized in that:
Bud inducing culture A described in step (1) is MS culture medium+3～6mgL-16-BA+0.1～0.2mg·L-1NAA+30g·L-1Sucrose+7gL-1Agar.
3. method according to claim 1, is characterized in that:
Described seedling culture medium is 1/2MS culture medium+0.5～1gL-1Active carbon+15gL-1Sucrose+7g·L-1Agar.
4. method according to claim 1, is characterized in that:
The time of the cultivation described in step (1) is 20～40 days;
The time of the cultivation described in step (2) is 15～45 days.
5. method according to claim 1, is characterized in that:
In step (3):
Described adventitious bud length is 1～2cm;
Described test-tube plantlet is that adventitious bud is transferred to the plant more than high 6cm obtaining on seedling culture medium;
The described time of cultivating in bud inducing culture B is 20～40 days;
The described time of cultivating on seedling culture medium of transferring to is 15～45 days.
6. method according to claim 1, is characterized in that:
In step (6), the time of described domestication is 3～7 days.
7. method according to claim 1, is characterized in that:
The pH of the described culture medium of step (1), (2), (3), (5) is 5.6～5.9, except TDZ adoptsWith joining after filtration sterilization outside the culture medium of autoclave sterilization, remaining culture medium all adopts HTHPSterilizing;
The condition of the described cultivation of step (1), (2), (3), (5) is all at 26～28 DEG C, 30 μ molm-2s-1Under light intensity, 16L/8D illumination condition, carry out.
8. method according to claim 1, is characterized in that: the chromosome number described in step (8)Order analytical method: choose liploid plant and process the candidate plant obtaining through colchicine, test lastIts 17:00 hole irrigation foot water, morning next day 9:00 point to 10:00 point, get the delicate sturdy tip of a root of root," dissociate-hypotonic-dye-compressing tablet-observation " method of employing is carried out Chromosome Analysis; Concrete steps are as follows:Scrape off tip of a root epidermis with scalpel, be soaked in dissociation solution, 5～8min dissociates under normal temperature; Take out the tip of a root,With distilled water flushing 3～4 times, then be put into hypotonic 25～30min in distilled water; The meristematic zone material that takes a morsel is usedDissecting needle is dialled loose, drips the pinkish red dye liquor dyeing of phenol 3～5min; Covered, sops up unnecessary dye liquor,Cover on cover glass with filter paper again, knock gently cover plate across filter paper along diagonal with pencil eraser head, make materialMaterial composition is uniformly dispersed; The slice, thin piece pressing is placed in to micro-Microscopic observation, qualification chromosome number; ObserveCell number is no less than 30, more than 85% is that constant consistent chromosome number is this plant in somatoblastChromosome number;
Described dissociation solution is 37%HCl and 95%C2H5OH 1:3～5 preparation by volume obtains.
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