CN101869052B - Method for building induction and cultivation system of alpine ash tetraploid plant - Google Patents
Method for building induction and cultivation system of alpine ash tetraploid plant Download PDFInfo
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- CN101869052B CN101869052B CN2010101713544A CN201010171354A CN101869052B CN 101869052 B CN101869052 B CN 101869052B CN 2010101713544 A CN2010101713544 A CN 2010101713544A CN 201010171354 A CN201010171354 A CN 201010171354A CN 101869052 B CN101869052 B CN 101869052B
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Abstract
The invention discloses a method for building the induction and the cultivation system of an alpine ash tetraploid plant. The method comprises the following steps that: (1) the stem segment with axillary bud or terminal bud of alpine ash aseptic group culture seedling is placed in a container containing processing liquid, the processing liquid contains colchicine and auxiliary agent SD, the container is sealed and placed on a shaking table to oscillate, double processing is carried out for 12 to 18 hours at 35 to 40 degrees centigrade, and then the stem segment is flushed with sterile water to remove colchicine residual liquid and obtain the stem segment after double processing, and (2) the stem segment after the double processing is inoculated in enrichment medium to culture and transferred into newly prepared enrichment medium at intervals of 15 to 20 days and then radication culture is carried out after 3 to 5 times of transfer. The innovation point of the invention is that the auxiliary agent SD is used in the induction process, which can use the low colchicine concentration, save use amount, reduce cost and obviously improve the induction rate from 20 to 30 percent to 60 to 65 percent.
Description
Technical field
The invention belongs to forestry biotechnology breeding field or plant cell engineering field, particularly proposed a kind of method of setting up alpine ash clone Eg5 multiploid induction and cultivation system.
Background technology
Alpine ash (Eucalyptus grandis) is that eucalyptus belongs to the high megaphanerophyte in the two opercule subgenus transverse vein group Liu An system, formerly is distributed in Australian east coast.Its growth rapidly afforests that height of tree year increment can reach 2-3m in back 10 years, and form is perfectly straight, and is satisfactory, can be widely used in purposes such as papermaking, building, wood-based plate and firewood forest.Therefore, it is one of the most frequently used seeds of fast-growing and high-yield plantation, also is one of maximum eucalyptus kind of area under cultivation.Alpine ash is extensively planted in the southern Fujian of China at present, the northern subtropical zone in Sichuan, Yunnan, Xiang Nan, Gan Nan and Guangdong and Guangxi Provinces.
Alpine ash Eg5 (breeding numbering: Fujian S-SC-EG-005-2007) be the alpine ash choiceness of Yongan, Fujian forestry group seed selection; Through the authorization of the Fujian Province forest variety certification committee; Announce by Fujian Province forestry department, and receive the protection of breeding protection rules.
The polyploid plant of breeding through ploidy breeding generally has following 2 characteristic features: 1) " the huge property " of organ, and all bigger like plant stem, blade, flower, fruit, cell, pore etc., therefore, can improve crop yield; 2) resistance strengthens, and disease-resistant, drought resisting, cold resistance like enhancing crop make crop adaptability become wide.Therefore, polyploid breeding has very positive meaning in agriculture and forestry are produced.
Eucalyptus is a kind of alien species, and according to the result of study of Hu Zhouhe, using simple colchicine to handle the after stain colour solid has only increased by 3～5 on original 22 basis, do not realize doubling.Use the abductive approach of Tan Deguan on No. 12 eucalyptus of the Congo, induce the polyploid of alpine ash bud with the colchicine (wherein having added the bleeding agent dimethyl sulfoxide (DMSO)) of variable concentrations, have to 20%～30% inductivity, inductivity is low excessively.
Summary of the invention
For the shortcoming that remedies prior art with not enough, primary and foremost purpose of the present invention is to provide a kind of inducing and the method for cultivation system of alpine ash tetraploid plant of setting up; This method reaches more than 60% inductivity.And, to handle the vitrification phenomenon that back stem section occurs at colchicine, designed special-purpose medium, can effectively suppress the appearance of vitrification phenomenon, obtained the healthy and strong field-transplanting seedling of taking root.
The object of the invention is realized through following technical proposals: a kind ofly set up inducing and the method for cultivation system of alpine ash tetraploid plant.This method comprises following operating procedure:
(1) the stem section that the aseptic tissue cultivating seedling of alpine ash is had an axillary-bud or top-bud places the container that treatment fluid is housed, and it is that 0.1～0.5% colchicine and mass percent concentration are 5～12% auxiliary agent SD that said treatment fluid contains mass percent concentration; Be placed on the shaking table with the hunting speed of 80～120r/min container is airtight, under 35～40 ℃ of conditions, doubled to handle 12～18 hours; After disposing, use aseptic water washing stem section,, obtain stem section through doubling to handle until removing the colchicine debris fully;
(2) will through the stem section that doubling to handle and be inoculated in the special-purpose proliferated culture medium and cultivate, and whenever in the proliferated culture medium that promptly was transferred to new preparation in 15～20 days, transfer and carry out culture of rootage again after 3～5 times;
Said proliferated culture medium is made up of following component by mass volume ratio: nitrate of lime (Ca (NO
3)
24H
2O) 537～576mg/L, potassium nitrate (KNO
3) 480～520mg/L, magnesium sulfate (MgSO
47H
2O) 355～390mg/L, potassium dihydrogen phosphate (KH
2PO
4) 150～190mg/L, calcium chloride (CaCl
22H
2O) 77～116mg/L, manganese sulphate (MnSO
44H
2O) 20.2～24.5mg/L, zinc sulphate (ZnSO
47H
2O) 7.7～11.6mg/L, boric acid (H
3BO
3) 5.4～7.2mg/L, copper sulphate (CuSO
45H
2O) 0.018～0.029mg/L, sodium molybdate (Na
2M0O
42H
2O) 0.19～0.38mg/L, ferrous sulfate (FeSO
47H
2O) 25.3～29.4mg/L, disodium ethylene diamine tetraacetate (Na
2-EDTA) 35.3～38.6mg/L, phloridzin 2～4mg/L, thiamine hydrochloride (VB
1) 0.092～0.112mg/L, pyridoxine hydrochloride (VB
6) 0.41～0.58mg/L, nicotinic acid (VB
5) 0.39～0.61mg/L, glycine 1.5～2.4mg/L, inositol 91～112mg/L, vitamin b3 (VB
2) 5.0～7.0mg/L, kinetin (KT) 0.4～0.6mg/L, methyl (NAA) 0.15～0.25mg/L, agar 6100～6800mg/L and sucrose 30000～45000mg/L, surplus is a distilled water.
Said step (1) and step (2) are under aseptic condition, to operate.
Said proliferated culture medium preferably is made up of following component by mass volume ratio: nitrate of lime 556mg/L, potassium nitrate 500mg/L, magnesium sulfate 370mg/L, potassium dihydrogen phosphate 170mg/L; Calcium chloride 96mg/L, manganese sulphate 22.5mg/L, zinc sulphate 8.6mg/L, boric acid 6.2mg/L, copper sulphate 0.025mg/L, sodium molybdate 0.25mg/L; Ferrous sulfate 27.3mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, phloridzin 2.5mg/L, thiamine hydrochloride 0.1mg/L; Pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, glycine 2.0mg/L; Inositol 100mg/L, vitamin b3 6.0mg/L, kinetin 0.5mg/L; Methyl 0.2mg/L, agar 6800mg/L and sucrose 45000mg/L, surplus is a distilled water.
The length of the said stem section of step (1) is 0.7～1.3cm.
The consumption of the said treatment fluid of step (1) is that every 100mL treatment fluid is handled 20～30 pieces of stem sections.
The medium that the said culture of rootage of step (2) adopts is made up of following component by mass volume ratio: potassium nitrate (KNO
3) 614～654mg/L, ammonium nitrate (NH
4NO
3) 530～570mg/L, potassium dihydrogen phosphate (KH
2PO
4) 47～67mg/L, magnesium sulfate (MgSO
47H
2O) 113～133mg/L, calcium chloride (CaCl
22H
2O) 137～157mg/L, KI (KI) 0.8～0.9mg/L, boric acid (H
3BO
3) 5.2～7.2mg/L, manganese sulphate (MnSO
44H
2O) 20.3～24.5mg/L, zinc sulphate (ZnSO
47H
2O) 7.6～9.4mg/L, sodium molybdate (Na
2MoO
42H
2O) 0.2～0.3mg/L, copper sulphate (CuSO
45H
2O) 0.02～0.03mg/L, cobalt chloride (CoCl
26H
2O) 0.02～0.03mg/L, disodium ethylene diamine tetraacetate (Na
2EDTA) 34.3～40.3mg/L, ferrous sulfate (FeSO
47H
2O) 25.8～29.8mg/L, inositol 90～110mg/L, glycine 1.6～2.6mg/L, thiamine hydrochloride (VB
1) 0.08～0.12mg/L, puridoxine hydrochloride (VB
6) 0.4～0.6mg/L, nicotinic acid (VB
5) 0.4～0.6mg/L, indolebutyric acid (IBA) 0.42～0.62mg/L, ABT root-inducing powder 0.39～0.69mg/L, sucrose 25000～40000mg/L and agar 6100～6800mg/L, surplus is a distilled water.
The said alpine ash of step (1) is authorization kind Eg5 clone.
The present invention serves as to handle material with the axillalry bud and the terminal bud of alpine ash clonal tissue culture seedling, handles with colchicine, carries out chromosome doubling; Axillalry bud after will handling then and terminal bud the stem section of giving birth to and are inserted in the special special culture media, make its healthy growing, and change field-transplanting over to after taking root, and can go out garden afforestation behind the refining seedling.
Use inducing in the process of colchicine at alpine ash,, just need to improve the concentration of colchicine or the soak time that prolongs colchicine solution if seek out higher inductivity; But, do like this and can cause pending tender shoots dead, and the death of tender shoots can cause the reduction of inductivity; And single use colchicine solution can cause after inducing processing, having more chimeric appearance.So, need when handling, add bleeding agent.Under the situation that bleeding agent and colchicine solution are used with, can use lower colchicine solution or shorten the processing time, thereby reduce lethality, simultaneously, this has also reduced chimeric appearance effectively.But, bleeding agents such as existing dimethyl sulfoxide (DMSO), dimethylacetylamide or azone, action effect is undesirable, on inductivity, does not increase significantly.
In conjunction with domestic and international newest research results, novelty of the present invention originally as the SD auxiliary agent (its active ingredient is a kind of Triterpene Glycosides compounds) of auxiliary agent of weedicide as bleeding agent, reached satisfied effect.Compare with using dimethyl sulfoxide (DMSO), make used additives SD, inductivity can rise to 60% significantly from 35%; And when using dimethyl sulfoxide (DMSO), the colchicine solution concentration when reaching the highest inductivity is 0.25%, if but use SD, the concentration of the colchicine solution that uses when reaching the highest inductivity is merely 0.15%; No matter be in the small scale experiments process; It still is the process of implementing in order to satisfy large-scale industrialized production needs of inducing; Often need inducing of a large amount of buds, this just needs to use more colchicine, but colchicine costs an arm and a leg and is generally 350～400 yuan/gram.Therefore, in order to reach maximum inductivity, the colchicine working concentration drops to 0.15% from 0.25%, and has saved production cost significantly.
With at present the colchicine tetraploid induction method of public reported compare, innovation below the inventive method also has: the temperature during processing does not adopt normal temperature, but 35～40 ℃ high temperature.Because the mitosis of alpine ash cell and temperature relation are comparatively close, in certain temperature range, temperature is high more; The mitosis of cell is vigorous more; The cell that is in prophase can increase, and colchicine only works to the cell that is in prophase, produces and doubles effect.So the temperature rising can make inductivity improve.In addition, the temperature appropriateness raises, and also can cause cell membrane active with extraneous mass exchange, and cell leakage has increase to a certain degree, impels colchicine solution to arrive pending position more easily, strengthens to induce effect.But temperature is too high, can cause the high temperature stress effect, causes the plant physiology activity to get muddled, and enzyme deactivation, even adverse consequences such as protein denaturation might cause stem section to be induced dead, thereby has caused the reduction of inductivity.
Because it is a kind of adverse circumstance that colchicine is handled plant; So, even double the induced bud of success, often demonstrate also that growth potential is weak, deformity, vitrifying, be difficult for becoming phenomenon such as work; The induced bud that causes doubling success is g and D normally, can not survive.It is strong that the medium of can normal growth before using alpine ash Eg5 to double growing can't realize doubling to handle holding up of back seedling, so, need the plant after the special medium of development be used to hold up strong the processing.Special culture media of the present invention has following advantage: (1) has added the phloridzin of 2～4mg/L; (2) do not contain NH
4 +Ion, the nitrogen content in the medium is low; (3) basic element of cell division uses kinetin rather than 6-benzyladenine; (4) sucrose concentration is brought up to 45000mg/L; (5) not the change of the simple material consumption on Ms or other medium base, but employed chemical reagent in the Ms medium has been carried out conversion.Use other medium, can't the vitrifying rate (vitrified seedling/seedling sum) of seedling be reduced to below 50%, and the special culture media that adopts the present invention to use then can be reduced to the vitrifying rate of inducing seedling below 5%; And, significantly hold up strong effect to inducing seedling also to have, weak seedling quantity is reduced to below 5% from 30%.
The present invention compared with prior art has following advantage:
(1) novelty of the present invention a kind of auxiliary agent of weedicide SD has been used inducing in the process of colchicine, can use lower colchicine concentration to reach maximum inductivity, save consumption; In addition, use SD to substitute traditional bleeding agents such as azone, dimethyl sulfoxide (DMSO), inductivity reaches 60～65%.
(2) technical scheme of the present invention has constituted one and has induced the technical system that is integrated with tissue-culturing rapid propagation, makes to have the polyploid seedling that growth rate significantly accelerates and not only can produce, and can cultivate into alively, forms the seedling of a stalwartness, is used for production of forestry.
(3) when doubling to handle, the stem segment length of use is 0.7～1.3cm, then; Through 3～5 times the switching, carried out culture of rootage and field-transplanting after, grown up to the high plant of 20～30cm; Get its newborn young tender tip of a root and the stem growing point on top, detect its chromosome number and be 4x, non-chimera and dliploid---at this moment; Carry out the statistics of inductivity, so the chromosome doubling of the inventive method is effective and reliable and stable.
Description of drawings
Fig. 1 is the microscopic examination figure of stem tip chromosome counting, and wherein a is that b is for to induce the plant of processing through the present invention without the plant of inducing processing.
Fig. 2 is the microscopic examination figure of root tip chromosomes counting, and wherein a is that b is for to induce the plant of processing through the present invention without the plant of inducing processing.
Embodiment
Below in conjunction with concrete instance and accompanying drawing the present invention is done further detailed narration, but implementation method of the present invention is flexible, is not limited only to this routine described concrete operations mode.
Embodiment 1
(1) (the stem segment length is 0.7～1.3cm) to place the container that the 100mL treatment fluid is housed, and it is that 0.1% colchicine and mass percent concentration are 5% auxiliary agent SD (Nantong flying apsaras chemical experiment Co., Ltd produces) that said treatment fluid contains mass percent concentration with the stem section that has axillary-bud or top-bud of 20 pieces of aseptic tissue cultivating seedling of alpine ash Eg5; Be placed on the shaking table with the hunting speed of 100r/min container is airtight, under 40 ℃ of conditions, doubled to handle 12 hours; After disposing, use aseptic water washing stem section,, obtain stem section through doubling to handle until removing the colchicine debris fully;
(2) will through the stem section that doubling to handle and be inoculated in the proliferated culture medium and cultivate, and whenever in the proliferated culture medium that promptly was transferred to new preparation in 18 days, transfer and carry out culture of rootage again after 3 times;
Said proliferated culture medium is made up of following component by mass volume ratio: nitrate of lime 556mg/L, potassium nitrate 500mg/L, magnesium sulfate 370mg/L, potassium dihydrogen phosphate 170mg/L; Calcium chloride 96mg/L, manganese sulphate 22.5mg/L, zinc sulphate 8.6mg/L, boric acid 6.2mg/L, copper sulphate 0.025mg/L, sodium molybdate 0.25mg/L; Ferrous sulfate 27.3mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, phloridzin 2.5mg/L, thiamine hydrochloride 0.1mg/L; Pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, glycine 2.0mg/L; Inositol 100mg/L, vitamin b3 6.0mg/L, kinetin 0.5mg/L; Methyl 0.2mg/L, agar 6800mg/L and sucrose 45000mg/L, surplus is a distilled water.
Said root media is made up of following component by mass volume ratio: potassium nitrate 634mg/L, ammonium nitrate 550mg/L, potassium dihydrogen phosphate 57mg/L, magnesium sulfate 123mg/L; Calcium chloride 147mg/L, KI 0.83mg/L, boric acid 6.2mg/L, manganese sulphate 22.3mg/L; Zinc sulphate 8.6mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L; Disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L; Thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, indolebutyric acid 0.5mg/L; ABT root-inducing powder (production of Beijing Ai Bidi research and development centre) 0.5mg/L, sucrose 30000mg/L and agar 6500mg/L, surplus is a distilled water;
More than operation is all carried out under aseptic condition, to prevent the pollution of pending tissue cultivating seedling.
Embodiment 2
(1) (the stem segment length is 0.7～1.3cm) to place the container that the 100mL treatment fluid is housed, and it is that 0.3% colchicine and mass percent concentration are 8% auxiliary agent SD (Nantong flying apsaras chemical experiment Co., Ltd produces) that said treatment fluid contains mass percent concentration with the stem section that has axillary-bud or top-bud of 30 pieces of aseptic tissue cultivating seedling of alpine ash Eg5; Be placed on the shaking table with the hunting speed of 80r/min container is airtight, under 35 ℃ of conditions, doubled to handle 18 hours; After disposing, use aseptic water washing stem section,, obtain stem section through doubling to handle until removing the colchicine debris fully;
(2) will through the stem section that doubling to handle and be inoculated in the proliferated culture medium and cultivate, and whenever in the proliferated culture medium that promptly was transferred to new preparation in 20 days, transfer and carry out culture of rootage again after 4 times;
Said proliferated culture medium is made up of following component by mass volume ratio: nitrate of lime 537mg/L, potassium nitrate 480mg/L, magnesium sulfate 355mg/L, potassium dihydrogen phosphate 150mg/L; Calcium chloride 77mg/L, manganese sulphate 20.2mg/L, zinc sulphate 7.7mg/L, boric acid 5.4mg/L; Copper sulphate 0.018mg/L, sodium molybdate 0.19mg/L, ferrous sulfate 25.3mg/L, disodium ethylene diamine tetraacetate 35.3mg/L; Phloridzin 2mg/L, thiamine hydrochloride 0.092mg/L, pyridoxine hydrochloride 0.41mg/L, nicotinic acid 0.39mg/L; Glycine 1.5mg/L, inositol 91mg/L, vitamin b3 5.0mg/L, kinetin 0.4mg/L; Methyl 0.15mg/L, agar 6100mg/L and sucrose 30000mg/L, surplus is a distilled water;
The root media that said culture of rootage adopts is made up of following component by mass volume ratio: potassium nitrate 614mg/L, ammonium nitrate 530mg/L, potassium dihydrogen phosphate 47mg/L, magnesium sulfate 113mg/L; Calcium chloride 137mg/L, KI 0.8mg/L, boric acid 5.2mg/L, manganese sulphate 20.3mg/L; Zinc sulphate 7.6mg/L, sodium molybdate 0.2mg/L, copper sulphate 0.02mg/L, cobalt chloride 0.02mg/L; Disodium ethylene diamine tetraacetate 34.3mg/L, ferrous sulfate 25.8mg/L, inositol 90mg/L, glycine 1.6mg/L; Thiamine hydrochloride 0.08mg/L, puridoxine hydrochloride 0.4mg/L, nicotinic acid 0.4mg/L, indolebutyric acid 0.42mg/L; ABT root-inducing powder (production of Beijing Ai Bidi research and development centre) 0.39mg/L, sucrose 25000mg/L and agar 6100mg/L, surplus is a distilled water;
More than operation is all carried out under aseptic condition, to prevent the pollution of pending tissue cultivating seedling.
Embodiment 3
(1) (the stem segment length is 0.7～1.3cm) to place the container that the 100mL treatment fluid is housed, and it is that 0.5% colchicine and mass percent concentration are 12% auxiliary agent SD (Nantong flying apsaras chemical experiment Co., Ltd produces) that said treatment fluid contains mass percent concentration with the stem section that has axillary-bud or top-bud of 25 pieces of aseptic tissue cultivating seedling of alpine ash Eg5; Be placed on the shaking table with the hunting speed of 120r/min container is airtight, under 38 ℃ of conditions, doubled to handle 15 hours; After disposing, use aseptic water washing stem section,, obtain stem section through doubling to handle until removing the colchicine debris fully;
(2) will through the stem section that doubling to handle and be inoculated in the proliferated culture medium and cultivate, and whenever in the proliferated culture medium that promptly was transferred to new preparation in 15 days, transfer and carry out culture of rootage again after 5 times;
Said proliferated culture medium is made up of following component by mass volume ratio: nitrate of lime 576mg/L, potassium nitrate 520mg/L, magnesium sulfate 390mg/L, potassium dihydrogen phosphate 190mg/L; Calcium chloride 116mg/L, manganese sulphate 24.5mg/L, zinc sulphate 11.6mg/L, boric acid 7.2mg/L; Copper sulphate 0.029mg/L, sodium molybdate 0.38mg/L, ferrous sulfate 29.4mg/L, disodium ethylene diamine tetraacetate 38.6mg/L; Phloridzin 4mg/L, thiamine hydrochloride 0.112mg/L, pyridoxine hydrochloride 0.58mg/L, nicotinic acid 0.61mg/L; Glycine 2.4mg/L, inositol 112mg/L, vitamin b3 7.0mg/L, kinetin 0.6mg/L; Methyl 0.25mg/L, agar 6800mg/L and sucrose 45000mg/L, surplus is a distilled water;
The root media that said culture of rootage adopts is made up of following component by mass volume ratio: potassium nitrate 654mg/L, ammonium nitrate 570mg/L, potassium dihydrogen phosphate 67mg/L, magnesium sulfate 133mg/L; Calcium chloride 157mg/L, KI 0.9mg/L, boric acid 7.2mg/L, manganese sulphate 24.5mg/L; Zinc sulphate 9.4mg/L, sodium molybdate 0.3mg/L, copper sulphate 0.03mg/L, cobalt chloride 0.03mg/L; Disodium ethylene diamine tetraacetate 40.3mg/L, ferrous sulfate 29.8mg/L, inositol 110mg/L, glycine 2.6mg/L; Thiamine hydrochloride 0.12mg/L, puridoxine hydrochloride 0.6mg/L, nicotinic acid 0.6mg/L, indolebutyric acid 0.62mg/L; ABT root-inducing powder (production of Beijing Ai Bidi research and development centre) 0.69mg/L, sucrose 40000mg/L and agar 6800mg/L, surplus is a distilled water;
More than operation is all carried out under aseptic condition, to prevent the pollution of pending tissue cultivating seedling.
Embodiment 4
Alpine ash seedling after the culture of rootage of embodiment 1 gained is carried out field-transplanting; After growing up to the high plant of 20～30cm, observe form morph (this modal variation has: with compare without the plant of inducing, it is big by 15%～40% that leaf area becomes; Dark green leaf color, blade thickening 5%～10%; Stem chap 7%～14%, internode shorten 10%～22%) plant, get its newborn young tender tip of a root and the stem apex on top and carry out following processing and microexamination:
Stem tip chromosome compressing tablet and counting method: 9:00 takes off the growing point on young tender stem top the morning, and places 18 ℃ immediately, and preliminary treatment is 3 hours in the oxine solution of 0.002mol/L; Use distilled water to clean 3 times, use ethanol again: the Ka Nuoshi fixer of glacial acetic acid=3: 1 volume ratio fixing 10h under 4 ℃ of conditions, take out the back with distilled water cleaning 3 times; Place the hydrochloric acid solution of 1mol/L under 60 ℃ of conditions, to dissociate 12 minutes, use distilled water to clean 5～6 times, use carbolfuchsin dyeing compressing tablet after 15 minutes; Use the optical microphotograph lens to observe chromosome number; The result is as shown in Figure 1, the microscopic examination figure of a for counting without the stem tip chromosome of 2 times of body plant inducing processing, and b is the microscopic examination figure that induces the stem tip chromosome of the plant of processing to count through the present invention; Can be observed chromosome and increase doubly, be 4 times of bodies.
Root tip chromosomes compressing tablet and counting method: the 12:00 tip of a root meristematic zone foremost of getting plant to be measured partly used 0.2% colchicine preliminary treatment 2 hours at noon, then, used the Camoy fixer fixedly 15-18 hour; The dilute hydrochloric acid solution that places 1mol/L then, places on the slide 60 ℃ of water bath with thermostatic control acidifyings 6 minutes; Drip the pinkish red dye liquor dyeing of improvement carbolic acid after 30 minutes, covered uses erasing rubber to rap cover plate; Then, use the optical perspective microscope to amplify 1000 times and observe chromosome number, the result is as shown in Figure 2; The microscopic examination figure of a for counting without the root tip chromosomes of 2 times of body plant inducing processing; B is the microscopic examination figure that induces the root tip chromosomes of the plant of processing to count through the present invention, can be observed chromosome to increase doubly, is 4 times of bodies.The preparation method of the improvement carbolic acid fuchsin solution described in the preceding text is: 1) preparation stoste A: get in the alcohol that the 3g basic fuchsin is dissolved in 100mL volume fraction 70%, but long preservation; 2) preparation stoste B: getting 10mL stoste A, to add the 90mL volume fraction be 5% aqua carbolisata solution; 3) preparation dyeing liquor: get the formaldehyde that 45mL stoste B adds 6mL glacial acetic acid and 6mL volume fraction 37%; 4) preparation improvement carbolfuchsin solution: get above-mentioned dyeing liquor 2～10mL, add the acetic acid and the 1.8g sorbierite of the volume fraction 45% of 90～98mL; 5) gained improvement carbolic acid fuchsin solution dye liquor is placed the back use of 2 weeks.
Embodiment 5 uses flow cytometer to confirm chromosome doubling:
Operating procedure was divided into for 3 steps:
(1) cell nucleus suspension preparation: the tender stem and the spire that take by weighing the alpine ash seedling after the culture of rootage of 100～500mg embodiment, 1 gained; In dripping the culture dish that 2ml extraction buffer solution is arranged, grind; 260 order nylon mesh screens filter; Centrifugal and rinsing is 3 times under the 1000r/min condition, collects deposition of cells, in order to dyeing.Said extracted buffer solution composition: 15mmol/L Tris-HCl (the pH value is 7.5), 80mmol/L KCl, 20mmol/L NaCl, 0.20mol/L EDTA-Na
2, the mercaptoethanol of 15mmol/L and 0.05% TritonX-100.
(2) chromosomal specific stain: discard supernatant liquor after the nuclear suspension of preparation is centrifugal, add 2mL dyeing buffer solution, dark normal temperature condition is dyeing 30min down.500 order nylon mesh screens filter, and after filtrating was collected with the special-purpose standard test tube of flow cytometer, the machine of going up was immediately measured.Above-mentioned dyeing buffer solution composition: extract buffer solution+3000U/mLRNA enzyme A+10ug/mL PI (ethidium bromide).
(3) use U.S. BECTON-DICKINSON company, model is that the flow cytometer of Racs vantageSE DIVA carries out ploidy mensuration.Use the dna content of dliploid alpine ash clone Eg5 to be contrast.The excitation source of this flow cytometer can excite the chromosomal DNA molecule through fluorescent staining to send fluorescence; Then; Determinator detects the fluorescence intensity that inspires; The Computer Analysis software that links to each other with determinator can be analyzed fluorescence intensity, draws the corresponding relation between fluorescence intensity and plant DNA relative amount.
Through detect finding, the DNA relative amount of cell is 2 times of adjoining tree DNA relative amount in the tetraploid plant to be measured.Can confirm that thus alpine ash seedling to be measured is a tetraploid plant.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. set up inducing and the method for cultivation system of alpine ash tetraploid plant for one kind, it is characterized in that comprising following operating procedure:
(1) the stem section that the aseptic tissue cultivating seedling of alpine ash is had an axillary-bud or top-bud places the container that treatment fluid is housed, and it is that 0.1～0.5% colchicine and mass percent concentration are 5～12% auxiliary agent SD that said treatment fluid contains mass percent concentration; Be placed on the shaking table with the hunting speed of 80～120r/min container is airtight, under 35～40 ℃ of conditions, doubled to handle 12～18 hours; After disposing, use aseptic water washing stem section,, obtain stem section through doubling to handle until removing the colchicine debris fully;
(2) will through the stem section that doubling to handle and be inoculated in the proliferated culture medium and cultivate, and whenever in the proliferated culture medium that promptly was transferred to new preparation in 15～20 days, transfer and carry out culture of rootage again after 3～5 times;
Said proliferated culture medium is made up of following component by mass volume ratio: nitrate of lime 537～576mg/L, potassium nitrate 480～520mg/L, magnesium sulfate 355～390mg/L, potassium dihydrogen phosphate 150～190mg/L; Calcium chloride 77～116mg/L, manganese sulphate 20.2～24.5mg/L, zinc sulphate 7.7～11.6mg/L, boric acid 5.4～7.2mg/L; Copper sulphate 0.018～0.029mg/L, sodium molybdate 0.19～0.38mg/L, ferrous sulfate 25.3～29.4mg/L; Disodium ethylene diamine tetraacetate 35.3～38.6mg/L, phloridzin 2～4mg/L, thiamine hydrochloride 0.092～0.112mg/L; Pyridoxine hydrochloride 0.41～0.58mg/L, nicotinic acid 0.39～0.61mg/L, glycine 1.5～2.4mg/L; Inositol 91～112mg/L, vitamin b3 5.0～7.0mg/L, kinetin 0.4～0.6mg/L; Methyl 0.15～0.25mg/L, agar 6100～6800mg/L and sucrose 30000～45000mg/L, surplus is a distilled water;
Said step (1) and step (2) are under aseptic condition, to operate;
The medium that the said culture of rootage of step (2) adopts is made up of following component by mass volume ratio: potassium nitrate 614～654mg/L, ammonium nitrate 530～570mg/L, potassium dihydrogen phosphate 47～67mg/L, magnesium sulfate 113～133mg/L; Calcium chloride 137～157mg/L, KI 0.8～0.9mg/L, boric acid 5.2～7.2mg/L, manganese sulphate 20.3～24.5mg/L; Zinc sulphate 7.6～9.4mg/L, sodium molybdate 0.2～0.3mg/L, copper sulphate 0.02～0.03mg/L; Cobalt chloride 0.02～0.03mg/L, disodium ethylene diamine tetraacetate 34.3～40.3mg/L, ferrous sulfate 25.8～29.8mg/L; Inositol 90～110mg/L, glycine 1.6～2.6mg/L, thiamine hydrochloride 0.08～0.12mg/L; Puridoxine hydrochloride 0.4～0.6mg/L, nicotinic acid 0.4～0.6mg/L, indolebutyric acid 0.42～0.62mg/L; ABT root-inducing powder 0.39～0.69mg/L, sucrose 25000～40000mg/L and agar 6100～6800mg/L, surplus is a distilled water.
2. a kind of inducing and the method for cultivation system of alpine ash tetraploid plant of setting up according to claim 1, it is characterized in that: the length of the said stem section of step (1) is 0.7～1.3cm.
3. a kind of inducing and the method for cultivation system of alpine ash tetraploid plant of setting up according to claim 1, it is characterized in that: the consumption of the said treatment fluid of step (1) is that every 100mL treatment fluid is handled 20～30 pieces of stem sections.
4. a kind of inducing and the method for cultivation system of alpine ash tetraploid plant of setting up according to claim 3, it is characterized in that: said proliferated culture medium is made up of following component by mass volume ratio: nitrate of lime 556mg/L, potassium nitrate 500mg/L, magnesium sulfate 370mg/L; Potassium dihydrogen phosphate 170mg/L, calcium chloride 96mg/L, manganese sulphate 22.5mg/L, zinc sulphate 8.6mg/L, boric acid 6.2mg/L, copper sulphate 0.025mg/L; Sodium molybdate 0.25mg/L, ferrous sulfate 27.3mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, phloridzin 2.5mg/L; Thiamine hydrochloride 0.10mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, glycine 2.0mg/L; Inositol 100mg/L, vitamin b3 6.0mg/L, kinetin 0.5mg/L; Methyl 0.2mg/L, agar 6800mg/L and sucrose 45000mg/L, surplus is a distilled water.
5. a kind of inducing and the method for cultivation system of alpine ash tetraploid plant of setting up according to claim 1 is characterized in that: the said alpine ash of step (1) is authorization kind Eg5 clone.
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| CN102823504B (en) * | 2012-09-26 | 2013-05-29 | 钦州市林业科学研究所 | Eucalypt tissue culture medium |
| CN103548688A (en) * | 2013-11-01 | 2014-02-05 | 重庆文理学院 | Polyploid breeding method for eucalyptus camaldulensis 201-2 |
| CN105123754A (en) * | 2015-08-13 | 2015-12-09 | 宁夏大学 | Nursery stock vitality protection agent |
| CN108935086A (en) * | 2018-08-14 | 2018-12-07 | 吉林农业科技学院 | A kind of chemical seed-soaking liquid and preparation method thereof for corn doubling monoploids |
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