CN104396759B - The method that ash tree tissue cultures is bred fast - Google Patents
The method that ash tree tissue cultures is bred fast Download PDFInfo
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- CN104396759B CN104396759B CN201410764750.6A CN201410764750A CN104396759B CN 104396759 B CN104396759 B CN 104396759B CN 201410764750 A CN201410764750 A CN 201410764750A CN 104396759 B CN104396759 B CN 104396759B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a kind of method that ash tree tissue cultures is bred fast, the method is by several steps such as materials disinfection, Primary culture, Proliferation, Differentiation cultivation, culture of rootage, transplanting, strong seedling culture, breed the axillalry bud of ash tree or stem apex into nursery seedling, the survival rate of test-tube seedling transplanting is more than 90%.The method breeds that speed is fast, and can carry out in the anniversary, that significantly can accelerate ash tree breeds process, effectively solves the problem in the required green source of planting seedlings of China's saline land large area greening, significantly can also reduce the cost of greening simultaneously, save public resource.
Description
Technical field
The present invention relates to a kind of method that trees breed, be specifically related to a kind of method that ash tree is bred in cultivation.
Background technology
Ash tree (Fraxinuschinensis), call blue or green bulky wood, white wattle, be Oleaceae Fraxinus deciduous tree, the whole world about has 70 kinds altogether, and there is kind more than 20 in China, mostly is commerical tree species and Landscape Trees, is mainly distributed in Temperate Region in China.Ash tree is with luxuriant foliage and spreading branches in leafy profusion, well developed root system, and rapidly, form is logical straight, and tree-like attractive in appearance, strong adaptability, has good salt-resistance, can plant in saline land, is the important species for saline land greening in growth.
For a long time, ash tree is based on seminal propagation, and because its juvenile stage is long, have obvious solid interval, therefore the breeding cycle is longer, and moreover, seminal propagation also exists many disadvantages such as provenance genetic variation.
There is large-area saline land in the areas such as China coast, the required nursery stock enormous amount of greening, and as the propagation method cycle that the ash tree of excellent salt-resistant plant is current is long, efficiency is low, cannot meet the demand of greening process, and this problems demand solves.
Summary of the invention
The object of this invention is to provide the fast breeding method of a kind of ash tree, the method adopts the axillalry bud of ash tree or stem-tip tissue to carry out cultivation and breeds, and that significantly can accelerate ash tree breeds process, and the survival rate of breeding seedling is more than 90%.
In order to realize object of the present invention, inventor provide following technical scheme.
The method that ash tree tissue cultures is bred fast, comprises following operating procedure:
A, materials disinfection: get ash tree axillalry bud or stem apex, rinse 2-4 hour with circulating water, absorb the water of surface attachment subsequently with filter paper, the alcohol with 75% carries out surface sterilization 20-30 second, then adopt the hypochlorite disinfectant 20-25 minute of 5%, then use aseptic water washing 5-6 time;
B, Primary culture: with the water of sterilized filter paper gettering step A pasteurization material surface attachment, subsequently material is intercepted, be inoculated in Primary culture base, be placed in the transfer room of 22-28 DEG C, illumination cultivation 20-25 days, obtain test-tube plantlet, Primary culture base is prepared from for adding Thidiazuron in 38000 mg/litre MS solid culture mediums;
C, Proliferation, Differentiation are cultivated: the stem section, the stem apex that the test-tube plantlet that step B cultivates are cut into 0.3-0.5 centimetre, be seeded in Proliferation, Differentiation medium and carry out Proliferation, Differentiation cultivation, cultivation cycle is 30-40 days, obtain propagation test-tube plantlet of growing thickly, proliferated culture medium be in the MS solid culture medium of 38000 mg/litre, add lactoalbumin hydrolysate, Thidiazuron, heteroauxin be prepared from;
D, culture of rootage: clip 2.5-3 centimetre of healthy and strong simple bud propagation test-tube plantlet, be inoculated in root media and carry out culture of rootage, cultivation cycle is 20-30 days, obtain seedling of taking root, root media is be prepared from add agar, indolebutyric acid, methyl α-naphthyl acetate in the MS medium of 19000 mg/litre after;
E, transplanting: choose the seedling of taking root with complete, the medium in foundation portion is washed away with clear water, transplant in moistening vermiculite, spray water saturated to vermiculite, be placed in temperature 22-25 DEG C, cultivate under illumination 500-600LX condition, cultivation cycle is 20-25 days, obtains transplanted seedling, in incubation, 1-9 days controlled humidities are that after the >=90%, 10th day, controlled humidity is 75%-85%;
F, strong seedling culture: under transplanted seedling is positioned over room temperature condition, water weekly one time of nutrition liquid, and concentration is 1/2MS nutrient solution or 0.2% urea, and carrying out strong seedling culture is 50-60 days, transplants to nursery.
The method that above-mentioned ash tree tissue cultures is bred fast, axillalry bud described in steps A or stem apex are axillalry bud or the stem apex of current growth.The age of tree and culture success ratio are inversely proportional to, and both the longer culture success ratio of the age of tree was less, the easy cultivation that the age of tree is short, and survival rate is high, so the sprouting of preferably current growth is cultivated.
The method that above-mentioned ash tree tissue cultures is bred fast, the length intercepted described in step B is 0.3-0.5 centimetre.Selected region is less relative to bacterium, and plant cell growth enlivens, and is easy to cultivate and late growing stage.
The method that above-mentioned ash tree tissue cultures is educated fast, the concentration of adding Thidiazuron described in step B is 2 mg/litre.The object of adding Thidiazuron promotes Bud polarization, and prove through test of many times, under this concentration conditions, Bud polarization quantity is many, and tufted seedling quality is good, sturdy, is preferred concentration.
The method that above-mentioned ash tree tissue cultures is bred fast, the concentration of adding lactoalbumin hydrolysate described in step C is 300-500 mg/litre, and the concentration of Thidiazuron is 1.5 mg/litre, heteroauxin 0.5 mg/litre.Add lactoalbumin hydrolysate and can promote that test-tube plantlet is more healthy and stronger.
The method that above-mentioned ash tree tissue cultures is bred fast, the concentration of adding agar described in step D is 5-6 grams per liter, indolebutyric acid 1-1.5 mg/litre, methyl α-naphthyl acetate 0.5-1 mg/litre.
The method that above-mentioned ash tree tissue cultures is bred fast, water content >=90% of vermiculite moistening described in step e.
Healthy and strong simple bud described in step D refers to that growth has 2-4 sheet leaf, and leaf dark green, open and flat simple bud.
Described in step e, complete refers to the long 0.2-0.6 of root centimetre, individual plant takes root the root system of several more than 3.
Select clip 2.5-3 centimetre of high simple bud propagation test-tube plantlet in step D, be consider to be not easy to test-tube seedling transplanting lower than during this value range, affect survival rate, be unfavorable for cultivation stage test-tube plantlet growth higher than this scope, easily occur that young plant is bending.Draw through test of many times, the comparatively suitable culture of rootage of this scope.
In the process of transplanting described in step e, controlled humidity can select the method for covering, can select the transparent plastic seedling disk cover being specifically designed to plant transplantation seedling, be beneficial to the moisturizing of transplanted seedling environment and printing opacity during covering.Test-tube plantlet is from closing completely and taking out aseptic environment, and being transplanted in matrix, is the crucial transitional processes from autotrophy to heterotrophism, humidity is covered more than 90%, after transplanted seedling grows new blade, generally at 7-10 days after transplanting, open covering, humid control is at 75%-85%.
The medium of Proliferation, Differentiation described in step C can reuse 1 time again after clip propagation test-tube plantlet, and concrete grammar is the liquid medium injecting 15-20 milliliter in used Proliferation, Differentiation medium, to ensure that test-tube plantlet proceeds effective differentiation.This liquid medium composition is based on MS, add the sucrose of 10000 mg/litre, the Thidiazuron of 1.5 mg/litre, the 6-benzylaminopurine of 0.5 mg/litre, the lactoalbumin hydrolysate of 300-500 mg/litre wherein, effectively break up cultivation, cultivation cycle 30-40 days, can obtain breeding test-tube plantlet.
The method of the invention is carried out in artificial culture environment, gives the required nutrition fully of plant, illumination, temperature.Do not disturb by natural environment, the anniversary can breed, and reproduction speed is fast, and seedling quality is good, can realize the preservation to ash tree fine germplasm resources and standardization breeding.
The present invention with ash tree axillalry bud or Shoot tip explants for material, set up ash tree tissue-culturing quick-propagation system, the test-tube seedling transplanting survival rate more than 90% cultivated, breeding cycle is short, accelerate the process that ash tree breeds, facilitate the application of biotechnology, effectively solve the problem in the required green source of planting seedlings of China's saline land large area greening.
Embodiment
Below in conjunction with specific embodiment, content of the present invention is further described in detail.
Embodiment
For breeding the plant that the ash tree of drawing materials can be field grown health, such as be grown on the ash tree grown in the heavy saline of Haixing County, Cangzhou, Hebei Province, for the age of tree ash tree of 8 years, choose the sprouting of current growth, remove unnecessary blade and inappropriate part.
A, materials disinfection: get ash tree axillalry bud or the stem apex part with growing point, length optional gets 2-3cm, 2-4 hour is rinsed with the circulating water of cleaning, the water of surface attachment is absorbed subsequently with filter paper, alcohol with 75% carries out surface sterilization 20-30 second, then adopt the hypochlorite disinfectant 20-25 minute of 5%, then use aseptic water washing 5-6 time;
B, Primary culture: with the water of sterilized filter paper gettering step A pasteurization material surface attachment, adopt scalpel (12 centimeter length subsequently, can more allowing blade replacement) cut into axillalry bud or stem apex, be inoculated in Primary culture base, be placed in the transfer room of 22-28 DEG C, illumination cultivation 20-25 days, obtains test-tube plantlet, and Primary culture base is prepared from for adding Thidiazuron 2 mg/litre in 38000 mg/litre MS solid culture mediums;
C, Proliferation, Differentiation are cultivated: the stem section, the stem apex that the test-tube plantlet that step B cultivates are cut into 0.3-0.5 centimetre, be seeded in Proliferation, Differentiation medium and carry out Proliferation, Differentiation cultivation, cultivation cycle is 30-40 days, obtain breeding test-tube plantlet, proliferated culture medium be in the MS solid culture medium of 38000 mg/litre, add lactoalbumin hydrolysate 300-500 mg/litre, Thidiazuron 1.5 mg/litre, heteroauxin 0.5 mg/litre be prepared from;
D, culture of rootage: clip 2.5-3 centimetre of healthy and strong simple bud propagation test-tube plantlet, be inoculated in root media and carry out culture of rootage, cultivation cycle is 20-30 days, obtain seedling of taking root, root media be in the MS medium of 19000 mg/litre, add agar 5-6 gram/L, indolebutyric acid 1-1.5 mg/litre, methyl α-naphthyl acetate 0.5-1 mg/litre be prepared from;
E, transplanting: the Miao Panzhong new vermiculite being loaded 35 caves, then water content more than 90% is reached with the moistening vermiculite of running water, seedling of taking root is taken out gently from culture vessel, choose the seedling of taking root with complete, the medium in foundation portion is washed away with clear water, transplant in moistening vermiculite, spray water saturated to vermiculite, cover with the transparent lid of special plastic, be placed in temperature 22-25 DEG C, cultivate under illumination 500-600LX condition, within 7-10 days, take off lid vinyl cover, controlled humidity is 75%-85%, cultivation cycle is 20-25 days, obtains transplanted seedling;
F, strong seedling culture: under transplanted seedling is positioned over room temperature condition, water weekly one time of nutrition liquid, and nutrient solution is 1/2MS nutrient solution or 0.2% urea, carries out strong seedling culture 50-60 days, and transplanted seedling robust growth, leaf dark green can be transplanted to nursery.
Draw materials three batches altogether, often criticize 100 examples of drawing materials, during transplanting, the transplanting survival rate of gained three batches of test-tube plantlets is respectively 90%, 94%, 95%.
MS culture medium prescription of the present invention is as shown in table 1.
Table 1
Composition (mg/L)
| Potassium nitrate | P0tassium Nitrate | 1900 |
| Potassium nitrate | Ammonium Nitrate | 1650 |
| Potassium dihydrogen phosphate | Potassium Phosphate Monobasic | 170 |
| Magnesium sulfate | Magnesium Sulfate | 370 |
| Calcium chloride | Calcium Chl0ride | 440 |
| Potassium iodide | Potassium Iodide | 0.83 |
| Boric acid | Boic Acid | 6.2 |
| Manganese sulphate | Manganese Sulfate | 20.3 |
| Zinc sulphate | Zinc Sulfate | 8.6 |
| Sodium molybdate | Sodium Molybdate | 0.25 |
| Copper sulphate | Cupric Sulfate | 0.025 |
| Cobalt chloride | Cobalt Chloride.6H2O | 0.025 |
| Sodium ethylene diamine tetracetate | Na2EDTA | 37.3 |
| Ferrous sulfate | Ferrous Suifate | 27.8 |
| Inositol | Myo-inositol | 200 |
| Glycine | Glycin | 2 |
| Thiamine hydrochloride | Thamine.HCl | 0.25 |
| Puridoxine hydrochloride | Pyridoxine.HCl | 0.5 |
| Nicotinic acid | Nicotinic Acid | 0.5 |
| Sucrose | Sucrose | 30000 |
| Agar | Agar | 7000 |
Claims (4)
1. the ash tree tissue cultures method of breeding fast, is characterized in that, comprise following operating procedure:
A, materials disinfection: get ash tree axillalry bud or stem apex, rinse 2-4 hour with circulating water, absorb the water of surface attachment subsequently with filter paper, the alcohol with 75% carries out surface sterilization 20-30 second, then adopt the hypochlorite disinfectant 20-25 minute of 5%, then use aseptic water washing 5-6 time;
B, Primary culture: with the water of sterilized filter paper gettering step A pasteurization material surface attachment, subsequently material is intercepted, be inoculated in Primary culture base, be placed in the transfer room of 22-28 DEG C, illumination cultivation 20-25 days, obtain test-tube plantlet, Primary culture base is prepared from for adding Thidiazuron in 38000mg/LMS solid culture medium;
C, Proliferation, Differentiation are cultivated: the stem section, the stem apex that the test-tube plantlet that step B cultivates are cut into 0.3-0.5 centimetre, be seeded in Proliferation, Differentiation medium and carry out Proliferation, Differentiation cultivation, cultivation cycle is 30-40 days, obtain propagation test-tube plantlet of growing thickly, proliferated culture medium be in the MS solid culture medium of 38000mg/L, add lactoalbumin hydrolysate, Thidiazuron, heteroauxin be prepared from;
D, culture of rootage: clip 2.5-3 centimetre of healthy and strong simple bud propagation test-tube plantlet, be inoculated in root media and carry out culture of rootage, cultivation cycle is 20-30 days, obtain seedling of taking root, root media is be prepared from add agar, indolebutyric acid, methyl α-naphthyl acetate in the MS medium of 19000mg/L after;
E, transplanting: choose the seedling of taking root with complete, the medium in foundation portion is washed away with clear water, transplant in moistening vermiculite, spray water saturated to vermiculite, be placed in temperature 22-25 DEG C, cultivate under illumination 500-600LX condition, cultivation cycle is 20-25 days, obtains transplanted seedling, in incubation, 1-9 days controlled humidities are >=90%, and after the 10th day, controlled humidity is 75%-85%;
F, strong seedling culture: under transplanted seedling is positioned over room temperature condition, water weekly one time of nutrition liquid, and concentration is 1/2MS nutrient solution or 0.2% urea, carries out strong seedling culture 50-60 days, transplants to nursery;
The concentration of adding Thidiazuron described in step B is 2 mg/litre;
The concentration of adding lactoalbumin hydrolysate described in step C is 300-500 mg/litre, and the concentration of Thidiazuron is 1.5 mg/litre, heteroauxin 0.5 mg/litre;
The concentration of adding agar described in step D is 5-6 grams per liter, indolebutyric acid 1-1.5 mg/litre, methyl α-naphthyl acetate 0.5-1 mg/litre.
2. the ash tree tissue cultures according to claim 1 method of breeding fast, it is characterized in that, axillalry bud described in steps A or stem apex are axillalry bud or the stem apex of current growth.
3. the ash tree tissue cultures according to claim 1 method of breeding fast, it is characterized in that, the length intercepted described in step B is 0.3-0.5 centimetre.
4. the ash tree tissue cultures according to claim 1 method of breeding fast, is characterized in that, water content >=90% of vermiculite moistening described in step e.
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| CN107047217B (en) * | 2017-06-09 | 2020-05-26 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Tissue culture seedling transplanting method for woody plant fraxinus chinensis |
| CN109362569A (en) * | 2018-12-11 | 2019-02-22 | 梁伟艺 | A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles |
| CN109757382A (en) * | 2019-03-21 | 2019-05-17 | 鲁东大学 | The rapid propagation method of Chinese wax male elite plant strain |
| CN109984038B (en) * | 2019-04-08 | 2022-04-12 | 鲁东大学 | Culture medium for rapidly promoting simultaneous completion of fraxinus chinensis shoot tip proliferation and rooting and application thereof |
| CN112075340B (en) * | 2019-06-12 | 2021-07-09 | 东北林业大学 | Method for rapidly breaking dormant buds of fraxinus mandshurica tissue culture seedlings and successfully propagating in vitro |
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| CN102197785A (en) * | 2010-03-24 | 2011-09-28 | 辽宁省林业科学研究院 | Medium for rapid breeding and propagation of fraxinus americana tissue culture and application thereof |
| CN102228001A (en) * | 2011-04-28 | 2011-11-02 | 东北林业大学 | Micropropagation method of fraxinus rhynchophylla |
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