CN104542281A - Tissue culture and propagation method of viburnum tinus - Google Patents
Tissue culture and propagation method of viburnum tinus Download PDFInfo
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Abstract
The invention provides a tissue culture and propagation method of viburnum tinus. The tissue culture and propagation method comprises the following steps: selection and treatment of explants: selecting stem segments with axillary buds of viburnum tinus as culture materials, rinsing with water, performing surface sterilization with alcohol on a super clean bench, rinsing with sterile water, sterilizing with mercuric chloride, rinsing with sterile water, and inoculating in an induction medium; induction culture: taking an MS medium to be mixed with 6-benzylamino adenine, naphthylacetic acid, sucrose and agar, using the mixture I as the induction medium, and inoculating the selected stem segments with axillary buds of viburnum tinus in the induction medium; proliferation culture: transferring the explants growing in the induction medium to the induction medium formed by mixing the MS medium with 6-benzylamino adenine, naphthylacetic acid, sucrose and agar, and performing continuous culture; rooting culture: selecting 1/2 MS medium to be mixed with indolebutyric acid, activated carbon, sucrose and agar, and using the mixtureII as a rooting medium; transplanting: taking out the rooted plants, and transplanting into a mixed medium containing humus soil, cinders and sawdust.
Description
Technical field
The present invention relates to technical field of agriculture, more specifically, particularly a kind of tissue culture propagation method of Mediterranean Jia Opulus (Viburnum tinus).
Background technology
Mediterranean Jia Opulus tree crown is spherical in shape, and crown diameter can reach 2.5 ~ 3 meters.Elliptic leaf shape, bottle green, leaf is long 10 centimetres, cyme, Dan Hua little, only 0.6 centimetre, bud pink, and bud stage is very long, can reach 5 wheat harvesting periods, floral white after in full bloom, and whole inflorescence diameter reaches 10 centimetres, the florescence in original producton location from November until next April in spring.The yellow green bud just tiny as seen at the beginning of 10 months in District of Shanghai, along with the elongation of inflorescence, bud is more and more intensive is covered in branch top, color is also progressively deepened in blackish red look, look far into the distance as blocks of red cloud, float on cyan tree crown, especially noticeable, for the winter adds warmness and anger.Full-bloom stage is at mid or late March, and the bud as red cloud bursts forth into snow-white a slice, cuts a conspicuous figure in the garden with hundreds of flowers of springtime.Fruit is avette, deep blue-black, 0.6 centimetre, footpath.Mediterranean Jia Opulus, is easier to break up bud, and 1 to two years raw treelets are common to bloom.If suitably control to nourish and grow, it also can be made at summer or fall flowering, and mass-planting then commonly in 1 year can have colored plant.Fast, with luxuriant foliage and spreading branches in leafy profusion, resistance to pruning, is suitable for making hedgerow, also can plant and view and admire in flower garden, is evergreen shrubs rare in the decorative flower in winter of Yangtze River Delta Region in growth.
But the quickly tissue culture propagation method of effective Mediterranean pod Opulus is not yet disclosed at present.
Summary of the invention
The present invention is intended at least solve one of Problems existing in background technology.
An object of the present invention is the tissue culture propagation method providing a kind of Mediterranean Jia Opulus, comprises the steps: step 101: choosing and process of explant: choose Mediterranean Jia Opulus stem segment with axillary bud as culture materials, with running water; Super-clean bench is used alcohol surface sterilizing, aseptic water washing, then use mercury chloride sterilizing, then use aseptic water washing, be inoculated in inducing culture; Step 102: Fiber differentiation: as inducing culture after using MS medium, 6-benzyl aminoadenine, methyl α-naphthyl acetate, sucrose, agar to mix, selected Mediterranean Jia Opulus stem segment with axillary bud is inoculated in inducing culture; Step 103: Multiplying culture: the explant be grown on inducing culture is transferred in MS medium, 6-benzyl aminoadenine, methyl α-naphthyl acetate, sucrose, agar and carry out continuous culture as inducing culture after mixing; Step 104: culture of rootage: as root media after selecting 1/2MS medium, indolebutyric acid, active carbon, sucrose, agar to mix; Step 105: transplant: the plant taken root is taken out, is transplanted to detritus soil: breeze: on wood sawdust mixed-matrix.
Further, be 20-30 minute with washing time in described step 101, alcohol ratio is 70%, is 30 seconds with the surface sterilizing time, and mercury chloride ratio is 0.1%, is 4-12 minute with the time of mercury chloride sterilizing.
Further, in described step 102, the ratio of 6-benzyl aminoadenine is 0.5-2.0mg/L, and the ratio of methyl α-naphthyl acetate is 0.1-0.5mg/L, and sucrose ratio is 2%, and agar ratio is 0.6%, and the pH value of inducing culture is 5.8.
Further, in described step 103, the ratio of 6-benzyl aminoadenine is 0.1-0.5mg/L, and methyl α-naphthyl acetate ratio is 0.1-0.3mg/L, and sucrose ratio is 2%, and agar ratio is 0.6%.
Further, the condition of culture of the medium of described step 104: light intensity is 1500-2000lx, temperature is 23 to 27 DEG C, light application time is 12h/d, indolebutyric acid ratio is 0.1-2.0mg/L, and active carbon ratio is 0.1-0.5%, and sucrose ratio is 2%, agar ratio is 0.6%, and the pH value of root media is 5.8.
Further, detritus soil in described step 105: breeze: the ratio of wood sawdust is 3: 1: 1.
Beneficial effect of the present invention is: the production of (1) Mediterranean Jia Opulus can be carried out under manual control condition, not by the impact of the factors such as season, weather conditions, soil, damage by disease and insect and agricultural chemicals, and the strict quality controlling Mediterranean Jia Opulus nursery stock.(2) growth rate is fast, with short production cycle, and equipment is simple, and floor space is few, saves man power and material etc., is convenient to factorial praluction.(3) in tissue culture procedures, carried out detoxification treatment, improve the quality of Mediterranean Jia Opulus nursery stock, this technical method solves Mediterranean Jia Opulus Fast-propagation and stable important technical links of taking root thereof simultaneously, can be applicable to factorial praluction.(4) Mediterranean Jia Opulus germ plasm resource can be preserved, enrich Landscape Trees and resource thereof.
Accompanying drawing explanation
Figure 1 shows that the flow chart of the tissue culture propagation method of a kind of Mediterranean of embodiment of the present invention Jia Opulus.
Embodiment
Hereafter will describe the present invention in detail in conjunction with specific embodiments.It should be noted that the combination of technical characteristic or the technical characteristic described in following embodiment should not be considered to isolated, they can mutually be combined thus be reached better technique effect.
Figure 1 shows that the flow chart of the tissue culture propagation method of a kind of Mediterranean of embodiment of the present invention Jia Opulus.
The tissue culture propagation method of the Mediterranean Jia Opulus that the embodiment of the present invention provides, comprises the steps:
Step 101: choosing and process of explant: choose Mediterranean Jia Opulus stem segment with axillary bud as culture materials, with running water; Super-clean bench is used alcohol surface sterilizing, aseptic water washing, then use mercury chloride sterilizing, then use aseptic water washing, be inoculated in inducing culture;
Step 102: Fiber differentiation: as inducing culture after using MS medium, 6-benzyl aminoadenine, methyl α-naphthyl acetate, sucrose, agar to mix, selected Mediterranean Jia Opulus stem segment with axillary bud is inoculated in inducing culture;
Step 103: Multiplying culture: the explant be grown on inducing culture is transferred in MS medium, 6-benzyl aminoadenine, methyl α-naphthyl acetate, sucrose, agar and carry out continuous culture as inducing culture after mixing;
Step 104: culture of rootage: as root media after selecting 1/2MS medium, indolebutyric acid, active carbon, sucrose, agar to mix;
Step 105: transplant: the plant taken root is taken out, is transplanted to detritus soil: breeze: on wood sawdust mixed-matrix.
Further, be 20-30 minute with washing time in described step 101, alcohol ratio is 70%, is 30 seconds with the surface sterilizing time, and mercury chloride ratio is 0.1%, is 4-12 minute with the time of mercury chloride sterilizing.
In the present invention, in described step 102, the ratio of 6-benzyl aminoadenine is 0.5-2.0mg/L, and the ratio of methyl α-naphthyl acetate is 0.1-0.5mg/L, and sucrose ratio is 2%, and agar ratio is 0.6%, and the pH value of inducing culture is 5.8.In described step 103, the ratio of 6-benzyl aminoadenine is 0.1-0.5mg/L, and methyl α-naphthyl acetate ratio is 0.1-0.3mg/L, and sucrose ratio is 2%, and agar ratio is 0.6%.The condition of culture of the medium of described step 104: light intensity is 1500-2000lx, temperature is 23 to 27 DEG C, light application time is 12h/d, indolebutyric acid ratio is 0.1-2.0mg/L, active carbon ratio is 0.1-0.5%, sucrose ratio is 2%, and agar ratio is 0.6%, and the pH value of root media is 5.8.Detritus soil in described step 105: breeze: the ratio of wood sawdust is 3: 1: 1.
Following object lesson of enumerating is described in detail:
First, for the startup of Mediterranean Jia Opulus in-vitro propagate and the process of Multiplying culture as follows:
(1) the choosing and process of explant
From plant, the branch of clip band axillalry bud, is cut into about 1cm stem segment with axillary bud, with running water 20 ~ 30min.With 70% alcohol surface sterilizing 30s on super-clean bench, aseptic water washing 1 time, then 0.1% mercury chloride sterilizing is used, process 4 respectively, 6,8,10,12min, then use aseptic water washing 5 times, be seeded in MS medium (containing sucrose 2%, agar 0.6%, pH5.8), each process inoculation 50 bottles, every bottle graft 1 explant.The lethality that 30d " Invest, Then Investigate " respectively processes, pollution rate and survival rate.Explant number/inoculation explant number × 100% of lethality (%)=death; Explant number/inoculation explant number × 100% of pollution rate (%)=pollution; Explant number/inoculation explant number × 100% of survival rate (%)=survive.
(2) Primary culture
Choose about 1cm stem segment with axillary bud for examination material.With MS medium for minimal medium, additional following hormone concentration (unit: mg/L) combination respectively: the fast 0.5+ methyl α-naphthyl acetate 0.1 of the amino gland of 6-benzyl; 6-benzyl aminoadenine 0.5+ methyl α-naphthyl acetate 0.3; 6-benzyl aminoadenine 0.5+ methyl α-naphthyl acetate 0.5; 6-benzyl aminoadenine 1.0+ methyl α-naphthyl acetate 0.1; 6-benzyl aminoadenine 1.0+ methyl α-naphthyl acetate 0.3; 6-benzyl aminoadenine 1.0+ methyl α-naphthyl acetate 0.5; 6-benzyl aminoadenine 2.0+ methyl α-naphthyl acetate 0.1; 6-benzyl aminoadenine 2.0+ methyl α-naphthyl acetate 0.3; 6-benzyl aminoadenine 2.0+ methyl α-naphthyl acetate 0.5.Often kind of medium all adds sucrose 2%, agar 0.6%, pH5.8.Often process inoculation 50 bottles, every bottle graft 1 explant, adds up the upgrowth situation of axillary bud sprouting rate and bud after 30d.Explant number × 100% of the explant number/inoculation of axillary bud sprouting rate=axillary bud sprouting.
(3) Multiplying culture
Choosing the sprout that Primary culture obtains is examination material.With MS medium for minimal medium, additional following hormone concentration (unit: mg/L) combination respectively: 6-benzyl aminoadenine 0.1+ methyl α-naphthyl acetate 0.1; 6-benzyl aminoadenine 0.1+ methyl α-naphthyl acetate 0.2; 6-benzyl aminoadenine 0.1+ methyl α-naphthyl acetate 0.3; 6-benzyl aminoadenine 0.3+ methyl α-naphthyl acetate 0.1; 6-benzyl aminoadenine 0.3+ methyl α-naphthyl acetate 0.2; 6-benzyl aminoadenine 0.3+ methyl α-naphthyl acetate 0.3; 6-benzyl aminoadenine 0.5+ methyl α-naphthyl acetate 0.1; 6-benzyl aminoadenine 0.5+ methyl α-naphthyl acetate 0.2; 6-benzyl aminoadenine 0.5+ methyl α-naphthyl acetate 0.3.Often kind of medium all adds sucrose 2%, agar 0.6%, pH5.8.Often process inoculation 30 bottles, every bottle graft 1, adds up growth coefficient after 30d, and observes indefinite bud growing state.Sprout number × 100% of growth coefficient=indefinite bud sum/inoculation.The condition of culture of above-mentioned medium: light intensity is about 1500lx, temperature (25 ± 2) DEG C, light application time 12h/d.
Result of the test about above-mentioned cultivation:
Result of study shows, best with 0.1% mercury chloride sterilization 8min effect, its survival rate is 70%; Best axillalry bud Primary culture base is medium+6-benzyl aminoadenine 1.0mg/L+ methyl α-naphthyl acetate 0.3mg/L+ sucrose 2%+ agar 0.6%, and axillary bud sprouting rate is 90%; The medium being conducive to most adventitious bud proliferation is MS medium+6-benzyl aminoadenine 0.5mg/L+ methyl α-naphthyl acetate 0.2mg/L+ sucrose 2%+ agar 0.6%, and cultivate 30d, growth coefficient is up to 4.57.
Secondly, experiment material used in the present invention, experimental design, and experimental result is as follows:
(1) experiment material
With Mediterranean Jia Opulus unrooted plantlet in vitro for material, height of seedling is about 3cm, robust growth, and leaf look dark green.
(2) experimental design
The impact that medium is taken root on Mediterranean pod Opulus plantlet in vitro: uniform Mediterranean pod Opulus plantlet in vitro is inoculated on medium, 1/2MS medium and 1/4MS medium respectively, 0.6% agar and 2% sucrose is all added in medium, pH is 5.8, in temperature (25 ± 2) DEG C, cultivate under illumination 12h/d, light intensity 1500-2000lx condition (lower same).Often process inoculation plantlet in vitro 15, arranges 3 repetitions.Record is taken root the time started, cultivates after 25d, calculates and often processes average rooting rate (seedling number of taking root/inoculation seedling number), on average to take root the average length of several (sum of taking root/inoculation seedling number) and root.
The impact that indolebutyric acid concentration is taken root on Mediterranean pod Opulus plantlet in vitro: with 1/2MS medium for minimal medium, add 0 wherein respectively, 0.1,0.5,1.0,1.5, the indolebutyric acid of 2.0mg/L, inoculate uniform Mediterranean Jia Opulus plantlet in vitro after sterilizing and cultivate.Often process inoculation plantlet in vitro 15, arranges 3 repetitions.Record is taken root the time started, cultivates after 25d, calculates and often processes average rooting rate, on average to take root the average length of number and root.
The impact that active carbon is taken root on Mediterranean pod Opulus plantlet in vitro: with 1/2MS medium for minimal medium, the indolebutyric acid solution of 1.0mg/L is added in medium, add the active carbon of 0,0.1%, 0.3% and 0.5% simultaneously wherein respectively, inoculate uniform Mediterranean Jia Opulus plantlet in vitro after sterilizing and cultivate.Often process inoculation plantlet in vitro 15, arranges 3 repetitions.Record is taken root the time started, cultivates after 25d, calculates and often processes average rooting rate, on average to take root the average length of number and root.
(3) result of the test
Result of study shows, adopt 1/2MS medium to add 1.0mg/L indolebutyric acid and 0.3% active carbon, Mediterranean Jia Opulus rooting efficiency is best, and average rooting rate reaches 95.6%, and number of on average taking root can reach 4.53, and the average length of root is 2.67cm.
Finally, the Mediterranean Jia Opulus cultivated for utilizing aforesaid way, the process of carrying out test-tube plantlet hardening and transplanting is as follows:
Bottle seedling is directly moved into booth hardening 3d from group training room, then opens bottle cap, in bottle, add a small amount of water hardening 2d of people.Healthy seedling after hardening is gone out from bottle inner clip, be placed on after soaking 1 ~ 2h in the carbendazim solution of 0.1% after cleaning medium, transplant on mixed-matrix (detritus soil 60%+ breeze 20%+ wood sawdust 20%), water sufficient water, set up Small plastic shed, build film, regularly ventilate, water and the management such as diseases prevention, survival rate is more than 85%.
Beneficial effect of the present invention: the production of (1) Mediterranean Jia Opulus can be carried out under manual control condition, not by the impact of the factors such as season, weather conditions, soil, damage by disease and insect and agricultural chemicals, the strict quality controlling Mediterranean Jia Opulus nursery stock.(2) growth rate is fast, with short production cycle, and equipment is simple, and floor space is few, saves man power and material etc., is convenient to factorial praluction.(3) in tissue culture procedures, carried out detoxification treatment, improve the quality of Mediterranean Jia Opulus nursery stock, this technical method solves Mediterranean Jia Opulus Fast-propagation and stable important technical links of taking root thereof simultaneously, can be applicable to factorial praluction.(4) Mediterranean Jia Opulus germ plasm resource can be preserved, enrich Landscape Trees and resource thereof.
Although give some embodiments of the present invention, it will be understood by those of skill in the art that without departing from the spirit of the invention herein, can change embodiment herein.Above-described embodiment is exemplary, should using embodiment herein as the restriction of interest field of the present invention.
Claims (6)
1. a tissue culture propagation method of Mediterranean pod Opulus, is characterized in that, comprises the steps:
Step 101: choosing and process of explant: choose Mediterranean Jia Opulus stem segment with axillary bud as culture materials, with running water; Super-clean bench is used alcohol surface sterilizing, aseptic water washing, then use mercury chloride sterilizing, then use aseptic water washing, be inoculated in inducing culture;
Step 102: Fiber differentiation: as inducing culture after using MS medium, 6-benzyl aminoadenine, methyl α-naphthyl acetate, sucrose, agar to mix, selected Mediterranean Jia Opulus stem segment with axillary bud is inoculated in inducing culture;
Step 103: Multiplying culture: the explant be grown on inducing culture is transferred in MS medium, 6-benzyl aminoadenine, methyl α-naphthyl acetate, sucrose, agar and carry out continuous culture as inducing culture after mixing;
Step 104: culture of rootage: as root media after selecting 1/2MS medium, indolebutyric acid, active carbon, sucrose, agar to mix;
Step 105: transplant: the plant taken root is taken out, is transplanted to detritus soil: breeze: on wood sawdust mixed-matrix.
2. the tissue culture propagation method of Mediterranean Jia Opulus as claimed in claim 1, is characterized in that, is 20-30 minute with washing time in described step 101, alcohol ratio is 70%, be 30 seconds with the surface sterilizing time, mercury chloride ratio is 0.1%, is 4-12 minute with the time of mercury chloride sterilizing.
3. the tissue culture propagation method of Mediterranean Jia Opulus as claimed in claim 2, it is characterized in that, in described step 102, the ratio of 6-benzyl aminoadenine is 0.5-2.0mg/L, the ratio of methyl α-naphthyl acetate is 0.1-0.5mg/L, sucrose ratio is 2%, agar ratio is 0.6%, and the pH value of inducing culture is 5.8.
4. the tissue culture propagation method of Mediterranean Jia Opulus as claimed in claim 3, it is characterized in that, in described step 103, the ratio of 6-benzyl aminoadenine is 0.1-0.5mg/L, and methyl α-naphthyl acetate ratio is 0.1-0.3mg/L, and sucrose ratio is 2%, and agar ratio is 0.6%.
5. the tissue culture propagation method of Mediterranean Jia Opulus as claimed in claim 4, it is characterized in that, the condition of culture of the medium of described step 104: light intensity is 1500-20001x, temperature is 23 to 27 DEG C, and light application time is 12h/d, and indolebutyric acid ratio is 0.1-2.0mg/L, active carbon ratio is 0.1-0.5%, sucrose ratio is 2%, and agar ratio is 0.6%, and the pH value of root media is 5.8.
6. the tissue culture propagation method of Mediterranean Jia Opulus as claimed in claim 5, is characterized in that, detritus soil in described step 105: breeze: the ratio of wood sawdust is 3: 1: 1.
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| CN105028152A (en) * | 2015-06-26 | 2015-11-11 | 江苏绿苑园林建设有限公司 | Tender branch cutting and seedling culturing method for evergreen viburnum opulus |
| CN105340756A (en) * | 2015-12-15 | 2016-02-24 | 扬州大学 | Method for Viburnum macrocephalum Fort. f. keteleeri in vitro culture and rapid propagation |
| CN105494097A (en) * | 2015-12-17 | 2016-04-20 | 北华大学 | In-vitro rapid propagation technology of viburnum sargentii koehne |
| CN109156361A (en) * | 2018-10-25 | 2019-01-08 | 吉林农业大学 | A kind of in vitro quick breeding by group culture method of pruning Jia Opulus |
| CN109964814A (en) * | 2018-10-10 | 2019-07-05 | 北华大学 | A kind of rapid propagation in vitro method of warm batten Jia Opulus |
| CN116472963A (en) * | 2023-04-27 | 2023-07-25 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Method for inducing callus by using viburnum sargenti leaves and establishing rapid propagation system |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105028152A (en) * | 2015-06-26 | 2015-11-11 | 江苏绿苑园林建设有限公司 | Tender branch cutting and seedling culturing method for evergreen viburnum opulus |
| CN105340756A (en) * | 2015-12-15 | 2016-02-24 | 扬州大学 | Method for Viburnum macrocephalum Fort. f. keteleeri in vitro culture and rapid propagation |
| CN105494097A (en) * | 2015-12-17 | 2016-04-20 | 北华大学 | In-vitro rapid propagation technology of viburnum sargentii koehne |
| CN109964814A (en) * | 2018-10-10 | 2019-07-05 | 北华大学 | A kind of rapid propagation in vitro method of warm batten Jia Opulus |
| CN109964814B (en) * | 2018-10-10 | 2022-06-24 | 北华大学 | In-vitro rapid propagation method of viburnum sargentii |
| CN109156361A (en) * | 2018-10-25 | 2019-01-08 | 吉林农业大学 | A kind of in vitro quick breeding by group culture method of pruning Jia Opulus |
| CN116472963A (en) * | 2023-04-27 | 2023-07-25 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Method for inducing callus by using viburnum sargenti leaves and establishing rapid propagation system |
| CN116472963B (en) * | 2023-04-27 | 2024-02-09 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Method for inducing callus by using viburnum sargenti leaves and establishing rapid propagation system |
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