CN104542281B - Mediterranean Jia Opulus tissue culture propagation method - Google Patents
Mediterranean Jia Opulus tissue culture propagation method Download PDFInfo
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- CN104542281B CN104542281B CN201410830457.5A CN201410830457A CN104542281B CN 104542281 B CN104542281 B CN 104542281B CN 201410830457 A CN201410830457 A CN 201410830457A CN 104542281 B CN104542281 B CN 104542281B
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Abstract
The invention provides a kind of Mediterranean Jia Opulus tissue culture propagation method, including：The selection and processing of explant：Mediterranean Jia Opulus stem segment with axillary bud is chosen as culture materials, is rinsed with flowing water；Alcohol surface sterilizing is used on super-clean bench, aseptic water washing is then sterilized with mercury chloride, then with aseptic water washing, is inoculated in inducing culture；Fiber differentiation：Using after MS culture mediums, 6 benzyl aminoadenines, methyl α-naphthyl acetate, sucrose, agar mixing as inducing culture, selected Mediterranean Jia Opulus stem segment with axillary bud is inoculated in inducing culture；Multiplying culture：The explant being grown on inducing culture is transferred and continuously cultivated as inducing culture after MS culture mediums, 6 benzyl aminoadenines, methyl α-naphthyl acetate, sucrose, agar are mixed；Culture of rootage：Root media is used as after selection 1/2MS culture mediums, indolebutyric acid, activated carbon, sucrose, agar mixing；Transplant：The plant taken root is taken out, detritus soil is transplanted to：Breeze：On wood sawdust mixed-matrix.
Description
Technical field
The present invention relates to technical field of agriculture, more specifically, more particularly to a kind of Mediterranean Jia Opulus (Viburnum
Tinus tissue culture propagation method).
Background technology
Mediterranean Jia Opulus tree crowns are spherical in shape, and crown diameter is up to 2.5~3 meters.Elliptic leaf shape, bottle green, 10 centimetres of leaf length, poly- umbrella
Inflorescence, single flower is small, only 0.6 centimetre, and bud pink, bud stage is very long, up to 5 wheat harvesting periods, floral white after blooming, whole inflorescence
Diameter is up to 10 centimetres, and the florescence is in original producton location from November until next April in spring.The just visible tiny yellow green at the beginning of District of Shanghai 10 months
Bud, with the elongation of inflorescence, bud is more and more intensive to be covered in branch top, and it is in blackish red color that color, which is also progressively deepened, is looked far into the distance
As blocks of red cloud, float on blackish green tree crown, it is especially noticeable, it is to add warmness and anger in winter.Full-bloom stage
In mid or late March, the bud as red cloud bursts forth into snow-white a piece of, is cut a conspicuous figure in the garden with hundreds of flowers of springtime.It is really avette, it is dark blue black
Color, 0.6 centimetre of footpath.Mediterranean Jia Opulus, are easier to break up bud, one blooms to biennial treelet is common.If suitable control is sought
Health is long, can also make it in summer or fall flowering, and mass-planting then common in 1 year can have colored plant.Growth is quick, and branches and leaves are numerous
Cyclopentadienyl, resistance to trimming, suitable for making hedgerow, can also be planted and viewed and admired in flower garden, be rare in the winter decorative flower of Yangtze River Delta Region
Evergreen shrubs.
But effective Mediterranean pod Opulus quickly tissue culture propagation method is not yet disclosed at present.
The content of the invention
It is contemplated that at least solving one of problem present in background technology.
An object of the present invention is to provide a kind of Mediterranean Jia Opulus tissue culture propagation method, comprised the following steps：Step
Rapid 101：The selection and processing of explant：Mediterranean Jia Opulus stem segment with axillary bud is chosen as culture materials, is rinsed with flowing water；Super
Alcohol surface sterilizing is used on net platform, aseptic water washing is then sterilized with mercury chloride, then with aseptic water washing, is inoculated in induction
In culture medium；Step 102：Fiber differentiation：After MS culture mediums, 6- benzyls aminoadenine, methyl α-naphthyl acetate, sucrose, agar mixing
As inducing culture, selected Mediterranean Jia Opulus stem segment with axillary bud is inoculated in inducing culture；Step 103：Propagation
Culture：The explant being grown on inducing culture is transferred in MS culture mediums, 6- benzyls aminoadenine, methyl α-naphthyl acetate, sucrose, fine jade
Continuously cultivated as inducing culture after fat mixing；Step 104：Culture of rootage：Select 1/2MS culture mediums, indolebutyric acid,
Root media is used as after activated carbon, sucrose, agar mixing；Step 105：Transplant：The plant taken root is taken out, corruption is transplanted to
Matter soil：Breeze：On wood sawdust mixed-matrix.
Further, it is 20-30 minutes with washing time in the step 101, alcohol ratio is 70%, is gone out with surface
The bacterium time is 30 seconds, and mercury chloride ratio is 0.1%, and the time sterilized with mercury chloride is 4-12 minutes.
Further, the ratio of 6- benzyl aminoadenines is 0.5-2.0mg/L, the ratio of methyl α-naphthyl acetate in the step 102
For 0.1-0.5mg/L, sucrose ratio is 2%, and agar ratio is 0.6%, and the pH value of inducing culture is 5.8.
Further, the ratio of 6- benzyl aminoadenines is 0.1-0.5mg/L in the step 103, and methyl α-naphthyl acetate ratio is
0.1-0.3mg/L, sucrose ratio is 2%, and agar ratio is 0.6%.
Further, the condition of culture of the culture medium of the step 104：Light intensity is 1500-2000lx, and temperature is 23 to 27
DEG C, light application time is 12h/d, and indolebutyric acid ratio is 0.1-2.0mg/L, and activated 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 the step 105：Breeze：The ratio of wood sawdust is 3: 1: 1.
The beneficial effects of the present invention are：(1) Mediterranean Jia Opulus production can be carried out under the conditions of manual control, not by
The influence of the factors such as season, weather conditions, soil, pest and disease damage and agricultural chemicals, the quality of strict control Mediterranean Jia Opulus nursery stocks.(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 easy to factorial praluction.(3)
Detoxification treatment has been carried out in tissue culture procedures, the quality of Mediterranean Jia Opulus nursery stocks is improved, while the technical method is solved
The important technical links that Mediterranean Jia Opulus quickly breed and its stably taken root, can be applied to factorial praluction.(4) it can preserve
Mediterranean Jia Opulus germ plasm resources, enrich Landscape Trees and its resource.
Brief description of the drawings
Fig. 1 show a kind of flow chart of Mediterranean Jia Opulus tissue culture propagation method of the embodiment of the present invention.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.It should be noted that the skill described in following embodiments
The combination of art feature or technical characteristic be not construed as it is isolated, they can be mutually combined so as to reach preferably
Technique effect.
Fig. 1 show a kind of flow chart of Mediterranean Jia Opulus tissue culture propagation method of the embodiment of the present invention.
Jia Opulus in Mediterranean provided in an embodiment of the present invention tissue culture propagation method, comprises the following steps：
Step 101：The selection and processing of explant：Mediterranean Jia Opulus stem segment with axillary bud is chosen as culture materials, with stream
Water is rinsed；Alcohol surface sterilizing is used on super-clean bench, aseptic water washing is then sterilized with mercury chloride, then with aseptic water washing,
It is inoculated in inducing culture；
Step 102：Fiber differentiation：Use work after MS culture mediums, 6- benzyls aminoadenine, methyl α-naphthyl acetate, sucrose, agar mixing
For inducing culture, selected Mediterranean Jia Opulus stem segment with axillary bud is inoculated in inducing culture；
Step 103：Multiplying culture：The explant being grown on inducing culture is transferred in MS culture mediums, 6- benzyl amino
Continuously cultivated as inducing culture after adenine, methyl α-naphthyl acetate, sucrose, agar mixing；
Step 104：Culture of rootage：Select conduct after 1/2MS culture mediums, indolebutyric acid, activated carbon, sucrose, agar mixing
Root media；
Step 105：Transplant：The plant taken root is taken out, detritus soil is transplanted to：Breeze：On wood sawdust mixed-matrix.
Further, it is 20-30 minutes with washing time in the step 101, alcohol ratio is 70%, is gone out with surface
The bacterium time is 30 seconds, and mercury chloride ratio is 0.1%, and the time sterilized with mercury chloride is 4-12 minutes.
In the present invention, the ratio of 6- benzyl aminoadenines is 0.5-2.0mg/L, the ratio of methyl α-naphthyl acetate in the step 102
Example 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.The step
The ratio of 6- benzyls aminoadenine is 0.1-0.5mg/L in 103, and methyl α-naphthyl acetate ratio is 0.1-0.3mg/L, and sucrose ratio is 2%,
Agar ratio is 0.6%.The condition of culture of the culture medium of the step 104：Light intensity is 1500-2000lx, and temperature is 23 to 27
DEG C, light application time is 12h/d, and indolebutyric acid ratio is 0.1-2.0mg/L, and activated 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.Detritus soil in the step 105：Breeze：The ratio of wood sawdust
Example is 3: 1: 1.
Specific example will be enumerated below to be described in detail：
Process firstly, for the Mediterranean Jia Opulus startups bred in vitro and Multiplying culture is as follows：
(1) selection and processing of explant
Branch of the clip with axillary bud from plant, is cut into 1cm or so stem segment with axillary bud, and 20~30min is rinsed with flowing water.
With 70% alcohol surface sterilizing 30s on super-clean bench, aseptic water washing 1 time, then with 0.1% mercury chloride sterilize, respectively handle 4,
6th, 8,10,12min, then with aseptic water washing 5 times, is seeded in MS culture mediums (containing sucrose 2%, agar 0.6%, pH5.8),
Each 50 bottles of processing inoculation, every bottle connects 1 explant.The death rate, pollution rate and the survival rate of each processing are investigated after 30d.It is dead
The explant number of rate (%)=death/inoculation explant number × 100%；Outside explant number/inoculation of pollution rate (%)=pollution
Implant number × 100%；Explant number/inoculation explant number × 100% for survival rate (%)=survive.
(2) Primary culture
It is examination material to choose 1cm or so stem segment with axillary bud.Using MS culture mediums as minimal medium, hormone is appended below respectively dense
Spend (unit：Mg/L) combine：The fast 0.5+ methyl α-naphthyl acetates 0.1 of 6- benzyl amino glands；6- benzyl aminoadenine 0.5+ methyl α-naphthyl acetates 0.3；6- benzyls
Aminoadenine 0.5+ methyl α-naphthyl acetates 0.5；6- benzyl aminoadenine 1.0+ methyl α-naphthyl acetates 0.1；6- benzyl aminoadenine 1.0+ methyl α-naphthyl acetates
0.3；6- benzyl aminoadenine 1.0+ methyl α-naphthyl acetates 0.5；6- benzyl aminoadenine 2.0+ methyl α-naphthyl acetates 0.1；6- benzyls aminoadenine 2.0
+ methyl α-naphthyl acetate 0.3；6- benzyl aminoadenine 2.0+ methyl α-naphthyl acetates 0.5.Every kind of culture medium adds sucrose 2%, agar 0.6%,
pH5.8.50 bottles of often processing inoculation, every bottle connects the upgrowth situation that axillary bud sprouting rate and bud are counted after 1 explant, 30d.Axillary bud is sprouted
Explant number × 100% of explant number/inoculation of hair rate=axillary bud sprouting.
(3) Multiplying culture
The sprout that selection Primary culture is obtained is examination material.Using MS culture mediums as minimal medium, hormone is appended below respectively
Concentration (unit：Mg/L) combine：6- benzyl aminoadenine 0.1+ methyl α-naphthyl acetates 0.1；6- benzyl aminoadenine 0.1+ methyl α-naphthyl acetates 0.2；
6- benzyl aminoadenine 0.1+ methyl α-naphthyl acetates 0.3；6- benzyl aminoadenine 0.3+ methyl α-naphthyl acetates 0.1；6- benzyl aminoadenine 0.3+ naphthalenes
Acetic acid 0.2；6- benzyl aminoadenine 0.3+ methyl α-naphthyl acetates 0.3；6- benzyl aminoadenine 0.5+ methyl α-naphthyl acetates 0.1；6- benzyl amino glands are fast
Purine 0.5+ methyl α-naphthyl acetates 0.2；6- benzyl aminoadenine 0.5+ methyl α-naphthyl acetates 0.3.Every kind of culture medium adds sucrose 2%, agar
0.6%, pH5.8.30 bottles of often processing inoculation, every bottle connects 1, growth coefficient is counted after 30d, and observe adventitious bud growing state.
Sprout number × 100% of growth coefficient=adventitious bud sum/inoculation.The condition of culture of above-mentioned culture medium：Light intensity about 1500lx, temperature
Spend (25 ± 2) DEG C, light application time 12h/d.
Result of the test on above-mentioned culture：
Result of study shows that with 0.1% mercury chloride sterilization 8min effects preferably, its survival rate is 70%；Optimal axillary bud is opened
Dynamic culture medium is culture medium+6- benzyl aminoadenine 1.0mg/L+ methyl α-naphthyl acetate 0.3mg/L+ sucrose 2%+ agar 0.6%, and axillary bud is sprouted
Hair rate is 90%；Culture medium most beneficial for adventitious bud proliferation is MS culture medium+6- benzyl aminoadenine 0.5mg/L+ methyl α-naphthyl acetates
0.2mg/L+ sucrose 2%+ agar 0.6%, cultivates 30d, and growth coefficient is up to 4.57.
Secondly, experiment material used in the present invention, experimental design, and experimental result are as follows：
(1) experiment material
Using Mediterranean Jia Opulus unrooteds tissue-cultured seedling as material, height of seedling about 3cm, robust growth, leaf color is dark green.
(2) experimental design
Influence of the culture medium to Mediterranean pod Opulus tissue culture seedling rootings：Uniform Mediterranean Jia Opulus tissue-cultured seedling is inoculated into respectively
On culture medium, 1/2MS culture mediums and 1/4MS culture mediums, 0.6% agar and 2% sucrose are added in culture medium, pH is 5.8, in
Cultivated (similarly hereinafter) under the conditions of temperature (25 ± 2) DEG C, illumination 12h/d, light intensity 1500-2000lx.Often processing inoculation tissue-cultured seedling 15,
3 repetitions are set.Record is taken root the time started, after culture 25d, is calculated and is often handled average rooting rate (rooted seedling number/inoculation seedling
Number), averagely take root number (sum of taking root/inoculation seedling number) and root average length.
Influence of the indolebutyric acid concentration to Mediterranean pod Opulus tissue culture seedling rootings：Using 1/2MS culture mediums as minimal medium,
Wherein respectively addition 0,0.1,0.5,1.0,1.5,2.0mg/L indolebutyric acid, uniform Mediterranean pod Opulus groups are inoculated with after sterilizing
Training seedling is cultivated.Often processing inoculation tissue-cultured seedling 15, sets 3 repetitions.Record is taken root the time started, after culture 25d, is calculated
Often handle average rooting rate, the average length of averagely take root number and root.
Influence of the activated carbon to Mediterranean pod Opulus tissue culture seedling rootings：Using 1/2MS culture mediums as minimal medium, in culture medium
1.0mg/L indolebutyric acid solution is added, while adding 0,0.1%, 0.3% and 0.5% activated carbon, sterilizing respectively wherein
After be inoculated with uniform Mediterranean pod Opulus tissue-cultured seedling and cultivated.Often processing inoculation tissue-cultured seedling 15, sets 3 repetitions.Record life
After root time started, culture 25d, calculate and often handle average rooting rate, the average length of averagely take root number and root.
(3) result of the test
Result of study shows, 1.0mg/L indolebutyric acids and 0.3% activated carbon, Mediterranean pod are added using 1/2MS culture mediums
Preferably, average rooting rate is up to 95.6%, and number of averagely taking root is up to 4.53, and the average length of root is 2.67cm for Opulus rooting efficiencies.
Finally, for the Mediterranean Jia Opulus cultivated using aforesaid way, the process of progress test tube seedling hardening and transplanting is such as
Under：
Bottle seedling is directly moved into greenhouse hardening 3d from tissue culture room, then opens bottle cap, a small amount of water hardening 2d of people is added in bottle.Will
Healthy seedling after hardening goes out from bottle inner clip, cleans to be placed on after culture medium in 0.1% carbendazim solution and soaks after 1~2h, transplants
On mixed-matrix (detritus soil 60%+ breeze 20%+ wood sawdusts 20%), water is poured, Small plastic shed is set up, film is covered, periodically
Divulge information, water and diseases prevention etc. is managed, survival rate is more than 85%.
Beneficial effects of the present invention：(1) Mediterranean Jia Opulus production can be carried out under the conditions of manual control, not by season
The influence of the factors such as section, weather conditions, soil, pest and disease damage and agricultural chemicals, the quality of strict control Mediterranean Jia Opulus nursery stocks.(2) increase
Grow speed fast, with short production cycle, equipment is simple, and floor space is few, saves man power and material etc., is easy to factorial praluction.(3) exist
Detoxification treatment has been carried out in tissue culture procedures, the quality of Mediterranean Jia Opulus nursery stocks is improved, while the technical method is solved
The important technical links that Mediterranean Jia Opulus quickly breed and its stably taken root, can be applied to factorial praluction.(4) it can preserve ground
Middle Hai Jia Opulus germ plasm resources, enrich Landscape Trees and its resource.
Although having been presented for some embodiments of the present invention herein, it will be appreciated by those of skill in the art that
Without departing from the spirit of the invention, the embodiments herein can be changed.Above-described embodiment be it is exemplary, no
The restriction of interest field of the present invention should be used as using the embodiments herein.
Claims (1)
1. a kind of Mediterranean Jia Opulus tissue culture propagation method, it is characterised in that comprise the following steps：
Step 101：The selection and processing of explant：Mediterranean Jia Opulus stem segment with axillary bud is chosen as culture materials, is rushed with flowing water
Wash；Alcohol surface sterilizing is used on super-clean bench, aseptic water washing is then sterilized with mercury chloride, the mercury chloride ratio is
0.1%, then with aseptic water washing, it is inoculated in inducing culture；
Step 102：Fiber differentiation：Use MS culture mediums, 6- benzyl aminoadenines 1.0mg/L, methyl α-naphthyl acetate 0.3mg/L, sucrose 2%
As inducing culture after being mixed with agar 0.6%, selected Mediterranean Jia Opulus stem segment with axillary bud is inoculated in Fiber differentiation
In base；The pH value of inducing culture is 5.8 in the step 102；
Step 103：Multiplying culture：The explant being grown on inducing culture is transferred fast in MS culture mediums, 6- benzyl amino glands
Purine 0.5mg/L, methyl α-naphthyl acetate 0.2mg/L, sucrose 2% and agar 0.6% are continuously cultivated after mixing as inducing culture；
Step 104：Culture of rootage：Select 1/2MS culture mediums, indolebutyric acid 1.0mg/L, activated carbon 0.3%, sucrose 2% and fine jade
Fat 0.6% is used as root media, the condition of culture of the culture medium of the step 104 after mixing：Light intensity is 1500-20001X,
Temperature is 23 to 27 DEG C, and light application time is 12h/d, and the pH value of root media is 5.8；
Step 105：Transplant：The plant taken root is taken out, is transplanted on detritus soil, breeze and wood sawdust mixed-matrix, wherein,
Detritus soil: breeze: the ratio of wood sawdust is 3: 1: 1；
Wherein, it is 20-30 minutes with washing time in the step 101, alcohol ratio is 70%, the surface sterilizing time is 30
Second, the time sterilized with mercury chloride is 4-12 minutes.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| 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 |
| 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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