CN107494266B - A kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis and tissue culture enrichment procedure - Google Patents
A kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis and tissue culture enrichment procedure Download PDFInfo
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- CN107494266B CN107494266B CN201710846163.5A CN201710846163A CN107494266B CN 107494266 B CN107494266 B CN 107494266B CN 201710846163 A CN201710846163 A CN 201710846163A CN 107494266 B CN107494266 B CN 107494266B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a kind of stabilization, the anti-browning of efficient Tokyo Dendronenthamia japonica var.chinensis and tissue culture enrichment procedures, include the following steps：1）Materials：4~May of spring, fair weather, took current year raw disease-free, robust growth nutrition branch；2）Sterilizing：Sterilizing methods are the combination of alcohol 30s+0.1% mercuric chloride 7min；3）Sterile anti-browning Establishing：Stem section after disinfection is divided in being dried on sterilized filter paper, is inoculated in containing 200mgL‑1 In the culture medium of the anti-browning agent of PVP, waits for that bud is grown to 3~4cm, carry out Multiplying culture；4）Multiplying culture：The bud of inductive formation is inoculated in proliferated culture medium（WPM+1.0 mg·L-16-BA+0.05 mg·L-1NAA）In, it is primary every 20~25 days subcultures.Present invention optimizes each stages of Tokyo Dendronenthamia japonica var.chinensis tissue culture proliferation, effectively reduce melting brown rate to 17.87%, improve growth coefficient and be up to 3.09.
Description
Technical field
The present invention relates to a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis and tissue culture enrichment procedures, belong to plant tissue culture technique neck
Domain.
Background technology
Tokyo Dendronenthamia japonica var.chinensis (Dendrobenthamia tonkinensis) belongs to Cornaceae (Cornaceae) Dendronenthamia japonica var.chinensis category
(Dendrobenthamia) plant is evergreen dungarunga or shrub.Its tree-like grace, branch is open and flat, well arranged (Hatta
Et al, 1999), the bright leaves of Ke Chunguan, the beautiful flower of summer reward, autumn see red autumnal leaves red fruit, are widely used in afforestation, scape is made in park, basin
Plant art；Its foliar application wound can subside a swelling, and root, seed decoct water clothes and can enrich blood, and have certain medical value；Its fruit containing it is high in fat with it is high
Calcium can be eaten raw, vinegar processed and wine brewing, be a kind of wild fruit of tool nutritive value (Han Weidong, 1993；Vareed, 2005)；Its wood
Material is hard, and texture straight is fine and smooth, and easy processing is good commerical tree species (Xin Lei, 2009).In conclusion seeds collection it is ornamental,
Edible, medicinal and timber-used value is the multipurpose rare tree for having very much exploitation prospect.
Dendronenthamia japonica var.chinensis current modes of reproduction in Tokyo is mainly Seedling propagation, grafting, cuttage and tissue culture propagation.Seedling propagation without
Method ensures the good characteristic of seeds, and Tokyo Dendronenthamia japonica var.chinensis seed has deep dormancy, and sowing could sprout for 2 years, extends nursery week
Phase increases seedling cost.Vegetative propagation can keep the merit of seeds, be that population expands numerous common method.But cuttage is numerous
The participation of the temperature for needing certain condition, humidity and nutriment, special active material is grown, complicated for operation, rooting rate is low；It transfers
Connect breeding breeding coefficient it is low, scion demand is big, and the breeding cycle is long, and low production efficiency, three of the above method cannot be satisfied city
Field demand, realizes Tokyo Dendronenthamia japonica var.chinensis large-scale production.
Invention content
The purpose of the present invention is to solve growing-seedling period length, breeding coefficients existing for traditional Tokyo Dendronenthamia japonica var.chinensis propagation method
The problems such as low, low production efficiency, provides a kind of stabilization, the anti-browning of efficient Tokyo Dendronenthamia japonica var.chinensis and tissue culture enrichment procedure, passes through
Optimize the behaviour in each stage such as Tokyo Dendronenthamia japonica var.chinensis materials, disinfection, sterile anti-browning Establishing, adventitious bud inducing, Multiplying culture
Make, effectively improve the anti-browning ability of Tokyo Dendronenthamia japonica var.chinensis adventitious bud, Tokyo Dendronenthamia japonica var.chinensis is promoted to generate a large amount of tools within a short period of time
The adventitious bud of parent's good characteristic lays the foundation for the foundation of later stage Tokyo Dendronenthamia japonica var.chinensis rapid propagation system.
In order to solve the above technical problems, a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis of present invention offer and tissue culture enrichment procedure, special
Sign is, including:
(1) selection of explant：Sample time is April in spring to May, chooses current year raw health, disease-free stem segment with bud
Section；
(2) sterilizing of explant：First 20~30s is impregnated with 70%~75% alcohol, after aseptic water washing 2~3 times, then use
0.08%~0.1% mercuric chloride impregnates 7~8min, with spare after aseptic water washing 4~5 times；
(3) sterile anti-browning Establishing：The stem section trimmed is inserted into the culture medium of sterilizing, after being sealed with sealed membrane
It is put into culturing room's culture, the culture medium is WPM minimal mediums, and hormone combinations are 6-BA and NAA, concentration is respectively 1.0~
2.0mg·L-1With 0.05~0.2mgL-1, 25~30gL of addition sucrose-1, 6.5~7gL of agar powder-1, anti-browning agent PVP
100~200mgL-1, and pH is adjusted to 5.70~5.78；
(4) Multiplying culture：It waits for that axillary bud is grown to 3~4cm, chooses the axillary bud material of robust growth, move it to adventitious bud increasing
Progress shoot proliferation culture in culture medium is grown, culturing room's culture is put into after being sealed with sealed membrane, it is primary every 20~25d subcultures,
The adventitious bud proliferation culture medium is WPM minimal mediums, and hormone combinations are 6-BA and NAA, concentration is respectively 1.0~
2.0mg·L-1With 0.05~0.2mgL-1, 25~30gL of addition sucrose-1, 6.5~7gL of agar powder-1, anti-browning agent PVP
100~200mgL-1, and pH is adjusted to 5.70~5.78.
Preferably, in the step (1), the specific method that explant is chosen is：Under fair weather, from Tokyo Dendronenthamia japonica var.chinensis
Current year raw health, disease-free nutrition branch are chosen in elite stand sunny slope tree body periphery, from clip length from top 1,2 sections be 3~
The stem with bud of 4cm retains 1 axillary bud, and axillary bud requires to have expanded but perula is not yet opened.The stem with bud of 3~4cm is easy to lure
Differentiation budding is led, the nutrient of stem section storage can provide nutrient, selected stem segment with axillary buds form of the invention at axillary bud initial stage
It has been basically formed that, and have perula protection, not easy to pollute, axillary bud growth speed is fast, adaptable, stabilization characteristics of genetics.
Preferably, in the step (2), alcohol concentration 70% impregnates 30s, and mercuric chloride a concentration of 0.1% impregnates 7min,
It is the optimization process combination of Tokyo Dendronenthamia japonica var.chinensis stem section disinfection, pollution rate and the death rate are all minimum.
Preferably, in the step (2), before explant sterilizing, the blade in stem section is trimmed, retains petiole and 1/5 blade,
It is rinsed 1~2 hour with tap water, then disinfects cleaned stem section on superclean bench.Sterilizing carries out stem
The appropriate trimming of section advantageously reduces workload of the experimenter on superclean bench, improves Disinfection Effect and working efficiency, stream
Water flushing can effectively remove stem section institute band soil, dust and bleeding sap, avoid the occurrence of because plant internal substance inhibits stem section
The phenomenon that growth.
Preferably, in the step (3), the Tokyo Dendronenthamia japonica var.chinensis stem section disinfected is laid on the filter paper sterilized and is dried
It is inoculated with again afterwards.Stem section body surface after disinfecting carries moisture, is placed in blot on the filter paper sterilized and dry, can be with
The moisture for removing stem section surface prevents bacterial adsorption caused by bacterium and the operation in air from influencing to try in stem section surface
Test success rate.
Preferably, in the step (3), pruning method is：Stem section head and the tail both ends and part are removed with sterilized scissors
Petiole avoids disinfectant from not washed completely from material wound, and remaining disinfectant can be transferred to outer by explant wound
Other positions of implant destroy explantation tissue structure, influence the growth conditions of explant, while after contacting disinfectant, explant
The protection structure that body wound surface is formed can hinder the nutritional ingredient in culture medium to enter in explant, influence the normal of explant
The success rate of growth and experiment.
When the stem section trimmed being inserted into the culture medium of sterilizing, insertion site depth accounts for the 1/4 of stem section length, every bottle of training
It supports base and is inserted into 2~3 stem sections, unsuitable too deep, the too deep then poor air permeability of insertion depth of stem section, stem section is easy death；Cross it is shallow then
Stem section is not easy to stablize, and is easy lodging.There is the particular matter of the axillary bud growth containing promotion in stem section, being inserted into 2~3 stem sections in every bottle has
Conducive to the accumulation of benefit materials.
Preferably, in the step (3), the culture medium is WPM minimal mediums, and hormone combinations are 6-BA and NAA, dense
Degree is respectively 1.5mgL-1And 0.05mgL-1, addition sucrose 30gL-1, agar powder 6.5gL-1, anti-browning agent PVP
200mg·L-1, and pH is adjusted to 5.75.
Preferably, in the step (4), the method that cuts of axillary bud material is：Use tissue culture knife by armpit on superclean bench
Bud is cut from explant inserted part, reject axillary bud on browning tissue, reject browning tissue can to avoid browning into
One step develops, and is conducive to normal growth of the axillary bud on proliferated culture medium.
When carrying out shoot proliferation culture, every bottle of culture medium accesses 3 axillary bud materials, there is the adventitious bud containing promotion in axillary bud material
The particular matter of growth is inserted into the accumulation that 3 axillary bud materials are conducive to benefit materials in every bottle.
Preferably, in the step (4), the adventitious bud proliferation culture medium is WPM minimal mediums, hormone combinations 6-
BA and NAA, concentration are respectively 1.0mgL-1And 0.05mgL-1, addition sucrose 30gL-1, agar powder 6.5gL-1, it is anti-brown
Agent PVP 200mgL-1, and pH is adjusted to 5.75.
Preferably, in the step (3) and step (4), culturing room's environmental condition is：Culturing room's light application time is 12h/
It, intensity of illumination 1500Lx, temperature is 21 ± 2 DEG C, and the photoperiod of the environment and intensity of illumination simulate Tokyo Dendronenthamia japonica var.chinensis and exist
Illumination condition under natural conditions, 21 ± 2 DEG C of environment temperature is suitable for the growth of Tokyo Dendronenthamia japonica var.chinensis group training material, while can press down
System cultivates indoor bacteria breed.
The advantageous effect that the present invention is reached：
Present invention optimizes the operating methods in Tokyo Dendronenthamia japonica var.chinensis tissue cultures each stage, effectively reduce melting brown rate, control
Pollution has been made, has improved value-added coefficient, specifically：
(1) handled by screening to sample time, sterilization method and anti-browning, it is determined that best sampling time and
The anti-browning agent of optimum concentration；It is 5.70~5.78 that the low pH of culture medium, which can inhibit browning, Medium's PH Value of the invention,.
(2) temperature can influence enzymatic activity in plant, and the induction of adventitious bud of the present invention and Multiplying culture are placed in 21 DEG C ± 2
Under DEG C temperature condition, inhibit aldehydes matter to generate under the premise of ensureing Tokyo Dendronenthamia japonica var.chinensis normal growth, mitigates browning hair
It is raw.
(3) long-term accumulation of Tokyo Dendronenthamia japonica var.chinensis wound aldehydes matter can cause browning, be shortened not in the present invention
The squamous subculture time is set to 20~25d by the time of normal bud squamous subculture.
(4) the axillary bud sprouting formula (WPM+1.5mgL that the present invention uses-1 6-BA+0.05mg·L-1NAA+30g·L-1
Sucrose+6.5gL-1Agar powder+200mgL-1) and adventitious bud proliferation formula (WPM+1.0mgL PVP-1 6-BA+
0.05mg·L-1NAA+30g·L-1Sucrose+6.5gL-1Agar powder+200mgL-1PVP), the Tokyo Dendronenthamia japonica var.chinensis of acquisition is cultivated
Axillary bud sprouting rate is up to 77.78%, and for adventitious bud proliferation coefficient up to 3.09, the Tokyo Dendronenthamia japonica var.chinensis adventitious bud speed of growth is fast, growing way compared with
Good, color is normal, without vitrification phenomenon.
(5) this method is easy to operate, repeatability is strong, is easily mastered, and reduces a large amount of manpower and materials consumption.
Specific implementation mode
With reference to specific embodiment, the invention will be further described.Following embodiment is only the preferred implementation of the present invention
Example does not constitute any restrictions for clearly illustrating technical scheme of the present invention to the practical range of the present invention.
Embodiment 1, the anti-browning of pollution are compared
The selection of explant：Respectively at 2014 4,5,7,8,10, mid-November, Nanjing Forestry University Baima base east
Capital Dendronenthamia japonica var.chinensis nursery acquisition current year raw health, disease-free nutrition branch, the stem segment with bud of 3~4cm is about from clip from top 1,2 sections
Section retains 1 axillary bud, is placed in ice chest that take back laboratory spare.
The sterilizing of explant：The blade in stem section is trimmed, petiole and 1/5 blade are retained, is rinsed 1~2 hour with tap water,
Then cleaned stem section is disinfected on superclean bench, first impregnates 30s, aseptic water washing 2 with 70% alcohol
After~3 times, then with 0.1% mercuric chloride impregnate 5,7,9,11min, with spare (see the table below 1) after aseptic water washing 4~5 times.
Sterile anti-browning Establishing：The Tokyo Dendronenthamia japonica var.chinensis stem section disinfected is placed in the filter paper (11 × 11cm) sterilized
On dry, remove part petiole at stem section head and the tail both ends and axil with sterilized scissors, the stem section sheared be inserted into sterilizing
Adventitious bud induction culture base (culture medium prescription WPM+1.5mgL-16-BA+0.3mg·L-1IBA), anti-browning processing is as follows
Table 2, pH adjust processing such as the following table 3, and stem section insertion site depth accounts for the 1/4 of stem section length, and every bottle of culture medium is inserted into 2~3 stems
Section, primary 20 bottles of inoculation, is repeated 3 times, and in the process, indoor environmental condition is cultivated in control, and cleaning is contaminated and brown in time
The stem section material and culture medium of change.
Table 1 samples the influence of season and disinfecting time to Tokyo Dendronenthamia japonica var.chinensis explant pollution rate and the death rate
Note：Lowercase letter note is indicated in the horizontal significant differences of α=0.05, similarly hereinafter in this table.
April in spring to May is suitble to acquire Tokyo Dendronenthamia japonica var.chinensis stem section, wherein April to May, alcohol it can be seen from upper table 1
30s is handled, 0.1% mercuric chloride processing 7min is the optimization process combination of Tokyo Dendronenthamia japonica var.chinensis stem section disinfection, pollution rate and other processing
Time, there are significant differences, only 12.86%.
The influence of 2 anti-browning agent type of table and concentration to Tokyo Dendronenthamia japonica var.chinensis stem section melting brown rate
It can be obtained by upper table 2, a concentration of 200mgL that this hair provides-1Anti- browning agent PVP to Tokyo Dendronenthamia japonica var.chinensis group
The anti-browning effect of training is best, can Tokyo Dendronenthamia japonica var.chinensis tissue culture melting brown rate be effectively reduced to 17.87%, different from anti-browning agent
Concentration compares that there are significant differences with different browning agent various concentrations.
Influence of 3 pH value of table to Tokyo Dendronenthamia japonica var.chinensis melting brown rate
| pH | Melting brown rate (%) | Germination rate (%) |
| 5.66 | 51.43a | 32.16c |
| 5.70 | 20.17c | 68.41a |
| 5.74 | 20.03c | 68.55a |
| 5.78 | 19.98c | 68.88a |
| 5.82 | 33.08b | 54.02b |
Can be obtained by upper table 3, range provided by the invention 5.70~5.78 pH value to Tokyo Dendronenthamia japonica var.chinensis tissue culture
Anti- browning effect is preferable, and explant is normally grown, break up influence it is smaller.Browning in tissue culture refers to explant
The quinones substance that contained phenolic compound and polyphenol oxidase are generated in the effect of oxygen, these quinones substances can be along culture medium
To external diffusion, inhibit other enzymatic activitys, influences plant differentiation growth.PH value it is excessively high it is too low culture medium can be caused really up to the mark and excessively soft,
Plant growth differentiation is obstructed, and plant growth gesture is low, is subject to browning influence, and suitable pH value can hold back to a certain extent
The activity of polyphenol oxidase processed improves the adaptability of plant, enhances the growth potential of plant, improves the anti-browning ability of plant.
Embodiment 2, sterile system germination rate compare
The selection of explant：In mid-April, 2015, acquired in base Tokyo, Nanjing Forestry University Baima Dendronenthamia japonica var.chinensis nursery
Current year raw health, disease-free nutrition branch, are about the stem with bud of 3~4cm from clip from top 1,2 sections, retain 1 axillary bud, set
It is spare in taking back laboratory in ice chest.
The sterilizing of explant：The blade in stem section is trimmed, petiole and 1/5 blade are retained, is rinsed 1~2 hour with tap water,
Then cleaned stem section is disinfected on superclean bench, first impregnates 30s, aseptic water washing 2 with 70% alcohol
After~3 times, then with 0.1% mercuric chloride 7min is impregnated, with spare after aseptic water washing 4~5 times.
Sterile anti-browning Establishing：The Tokyo Dendronenthamia japonica var.chinensis stem section disinfected is placed in the filter paper (11 × 11cm) sterilized
On dry, remove part petiole at stem section first place both ends and axil with sterilized scissors, the stem section sheared be inserted into sterilizing
Axillary bud deriving culture medium, culture medium is WPM minimal mediums in axillary bud deriving culture medium, and 30gL is added in culture medium-1Sucrose,
6.5g·L-1Agar powder and 200mgL-1PVP, hormone combinations see the table below 4, and insertion site depth accounts for the 1/4 of stem section length, often
Bottle culture medium is inserted into 2~3 stem sections, and primary 20 bottles of inoculation is repeated 3 times, and in the process, indoor environment item is cultivated in control
Part, cleaning in time is contaminated and the stem section material and culture medium of browning.
4 hormon of table is with the influence (concentration for comparing Tokyo Dendronenthamia japonica var.chinensis explant germination rate：mg·L-1)
Tokyo Dendronenthamia japonica var.chinensis sterile system establishes culture medium WPM+1.5mg used in the present invention it can be seen from upper table 4
L-1 6-BA+0.05mg·L-1NAA+30g·L-1Sucrose+6.5gL-1Agar powder+200mgL-1PVP and other hormone combinations
Compared to there are significant difference, explant germination rate can be up to 77.78%.
Embodiment 3, Multiplying culture formula compare
The selection of explant：Respectively at 2014 4,5,7,8,10, mid-November, Nanjing Forestry University Baima base east
Capital Dendronenthamia japonica var.chinensis nursery acquisition current year raw health, disease-free nutrition branch, the stem segment with bud of 3~4cm is about from clip from top 1,2 sections
Section retains 1 axillary bud, is placed in ice chest that take back laboratory spare.
The sterilizing of explant：The blade in stem section is trimmed, petiole and 1/5 blade are retained, is rinsed 1~2 hour with tap water,
Then cleaned stem section is disinfected on superclean bench, first impregnates 30s, aseptic water washing 2 with 70% alcohol
After~3 times, then with 0.1% mercuric chloride impregnate 5,7,9,11min, with spare after aseptic water washing 4~5 times.
Sterile anti-browning Establishing：The Tokyo Dendronenthamia japonica var.chinensis stem section disinfected is placed in the filter paper (11 × 11cm) sterilized
On dry, remove part petiole at stem section first place both ends and axil with sterilized scissors, the stem section sheared be inserted into sterilizing
Adventitious bud induction culture base (culture medium prescription WPM+1.5mgL-16-BA+0.05mg·L-1NAA+30g·L-1Sucrose+
6.5g·L-1Agar powder+200mgL-1PVP), insertion site depth accounts for the 1/4 of stem section length, and every bottle of culture medium is inserted into 2~3
A stem section, primary 20 bottles of inoculation, is repeated 3 times, and in the process, indoor environmental condition is cultivated in control, and cleaning is contaminated in time
With the stem section material and culture medium of browning.
Multiplying culture：Tokyo Dendronenthamia japonica var.chinensis stem section is proliferated through culture after a period of time, induction produces bud, waits for that bud is grown to 3
~4cm chooses robust growth, the normal bud material of color, cuts it from explant inserted part with tissue culture knife, can carry small
The tissue of browning on adventitious bud is rejected by part explantation tissue, and moving it to adventitious bud proliferation culture medium, (proliferated culture medium is shown in Table
5) shoot proliferation culture is carried out in, 3 stem sections of every bottle of access are put into culturing room's culture after sealing, every 20~25d subcultures are primary,
Primary 20 bottles of inoculation, is repeated 3 times, and value-added coefficient=single explant regenerates summation/60 for the adventitious bud to be formed.
Influence of the different proliferated culture mediums of table 5 to Tokyo Dendronenthamia japonica var.chinensis adventitious bud proliferation
The proliferation formula WPM+1.0mgL provided by the invention it can be seen from upper table 5-1 6-BA+0.05mg·L-1NAA
Compared to other three kinds of proliferated culture mediums there are significant difference, growth coefficient can reach 3.09.
Culture medium adds 30gL in above example-1Sucrose and 6.5gL-1Agar powder, pH value is adjusted to 5.70~
In 5.78 ranges, condition of culture is that culturing room's light application time is 12h/ days, intensity of illumination 1500Lx, and temperature is 21 DEG C ± 2 DEG C.
By embodiment 1,2,3 it is found that a kind of stabilization, the anti-browning of efficient Dendronenthamia japonica var.chinensis and tissue culture enrichment procedure are：Most preferably take
The material time is 4~May of spring, and best sterilization method is 70% alcohol 30s+0.1% mercuric chloride 7min, optimum anti-browning type
With a concentration of 200mgL-1PVP, the optimum formula of axillary bud deriving：WPM+1.5mg·L-1 6-BA+0.05mg·L-1NAA+
30gL-1 sucrose+6.5gL-1 agar powder+200mgL-1PVP, the optimum formula of adventitious bud proliferation：Minimal medium is
WPM, hormone combinations are 6-BA and NAA, and concentration is respectively 1.0mgL-1And 0.05mgL-1。
Compared to traditional propagation method, present invention reduces seedling costs, improve breeding efficiency, through the invention can
Enough effectively control Tokyo Dendronenthamia japonica var.chinensis tissue culture pollutions, reduce tissue culture melting brown rate up to 17.87%, and value-added coefficient is up to 3.09, is the later stage
The further investigation of Tokyo Dendronenthamia japonica var.chinensis provides a large amount of group training material, to realize that Dendronenthamia japonica var.chinensis factorial praluction in Tokyo lays the foundation.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvement and deformations can also be made, these improvement and deformations
Also it should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis and tissue culture enrichment procedure, characterized in that including:
（1）The selection of explant：Sample time is April in spring to May, chooses current year raw health, disease-free stem with bud；
（2）The sterilizing of explant：First 20 ~ 30s is impregnated with 70% ~ 75% alcohol, after aseptic water washing 2~3 times, then with 0.08% ~
0.1% mercuric chloride impregnates 7 ~ 8min, with spare after aseptic water washing 4~5 times；
（3）Sterile anti-browning Establishing：The stem section trimmed is inserted into the culture medium of sterilizing, is put into after being sealed with sealed membrane
Culturing room cultivates, and the culture medium is WPM minimal mediums, and hormone combinations are 6-BA and NAA, and concentration is respectively 1.0~
2.0mg·L-1With 0.05~0.2mgL-1, 25 ~ 30gL of addition sucrose-1, 6.5 ~ 7gL of agar powder-1, anti-browning agent PVP
100~200mgL-1, and pH is adjusted to 5.70~5.78；It is 21 ± 2 DEG C to cultivate room temperature；
（4）Multiplying culture：It waits for that axillary bud is grown to 3~4cm, chooses the axillary bud material of robust growth, move it to adventitious bud proliferation training
It supports and carries out shoot proliferation culture in base, culturing room's culture is put into after being sealed with sealed membrane, it is primary every 20~25d subcultures, it is described
Adventitious bud proliferation culture medium is WPM minimal mediums, and hormone combinations are 6-BA and NAA, and concentration is respectively 1.0~2.0mgL-1
With 0.05~0.2mgL-1, 25 ~ 30gL of addition sucrose-1, 6.5 ~ 7gL of agar powder-1, anti-browning agent PVP 100~
200mg·L-1, and pH to 5.70~5.78 is adjusted, culture room temperature is 21 ± 2 DEG C.
2. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
（1）In, the specific method that explant is chosen is：Under fair weather, from Tokyo Dendronenthamia japonica var.chinensis elite stand sunny slope tree body periphery, choose
Current year raw health, disease-free nutrition branch retain 1 armpit from the stem with bud that clip length from top 1,2 sections is 3~4cm
Bud.
3. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
（2）In, alcohol concentration 70% impregnates 30s, and mercuric chloride a concentration of 0.1% impregnates 7min.
4. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
（2）In, before explant sterilizing, the blade in stem section is trimmed, retains petiole and 1/5 blade, rinses 1~2 hour with tap water, so
Cleaned stem section is disinfected on superclean bench afterwards.
5. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
（3）In, the Tokyo Dendronenthamia japonica var.chinensis stem section disinfected is laid in after being dried on the filter paper sterilized and is inoculated with again.
6. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
（3）In, pruning method is：Stem section head and the tail both ends and part petiole are removed with sterilized scissors；The stem section trimmed is inserted into
When in the culture medium of sterilizing, insertion site depth accounts for the 1/4 of stem section length, and every bottle of culture medium is inserted into 2~3 stem sections.
7. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
（3）In, the culture medium is WPM minimal mediums, and hormone combinations are 6-BA and NAA, and concentration is respectively 1.5mgL-1With
0.05mg·L-1, addition sucrose 30gL-1, agar powder 6.5gL-1, anti-browning agent PVP 200mgL-1, and adjust pH to
5.75。
8. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
（4）In, the method that cuts of axillary bud material is：Axillary bud is cut from explant inserted part with tissue culture knife on superclean bench,
Reject the browning tissue on axillary bud；When carrying out shoot proliferation culture, every bottle of culture medium accesses 3 axillary bud materials.
9. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
（4）In, the adventitious bud proliferation culture medium is WPM minimal mediums, and hormone combinations are 6-BA and NAA, and concentration is respectively
1.0mg·L-1And 0.05mgL-1, addition sucrose 30gL-1, agar powder 6.5gL-1, anti-browning agent PVP 200mgL-1,
And pH is adjusted to 5.75.
10. a kind of anti-browning of Tokyo Dendronenthamia japonica var.chinensis according to claim 1 and tissue culture enrichment procedure, characterized in that the step
Suddenly（3）And step（4）In, culturing room's environmental condition is：Culturing room's light application time is 12h/ days, intensity of illumination 1500Lx.
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| CN104719134A (en) * | 2014-10-08 | 2015-06-24 | 江苏碧云天农林科技有限公司 | Dendrobenthemia japonica tissue culture method |
| CN104982334A (en) * | 2015-06-26 | 2015-10-21 | 江苏绿苑园林建设有限公司 | Leaf culturing and plant regeneration method for dendrobenthamia hongkongensis |
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| CN104982334A (en) * | 2015-06-26 | 2015-10-21 | 江苏绿苑园林建设有限公司 | Leaf culturing and plant regeneration method for dendrobenthamia hongkongensis |
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