CN109006473A - A kind of Kiwifruit Tissue Culture fast propagating culture medium and method - Google Patents
A kind of Kiwifruit Tissue Culture fast propagating culture medium and method Download PDFInfo
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- CN109006473A CN109006473A CN201810748851.2A CN201810748851A CN109006473A CN 109006473 A CN109006473 A CN 109006473A CN 201810748851 A CN201810748851 A CN 201810748851A CN 109006473 A CN109006473 A CN 109006473A
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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 Kiwifruit Tissue Culture fast propagating culture medium and methods, break up adventitious shoots culture base, adventitious buds proliferation culture medium, sugar-free tissue culture and inducement adventitious root culture medium including inducer blade differentiation callus tissue culture base, induction stem apex or axillary bud sprouting growth medium, evoked callus, by using endogenous hormones rooting method, sugar-free nutrition liquid is placed in specific culture vessel, manual control microenvironment, both it can shorten the experienced seedling period of taking root, transplanting survival rate can also be improved.Grow vigorous, root system is huge, without practicing seedling, directly transplants to nutritive cube or big Tanaka.The production efficiency of sugar-free rooting method of the invention is that tradition has 18 times of sugared rooting method or more, substantially increases the production efficiency of Kiwi berry detoxic seedling large-scale production.
Description
Technical field
The present invention relates to field of plant tissue culture technique, it particularly relates to which a kind of Kiwifruit Tissue Culture quickly breeds training
Support base and method.
Background technique
Actinidia Actinidiaceae, Actinidia defoliation liana are a kind of important commodity trees and fruit tree.Macaque
For peach rich in nutriments such as Vc, minerals and anthocyanins, nutritive value and medical value are high.In recent years, be widely used in fresh fruit,
The edible form such as beverage, health treatment, market demand are larger.Popularizing planting can meet more consumer demands, increase fruit
The economic well-being of workers and staff of agriculture.However, its seed propagation growth cycle is long, begins to spend the age long, it is unfavorable for quickly forming the market of Kiwi berry
Value.
Tissue cultures are a kind of Clonal Rapid Propagation methods, with the breeding cycle is short, genetic stability is good, breeding coefficient
The features such as big.The difficulty but Kiwi berry tradition tissue culture is taken root, is also easy to produce abnormal root, aerial root, and the easily long callus in root causes
It is extremely low to practice shoot survival percent.
For the problems in the relevant technologies, currently no effective solution has been proposed.
Summary of the invention
For above-mentioned technical problem in the related technology, the present invention propose a kind of Kiwifruit Tissue Culture fast propagating culture medium and
Method is able to solve above-mentioned technical problem.
To realize the above-mentioned technical purpose, the technical scheme of the present invention is realized as follows:
A kind of Kiwifruit Tissue Culture rapid propagation method, the following steps are included:
S1. the branch and/or young leaflet tablet for selecting Kiwi berry current year life are as explant；
S2. explant is handled, specifically includes the following steps:
When selecting branch as explant, removes the blade in branch stem section and retain axillary bud or strip stem apex by disinfection
After processing, in access induction stem apex or axillary bud sprouting growth medium, it is placed on culturing room's culture；
When selecting young leaflet tablet as explant, after young leaflet tablet is disinfected, access inducer blade breaks up callus
In tissue culture medium (TCM), it is placed on culturing room's culture；The callus size differentiated after inoculation is 0.8cm2-2.5cm2When, switching
Into evoked callus differentiation adventitious shoots culture base；
S3. shoot proliferation culture, specifically includes the following steps:
When selecting branch as explant, explant axillary bud, stem apex grow to 1.0cm- 2.5cm, and with 2-5 piece
It when young leaves generates, cuts sprouting and is forwarded to adventitious buds proliferation culture medium and expand numerous, obtain subculture Multiple Buds；
When selecting young leaflet tablet as explant, the callus of young leaflet tablet first differentiates adventitious bud, and adventitious bud is long
Test tube seedling is forwarded to adventitious buds proliferation culture medium and expand numerous, obtains subculture Multiple Buds by the test tube seedling of Cheng Chenggao 2-4cm；
S4. it takes root, specifically includes the following steps:
The seedling that subculture Multiple Buds are grown into is forwarded in sugar-free tissue culture and inducement adventitious root culture medium, culturing room CO2It is dense
Degree is 1000ppm-1500ppm, sugar-free tissue culture and inducement adventitious root culture medium prescription: NH4NO316.5mg/L,KNO319mg/L,
MgSO4·7H2O3.7mg /L,KH2PO41.7mg/L,CaCl2·2H2O4.4mg/L,KI0.083mg/L,H3BO3 0.62mg/L,
MnSO4·4H2O2.23mg/L,ZnSO4·7H2O0.86mg/L,Na2MnO4·2H 2O0.025mg/L,CuSO4·
5H2O0.0025mg/L,CoCl2·6H2O0.0025mg/ L,FeSO4·7H2O2.78mg/L,Na2-EDTA·2H2O3.73mg/
L；
S5. young seedling direct transplantation after taking root is to nutritive cube or big Tanaka.
Further, the formula of inducer blade differentiation callus tissue culture base are as follows: 1 liter of MS culture medium, 6- benzyl amino gland is fast
Purine 0.8-1.2mg/L, 2,4- dichlorphenoxyacetic acid 0.3-0.6m g/L, sucrose 25-30g/L, agar 5.8-6g/L.
Further, stem apex or axillary bud sprouting grown cultures based formulas are induced are as follows: 1 liter of MS culture medium, 6- benzyl amino gland is fast
Purine 0-0.5mg/L, methyl α-naphthyl acetate 0.1-0.3mg/L, sucrose 2 5-30g/L, agar 5.8-6g/L.
Further, evoked callus breaks up adventitious shoots culture based formulas are as follows: and 1 liter of MS culture medium, zeatin 2.0-
2.5mg/L, methyl α-naphthyl acetate 0.1mg/L, sucrose 25-30g/L, agar 5.8-6g/L.
Further, adventitious buds proliferation culture medium prescription are as follows: 1 liter of MS culture medium, methyl α-naphthyl acetate 0.1-0.4mg/L, 6- benzyl ammonia
Base adenine 0.6-1.0mg/L, sucrose 25-30g/L, agar 5.8-6g/L.
A kind of Kiwifruit Tissue Culture rapid propagation method according to claim 1, which is characterized in that training described in step S4
Indoor illumination 20000Lx, the humidity 50%-9 0% of room are supported, 25 DEG C -28 DEG C of temperature, cultivates indoor air-changing 15min/h, throughput
90mL/min- 180mL/min。
A kind of Kiwifruit Tissue Culture fast propagating culture medium, including inducer blade differentiation callus tissue culture base, induction stem apex
Or axillary bud sprouting growth medium, evoked callus differentiation adventitious shoots culture base, adventitious buds proliferation culture medium, sugar-free tissue culture lure
Lead adventitious root culture medium, wherein
The formula of inducer blade differentiation callus tissue culture base are as follows: 1 liter of MS culture medium, 6- benzyl aminoadenine 0.8-
1.2mg/L, 2,4- dichlorphenoxyacetic acid 0.3-0.6mg/L, sucrose 2 5-30g/L, agar 5.8-6g/L；
Induce stem apex or axillary bud sprouting grown cultures based formulas are as follows: 1 liter of MS culture medium, 6- benzyl aminoadenine 0-
0.5mg/L, methyl α-naphthyl acetate 0.1-0.3mg/L, sucrose 25-30g/L, agar 5.8-6g/L；
Evoked callus breaks up adventitious shoots culture based formulas are as follows: and 1 liter of MS culture medium, zeatin 2.0-2.5mg/L, naphthalene second
Sour 0.1mg/L, sucrose 25-30g/L, agar 5.8-6g/ L；
Adventitious buds proliferation culture medium prescription are as follows: 1 liter of MS culture medium, methyl α-naphthyl acetate 0.1-0.4m g/L, 6- benzyl aminoadenine
0.6-1.0mg/L, sucrose 25-30g/L, agar 5.8-6g/ L；
Sugar-free tissue culture and inducement adventitious root culture medium prescription are as follows: NH4NO316.5mg/L,KNO3 19mg/L,MgSO4·
7H2O3.7mg/L,KH2PO41.7mg/L,CaCl2·2H2O4.4mg/L, KI0.083mg/L,H3BO30.62mg/L,MnSO4·
4H2O2.23mg/L,ZnSO4·7H2O0.86 mg/L,Na2MnO4·2H2O0.025mg/L,CuSO4·5H2O0.0025mg/L,
CoC l2·6H2O0.0025mg/L,FeSO4·7H2O2.78mg/L,Na2-EDTA·2H2O3.73 mg/L。
Further, including inducer blade differentiation callus tissue culture base, induction stem apex or axillary bud sprouting growth medium,
Evoked callus breaks up adventitious shoots culture base, adventitious buds proliferation culture medium, sugar-free tissue culture and inducement adventitious root culture medium, wherein
The formula of inducer blade differentiation callus tissue culture base: 1 liter of MS culture medium, 6- benzyl aminoadenine 1.0mg/L, 2,
4- dichlorphenoxyacetic acid 0.4mg/L, sucrose 30g/L, agar 5.8g/L；
Induction stem apex or axillary bud sprouting grown cultures based formulas: 1 liter of MS culture medium, 6- benzyl aminoadenine 0.4mg/L, naphthalene
Acetic acid 0.1mg/L, sucrose 30g/L, agar 5.8g/ L；
Evoked callus differentiation adventitious shoots culture based formulas: 1 liter of MS culture medium, zeatin 2.0mg/L, methyl α-naphthyl acetate
0.1mg/L, sucrose 25-30g/L, agar 5.8-6g/L；
Adventitious buds proliferation culture medium prescription: 1 liter of MS culture medium, methyl α-naphthyl acetate 0.2mg/L, 6-benzyl aminoadenine 0.8-
1.0mg/L, sucrose 30g/L, agar 5.8g/L；
Sugar-free tissue culture and inducement adventitious root culture medium prescription: NH4NO316.5mg/L,KNO319 mg/L,MgSO4·
7H2O3.7mg/L,KH2PO41.7mg/L,CaCl2·2H2O4.4mg/L,KI 0.083mg/L,H3BO30.62mg/L,MnSO4·
4H2O2.23mg/L,ZnSO4·7H2O0.86m g/L,Na2MnO4·2H2O0.025mg/L,CuSO4·5H2O0.0025mg/L,
CoCl 2·6H2O0.0025mg/L,FeSO4·7H2O2.78mg/L,Na2-EDTA·2H2O3.73 mg/L。
Beneficial effects of the present invention: by using endogenous hormones rooting method, sugar-free nutrition liquid is placed in specific culture vessel
In, manual control microenvironment can both shorten the experienced seedling period of taking root, and can also improve transplanting survival rate.Grow vigorous, root system Pang
Greatly, without practicing seedling, directly transplanting to nutritive cube or big Tanaka.The production efficiency of sugar-free rooting method of the invention is that tradition has sugar
18 times or more of rooting method, substantially increase the production efficiency of Kiwi berry detoxic seedling large-scale production.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained, shall fall within the protection scope of the present invention.
MS culture medium is the common knowledge of those skilled in the art, contained group as minimal medium of the invention
Point and content such as table 1:
1 MS culture medium of table
Kiwifruit Tissue Culture rapid propagation method, the following steps are included:
S1. spring and summer (August in March -) takes the Kiwi berry current year raw branch or young leaflet tablet as explant around high noon,
Or simultaneous selection branch and young leaflet tablet are as explant；
S2. explant is handled, specifically includes the following steps:
When selecting branch as explant, removes the blade (retaining axillary bud) in stem section or strip stem apex, first with originally
Water is rinsed well, then washes 15min-20 min with dish washing liquid immersion.Then 2h or so is rinsed under flowing water.Super after rinsing well
It is disinfected on net workbench.Disinfection treatment first sterilizes 8min-10min with 0.1% mercuric chloride, with aseptic water washing 4-5 times.
The alcohol disinfecting 30s of 70%-75% is used again, it is rear to use aseptic water washing 3-4 times.It is subsequently placed on aseptic filter paper and blots.Ultra-clean
Stem section is cut into the segment of 0.8-1.5cm with the scissors after sterilizing, every segment contains at least one axillary bud on platform；With the tweezer after sterilizing
Son peels stem apex outermost layer blade off.Then it is accessed in the induction stem apex or axillary bud sprouting growth medium prepared.Every bottle
(volume 500ml) connects 2-4.It is placed on culturing room's culture of manual control microenvironment.Initial-stage culture illumination not above
5000Lx, the otherwise easily withered death of explant, wherein the formula of induction stem apex or axillary bud sprouting growth medium is MS culture medium
1 liter, 6- benzyl aminoadenine 0-0.5mg/L, methyl α-naphthyl acetate 0.1-0.3mg/ L, sucrose 25-30g/L, agar 5.8-6g/L；Its
In, induction stem apex or axillary bud sprouting grown cultures based formulas: 1 liter of MS culture medium, 6- benzyl aminoadenine 0-0.5mg/L, naphthalene second
Sour 0.1-0.3mg/L, sucrose 25-30g/L, agar 5.8-6g/L.
When selecting young leaflet tablet as explant, young leaflet tablet is chosen, is first scrubbed clean under tap water with banister brush,
10min-15min is washed with dish washing liquid immersion again.Then 2h or so is rinsed under flowing water.It is enterprising in superclean bench after rinsing well
Row disinfection treatment.Disinfection treatment first sterilizes 6min-9min with 0.1% mercuric chloride, with aseptic water washing 4-5 times.70%-75% is used again
Alcohol disinfecting 30S, it is rear to use aseptic water washing 3-4 times.It is subsequently placed on aseptic filter paper and blots.On the super-clean bench with after sterilizing
Blade is cut into 0.8cm by scissors2-1. 2cm2Fritter, cut leaf margin, blade tip, yellow leaf etc. height differentiation or stale part.
Then it is accessed in the inducer blade differentiation callus tissue culture base prepared.Face of blade is paid attention to when inoculation upward, is laid flat
Or partial insertion culture medium, every bottle connects 3-4.It is then placed within culturing room's culture of manual control microenvironment.Initial-stage culture without
Light requirement shines or illumination is not above 5000Lx, otherwise explant or the easy browning of callus, withered, and wherein inducer blade differentiation is cured
The formula of injured tissue culture medium is 1 liter of MS culture medium, 6- benzyl aminoadenine 0.8-1.2mg/L, 2,4- dichlorphenoxyacetic acids
0.3-0.6mg/L, sucrose 25-30g/L, agar 5.8-6g/L；10-20d after blade inoculation starts to differentiate callus,
20d-4 0d callus size reaches 0.8cm2-2.5cm2, it is forwarded to evoked callus differentiation on superclean bench and grows thickly
In bud culture medium.15-20d or so callus starts to become green from yellow green, and it is different degrees of that surface starts simultaneously at appearance
Graininess protrusion, these protrusions of 30d-4 0d start to differentiate adventitious bud in varying numbers successively, and the differentiation rate of bud is reachable
90% or more (every piece of callus can produce the 2-8 effective buds that can finally develop seedling).Adventitious bud production postnatal growth is very fast,
50d can grow up to the test tube seedling of high 2-4cm, differentiate 3-5 piece spire.Wherein, evoked callus breaks up adventitious shoots culture base
Formula: 1 liter of MS culture medium, zeatin 2.0-2.5mg/L, methyl α-naphthyl acetate 0.1mg/L, sucrose 25-30g/L, agar 5.8-6g/L.
S3. shoot proliferation culture, specifically includes the following steps:
When selecting branch as explant, 7-10d after explant inoculation, axillary bud starts sprouting, stem apex starts to grow.
18d or so axillary bud/stem apex grows to 1.0cm-2.5cm, and generates with 2-5 piece young leaves.On superclean bench, sprouting is cut
Be forwarded on adventitious buds proliferation culture medium expand it is numerous, 10 plants every bottle.The each sprouting of 7d-10d can be proliferated out 2-5 test tube seedling.
After the long height to differ in size to 1cm-5cm of 20d or so proliferation seedling, it is forwarded to same medium shoot proliferation.Every bottle can
2-4 bottles of amplification；Wherein, adventitious buds proliferation culture medium prescription: 1 liter of MS culture medium, methyl α-naphthyl acetate 0.1-0.4mg/L, 6- benzyl amino gland
Purine 0.6-1.0mg/L, sucrose 25-30g/L, agar 5.8-6g/L.
When selecting young leaflet tablet as explant, the test tube seedling in step 2 is forwarded in adventitious buds proliferation culture medium,
10 plants every bottle, each sprouting of 20d or so can be proliferated out the test tube seedling that 5 1cm-5cm of 2- differ in size, and then be forwarded to identical
Culture medium shoot proliferation, every bottle 2-4 bottles amplifiable；Wherein, adventitious buds proliferation culture medium prescription: 1 liter of MS culture medium, naphthalene
Acetic acid 0.1-0.4mg/L, 6- benzyl aminoadenine 0.6-1.0mg/L, sucrose 25-30g/L, agar 5.8-6g/L.
S4. it takes root, specifically includes the following steps:
The seedling that subculture Multiple Buds are grown into is forwarded in sugar-free tissue culture and inducement adventitious root culture medium, sugar-free tissue culture and inducement
Adventitious root culture medium prescription: NH4NO316.5mg/L,KNO3 19mg/L,MgSO4·7H2O3.7mg/L,KH2PO41.7mg/L,
CaCl2·2H2O4. 4mg/L,KI0.083mg/L,H3BO30.62mg/L,MnSO4·4H2O2.23mg/L,ZnS O4·
7H2O0.86mg/L,Na2MnO4·2H2O0.025mg/L,CuSO4·5H2O 0.0025 mg/L,CoCl2·6H2O
0.0025mg/L,FeSO4·7H2O2.78mg/L,Na2-E DTA·2H2O3.73mg/L；Culturing room CO2Concentration is 1000ppm-
1500ppm, indoor illumination 20000Lx, humidity 50%-90%, 25 DEG C -28 DEG C of temperature, CO2100 0ppm-1500ppm of concentration,
Cultivate forced ventilation 15min/h, throughput 90ml/min-18 0ml/min in box.
S5. the young seedling direct transplantation after taking root is to nutritive cube or big Tanaka, without practicing seedling.
Embodiment 1:
A kind of Kiwi berry fast propagating culture medium, including inducer blade differentiation callus tissue culture base, induction stem apex or armpit
Bud germination and growth culture medium, evoked callus differentiation adventitious shoots culture base, adventitious buds proliferation culture medium, sugar-free tissue culture and inducement are not
Determine root culture medium, wherein
Inducer blade breaks up callus tissue culture base: 1 liter of MS culture medium, 6- benzyl aminoadenine 0.8-1.2mg/L, 2,4-
Dichlorphenoxyacetic acid 0.3-0.6mg/L, sucrose 25-30g/L, agar 5.8-6g/L；When preparation, MS culture medium, addition are first measured
Sucrose is added in 6- benzyl aminoadenine, 2,4- dichlorphenoxyacetic acid, after sucrose dissolution, adjusts between pH to 5. 8-5.9, adds
Entering agar, heating melts agar, the autoclave sterilization 21min at 121 DEG C, in use, cultivated under illumination (12h,
2000Lx), cultivation temperature is 21-25 DEG C.
Embodiment 1-1:
Inducer blade breaks up callus tissue culture base: first measuring 1 liter of MS culture medium, adds 6- benzyl aminoadenine
Sucrose 25g is added in 0.8mg, 2,4- dichlorphenoxyacetic acid 0.3mg, after sucrose dissolution, adjusts pH to 5.8, agar is added
5.8g heating melts agar, the autoclave sterilization 21min at 121 DEG C.
Embodiment 1-2:
Inducer blade breaks up callus tissue culture base: 1 liter of MS culture medium first measured, 6- benzyl aminoadenine 1.2mg is added,
Sucrose 30g is added in 2,4- dichlorphenoxyacetic acid 0.6mg, after sucrose dissolution, adjusts pH to 5.9, agar 6g is added, heating makes
Agar melts, the autoclave sterilization 21min at 121 DEG C.
Embodiment 1-3:
Inducer blade breaks up callus tissue culture base: first measuring 1 liter of MS culture medium, adds 6- benzyl aminoadenine
Sucrose 27g is added in 1.0mg, 2,4- dichlorphenoxyacetic acid 0.5mg, after sucrose dissolution, adjusts pH to 5.9, agar is added
5.9g, heating melt agar, the autoclave sterilization 21min at 121 DEG C.
Experiment one: break up callus to blade is compared by different exogenous plant hormones or plant growth regulator analysis
The influence of growth:
10 groups of comparative tests are done according to different exogenous plant hormones or plant growth regulator and different usage amounts, such as table 2
Shown, every group is done 3 parallel samples, MS culture medium, sucrose addition, agar addition sugarcane all the same in 10 groups of comparative tests
Sugared 30g/L, agar 5.8g/ L use identical preparation process.
Table 2:10 group inducer blade breaks up callus tissue culture base
Obtain after experimental observation and data statistic analysis: C group processing callus growth is best, mean size 1.8cm2With
On, differentiation rate is up to 95% or more, and for blade in yellow green to light green color, melting brown rate, glass rate are low.
Embodiment 2:
A kind of Kiwi berry fast propagating culture medium, including inducer blade differentiation callus tissue culture base, induction stem apex or armpit
Bud germination and growth culture medium, evoked callus differentiation adventitious shoots culture base, adventitious buds proliferation culture medium, sugar-free tissue culture and inducement are not
Determine root culture medium, wherein
Evoked callus differentiation adventitious shoots culture based formulas: 1 liter of MS culture medium, zeatin 2.0-2.5mg/L, methyl α-naphthyl acetate
0.1mg/L, sucrose 25-30g/L, agar 5.8-6g/L；When preparation, MS culture medium is first measured, zeatin, methyl α-naphthyl acetate is added, adds
Enter sucrose, after sucrose dissolution, adjust between pH to 5.8-5.9, agar is added, heating melts agar, the high temperature at 121 DEG C
High pressure sterilization 21min, in use, cultivating (12h, 2000LX) under illumination, cultivation temperature is 21-25 DEG C.
Embodiment 2-1:
Evoked callus breaks up adventitious shoots culture based formulas: 1 liter of MS culture medium first measured, zeatin 2.0mg is added,
Sucrose 25g is added in methyl α-naphthyl acetate 0.1mg, after sucrose dissolution, adjusts pH to 5.8, and agar 5.8g is added, and heating melts agar,
The autoclave sterilization 21min at 121 DEG C.
Embodiment 2-2:
Evoked callus breaks up adventitious shoots culture based formulas: 1 liter of MS culture medium first measured, zeatin 2.5mg is added,
Sucrose 30g is added in methyl α-naphthyl acetate 0.1mg, after sucrose dissolution, adjusts pH to 5.9, and agar 6.0g is added, and heating melts agar,
The autoclave sterilization 21min at 121 DEG C.
Embodiment 2-3:
Evoked callus breaks up adventitious shoots culture based formulas: 1 liter of MS culture medium first measured, zeatin 2.3mg is added,
Sucrose 26g is added in methyl α-naphthyl acetate 0.1mg, after sucrose dissolution, adjusts pH to 5.9, and agar 5.9g is added, and heating melts agar,
The autoclave sterilization 21min at 121 DEG C.
Experiment two: it is grown thickly by different exogenous plant hormones or plant growth regulator analysis to callus differentiation is compared
The influence of bud:
8 groups of comparative tests are done according to different exogenous plant hormones or plant growth regulator and different usage amounts, such as 3 institute of table
Show, every group is done 3 parallel samples, MS culture medium, sucrose addition, agar addition sucrose all the same in 8 groups of comparative tests
30g/L, agar 5.8g/ L use identical preparation process.The autoclave sterilization 21min at 121 DEG C, is cultivated under illumination
(12h, 10000Lx-20000Lx), cultivation temperature are 21-25 DEG C.
Table 3:8 group evoked callus breaks up adventitious shoots culture base
Obtain after experimental observation and data statistic analysis: Y2 group processing callus induction is best from sprouting, every callus block
It can induce 2-6 plants of buds for the first time.Differentiation rate is up to 90% or more again.Multiple Buds are forwarded to adventitious buds proliferation culture medium by 4 0d or so
In, remaining callus is continued to be forwarded in evoked callus differentiation adventitious shoots culture base, second of every callus block is average
It can induce 3 plants of buds.Differentiation rate is equally up to 90% or more again.After 40d, continue to transfer with the method, until callus no longer divides
Until changing Multiple Buds.
Embodiment 3:
A kind of Kiwi berry fast propagating culture medium, including inducer blade differentiation callus tissue culture base, induction stem apex or armpit
Bud germination and growth culture medium, evoked callus differentiation adventitious shoots culture base, adventitious buds proliferation culture medium, sugar-free tissue culture and inducement are not
Determine root culture medium, wherein
Induction stem apex or axillary bud sprouting grown cultures based formulas: 1 liter of MS culture medium, 6- benzyl aminoadenine 0-0.5mg/L,
Methyl α-naphthyl acetate 0.1-0.3mg/L, sucrose 25-30g/L, agar 5.8-6g/L；When preparation, MS culture medium is first measured, adds 6- benzyl ammonia
Sucrose is added in base adenine, methyl α-naphthyl acetate, after sucrose dissolution, adjusts between pH to 5.8-5.9, and agar is added, and heating makes agar
Melt, the autoclave sterilization 21min at 121 DEG C, in use, (12h, 2000Lx) is cultivated under illumination, cultivation temperature 21-25
℃。
Embodiment 3-1:
It induces stem apex or axillary bud sprouting grown cultures based formulas: first measuring 1 liter of MS culture medium, addition 6- benzyl amino gland is fast
Sucrose 25g is added in purine 0mg, methyl α-naphthyl acetate 0.1mg, after sucrose dissolution, adjusts pH to 5.8, and agar 5.8g is added, and heating makes fine jade
Rouge melts, the autoclave sterilization 21min at 121 DEG C.
Embodiment 3-2:
It induces stem apex or axillary bud sprouting grown cultures based formulas: first measuring 1 liter of MS culture medium, addition 6- benzyl amino gland is fast
Sucrose 30g is added in purine 0.5mg, methyl α-naphthyl acetate 0.3mg, after sucrose dissolution, adjusts pH to 5.9, and agar 6g is added, and heating makes fine jade
Rouge melts, the autoclave sterilization 21min at 121 DEG C.
Embodiment 3-3:
It induces stem apex or axillary bud sprouting grown cultures based formulas: first measuring 1 liter of MS culture medium, addition 6- benzyl amino gland is fast
Sucrose 27g is added in purine 0.3mg, methyl α-naphthyl acetate 0.2mg, after sucrose dissolution, adjusts pH to 5.9, agar 5.9g is added, heating makes
Agar melts, the autoclave sterilization 21min at 121 DEG C.
Experiment three: raw to comparison stem apex or axillary bud sprouting by different exogenous plant hormones or plant growth regulator analysis
Long influence:
16 groups of comparative tests are done according to different exogenous plant hormones or plant growth regulator and different usage amounts, such as table 4
Shown, every group is done 3 parallel samples, MS culture medium, sucrose addition, agar addition sugarcane all the same in 16 groups of comparative tests
Sugared 30g/L, agar 5.8g/ L use identical preparation process.
Table 4:16 group induces stem apex or axillary bud sprouting growth medium
Wherein, half stem apex half stem section is selected in each processing, processing 4 is blank control group, experimental observation and data
It is obtained after statistical analysis: No. 2 processing group axillary bud sprouting rates and stem apex growth rate highest (containing pollution).No. 2 processing group axillary bud sproutings
Rate 28%, stem apex germination rate 34%.
Embodiment 4:
A kind of Kiwi berry fast propagating culture medium, including inducer blade differentiation callus tissue culture base, induction stem apex or armpit
Bud germination and growth culture medium, evoked callus differentiation adventitious shoots culture base, adventitious buds proliferation culture medium, sugar-free tissue culture and inducement are not
Determine root culture medium, wherein
Adventitious buds proliferation culture medium prescription: 1 liter of MS culture medium, methyl α-naphthyl acetate 0.1-0.4mg/ L, 6- benzyl aminoadenine
0.6-1.0mg/L, sucrose 25-30g/L, agar 5.8-6g/L；When preparation, MS culture medium is first measured, adds methyl α-naphthyl acetate, 6- benzyl ammonia
Sucrose is added in base adenine, after sucrose dissolution, adjusts between pH to 5.8-5.9, and agar is added, and heating melts agar,
Autoclave sterilization 21min at 121 DEG C, in use, cultivating (12h, 2000LX) under illumination, cultivation temperature is 21-25 DEG C.
Embodiment 4-1:
Adventitious buds proliferation culture medium prescription: first measuring 1 liter of MS culture medium, adds methyl α-naphthyl acetate 0.1mg, and 6- benzyl amino gland is fast
Sucrose 25g is added in purine 0.6mg, after sucrose dissolution, adjusts pH to 5.8, and agar 5.8g is added, and heating melts agar,
Autoclave sterilization 21min at 121 DEG C.
Embodiment 4-2:
Adventitious buds proliferation culture medium prescription: first measuring 1 liter of MS culture medium, adds methyl α-naphthyl acetate 0.4mg, and 6- benzyl amino gland is fast
Sucrose 30g is added in purine 1.0mg, after sucrose dissolution, adjusts pH to 5.9, agar 6g is added, heating melts agar, 121
Autoclave sterilization 21min at DEG C.
Embodiment 4-3:
Adventitious buds proliferation culture medium prescription: first measuring 1 liter of MS culture medium, adds methyl α-naphthyl acetate 0.2mg, and 6- benzyl amino gland is fast
Sucrose 28g is added in purine 0.8mg, after sucrose dissolution, adjusts pH to 5.9, and agar 5.9g is added, and heating melts agar,
Autoclave sterilization 21min at 121 DEG C.
Experiment four: by different exogenous plant hormones or plant growth regulator analysis to the shadow for comparing adventitious buds proliferation
It rings:
8 groups of comparative tests are done according to different exogenous plant hormones or plant growth regulator and different usage amounts, such as 5 institute of table
Show, every group is done 3 parallel samples, MS culture medium, sucrose addition, agar addition sucrose all the same in 8 groups of comparative tests
30g/L, agar 5.8g/ L use identical preparation process.The autoclave sterilization 21min at 121 DEG C, is cultivated under illumination
(12h, 10000Lx-20000Lx), cultivation temperature are 23-27 DEG C.
Table 5:8 group adventitious buds proliferation culture medium
Obtain after experimental observation and data statistic analysis: J2, J3 group processing proliferation seedling growing way are best, proliferation times 5.
It is proliferated phenomena such as seedling is without vitrifying, browning.
In adventitious buds proliferation incubation, with the increase of subculture number, gradually decreases plant exogenous hormone and plant is raw
The dosage of long regulator.Wherein after adventitious buds proliferation culture medium subculture 5-7 times, formula is by original 1 liter of MS culture medium, naphthalene second
Sour 0.2mg/L, 6-benzyl aminoadenine 1.0mg/L, sucrose 30g/L, agar 5.8g/L, by for successive adjustment be MS culture medium 1
It rises, methyl α-naphthyl acetate 0.2mg/L, 6- benzyl aminoadenine 0.7mg/L, sucrose 20g/L, agar 5.8g/L.
Embodiment 5:
A kind of Kiwi berry fast propagating culture medium, including inducer blade differentiation callus tissue culture base, induction stem apex or armpit
Bud germination and growth culture medium, evoked callus differentiation adventitious shoots culture base, adventitious buds proliferation culture medium, sugar-free tissue culture and inducement are not
Determine root culture medium, wherein
Sugar-free tissue culture and inducement adventitious root culture medium prescription: NH4NO316.5mg/L,KNO319 mg/L,MgSO4·
7H2O3.7mg/L,KH2PO41.7mg/L,CaCl2·2H2O4.4 mg/L,KI0.083mg/L,H3BO30.62mg/L,MnSO4·
4H2O2.23mg/L,ZnSO 4·7H2O0.86mg/L,Na2MnO4·2H2O0.025mg/L,CuSO4·5H2O0.0025 mg/L,
CoCl2·6H2O0.0025mg/L,FeSO4·7H2O2.78mg/L,Na2-EDT A·2H2O3.73mg/L, Kiwi berry traditional group
Difficulty of taking root is trained, abnormal root, aerial root are also easy to produce, the easily long callus in root causes experienced shoot survival percent extremely low.The present invention uses
Sugar-free tissue culture rooting method, i.e., be not added that sucrose, organic matter, hormone, agar etc. are any to be had in sugar-free tissue culture and inducement adventitious root culture medium
Machine substance.Using endogenous hormones rooting method, sugar-free tissue culture and inducement adventitious root culture medium is placed in specific culture vessel, it is artificial to control
Microenvironment processed can both shorten the experienced seedling period of taking root, and can also improve transplanting survival rate.
When culture, cultivate indoor illumination 20000Lx, humidity 50%-90%, 25 DEG C -28 DEG C of temperature, CO2Concentration
1000ppm-1500ppm cultivates forced ventilation 15min/h, throughput 90ml/min-180ml/min in box.
Sugar-free tissue culture and inducement adventitious root culture medium and tradition through the invention has sugared root media to compare test,
Test result part table 6.
6 sugar-free tissue culture and inducement adventitious root culture medium of table and tradition have sugared root media production efficiency to compare
Above data is shown, using endogenous hormones rooting method, sugar-free nutrition liquid is placed in specific culture vessel, artificial to control
Microenvironment processed can both shorten the experienced seedling period of taking root, and can also improve transplanting survival rate.Start within the culture of sugar-free rooting method the 7th day
It takes root, takes root within the 10th day 30%, take root within 15 days 98% or more.- 30 days 20 days, height of seedling 5cm-8cm, grow vigorous, root system Pang
Greatly, without practicing seedling, directly transplanting to nutritive cube or big Tanaka.The production efficiency of sugar-free rooting method of the present invention is that tradition has sugared life
18 times or more of method for root, substantially increase the production efficiency of Kiwi berry detoxic seedling large-scale production.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of Kiwifruit Tissue Culture rapid propagation method, which comprises the following steps:
S1. the branch and/or young leaflet tablet for selecting Kiwi berry current year life are as explant；
S2. explant is handled, specifically includes the following steps:
When selecting branch as explant, removes the blade in branch stem section and retain axillary bud or strip stem apex by disinfection treatment
Afterwards, access induces in stem apex or axillary bud sprouting growth medium, is placed on culturing room's culture；
When selecting young leaflet tablet as explant, after young leaflet tablet is disinfected, access inducer blade breaks up callus
In culture medium, it is placed on culturing room's culture；The callus size differentiated after inoculation is 0.8cm2-2.5cm2When, it is forwarded to and lures
It leads in callus differentiation adventitious shoots culture base；
S3. shoot proliferation culture, specifically includes the following steps:
When selecting branch as explant, explant axillary bud, stem apex grow to 1.0cm-2.5cm, and produce with 2-5 piece young leaves
It when raw, cut sprouting and is forwarded to adventitious buds proliferation culture medium and expand numerous, obtain subculture Multiple Buds；
When selecting young leaflet tablet as explant, the callus of young leaflet tablet first differentiates adventitious bud, and adventitious bud grows up into
Test tube seedling is forwarded to adventitious buds proliferation culture medium and expand numerous, obtains subculture Multiple Buds by the test tube seedling of high 2-4cm；
S4. it takes root, specifically includes the following steps:
The seedling that subculture Multiple Buds are grown into is forwarded in sugar-free tissue culture and inducement adventitious root culture medium, culturing room CO2Concentration is
1000ppm-1500ppm, sugar-free tissue culture and inducement adventitious root culture medium prescription: NH4NO316.5 mg/L, KNO319 mg/L,
MgSO4·7H2O3.7mg/L, KH2PO41.7mg/L, CaCl2·2H2O4.4mg/L, KI0.083 mg/L,H3BO30.62mg/
L,MnSO4·4H2O2.23mg/L,ZnSO4·7H2O0.86mg/L, Na2MnO4·2H2O0.025 mg /L, CuSO4·
5H2O0.0025 mg /L, CoCl2·6H2O0.0025 mg /L, FeSO4·7H2O2.78 mg /L, Na2-EDTA·
2H2O3.73 mg /L;
S5. young seedling direct transplantation after taking root is to nutritive cube or big Tanaka.
2. a kind of Kiwifruit Tissue Culture rapid propagation method according to claim 1, which is characterized in that inducer blade breaks up callus
The formula of tissue culture medium (TCM) are as follows: 1 liter of MS culture medium, 6- benzyl aminoadenine 0.8-1.2mg/L, 2,4- dichlorphenoxyacetic acid 0.3-
0.6mg/L, sucrose 25-30g/L, agar 5.8-6g/L.
3. a kind of Kiwifruit Tissue Culture rapid propagation method according to claim 1 or claim 2, which is characterized in that induction stem apex or armpit
Bud germination and growth culture medium prescription are as follows: 1 liter of MS culture medium, 6- benzyl aminoadenine 0-0.5mg/L, methyl α-naphthyl acetate 0.1-0.3mg/
L, sucrose 25-30g/L, agar 5.8-6g/L.
4. a kind of Kiwifruit Tissue Culture rapid propagation method according to claim 1, which is characterized in that evoked callus differentiation
Adventitious shoots culture based formulas are as follows: 1 liter of MS culture medium, zeatin 2.0-2.5mg/L, methyl α-naphthyl acetate 0.1mg/L, sucrose 25-30g/L,
Agar 5.8-6g/L.
5. a kind of Kiwifruit Tissue Culture rapid propagation method according to claim 1, which is characterized in that adventitious buds proliferation culture medium
Formula are as follows: 1 liter of MS culture medium, methyl α-naphthyl acetate 0.1-0.4mg/L, 6- benzyl aminoadenine 0.6-1.0mg/L, sucrose 25-30g/L,
Agar 5.8-6g/L.
6. a kind of Kiwifruit Tissue Culture rapid propagation method according to claim 1, which is characterized in that culture described in step S4
The indoor illumination 20000Lx of room, humidity 50%-90%, cultivate indoor air-changing 15min/h, throughput 90mL/ by 25 DEG C -28 DEG C of temperature
min-180mL/min。
7. a kind of Kiwifruit Tissue Culture fast propagating culture medium, which is characterized in that including inducer blade differentiation callus tissue culture base,
Stem apex or axillary bud sprouting growth medium, evoked callus is induced to break up adventitious shoots culture base, adventitious buds proliferation culture medium, nothing
Sugared tissue culture and inducement adventitious root culture medium, wherein
The formula of inducer blade differentiation callus tissue culture base are as follows: 1 liter of MS culture medium, 6- benzyl aminoadenine 0.8-1.2mg/L,
2,4- dichlorphenoxyacetic acid 0.3-0.6mg/L, sucrose 25-30g/L, agar 5.8-6g/L；
Induce stem apex or axillary bud sprouting grown cultures based formulas are as follows: 1 liter of MS culture medium, 6- benzyl aminoadenine 0-0.5mg/L,
Methyl α-naphthyl acetate 0.1-0.3mg/L, sucrose 25-30g/L, agar 5.8-6g/L；
Evoked callus breaks up adventitious shoots culture based formulas are as follows: and 1 liter of MS culture medium, zeatin 2.0-2.5mg/L, methyl α-naphthyl acetate
0.1mg/L, sucrose 25-30g/L, agar 5.8-6g/L；
Adventitious buds proliferation culture medium prescription are as follows: 1 liter of MS culture medium, methyl α-naphthyl acetate 0.1-0.4mg/L, 6- benzyl aminoadenine 0.6-
1.0mg/L, sucrose 25-30g/L, agar 5.8-6g/L；
Sugar-free tissue culture and inducement adventitious root culture medium prescription are as follows: NH4NO316.5mg/L, KNO319mg/L, MgSO4·
7H2O3.7mg/L,KH2PO41.7mg/L,CaCl2·2H2O4.4mg/L, KI0.083mg/L,H3BO30.62mg/L,MnSO4·
4H2O2.23mg/L,ZnSO4·7H2O0.86mg/L, Na2MnO4·2H2O0.025mg/L, CuSO4·5H2O0.0025mg/L,
CoCl2·6H2O0.0025mg/L, FeSO4·7H2O2.78 mg /L, Na2-EDTA·2H2O3.73 mg /L。
8. a kind of Kiwifruit Tissue Culture fast propagating culture medium according to claim 7, which is characterized in that including inducer blade point
Change callus tissue culture base, induction stem apex or axillary bud sprouting growth medium, evoked callus breaks up adventitious shoots culture base, clump
It sprouts proliferated culture medium, sugar-free tissue culture and inducement adventitious root culture medium, wherein
The formula of inducer blade differentiation callus tissue culture base: 1 liter of MS culture medium, 6- benzyl aminoadenine 1.0mg/L, 2,4- bis-
Chlorophenoxyacetic acid 0.4mg/L, sucrose 30g/L, agar 5.8g/L；
Induction stem apex or axillary bud sprouting grown cultures based formulas: 1 liter of MS culture medium, 6- benzyl aminoadenine 0.4mg/L, naphthalene second
Sour 0.1mg/L, sucrose 30g/L, agar 5.8g/L；
Evoked callus differentiation adventitious shoots culture based formulas: 1 liter of MS culture medium, zeatin 2.0mg/L, methyl α-naphthyl acetate 0.1mg/L,
Sucrose 25-30g/L, agar 5.8-6g/L；
Adventitious buds proliferation culture medium prescription: 1 liter of MS culture medium, methyl α-naphthyl acetate 0.2mg/L, 6- benzyl aminoadenine 0.8-1.0mg/L,
Sucrose 30g/L, agar 5.8g/L；
Sugar-free tissue culture and inducement adventitious root culture medium prescription: NH4NO316.5mg/L, KNO319mg/L, MgSO4·7H2O3.7mg/
L,KH2PO41.7mg/L,CaCl2·2H2O4.4mg/L, KI0.083mg/L,H3BO30.62mg/L,MnSO4·4H2O2.23mg/
L,ZnSO4·7H2O0.86mg/L, Na2MnO4·2H2O0.025 mg /L, CuSO4·5H2O0.0025 mg /L,
CoCl2·6H2O0.0025 mg /L, FeSO4·7H2O2.78 mg /L, Na2-EDTA·2H2O3.73 mg /L。
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| CN110140658A (en) * | 2019-06-26 | 2019-08-20 | 云南师范大学 | A method of Kiwi berry seedling is cultivated using Stem Segments In Chinese Gooseberry forming layer stem cell |
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| CN110651710B (en) * | 2019-09-17 | 2021-06-04 | 湖北大学 | Production method of kiwi fruit seedlings without canker pathogenic bacteria |
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| CN111149699A (en) * | 2020-01-19 | 2020-05-15 | 华中农业大学 | Culture medium and genetic transformation method of red-yang kiwi fruit |
| CN111149699B (en) * | 2020-01-19 | 2021-11-30 | 华中农业大学 | Culture medium and genetic transformation method of red-yang kiwi fruit |
| CN111345234A (en) * | 2020-04-13 | 2020-06-30 | 井冈山大学 | In-vitro rapid seedling culture method for kiwi fruits |
| CN111713410A (en) * | 2020-07-03 | 2020-09-29 | 四川农业大学 | Kiwi explant detoxification method |
| CN114766367A (en) * | 2022-05-18 | 2022-07-22 | 西北农林科技大学 | Kiwi fruit tissue culture method |
| CN115136893A (en) * | 2022-08-05 | 2022-10-04 | 江苏省农业科学院 | Callus regeneration system establishment method for picking of macaque macrosperma |
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