CN104126511B - The method for tissue culture of a kind of precocious stem of Radix pyri section and culture medium - Google Patents
The method for tissue culture of a kind of precocious stem of Radix pyri section and culture medium Download PDFInfo
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Abstract
The present invention provides method for tissue culture and the culture medium of a kind of precocious stem of Radix pyri section, belongs to molecular biology of plant cells technical field.Described method for tissue culture, comprises the steps: to carry out inducing differentiation culture by outer implant employing initial inoculation culture medium, obtains just for tissue cultured seedling；Just carrying out enrichment culture for tissue cultured seedling proliferated culture medium by described, then cutting newly slightly transfers carries out successive transfer culture into strong seedling culture base, then proceeds to root media A and root media B successively and carry out root culture.The described culture medium for precocious stem of Radix pyri section tissue culture, by one or more combinations in initial inoculation culture medium, proliferated culture medium, strong seedling culture base, root media.Method for tissue culture of the present invention, sterilization method safety, time are short, significantly improve tissue cultured seedling growth coefficient, rooting rate, average root length peace all radicals.
Description
Technical field
The invention belongs to molecular biology of plant cells field, be specifically related to method for tissue culture and the culture medium of stem of Radix pyri section.
Background technology
Su Cui 1 is high-quality early ripening pear, and parent is crisp × emerald green hat of China, and hybridization in 2003 is cultivated cross hybrid seedling, within 2011, passed through Jiangsu Province for 2004
Crop breed audit committee kind accreditation (Su Jian fruit 201107).Pulp white, meat is delicate, and stone cell is few or nothing, juice multi-flavor is sweet,
Raciness, quality are good.Bud is easily formed, and continuous bearing capacity is strong, and fruit-setting rate is high, and the fruit maturation phase is at the beginning of 7 months.
Su Cui 2 falls within early ripening pear, and parent is green × emerald green hat of Xizi, a famous beauty in the late Spring and Autumn Period, and hybridization in 2003 is cultivated cross hybrid seedling, within 2011, passed through Jiangsu for 2004
Save variety certification.Fruit falls oval, and peel yellow green is rustless, pulp white, and meat is delicate, and stone cell is few or nothing, and bud is easily formed,
Bearing capacity is strong continuously, and fruit-setting rate is high, and the fruit maturation phase is mid-July.
When Su Cui 1 and Su Cui 2 use conventional cottage method to breed, breeding coefficient is low, highly heterozygosis；Use conventional tissue culture method
When breeding, test tube seedling proliferation coefficient is low, and average rooting rate, mean elements are relatively low, and root length is shorter.
Summary of the invention
It is an object of the invention to provide the method for tissue culture of a kind of stem of Radix pyri section, the method significantly improves the rate of increase of test tube Seedling, growth coefficient, life
Root rate, average root length peace all radicals.
It is a further object of the present invention to provide the culture medium for stem of Radix pyri section tissue culture.
The purpose of the present invention adopts the following technical scheme that realization.
The method for tissue culture of a kind of precocious stem of Radix pyri section, comprises the steps: to carry out inducing differentiation culture by outer implant employing initial inoculation culture medium,
Obtain just for tissue cultured seedling；Just carrying out enrichment culture for tissue cultured seedling proliferated culture medium by described, then cutting newly slightly transfers continues into strong seedling culture base
Culture, then proceed to root media A and root media B successively and carry out root culture；
Described initial inoculation culture medium is interpolation 6-BA, NAA and GA in 1/2MS or AS minimal medium3After the culture medium that obtains, described
The final concentration of 0.05-0.5mg/L of the final concentration of 0.5-1.0mg/L of 6-BA, described NAA, described GA3Final concentration of 0.2-3mg/L, institute
The pH stating initial inoculation culture medium is 5.5-5.8；
Described proliferated culture medium is interpolation 6-BA, NAA and GA in MS culture medium3After the culture medium that obtains, described 6-BA's is final concentration of
0.5～1.0mg/L, final concentration of the 0.1 of described NAA～0.2mg/L, described GA3Final concentration of 0.5-3mg/L, described proliferated culture medium
PH is 5.5-5.8；
Described strong seedling culture base is 1/3MS culture medium or 1/4QL culture medium, and the pH of described strong seedling culture base is 5.5-5.8；
Described root media A is the culture medium obtained after interpolation IAA, 6-BA and NAA in 1/4QL culture medium, and the end of described IAA is dense
Degree is the final concentration of 0.05-0.15mg/L of the final concentration of 0.8-1.2mg/L, described NAA of 0.4-0.6mg/L, described 6-BA, described training of taking root
The pH supporting base is 5.5-5.8；
Described root media B is 1/4QL culture medium.
In the present invention, described initial inoculation culture medium, proliferated culture medium, strong seedling culture base, root media A and root media B all add
Added with sucrose and agar, final concentration of the 20 of described sucrose～30g/L, the final concentration of 5-7g/L of described agar.
In the present invention, the condition of culture in described initial inoculation culture medium is: cultivation temperature 22～25 DEG C, illumination every day 15-16h, dark 8-9h,
Intensity of illumination is 1500-2000lx, and every 25-32d changes an initial inoculation culture medium.
In the present invention, the condition of culture in described proliferated culture medium is: cultivation temperature 22～25 DEG C, illumination every day 15-17h, dark 7-9h, light
Being 1500-2000lx according to intensity, incubation time is 25-32d.
In the present invention, the condition of culture in described strong seedling culture base is: cultivation temperature 22～25 DEG C, illumination every day 15-17h, dark 7-9h, light
Being 1500-2000lx according to intensity, once, subculture number is 2-3 time to every 13-16 days subcultures.
In the present invention, the condition of culture in described root media A is: cultivation temperature 24～26 DEG C, dark culturing 7-12 days.
In the present invention, the condition of culture in described root media B is: cultivation temperature 22～25 DEG C, illumination every day 15-16h, dark 8-9h,
Intensity of illumination is 1500-2000lx, and incubation time is 50-60 days.
In the present invention, described outer implant is for becoming maternal plant stem section in age；It is preferably green tape leaf stem section then.
In the present invention, described outer implant is handled as follows before induction differentiation culture: use outer implant described in Carbenicillin aqueous solution soaking, so
Rear employing contains love makes every effort to overcomeTMDescribed outer implant is carried out disinfection by the solution of A, B and polysorbas20.
The present invention is also claimed the culture medium for precocious stem of Radix pyri section tissue culture, by completely or partially forming in following culture medium:
(a) initial inoculation culture medium；
Described initial inoculation culture medium is interpolation 6-BA, NAA and GA in 1/2MS or AS minimal medium3After the culture medium that obtains, described
The final concentration of 0.05-0.5mg/L of the final concentration of 0.5-1.0mg/L of 6-BA, described NAA, described GA3Final concentration of 0.2-3mg/L, institute
The pH stating initial inoculation culture medium is 5.5-5.8；
(b) proliferated culture medium；
Described proliferated culture medium is interpolation 6-BA, NAA and GA in MS culture medium3After the culture medium that obtains, described 6-BA's is final concentration of
The final concentration of 0.1-0.2mg/L of 0.5-1.0mg/L, described NAA, described GA3Final concentration of 0.5-3mg/L, the pH of described proliferated culture medium
For 5.5-5.8；
(c) strong seedling culture base；
Described strong seedling culture base is 1/3MS culture medium or 1/4QL culture medium, and the pH of described strong seedling culture base is 5.5-5.8；
(d) root media；
Described root media includes root media A and root media B；
Described root media A is the culture medium obtained after interpolation IAA, 6-BA and NAA in 1/4QL culture medium, and the end of described IAA is dense
Degree is the final concentration of 0.05-0.15mg/L of the final concentration of 0.8-1.2mg/L, described NAA of 0.4-0.6mg/L, described 6-BA；
Described root media B is 1/4QL culture medium.
The method for tissue culture of stem of Radix pyri section of the present invention, compared with normal cutting propagation propagation method, overcomes pears Seedling cottage propagation coefficient shortcoming low, highly heterozygosis,
Obtaining a large amount of clone test tube Seedling, its stabilization characteristics of genetics, the rate of increase are high, provide substantial amounts of test tube Seedling for genetic transformation and expansion cultivated area.
The method for tissue culture of stem of Radix pyri section of the present invention compared with conventional tissue culture method,
(A) disinfection way utilizes love to make every effort to overcomeTMA, B replace mercuric chloride, safety non-toxic.Use stem section as outer implant, shorten Multiple Buds growth
Time；Cancel alcohol disinfecting, shorten love and make every effort to overcomeTMThe disinfecting time of A, B, improves two kind survival rates, reduces melting brown rate；At vortex
Reason reduces pollution rate.
(B) initial inoculation culture medium selects AS culture medium, plant strain growth gesture and grow up gesture and be better than MS and 1/2MS culture medium.
(C) tissue cultured seedling growth coefficient significantly improves, and after strong seedling culture, plant strain growth is healthy, and blade is open and flat, leaf green, without vitrification phenomenon.
(D) rooting method of the present invention significantly improves tissue cultured seedling growth coefficient, rooting rate, average root length peace all radicals.
Detailed description of the invention
6-BA:6-benzyl aminoadenine；NAA:1-naphthalene acetic acid；GA3: gibberellins；IAA: indole-3-acetic acid；IBA:3-indolebutyric acid.
The composition of MS culture medium: (1) a great number of elements: NH4NO31650mg/L, KNO31900mg/L, CaCl2·2H2O440mg/L,
MgSO4·7H2O370mg/L, KH2PO4170mg/L；(2) trace element: MnSO4·4H2O22.3mg/L, ZnSO4·7H2O8.6mg/L,
CoCl2·6H2O0.025mg/L, CuSO4·5H2O0.025mg/L, Na2MoO4·2H2O0.25mg/L, KI0.083mg/L, H3BO36.2mg/L；
(3) iron salt: Na2EDTA37.3mg/L, FeSO4·7H2O27.8mg/L；(4) organic substance: nicotinic acid 0.5mg/L, thiamine hydrochloride 0.1mg/L,
Pyridoxine hydrochloride 0.5mg/L, inositol 100mg/L, glycine 2mg/L.The solvent of MS culture medium is water.
1/2MS culture medium: composition is identical with MS culture medium, the half of identical component during wherein a great number of elements concentration takes MS culture medium, other become
The content divided is with MS culture medium.
1/3MS culture medium: composition is identical with MS culture medium, during wherein a great number of elements concentration takes MS culture medium the 1/3 of identical component, other compositions
Content with MS culture medium.
The composition of AS culture medium: (1) a great number of elements: NH4NO3525mg/L, KNO3950mg/L, CaCl2·2H2O220mg/L,
MgSO4·7H2O185mg/L, KH2PO485mg/L；(2) trace element: MnSO4·4H2O2.23mg/L, ZnSO4·7H2O1.05mg/L,
CoCl2·6H2O0.0025mg/L, CuSO4·5H2O0.0025mg/L, Na2MoO4·2H2O0.025mg/L, KI0.083mg/L, H3BO3
0.62mg/L；(3) iron salt: Na2EDTA27.7mg/L, FeSO4·7H2O20.7mg/L；(4) organic substance: nicotinic acid 0.5mg/L, hydrochloric acid sulfur
Amine element 0.4mg/L, pyridoxine hydrochloride 0.5mg/L, inositol 10mg/L, glycine 0.2mg/L.The solvent of AS culture medium is water.
QL culture medium composition: (1) a great number of elements: NH4NO3400mg/L, KH2PO4270mg/L, CaNO3833.77mg/L, MgSO4
175.79mg/L；(2) trace element: H3BO36.2mg/L, CoCl2·6H2O0.025mg/L, CuSO4·5H2O0.025mg/L, MnSO4·H2O
0.76mg/L, Na2MoO4·2H2O0.25mg/L, KI0.08mg/L, ZnSO4·7H2O8.6mg/L；(3) iron salt: Na2EDTA37.3mg/L,
FeSO4·7H2O27.8mg/L；(4) organic substance: inositol 29.61mg/L, thiamine hydrochloride 0.118mg/L.The solvent of QL culture medium is water.
1/4QL culture medium: composition is identical with QL culture medium, during wherein a great number of elements concentration takes QL culture medium the 1/4 of identical component, other compositions
Concentration with QL culture medium.
Love is made every effort to overcomeTMA, B: purchased from Yu Han bio tech ltd, Shanghai, article No. E0015.
Embodiment 1
Adopt and with the following method ' Su Cui 1 ' and ' Su Cui 2 ' stem of Radix pyri section is carried out tissue culture.
Outer implant collection and sterilization: October the previous year to December, gather annotinous branch, be stored in 4 DEG C of refrigerators from orchard.February then
The last ten-days period, to early March, take branch and insert in sterilized water, 22 DEG C-25 DEG C, illumination every day 8h, dark 16h, and intensity of illumination 1500-2000lx is urged
Bud, every 7d changes a fresh sterile water.April, early and middle ten days, after bud is sprouted, took multiple stem with bud, was cut into 1-1.5cm height, one tender shoots of band
Stem section, disinfect as follows: stem with bud with after tap water immersion treatment 2.5h containing 100mg/L Carbenicillin, uses mixing to disappear
Venom vortex 5-8 minute, then peels off bract, mixing disinfectant solution secondary sterilization 5-8 minute.The preparation method of mixing disinfectant solution is: make every effort to overcome from loveTMIn A, B, take a piece of A sheet and a piece of B sheet be dissolved in 500ml sterilized water, add the polysorbas20 of final concentration of 0.01% (mass percentage concentration),
Obtain mixing disinfectant solution.Mixing disinfectant solution is liked to make every effort to overcomeTMThe concentration of A, B is 500mg/L.
1) initial inoculation: in initial inoculation culture medium 1 and 2, the stem with bud after inoculation 20 is disinfected respectively, carry out induction differentiation
Cultivate, obtain just for tissue cultured seedling.Induction differentiation culture condition be: cultivation temperature 22 DEG C-25 DEG C, every day light application time 16h, 8h interlunation,
Intensity of illumination 1500-2000lx, cultivates 25d.
Initial inoculation culture medium 1: add 6-BA, NAA, GA in 1/2MS3, sucrose and agar, the final concentration of 1.0mg/L of 6-BA,
Final concentration of 0.2mg/L, GA of NAA3Final concentration of 0.5mg/L, the final concentration of 30g/L of sucrose, the final concentration of 5-7g/L of agar,
PH=5.8.
Initial inoculation culture medium 2: add 6-BA, NAA, GA in AS3, sucrose and agar, final concentration of 1.0mg/L, NAA of 6-BA
Final concentration of 0.2mg/L, GA3Final concentration of 0.5mg/L, the final concentration of 30g/L of sucrose, the final concentration of 5-7g/L, pH=5.8 of agar.
Statistics ' Su Cui 1 ' and ' Su Cui 2 ' is just for average melting brown rate, pollution rate and the survival rate of tissue cultured seedling, and concrete outcome is: ' Su Cui
No. 1 ' average melting brown rate 11.2%, pollution rate 19.9%, survival rate 68.9%；' Su Cui 2 ' average melting brown rate 10.5%, pollution rate 13.6%,
Survival rate 75.9%.
Observe and record induction differentiation culture in 25 days ' Su Cui 1 ' and ' Su Cui 2 ' just for the growing state of tissue cultured seedling, specifically such as table 1
With table 2.In initial inoculation culture medium 2, each plant strain growth gesture is good as can be seen from Table 1 and Table 2, and melting brown rate is low, and Bud polarization is good, has bright
The aobvious advantage that grows up, blade is open and flat, in green.
Growing state after the cultivation of table 1 ' Su Cui 1 ' initial inoculation
| Type of culture medium | Leaf growth situation | Plant strain growth situation |
| Initial inoculation culture medium 1 | Open and flat, grow normal, green | Growth potential is general, and growing up property of bud is weaker than plant in culture medium 2 |
| Initial inoculation culture medium 2 | Open and flat, grow normal, green | Growth potential is good, and growing up property of bud is good |
Growing state after the cultivation of table 2 ' Su Cui 2 ' initial inoculation
| Type of culture medium | Leaf growth situation | Plant strain growth situation |
| Initial inoculation culture medium 1 | Open and flat, grow normal, green | Growth potential is general, and growing up property of bud is weaker than plant in culture medium 2 |
| Initial inoculation culture medium 2 | Open and flat, grow normal, green | Growth potential is good, and growing up property of bud is good |
It should be noted that if inducing differentiation culture natural law to increase, then every 25-32d changes an initial inoculation culture medium.
2) enrichment culture: take 2 survived～high first for tissue cultured seedling of 3cm, be inoculated in containing variable concentrations 6-BA, NAA and GA3's
In proliferated culture medium, use following condition to carry out enrichment culture: cultivation temperature 22 DEG C-25 DEG C, every day light application time 16h, 8h interlunation, light
According to intensity 1500-2000lx, incubation time is 30d.After enrichment culture 30d, add up the growth coefficient of two kinds, and observe plant growth condition,
Concrete outcome is shown in Table 3 and 4.From table 3 and 4 it can be seen that 6-BA concentration is between 0.5-1.0mg/L, NAA concentration 0.1-0.2mg/L it
Between, the good differentiation of bud, growth coefficient is all more than 3；Add GA3, stem apex substantially extends, and growth coefficient improves, and reaches to be not added with GA3
About 2 times, GA3Concentration affects no significant difference at 0.5-3mg/L to growth coefficient；In all culture medium, plant strain growth is good, without vitrification
Phenomenon.
Proliferated culture medium 1: add 6-BA, NAA, sucrose and agar, final concentration of 0.5mg/L, NAA of 6-BA in MS culture medium
Final concentration of 0.1mg/L, the final concentration of 30g/L of sucrose, the final concentration of 5-7g/L, pH=5.8 of agar.
Proliferated culture medium 2: interpolation 6-BA, NAA, sucrose and agar in MS culture medium, the final concentration of 0.5mg/L of 6-BA, NAA's
Final concentration of 0.2mg/L, the final concentration of 30g/L of sucrose, the final concentration of 5-7g/L, pH=5.8 of agar.
Proliferated culture medium 3: add 6-BA, NAA, sucrose and agar, final concentration of 1.0mg/L, NAA of 6-BA in MS culture medium
Final concentration of 0.1mg/L, the final concentration of 30g/L of sucrose, the final concentration of 5-7g/L, pH=5.8 of agar.
Proliferated culture medium 4: add 6-BA, NAA, sucrose and agar, final concentration of 1.0mg/L, NAA of 6-BA in MS culture medium
Final concentration of 0.2mg/L, sucrose 30g/L, the final concentration of 5-7g/L, pH=5.8 of agar.
Proliferated culture medium 5: add 6-BA, NAA, GA in MS culture medium3, sucrose and agar, the final concentration of 1.0mg/L of 6-BA,
Final concentration of 0.2mg/L, GA of NAA3Final concentration of 0.5mg/L, the final concentration of 30g/L of sucrose, the final concentration of 5-7g/L of agar,
PH=5.8.
Proliferated culture medium 6: add 6-BA, NAA, GA in MS culture medium3, sucrose and agar, the final concentration of 1.0mg/L of 6-BA,
Final concentration of 0.2mg/L, GA of NAA3Final concentration of 3mg/L, the final concentration of 30g/L of sucrose, the final concentration of 5-7g/L, pH of agar
=5.8.
Parameter after table 3 ' Su Cui 1 ' enrichment culture
| Type of culture medium | Just for tissue culture plant inoculation number (individual) | Growth coefficient | Vitrification situation (%) |
| Proliferated culture medium 1 | 30 | 3.04 | 0 |
| Proliferated culture medium 2 | 30 | 3.89 | 0 |
| Proliferated culture medium 3 | 30 | 4.33 | 0 |
| Proliferated culture medium 4 | 30 | 5.95 | 0 |
| Proliferated culture medium 5 | 30 | 10.89 | 0 |
| Proliferated culture medium 6 | 30 | 10.95 | 0 |
Parameter after table 4 ' Su Cui 2 ' enrichment culture
| Type of culture medium | Just inoculate (individual) for tissue cultured seedling number | Growth coefficient | Vitrification situation (%) |
| Proliferated culture medium 1 | 50 | 3.89 | 0 |
| Proliferated culture medium 2 | 50 | 4.56 | 0 |
| Proliferated culture medium 3 | 50 | 5.96 | 0 |
| Proliferated culture medium 4 | 50 | 6.45 | 0 |
| Proliferated culture medium 5 | 50 | 11.9 | 0 |
| Proliferated culture medium 6 | 50 | 12.95 | 0 |
3) successive transfer culture: first for tissue cultured seedling to after enrichment culture, cut 1.5-2.0cm robust growth new the most slightly, be transferred to strong seedling culture base 1 He
In 2, carry out successive transfer culture under the following conditions: cultivation temperature 22 DEG C-25 DEG C, every day light application time 16h, 8h interlunation, intensity of illumination
1500-2000lx, every 15d subculture once, subculture 2 times, obtain Regenerated plant.Observe the growing state after each kind strong seedling culture, concrete such as table 5
Shown in 6.Can be seen that each kind stem is sturdy from table 5 and 6, blade is open and flat, and growth is normal, in green.
Strong seedling culture base 1:1/3MS culture medium is added sucrose and the agar of final concentration of 5g/L, the pH=5.8 of final concentration of 30g/L.
Strong seedling culture base 2:1/4QL culture medium adds final concentration of 30g/L sucrose and the agar of final concentration of 7g/L, pH=5.8.
Parameter after table 5 ' Su Cui 1 ' strong seedling culture
| Type of culture medium | Height of seedling (cm) | Blade indulges footpath/transverse diameter | Plant strain growth situation |
| Strong seedling culture base 1 | 3.40 | 1.45 | Stem is sturdy, blade is open and flat, green |
| Strong seedling culture base 2 | 3.31 | 1.34 | Stem is sturdy, blade is open and flat, green |
Parameter after table 6 ' Su Cui 2 ' strong seedling culture
| Type of culture medium | Height of seedling (cm) | Blade indulges footpath/transverse diameter | Plant strain growth situation |
| Strong seedling culture base 1 | 3.21 | 1.65 | Stem is sturdy, blade is open and flat, green |
| Strong seedling culture base 2 | 3.15 | 1.43 | Stem is sturdy, blade is open and flat, green |
4) root culture: take growth potential consistent, the Regenerated plant of high 3.0-4.0cm, it is inoculated in root media A.Root media A formula: 1/4QL
Culture medium is added IAA, 6-BA, NAA, sucrose and agar, the final concentration of 1.0mg/L of final concentration of 0.5mg/L, 6-BA of IAA,
The final concentration of 0.1mg/L of NAA, the final concentration of 25g/L of sucrose, the final concentration of 6g/L, pH=5.8 of agar.Condition of culture is: cultivate
Temperature is 25 DEG C, dark culturing 10d.
Regenerated plant after cultivating using root media A proceeds to root media B, cultivates 60d, obtains Seedling of taking root.Root media B formula:
1/4QL adds sucrose and agar, the final concentration of 20-30g/L of sucrose, the final concentration of 5-7g/L of agar.Condition of culture is: cultivation temperature
22 DEG C-25 DEG C, every day light application time 16h, 8h interlunation, intensity of illumination 1500-2000lx.
2 kinds are taken root as stated above, ' Su Cui 1 ' rooting rate 95.13%, average root length 8.46cm, mean elements 8.9；' Su Cui 2
Number ' rooting rate 93.25%, average root length 9.65cm, mean elements 9.2.
Embodiment 2 uses conventional tissue culture and rapid propagation method to cultivate ' Su Cui 1 ' and ' Su Cui 2 '
Adopt and cultivate ' Su Cui 1 ' and ' Su Cui 2 ' with the following method:
1) outer implant collection and sterilization: choose the annotinous branch of robust growth from field, clip edible tender branch, as outer implant, uses detergent
Rinsing, tap water rinses 30min.On superclean bench, will process after outer implant with 75% ethanol sterilizing 20s, the mercuric chloride sterilizing of 0.1%
8-10min, aseptic water washing 4-5 time.Scale removed by dissecting knife, cuts naked bub and is inoculated on inducing culture, 3 outer implant of every bottle graft kind.Lure
Leading culture medium is: add 6-BA, IBA, GA in 1/2MS culture medium3, sucrose and agar, the wherein final concentration of 1.0mg/L of 6-BA,
The final concentration of 0.2mg/L, GA of IBA3Final concentration of 2.0mg/L, the final concentration of 30g/L of sucrose, the final concentration of 6g/L, pH=5.8 of agar.
Condition of culture is: illumination every day 16h, dark 8h, temperature 25 DEG C, intensity of illumination 1500-2000lx.
The aseptic seedling of 2 kinds is sterilized as stated above, and ' Su Cui 1 ' average melting brown rate is 93.5%, and average survival is 5.6%；' Su Cui
No. 2 ' average melting brown rate is 95.2%, average survival is 6.5%.
2) inducing clumping bud: use inducing culture to cultivate about 15d and treat that bud grows to about 1cm, cut and be newly slightly transferred to new inducing culture
On, forming sprouting after 30-40d, every 21d subculture once, can obtain Multiple Buds in 60 days.Condition of culture is: illumination every day 16h, dark 8h,
Temperature 25 DEG C-28 DEG C, intensity of illumination 1500-2000lx.
3) successive transfer culture: be newly slightly transferred to subculture medium and cultivate, successive transfer culture based formulas: 1/2MS adds 6-BA, IBA, sucrose and
Agar, the final concentration of 0.2mg/L of final concentration of 1.5mg/L, IBA of 6-BA, the final concentration of 30g/L of sucrose and agar final concentration of
6g/L, pH=5.8.Condition of culture is: illumination every day 16h, dark 8h, temperature 25 DEG C-28 DEG C, intensity of illumination 1500-2000lx.
Two kind every 21d subcultures according to the method described above once, are total to subculture 4 times.' Su Cui 1 ' rate of increase is 100%, and growth coefficient is 2.5%；
' Su Cui 2 ' rate of increase is 100%, and growth coefficient is 3.4.Two kinds of glass phenomenons are serious, and vitrification ratio accounts for 40%, blade little and
Thin, in curling, transparence, light green, stem is thin and delicate.
4) root culture: cut the new of 1-1.5cm robust growth and be slightly transferred on root media, after dark treatment 5d, transfers to cultivate under light 60d,
Obtain Seedling of taking root.Prescription of rooting medium is: add IBA, sucrose and agar in 1/2MS, the final concentration of 0.6mg/L of IBA, sucrose
Final concentration of 60g/L, the final concentration of 6g/L, pH=5.8 of agar.Condition of culture is: illumination every day 16h, dark 8h, temperature 25 DEG C, illumination
Intensity 1500-2000lx.
' Su Cui 1 ' rooting rate 39.6% in root media, average root length 2.3cm, mean elements 4.
' Su Cui 2 ' rooting rate 29.86% in root media, average root length 3.65cm, mean elements 4.
The inventive method is compared with the method for embodiment 2, and (A) disinfection way utilizes love to make every effort to overcomeTMA, B replace mercuric chloride, safety non-toxic.Use
Stem with bud, as outer implant, shortens Multiple Buds growth time；Cancel alcohol disinfecting, shorten the disinfecting time of antibacterial, improve two kinds
Survival rate, reduces melting brown rate；Vortex processes and reduces pollution rate.(B) initial inoculation culture medium select AS culture medium, plant strain growth gesture and to
Upper growth type is better than MS and 1/2MS and cultivates.(C) tissue cultured seedling growth coefficient significantly improves, and after strong seedling culture, plant strain growth is healthy, blade
Open and flat, leaf green, without vitrification phenomenon.(D) rooting method of the present invention significantly improves the rooting rate of tissue cultured seedling, average root length peace all radicals.
Claims (9)
1. the method for tissue culture of a precocious stem of Radix pyri section, it is characterised in that comprise the steps: to use outer implant
Initial inoculation culture medium carries out inducing differentiation culture, obtains just for tissue cultured seedling；Just breed described for tissue cultured seedling
Culture medium carries out enrichment culture, and then cutting newly slightly transfers carries out successive transfer culture into strong seedling culture base, then turns successively
Enter root media A and root media B and carry out root culture；Described outer implant is green tape leaf stem section then；
Described initial inoculation culture medium is interpolation 6-BA, NAA and GA in 1/2MS or AS minimal medium3After
The culture medium arrived, the final concentration of 0.5-1.0mg/L of described 6-BA, described NAA's is final concentration of
0.05-0.5mg/L, described GA3Final concentration of 0.2-3mg/L, the pH of described initial inoculation culture medium be
5.5-5.8；
Described proliferated culture medium is the culture medium obtained after interpolation 6-BA, NAA in MS culture medium, described 6-BA
Final concentration of 0.5～1.0mg/L, final concentration of the 0.1 of described NAA～0.2mg/L, described proliferated culture medium
PH is 5.5-5.8；
Described strong seedling culture base is 1/3MS culture medium or 1/4QL culture medium, and the pH of described strong seedling culture base is
5.5-5.8；
Described root media A is the culture medium obtained after interpolation IAA, 6-BA and NAA in 1/4QL culture medium,
The final concentration of 0.8-1.2mg/L of the final concentration of 0.4-0.6mg/L of described IAA, described 6-BA, described NAA
Final concentration of 0.05-0.15mg/L, the pH of described root media A be 5.5-5.8；
Described root media B is 1/4QL culture medium；
Condition of culture in described root media A is: cultivation temperature 24～26 DEG C, dark culturing 7-12 days；
Condition of culture in described root media B is: cultivation temperature 22～25 DEG C, illumination every day 15-16 hour,
Dark 8-9 hour, intensity of illumination was 1500-2000lx, and incubation time is 50-60 days.
The method for tissue culture of precocious stem of Radix pyri section the most according to claim 1, it is characterised in that described initial inoculation
Culture medium, proliferated culture medium, strong seedling culture base, root media A and root media B are all added with
Sucrose and agar, final concentration of the 20 of described sucrose～30g/L, the final concentration of 5-7g/L of described agar.
The method for tissue culture of precocious stem of Radix pyri section the most according to claim 2, it is characterised in that described enrichment culture
Base is added with the GA of final concentration of 0.5-3mg/L3。
4. according to the method for tissue culture of stem of Radix pyri section precocious described in claim 1 or 2 or 3, it is characterised in that described
Condition of culture in initial inoculation culture medium is: cultivation temperature 22～25 DEG C, illumination every day 15-16 hour, black
Dark 8-9 hour, intensity of illumination was 1500-2000lx, within every 25-32 days, changed an initial inoculation culture medium.
The method for tissue culture of precocious stem of Radix pyri section the most according to claim 4, it is characterised in that described enrichment culture
Condition of culture in base is: cultivation temperature 22～25 DEG C, illumination every day 15-17 hour, dark 7-9 hour,
Intensity of illumination is 1500-2000lx, and incubation time is 25-32 days.
The method for tissue culture of precocious stem of Radix pyri section the most according to claim 5, it is characterised in that described strong seedling culture
Condition of culture in base is: cultivation temperature 22～25 DEG C, illumination every day 15-17 hour, dark 7-9 hour,
Intensity of illumination is 1500-2000lx, and once, subculture number is 2-3 time to every 13-16 days subcultures.
The method for tissue culture of precocious stem of Radix pyri section the most according to claim 6, it is characterised in that described outer implant exists
It is handled as follows before induction differentiation culture: by outer implant described in Carbenicillin aqueous solution soaking, then use
Make every effort to overcome containing loveTMDescribed outer implant is carried out disinfection by the solution of A, B and polysorbas20.
8., for the culture medium of precocious stem of Radix pyri section tissue culture, it is made up of following culture medium:
(a) initial inoculation culture medium；
Described initial inoculation culture medium is interpolation 6-BA, NAA and GA in 1/2MS or AS minimal medium3After
The culture medium arrived, the final concentration of 0.5-1.0mg/L of described 6-BA, described NAA's is final concentration of
0.05-0.5mg/L, described GA3Final concentration of 0.2-3mg/L, the pH of described initial inoculation culture medium be
5.5-5.8；
(b) proliferated culture medium；
Described proliferated culture medium is the culture medium obtained after interpolation 6-BA, NAA in MS culture medium, described 6-BA
Final concentration of 0.5～1.0mg/L, final concentration of the 0.1 of described NAA～0.2mg/L, described proliferated culture medium
PH be 5.5-5.8；
(c) strong seedling culture base；
Described strong seedling culture base is 1/3MS culture medium or 1/4QL culture medium, and the pH of described strong seedling culture base is
5.5-5.8；
(d) root media；
Described root media includes root media A and root media B；
Described root media A is the culture medium obtained after interpolation IAA, 6-BA and NAA in 1/4QL culture medium,
The final concentration of 0.8-1.2mg/L of the final concentration of 0.4-0.6mg/L of described IAA, described 6-BA, described NAA
Final concentration of 0.05-0.15mg/L；
Described root media B is 1/4QL culture medium.
The most according to claim 8 for the culture medium of precocious stem of Radix pyri section tissue culture, it is characterised in that described propagation
Culture medium is added with the GA of final concentration of 0.5-3mg/L3。
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| CN105850740A (en) * | 2016-04-19 | 2016-08-17 | 青岛农业大学 | Screening method for pear homologous transgene functional verification material |
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