CN104126511A - Tissue culture method for early-maturing pear stem section and culture medium - Google Patents
Tissue culture method for early-maturing pear stem section and culture medium Download PDFInfo
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Abstract
The invention provides a tissue culture method for an early-maturing pear stem section and a culture medium, and belongs to the technical field of plant cell molecule biology. The tissue culture method comprises the following steps of performing induced differentiation culture on an explant through an initial inoculation culture medium to obtain a primary tissue culture seedling; performing multiplication culture on the primary tissue culture seedling through a multiplication culture medium, then cutting off new branches, transferring into a seedling enhancing culture medium for subculture, and transferring into a rooting culture medium A and a rooting culture medium B for rooting culture. The culture medium for the tissue culture of the early-maturing pear stem section is formed by one or more than two of the initial inoculation culture medium, the multiplication culture medium, the seedling enhancing culture medium and the rooting culture medium. According to the tissue culture method disclosed by the invention, a disinfection method is safe and short in time; the multiplication coefficient, the rooting rate, the average root length and the average root number of the tissue culture seedlings are obviously increased.
Description
Technical field
The invention belongs to molecular biology of plant cells field, be specifically related to method for tissue culture and the medium of pears stem section.
Background technology
Su Cui is for No. 1 high-quality early ripening pear, and parent is crisp × emerald green hat of China, and hybridization in 2003, cultivates cross hybrid seedling for 2004, and 2011 by the kind accreditation of the Crop breed audit committee of Jiangsu Province (Su Jian fruit 201107).Pulp white, meat is delicate, the few or nothing of lithocyte, juice multi-flavor is sweet, raciness, quality are good.Bud easily forms, and bearing capacity is strong continuously, and fruit-setting rate is high, and the fructescence is at the beginning of 7 months.
Su Cui also belongs to early ripening pear No. 2, and parent is green × emerald green hat of Xizi, a famous beauty in the late Spring and Autumn Period, and hybridization in 2003, cultivates cross hybrid seedling for 2004, and 2011 by Jiangsu Province's variety certification.Fruit falls oval, and pericarp yellow green is rustless, pulp white, and meat is delicate, the few or nothing of lithocyte, bud easily forms, and bearing capacity is strong continuously, and fruit-setting rate is high, and the fructescence is mid-July.
When No. 1, Su Cui and Su Cui adopt for No. 2 conventional cottage method to breed, reproduction coefficient is low, height heterozygosis; While adopting conventional tissue culture method to breed, test-tube plantlet growth coefficient is low, and average rooting rate, mean elements are lower, and root is long shorter.
Summary of the invention
The object of this invention is to provide a kind of method for tissue culture of pears stem section, the method has significantly improved the rate of increase, growth coefficient, rooting rate, average root length and the mean elements of test-tube plantlet.
Another object of the present invention is to provide the medium of cultivating for pears stem section tissue.
Object of the present invention adopts following technical scheme to realize.
A method for tissue culture for jargonel stem section, comprises the steps: explant to adopt initial inoculation medium to induce differentiation to cultivate, and obtains just generation group training seedling; Described just generation group training seedling is bred to cultivation with proliferated culture medium, then cut newly and slightly transfer and carry out subculture cultivation into strong seedling culture base, then proceed to successively root media A and root media B carries out culture of rootage;
Described initial inoculation medium is to add 6-BA, NAA and GA in 1/2MS or AS minimal medium
3after the medium that obtains, the final concentration of described 6-BA is 0.5-1.0mg/L, the final concentration of described NAA is 0.05-0.5mg/L, described GA
3final concentration be 0.2-3mg/L, the pH of described initial inoculation medium is 5.5-5.8;
Described proliferated culture medium is in MS medium, to add 6-BA, NAA and GA
3after the medium that obtains, the final concentration of described 6-BA is 0.5～1.0mg/L, the final concentration of described NAA is 0.1～0.2mg/L, described GA
3final concentration be 0.5-3mg/L, the pH of described proliferated culture medium is 5.5-5.8;
Described strong seedling culture base is 1/3MS medium or 1/4QL medium, and the pH of described strong seedling culture base is 5.5-5.8;
Described root media A adds the medium obtaining after IAA, 6-BA and NAA in 1/4QL medium, the final concentration of described IAA is 0.4-0.6mg/L, the final concentration of described 6-BA is 0.8-1.2mg/L, the final concentration of described NAA is 0.05-0.15mg/L, and the pH of described root media is 5.5-5.8;
Described root media B is 1/4QL medium.
In the present invention, in described initial inoculation medium, proliferated culture medium, strong seedling culture base, root media A and root media B, be all added with sucrose and agar, the final concentration of described sucrose is 20～30g/L, and the final concentration of described agar is 5-7g/L.
In the present invention, the condition of culture in described initial inoculation medium is: 22～25 DEG C of cultivation temperature, and illumination every day 15-16h, dark 8-9h, intensity of illumination is 1500-2000lx, every 25-32d changes an initial inoculation medium.
In the present invention, the condition of culture in described proliferated culture medium is: 22～25 DEG C of cultivation temperature, and illumination every day 15-17h, dark 7-9h, intensity of illumination is 1500-2000lx, incubation time is 25-32d.
In the present invention, the condition of culture in described strong seedling culture base is: 22～25 DEG C of cultivation temperature, and illumination every day 15-17h, dark 7-9h, intensity of illumination is 1500-2000lx, once, subculture number is 2-3 time to every 13-16 days subculture.
In the present invention, the condition of culture in described root media A is: 24～26 DEG C of cultivation temperature, dark culturing 7-12 days.
In the present invention, the condition of culture in described root media B is: 22～25 DEG C of cultivation temperature, and illumination every day 15-16h, dark 8-9h, intensity of illumination is 1500-2000lx, incubation time is 50-60 days.
In the present invention, described explant is for becoming maternal plant stem section in age; Be preferably raw stem with bud then.
In the present invention, described explant is handled as follows before induction differentiation is cultivated: with explant described in carbenicillin aqueous solution soaking, then adopt to contain and like to make every effort to overcome
tMthe solution of A, B and polysorbas20 carries out disinfection to described explant.
The present invention is the claimed medium of cultivating for jargonel stem section tissue also, all or part of composition by following medium:
(a) initial inoculation medium;
Described initial inoculation medium is to add 6-BA, NAA and GA in 1/2MS or AS minimal medium
3after the medium that obtains, the final concentration of described 6-BA is 0.5-1.0mg/L, the final concentration of described NAA is 0.05-0.5mg/L, described GA
3final concentration be 0.2-3mg/L, the pH of described initial inoculation medium is 5.5-5.8;
(b) proliferated culture medium;
Described proliferated culture medium is in MS medium, to add 6-BA, NAA and GA
3after the medium that obtains, the final concentration of described 6-BA is 0.5-1.0mg/L, the final concentration of described NAA is 0.1-0.2mg/L, described GA
3final concentration be 0.5-3mg/L, the pH of described proliferated culture medium is 5.5-5.8;
(c) strong seedling culture base;
Described strong seedling culture base is 1/3MS medium or 1/4QL medium, and the pH of described strong seedling culture base is 5.5-5.8;
(d) root media;
Described root media comprises root media A and root media B;
Described root media A adds the medium obtaining after IAA, 6-BA and NAA in 1/4QL medium, and the final concentration of described IAA is 0.4-0.6mg/L, and the final concentration of described 6-BA is 0.8-1.2mg/L, and the final concentration of described NAA is 0.05-0.15mg/L;
Described root media B is 1/4QL medium.
The method for tissue culture of pears stem section of the present invention is compared with normal cutting propagation propagation method, overcome pears seedling cottage propagation coefficient low, height heterozygosis shortcoming, obtain a large amount of clone test-tube plantlets, its stabilization characteristics of genetics, the rate of increase are high, for genetic transformation with expand area under cultivation a large amount of test-tube plantlets is provided.
The method for tissue culture of pears stem section of the present invention compared with conventional tissue culture method,
(A) disinfection way utilization is liked to make every effort to overcome
tMa, B replace mercuric chloride, safety non-toxic.Adopt stem section as explant, shortened Multiple Buds growth time; Cancel alcohol disinfecting, shorten and like to make every effort to overcome
tMthe disinfecting time of A, B, has improved two kind survival rates, has reduced brown rate; Vortex processing has reduced pollution rate.
(B) initial inoculation medium is selected AS medium, and plant strain growth gesture and upwards growth potential are better than MS and 1/2MS medium.
(C) group training seedling proliferation coefficient significantly improves, after strong seedling culture, and plant strain growth health, blade is open and flat, and leaf green, without vitrification phenomenon.
(D) rooting method of the present invention significantly improves group training seedling proliferation coefficient, rooting rate, average root length and mean elements.
Embodiment
6-BA:6-benzyl aminoadenine; NAA:1-methyl α-naphthyl acetate; GA
3: gibberellin; IAA: indole-3-acetic acid; IBA:3-indolebutyric acid.
The composition of MS medium: (1) macroelement: NH
4nO
31650mg/L, KNO
31900mg/L, CaCl
22H
2o440mg/L, MgSO
47H
2o370mg/L, KH
2pO
4170mg/L; (2) trace element: MnSO
44H
2o22.3mg/L, ZnSO
47H
2o8.6mg/L, CoCl
26H
2o0.025mg/L, CuSO
45H
2o0.025mg/L, Na
2moO
42H
2o0.25mg/L, KI0.083mg/L, H
3bO
36.2mg/L; (3) molysite: Na
2eDTA37.3mg/L, FeSO
47H
2o27.8mg/L; (4) organic substance: nicotinic acid 0.5mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, inositol 100mg/L, glycine 2mg/L.The solvent of MS medium is water.
1/2MS medium: composition is identical with MS medium, wherein macroelement concentration is got the half of identical component in MS medium, and the content of other compositions is with MS medium.
1/3MS medium: composition is identical with MS medium, wherein macroelement concentration is got 1/3 of identical component in MS medium, and the content of other compositions is with MS medium.
The composition of AS medium: (1) macroelement: NH
4nO
3525mg/L, KNO
3950mg/L, CaCl
22H
2o220mg/L, MgSO
47H
2o185mg/L, KH
2pO
485mg/L; (2) trace element: MnSO
44H
2o2.23mg/L, ZnSO
47H
2o1.05mg/L, CoCl
26H
2o0.0025mg/L, CuSO
45H
2o0.0025mg/L, Na
2moO
42H
2o0.025mg/L, KI0.083mg/L, H
3bO
30.62mg/L; (3) molysite: Na
2eDTA27.7mg/L, FeSO
47H
2o20.7mg/L; (4) organic substance: nicotinic acid 0.5mg/L, thiamine hydrochloride 0.4mg/L, puridoxine hydrochloride 0.5mg/L, inositol 10mg/L, glycine 0.2mg/L.The solvent of AS medium is water.
QL medium composition: (1) macroelement: NH
4nO
3400mg/L, KH
2pO
4270mg/L, CaNO
3833.77mg/L, MgSO
4175.79mg/L; (2) trace element: H
3bO
36.2mg/L, CoCl
26H
2o0.025mg/L, CuSO
45H
2o0.025mg/L, MnSO
4h
2o0.76mg/L, Na
2moO
42H
2o0.25mg/L, KI0.08mg/L, ZnSO
47H
2o8.6mg/L; (3) molysite: Na
2eDTA37.3mg/L, FeSO
47H
2o27.8mg/L; (4) organic substance: inositol 29.61mg/L, thiamine hydrochloride 0.118mg/L.The solvent of QL medium is water.
1/4QL medium: composition is identical with QL medium, wherein macroelement concentration is got 1/4 of identical component in QL medium, and the concentration of other compositions is with QL medium.
Love is made every effort to overcome
tMa, B: purchased from Yu Han bio tech ltd, Shanghai, article No. E0015.
Embodiment 1
Adopt with the following method ' No. 1, Su Cui ' and ' No. 2, Su Cui ' pears stem section is organized to cultivation.
Explant gathers and sterilization: to December, gather annotinous branch October the previous year from orchard, be stored in 4 DEG C of refrigerators.Late Febuary, to early March, gets branch and inserts in sterile water then, and 22 DEG C-25 DEG C, illumination every day 8h, dark 16h, intensity of illumination 1500-2000lx, vernalization, every 7d changes fresh sterile water one time.April early and middle ten days, after bud is sprouted, get multiple stem with bud, be cut into that 1-1.5cm is high, stem section with a tender shoots, disinfect as follows: stem with bud is with containing after the running water immersion treatment 2.5h of 100mg/L carbenicillin, adopt and mix thimerosal vortex 5-8 minute, then peel off bract, mix thimerosal secondary sterilization 5-8 minute.The preparation method who mixes thimerosal is: make every effort to overcome from love
tMin A, B, get a slice A sheet and a slice B sheet is dissolved in 500ml sterile water, adding final concentration is the polysorbas20 of 0.01% (mass percentage concentration), obtains mixing thimerosal.Mix in thimerosal and like to make every effort to overcome
tMthe concentration of A, B is 500mg/L.
1) initial inoculation: in initial inoculation medium 1 and 2, inoculate respectively 20 stem with bud after disinfecting, induce differentiation to cultivate, obtain just generation group training seedling.Induction differentiation condition of culture is: 22 DEG C-25 DEG C of cultivation temperature, every day light application time 16h, interlunation 8h, intensity of illumination 1500-2000lx, cultivates 25d.
Initial inoculation medium 1: add 6-BA, NAA, GA in 1/2MS
3, sucrose and agar, the final concentration of 6-BA is that the final concentration of 1.0mg/L, NAA is 0.2mg/L, GA
3final concentration be that the final concentration of 0.5mg/L, sucrose is that the final concentration of 30g/L, agar is 5-7g/L, pH=5.8.
Initial inoculation medium 2: add 6-BA, NAA, GA in AS
3, sucrose and agar, the final concentration of 6-BA is that the final concentration of 1.0mg/L, NAA is 0.2mg/L, GA
3final concentration be that the final concentration of 0.5mg/L, sucrose is that the final concentration of 30g/L, agar is 5-7g/L, pH=5.8.
Statistics ' No. 1, Su Cui ' and ' No. 2, Su Cui ' is average brown rate, pollution rate and the survival rate of generation group training seedling just, and concrete outcome is: ' No. 1, Su Cui ' average brown rate 11.2%, pollution rate 19.9%, survival rate 68.9%; ' No. 2, Su Cui ' average brown rate 10.5%, pollution rate 13.6%, survival rate 75.9%.
Observe and record induction differentiation and cultivate ' No. 1, Su Cui ' and ' No. 2, Su Cui ' just growing state of generation group training seedling in 25 days, concrete as table 1 and table 2.On initial inoculation medium 2, each plant strain growth gesture is good as can be seen from Table 1 and Table 2, and brown rate is low, and bud well differentiated has significantly upwards growth vigor, and blade is open and flat, is green.
Growing state after table 1 ' No. 1, Su Cui ' initial inoculation is cultivated
| Type of culture medium | Leaf growth situation | Plant strain growth situation |
| Initial inoculation medium 1 | Open and flat, grow normal, green | Growth potential is general, and bud upwards growth property is weaker than plant in medium 2 |
| Initial inoculation medium 2 | Open and flat, grow normal, green | Growth potential is good, and bud upwards growth property is good |
Growing state after table 2 ' No. 2, Su Cui ' initial inoculation is cultivated
| Type of culture medium | Leaf growth situation | Plant strain growth situation |
| Initial inoculation medium 1 | Open and flat, grow normal, green | Growth potential is general, and bud upwards growth property is weaker than plant in medium 2 |
| Initial inoculation medium 2 | Open and flat, grow normal, green | Growth potential is good, and bud upwards growth property is good |
It should be noted that so every 25-32d changes an initial inoculation medium if induce differentiation cultivated days to increase.
2) propagation is cultivated: get the high first generation group training seedling of 2～3cm having survived, be inoculated in and contain variable concentrations 6-BA, NAA and GA
3proliferated culture medium in, adopt following condition to breed cultivation: 22 DEG C-25 DEG C of cultivation temperature, every day light application time 16h, interlunation 8h, intensity of illumination 1500-2000lx, incubation time is 30d.Propagation is cultivated after 30d, the growth coefficient of statistics two kinds, and observe plant growth condition, concrete outcome is in table 3 and 4.Can find out from table 3 and 4,6-BA concentration between 0.5-1.0mg/L, NAA concentration between 0.1-0.2mg/L, the good differentiation of bud, growth coefficient is all more than 3; Add GA
3, stem apex obviously extends, and growth coefficient improves, and reaches and does not add GA
32 times of left and right, GA
3concentration affects no significant difference at 0.5-3mg/L to growth coefficient; On all medium, plant strain growth is good, without vitrification phenomenon.
Proliferated culture medium 1: add 6-BA, NAA, sucrose and agar in MS medium, the final concentration of 6-BA is that the final concentration of 0.5mg/L, NAA is that the final concentration of 0.1mg/L, sucrose is that the final concentration of 30g/L, agar is 5-7g/L, pH=5.8.
Proliferated culture medium 2: add 6-BA, NAA, sucrose and agar in MS medium, the final concentration of 6-BA is 0.5mg/L, the final concentration of NAA is that the final concentration of 0.2mg/L, sucrose is that the final concentration of 30g/L, agar is 5-7g/L, pH=5.8.
Proliferated culture medium 3: add 6-BA, NAA, sucrose and agar in MS medium, the final concentration of 6-BA is that the final concentration of 1.0mg/L, NAA is that the final concentration of 0.1mg/L, sucrose is that the final concentration of 30g/L, agar is 5-7g/L, pH=5.8.
Proliferated culture medium 4: add 6-BA, NAA, sucrose and agar in MS medium, the final concentration of 6-BA is that the final concentration of 1.0mg/L, NAA is that the final concentration of 0.2mg/L, sucrose 30g/L, agar is 5-7g/L, pH=5.8.
Proliferated culture medium 5: add 6-BA, NAA, GA in MS medium
3, sucrose and agar, the final concentration of 6-BA is that the final concentration of 1.0mg/L, NAA is 0.2mg/L, GA
3final concentration be that the final concentration of 0.5mg/L, sucrose is that the final concentration of 30g/L, agar is 5-7g/L, pH=5.8.
Proliferated culture medium 6: add 6-BA, NAA, GA in MS medium
3, sucrose and agar, the final concentration of 6-BA is that the final concentration of 1.0mg/L, NAA is 0.2mg/L, GA
3final concentration be that the final concentration of 3mg/L, sucrose is that the final concentration of 30g/L, agar is 5-7g/L, pH=5.8.
Parameter after table 3 ' No. 1, Su Cui ' propagation is cultivated
| Type of culture medium | Just for tissue culture plant inoculation number (individual) | Growth coefficient | Vitrifying 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 ' No. 2, Su Cui ' propagation is cultivated
| Type of culture medium | Just generation group training seedling number inoculation (individual) | Growth coefficient | Vitrifying 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) subculture is cultivated: the first generation group training seedling after propagation is cultivated, cut 1.5-2.0cm robust growth newly slightly, be transferred in strong seedling culture base 1 and 2, carry out under the following conditions subculture cultivation: 22 DEG C-25 DEG C of cultivation temperature, every day light application time 16h, interlunation 8h, intensity of illumination 1500-2000lx, every 15d subculture once, subculture 2 times, obtains subculture seedling.Observe the growing state after each kind strong seedling culture, specifically as shown in Table 5 and 6.Can find out that from table 5 and 6 each kind stem is sturdy, blade is open and flat, and growth is normal, is green.
In strong seedling culture base 1:1/3MS medium, add the agar that sucrose that final concentration is 30g/L and final concentration are 5g/L, pH=5.8.
In strong seedling culture base 2:1/4QL medium, adding final concentration is the agar that 30g/L sucrose and final concentration are 7g/L, pH=5.8.
Parameter after table 5 ' No. 1, Su Cui ' strong seedling culture
| Type of culture medium | Height of seedling (cm) | Blade is indulged 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 ' No. 2, Su Cui ' strong seedling culture
| Type of culture medium | Height of seedling (cm) | Blade is indulged 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) culture of rootage: it is consistent to get growth potential, and the subculture seedling of high 3.0-4.0cm, is inoculated in root media A.Root media A formula: add IAA, 6-BA, NAA, sucrose and agar in 1/4QL medium, the final concentration of IAA is that the final concentration of 0.5mg/L, 6-BA is that the final concentration of 1.0mg/L, NAA is that the final concentration of 0.1mg/L, sucrose is that the final concentration of 25g/L, agar is 6g/L, pH=5.8.Condition of culture is: cultivation temperature is 25 DEG C, dark culturing 10d.
By adopting the subculture seedling after root media A cultivates to proceed to root media B, cultivate 60d, obtain the seedling of taking root.Root media B formula: add sucrose and agar in 1/4QL, the final concentration of sucrose is 20-30g/L, and the final concentration of agar is 5-7g/L.Condition of culture is: 22 DEG C-25 DEG C of cultivation temperature, every day light application time 16h, interlunation 8h, intensity of illumination 1500-2000lx.
2 kinds are taken root as stated above, ' No. 1, Su Cui ' rooting rate 95.13%, the long 8.46cm of average root, mean elements 8.9; ' No. 2, Su Cui ' rooting rate 93.25%, the long 9.65cm of average root, mean elements 9.2.
Embodiment 2 adopts conventional tissue culture and rapid propagation method to cultivate ' No. 1, Su Cui ' and ' No. 2, Su Cui '
Adopt and cultivate with the following method ' No. 1, Su Cui ' and ' No. 2, Su Cui ':
1) explant collection and sterilization: choose the annotinous branch of robust growth from field, clip children shoot, as explant, adopts washing powder rinsing, and running water rinses 30min.On superclean bench, by 75% alcohol sterilizing 20s for explant after treatment, 0.1% mercuric chloride sterilizing 8-10min, aseptic water washing 4-5 time.Scalpel is removed scale, cuts naked bub and is inoculated on inducing culture, 3 explants of every bottle graft kind.Inducing culture is: in 1/2MS medium, add 6-BA, IBA, GA
3, sucrose and agar, wherein the final concentration of 6-BA is 1.0mg/L, the final concentration of IBA is 0.2mg/L, GA
3final concentration be 2.0mg/L, the final concentration of sucrose is 30g/L, the final concentration of agar is 6g/L, pH=5.8.Condition of culture is: illumination every day 16h, dark 8h, 25 DEG C of temperature, intensity of illumination 1500-2000lx.
The aseptic seedling of 2 kinds is sterilized as stated above, and ' No. 1, Su Cui ' average brown rate is 93.5%, and average survival is 5.6%; ' No. 2, Su Cui ' average brown rate is 95.2%, and average survival is 6.5%.
2) inducing clumping bud: adopt inducing culture to cultivate 15d left and right and treat that bud grows to 1cm left and right, cut and be newly slightly transferred on new inducing culture, form 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, 25 DEG C-28 DEG C of temperature, intensity of illumination 1500-2000lx.
3) subculture is cultivated: be newly slightly transferred to subculture medium and cultivate, subculture medium formula: add 6-BA, IBA, sucrose and agar in 1/2MS, the final concentration of 6-BA is that the final concentration of 1.5mg/L, IBA is that the final concentration of 0.2mg/L, sucrose is that the final concentration of 30g/L and agar is 6g/L, pH=5.8.Condition of culture is: illumination every day 16h, dark 8h, 25 DEG C-28 DEG C of temperature, intensity of illumination 1500-2000lx.
Two kinds according to the method described above every 21d subculture once, subculture 4 times altogether.' No. 1, Su Cui ' rate of increase is 100%, and growth coefficient is 2.5%; ' No. 2, Su Cui ' rate of increase is 100%, and growth coefficient is 3.4.Two kinds of glass phenomenons are serious, and vitrifying ratio accounts for 40%, and blade is little and thin, are curling, transparence, light green, and stem is thin and delicate.
4) culture of rootage: cut being newly slightly transferred on root media of 1-1.5cm robust growth, after the reason 5d of dark place, transfer under light and cultivate 60d, obtain the seedling of taking root.Prescription of rooting medium is: in 1/2MS, add IBA, sucrose and agar, the final concentration of IBA is that the final concentration of 0.6mg/L, sucrose is that the final concentration of 60g/L, agar is 6g/L, pH=5.8.Condition of culture is: illumination every day 16h, dark 8h, 25 DEG C of temperature, intensity of illumination 1500-2000lx.
' No. 1, Su Cui ' rooting rate 39.6% in root media, the long 2.3cm of average root, 4 of mean elements.
' No. 2, Su Cui ' rooting rate 29.86% in root media, the long 3.65cm of average root, 4 of mean elements.
Compared with the method for the inventive method and embodiment 2, (A) disinfection way utilization is liked to make every effort to overcome
tMa, B replace mercuric chloride, safety non-toxic.Adopt stem with bud as explant, shortened Multiple Buds growth time; Cancel alcohol disinfecting, shorten the disinfecting time of bactericide, improved two kind survival rates, reduced brown rate; Vortex processing has reduced pollution rate.(B) initial inoculation medium is selected AS medium, and plant strain growth gesture and upwards vegetative form are better than MS and 1/2MS cultivates.(C) group training seedling proliferation coefficient significantly improves, after strong seedling culture, and plant strain growth health, blade is open and flat, and leaf green, without vitrification phenomenon.(D) rooting method of the present invention has significantly improved rooting rate, average root length and the mean elements of group training seedling.
Claims (10)
1. a method for tissue culture for jargonel stem section, is characterized in that comprising the steps: adopting initial inoculation medium to induce differentiation to cultivate explant, obtains just generation group training seedling; Described just generation group training seedling is bred to cultivation with proliferated culture medium, then cut newly and slightly transfer and carry out subculture cultivation into strong seedling culture base, then proceed to successively root media A and root media B carries out culture of rootage;
Described initial inoculation medium is to add 6-BA, NAA and GA in 1/2MS or AS minimal medium
3after the medium that obtains, the final concentration of described 6-BA is 0.5-1.0mg/L, the final concentration of described NAA is 0.05-0.5mg/L, described GA
3final concentration be 0.2-3mg/L, the pH of described initial inoculation medium is 5.5-5.8;
Described proliferated culture medium is in MS medium, to add the medium obtaining after 6-BA, NAA, and the final concentration of described 6-BA is 0.5 ~ 1.0mg/L, and the final concentration of described NAA is 0.1 ~ 0.2mg/L, and the pH of described proliferated culture medium is 5.5-5.8;
Described strong seedling culture base is 1/3MS medium or 1/4QL medium, and the pH of described strong seedling culture base is 5.5-5.8;
Described root media A adds the medium obtaining after IAA, 6-BA and NAA in 1/4QL medium, the final concentration of described IAA is 0.4-0.6mg/L, the final concentration of described 6-BA is 0.8-1.2mg/L, the final concentration of described NAA is 0.05-0.15mg/L, and the pH of described root media is 5.5-5.8;
Described root media B is 1/4QL medium.
2. the method for tissue culture of jargonel stem section according to claim 1, it is characterized in that being all added with sucrose and agar in described initial inoculation medium, proliferated culture medium, strong seedling culture base, root media A and root media B, the final concentration of described sucrose is 20 ~ 30g/L, and the final concentration of described agar is 5-7g/L; In preferred technical scheme, in described proliferated culture medium, be added with the GA that final concentration is 0.5-3mg/L
3.
3. according to the method for tissue culture of jargonel stem section described in claim 1 or 2, it is characterized in that the condition of culture in described initial inoculation medium is: 22 ~ 25 DEG C of cultivation temperature, illumination every day 15-16 hour, dark 8-9 hour, intensity of illumination is 1500-2000lx, within every 25-32 days, changes an initial inoculation medium.
4. the method for tissue culture of jargonel stem section according to claim 3, it is characterized in that the condition of culture in described proliferated culture medium is: 22 ~ 25 DEG C of cultivation temperature, illumination every day 15-17 hour, dark 7-9 hour, intensity of illumination is 1500-2000lx, incubation time is 25-32 days.
5. the method for tissue culture of jargonel stem section according to claim 4, it is characterized in that the condition of culture in described strong seedling culture base is: 22 ~ 25 DEG C of cultivation temperature, illumination every day 15-17 hour, dark 7-9 hour, intensity of illumination is 1500-2000lx, once, subculture number is 2-3 time to every 13-16 days subculture.
6. the method for tissue culture of jargonel stem section according to claim 5, is characterized in that the condition of culture in described root media A is: 24 ~ 26 DEG C of cultivation temperature, dark culturing 7-12 days.
7. the method for tissue culture of jargonel stem section according to claim 6, it is characterized in that the condition of culture in described root media B is: 22 ~ 25 DEG C of cultivation temperature, illumination every day 15-16 hour, dark 8-9 hour, intensity of illumination is 1500-2000lx, incubation time is 50-60 days.
8. the method for tissue culture of jargonel stem section according to claim 7, is characterized in that described explant is for becoming maternal plant stem section in age; Be preferably raw stem with bud then.
9. the method for tissue culture of jargonel stem section according to claim 8, is characterized in that described explant is handled as follows before induction differentiation is cultivated: with explant described in carbenicillin aqueous solution soaking, then adopt to contain and like to make every effort to overcome
tMthe solution of A, B and polysorbas20 carries out disinfection to described explant.
10. the medium of cultivating for jargonel stem section tissue, all or part of composition by following medium:
(a) initial inoculation medium;
Described initial inoculation medium is to add 6-BA, NAA and GA in 1/2MS or AS minimal medium
3after the medium that obtains, the final concentration of described 6-BA is 0.5-1.0mg/L, the final concentration of described NAA is 0.05-0.5mg/L, described GA
3final concentration be 0.2-3mg/L, the pH of described initial inoculation medium is 5.5-5.8;
(b) proliferated culture medium;
Described proliferated culture medium is in MS medium, to add the medium obtaining after 6-BA, NAA, and the final concentration of described 6-BA is 0.5 ~ 1.0 mg/L, and the final concentration of described NAA is 0.1 ~ 0.2 mg/L, and the pH of described proliferated culture medium is 5.5-5.8; In preferred technical scheme, in described proliferated culture medium, be added with the GA that final concentration is 0.5-3mg/L
3;
(c) strong seedling culture base;
Described strong seedling culture base is 1/3MS medium or 1/4QL medium, and the pH of described strong seedling culture base is 5.5-5.8;
(d) root media;
Described root media comprises root media A and root media B;
Described root media A adds the medium obtaining after IAA, 6-BA and NAA in 1/4QL medium, and the final concentration of described IAA is 0.4-0.6mg/L, and the final concentration of described 6-BA is 0.8-1.2mg/L, and the final concentration of described NAA is 0.05-0.15mg/L;
Described root media B is 1/4QL medium.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105557519A (en) * | 2015-12-16 | 2016-05-11 | 青岛百瑞吉生物工程有限公司 | Test-tube plantlet proliferation culture medium of pear tree somatic embryos |
| CN105557520A (en) * | 2015-12-16 | 2016-05-11 | 青岛百瑞吉生物工程有限公司 | Bud initiation culture medium of pear tree somatic embryos |
| 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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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101965797A (en) * | 2010-10-25 | 2011-02-09 | 江苏省农业科学院 | Quick propagation seedling-breeding method for reducing vitrifaction in multiple times of subculture of rosaceous plant |
| CN103380730A (en) * | 2013-08-05 | 2013-11-06 | 江苏省农业科学院 | Tissue-culture rapid propagation method for pyrus betulaefolia bunge |
| CN103444522A (en) * | 2012-06-04 | 2013-12-18 | 华中农业大学 | Preparation method for rooting of test-tube plantlet of ample flow pear |
-
2014
- 2014-08-22 CN CN201410420032.7A patent/CN104126511B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101965797A (en) * | 2010-10-25 | 2011-02-09 | 江苏省农业科学院 | Quick propagation seedling-breeding method for reducing vitrifaction in multiple times of subculture of rosaceous plant |
| CN103444522A (en) * | 2012-06-04 | 2013-12-18 | 华中农业大学 | Preparation method for rooting of test-tube plantlet of ample flow pear |
| CN103380730A (en) * | 2013-08-05 | 2013-11-06 | 江苏省农业科学院 | Tissue-culture rapid propagation method for pyrus betulaefolia bunge |
Non-Patent Citations (5)
| Title |
|---|
| H. TANG ETAL: "Plant regeneration from in vitro leaves of four commercial Pyrus species", 《PLANT SOIL ENVIRON.》, no. 4, 31 December 2008 (2008-12-31), pages 140 - 148 * |
| SABAH A. HASSANEN ETAL: "In vitro propagation of Pear Pyrus betulaefolia Rootstock", 《AMERICAN-EURASIAN J.AGRIC.&ENVIRON.SCI.》, vol. 12, no. 4, 31 December 2012 (2012-12-31), pages 484 - 489 * |
| 张玉娇等: "黄冠梨组织培养与快繁技术研究", 《江苏农业科学》, no. 3, 31 December 2009 (2009-12-31), pages 35 - 36 * |
| 李晓刚等: "豆梨低玻璃化组培快繁技术", 《江苏农业科学》, vol. 40, no. 2, 31 December 2012 (2012-12-31), pages 54 - 56 * |
| 黄天带等: "降低巴西橡胶树茎段培养污染率的方法研究", 《热带农业科学》, vol. 25, no. 6, 31 December 2005 (2005-12-31), pages 1 - 3 * |
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