CN108094198A - A kind of Strawberry Leaves in-vitro regeneration method - Google Patents
A kind of Strawberry Leaves in-vitro regeneration method Download PDFInfo
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- CN108094198A CN108094198A CN201711287169.XA CN201711287169A CN108094198A CN 108094198 A CN108094198 A CN 108094198A CN 201711287169 A CN201711287169 A CN 201711287169A CN 108094198 A CN108094198 A CN 108094198A
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- strawberry
- culture
- regeneration method
- vitro regeneration
- strawberry leaves
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Classifications
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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
-
- 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 present invention provides a kind of Strawberry Leaves in-vitro regeneration methods, using Strawberry Leaves as explant, through surface sterilization, inoculation, light culture, evoked callus, and the callus of induction is proliferated, is germinateed, is taken root and tissue culture seedling and propagating, establish the vitro Regeneration System of Strawberry Leaves, efficiently solve the problems such as strawberry explant materials are difficult, surface sterilization is not thorough, easy browning, and it can quickly and effectively induce callus, inductivity is up to 98%, significantly shorten the recovery time, micropropagation efficiency is improved, suitable for a large amount of acquisitions of nontoxic Strawberry Seedlings.
Description
Technical field
The invention belongs to Plant Tissue Breeding and rapid propagation in vitro field, and in particular to the in-vitro regeneration method of strawberry.
Background technology
Strawberry (Fragaria ananassaDuchesne) is full of nutrition, cultivates extensively, deep favorite for many years by the people
This fruit tree of sward.In recent years, be badly in need of large quantities of excellent Strawberry Seedlings in the production of various regions, but using conventional seedling propagation, breeding coefficient compared with
It is low, cost is higher, now mostly using tissue culture technology breed Strawberry Seedlings.The Study on tissue culture of strawberry is started from the 1960s, most
Early progress strawberry cultured in vitro research is lower village of Japan etc., their research shows with heat treatment and growing point culture phase
With reference to method exclude Strawberry Virus effect it is preferable.Miller reported Shoot Tip Culture in 1963 and successfully obtains strawberry for the first time
Tissue culture plant, Rosati etc. carry out cultured in vitro with the flower pesticide of 4 kinds such as pineapple strawberry and successfully obtain plant.The strawberry in China
Study on tissue culture is started late, and Qin Lanying etc. establishes the in vitro amount reproduction technology of strawberry stem tip, Xue Guangrong etc. with east grass
Certain kind of berries monokaryotic stage flower pesticide inoculated and cultured successfully obtains the haplobiont that chromosome number is 4.
Red-color strawberries, also known as " red cheek " strawberry, it is adaptable it is strong, growing way is prosperous, yield is high, fruit is big and mouthfeel is good etc.
Feature, it has also become the principal item of strawberry popularizing planting.In terms of the tissue cultures of " red cheek " strawberry, forefathers have been carried out one
A little research reports, and some have also been carried out to its large-scale production and has groped and puts into practice, but it is big to " red cheek " Plantlets of Strawberry
Operating process in scale processes is standardized still without detailed technology explanation.The problem of being primarily present is：Explant
Materials are difficult, surface sterilization is not thorough, and lead to problems such as easy browning and the callus induction time is long, efficiency is low.
The content of the invention
It is an object of the invention to provide a kind of Strawberry Leaves in-vitro regeneration methods.
In order to achieve the above objectives, present invention employs following technical schemes：
1) using Strawberry Leaves as explant, it will be seeded on regeneration culture medium after explant surface sterilization, then pass through successively
It crosses under light culture 8~10 days, illumination and cultivates 1~2 day, obtain callus；The regeneration culture medium is by the organic culture mediums of MS+B5
And 0.5~1.0mg/L 6-BA and 0.1~0.5mg/L NAA compositions, the organic culture mediums of MS+B5 use for organic principle
The MS culture mediums of the organic principle of B5 medium；
2) callus is expanded into numerous, bud elongation and root induction culture by multiplication, adventitious bud inducing, adventitious bud successively,
Obtain Plantlets of Strawberry；
3) by Strawberry Plantlets seedling and propagating.
Preferably, the explant is selected from the nearly top blade of strawberry, and the explant size of inoculation is 2~3cm × 2~3cm.
Preferably, the surface sterilization comprises the following steps：It is rushed prior to sterile water after 20~25s is impregnated in 75% alcohol
It washes, aseptic water washing after 10~15s is then impregnated in 3~5%NaClO, 8~10min is then impregnated in 0.1% mercuric chloride,
Last aseptic water washing.
Preferably, cultivated under the illumination and callus proliferation, adventitious bud inducing, adventitious bud expand the elongation of numerous, bud and
The condition of culture of root induction culture and tissue culture seedling and propagating is：20~25 DEG C, 1000~1500lx and 14~16h/d illumination.
Preferably, the culture medium that the callus proliferation uses is regeneration culture medium.Every 10~12 days in incubation
Switching once, is transferred 1~2 time.
Preferably, the culture medium that the adventitious bud inducing uses for additionally added with 1.5~2.0mg/L TDZ, 1.5~
The MS culture mediums of 2.0mg/L6-BA and 0.3~0.5mg/L IBA.Incubation time is 5~7 days.
Preferably, the adventitious bud expand numerous culture medium used for be additionally added with 1.5~2.0mg/L TDZ and 0.4~
The MS culture mediums of 0.6mg/LIBA.Incubation time is 5~7 days.
Preferably, the culture medium that the bud elongation uses is the additional MS culture medium for being added with 0.3~0.5mg/L 6-BA.
Incubation time is 5~7 days.
Preferably, the culture medium that the root induction uses is trained for the additional 1/2MS added with 0.3~0.5mg/L IBA
Support base.Incubation time is 5~7 days.
Preferably, the kind of the strawberry is selected from red-color strawberries.
Beneficial effects of the present invention are embodied in：
The present invention solves explant selection presence limitation in strawberry Regeneration in Vitro, surface sterilization is not thorough, easy browning,
The problems such as callus induction rate is low, and the recovery time is long, suitable for a large amount of acquisitions of nontoxic Strawberry Seedlings.
Further, present invention determine that a kind of in-vitro regeneration method of efficient " beauty " Strawberry Leaves, regenerating system
In culture medium needed for each stage it is clear and definite, can produce that growing way is vigorous, form is homogeneous, " red cheek " Strawberry Plantlets of stable quality
Seedling can be that industrialization large-scale production " red cheek " strawberry establishes reliable and stable basis.
Description of the drawings
Fig. 1 is blade inoculation state diagram；
Fig. 2 is callus induction state diagram；
Fig. 3 is callus proliferation state diagram；
Fig. 4 is adventitious buds differentiation state diagram；
Fig. 5 is adventitious bud proliferation state diagram；
Fig. 6 is the elongation state figure of bud；
Fig. 7 is state diagram of taking root；
Fig. 8 is tissue-cultured seedling reproductive status figure.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.
1. basal medium, optimization and additive
1.1st, basal medium includes MS culture mediums, 1/2MS culture mediums and the organic culture mediums of MS+B5, is formulated concrete composition
It is described as follows.
Table 1.MS culture medium prescriptions
Organic principle in above-mentioned MS culture mediums is substituted for the organic principle (table 2) of B5 medium, it is organic to obtain MS+B5
Culture medium：
Table 2.B5 culture medium organic principle formulas
The micro- dosage of above-mentioned MS culture mediums is reduced to 1/2 original culture medium by 1/2MS culture mediums.
Technical advantage explanation：The basal medium that existing strawberry cultured in vitro uses only includes a kind of common culture medium, and
Two kinds of common culture mediums are effectively combined by the present invention so that the culture medium of evoked callus is optimized, and shortens strawberry
The time of explant evoked callus, improve induced efficiency.
1.2nd, additive
6-BA (6- benzyls aminoadenine)；NAA (methyl α-naphthyl acetate)；TDZ (phenyl thiadiazolyl group urea)；IBA (indolebutyric acid) is purchased
From Xi'an Jing Bo bio tech ltd.
2. the selection of explant
" " the three pieces tender leaf on the nearly top of strawberry (takes blade too old or not deployed, too always to beauty to clip health
Leaf regeneration energy force difference, not deployed blade are not easy surface sterilization).
Technical advantage explanation：Explant selected by the present invention is blade, for flower pesticide and stolon, no growth period
Limitation, materials are more convenient, and with reference to condition of culture, the efficiency with higher evoked callus, induction time significantly shortens.
3. surface sterilization
After strawberry tender leaf is rinsed 2h with clear water, surface sterilization is carried out.Optimal sterilization method is：Prior to volume fraction 75%
Alcohol in impregnate 20s, then aseptic water washing 3 times impregnates 10s, sterile water in the NaClO aqueous solutions of mass fraction 3%
It rinses 5 times, most after immersion 8min, aseptic water washing 6 times in the mercuric chloride (mercuric chloride solution) of mass fraction 0.1%.
Technical advantage explanation：The sterilization method is used in combination using a variety of disinfectants, strictly controls disinfecting time so that more
Injured tissue pollution rate is low, browning is few, and blade can keep bright-coloured, survival rate is high, restoration ecosystem is fast.
4. it is inoculated with blade
Will " beauty " strawberry disinfection blade blade tip and leaf margin removal after be cut into inoculation needed for size leaf block, by than
Compared with present invention determine that the optimal size of blade inoculation is 3cm × 3cm, the leaf block back side lies against regeneration culture medium (MS+B5 downward
Organic culture medium+0.5mg/L 6-BA+0.1mg/L NAA) in.
Technical advantage explanation：The present invention is defined (3cm × 3cm) to the size for being inoculated with blade, the favourable leaf of this size
Piece edge tilts, convenient for the induction of callus (see Fig. 1).
5. dark reaction
The blade first light culture processing (being protected from light, 20 DEG C) through 10d after inoculation.
Technical advantage explanation：Invention increases dark treatments, substantially reduce callus induction time, dark treatment 10 days
Left and right is grown afterwards by shorter photo-irradiation treatment, that is, visible callus.
6. inducing strawberry Callus of Leaf
Using the organic culture mediums of MS+B5 as minimal medium, by experiment, the present invention finally determines that inducing strawberry blade is cured
The optimum medium of injured tissue is：Organic culture medium+0.5mg/L 6-BA+0.1mg/L the NAA of MS+B5.
It is placed under room temperature (25 DEG C), 1000~1500lx, 14h/d illumination and cultivates again after light culture processing, it can after a couple of days
See that callus is grown, realize the regeneration of evoked callus.
Technical advantage explanation：The optimum medium (regeneration culture medium) using the organic culture mediums of MS+B5 as minimal medium, and
Plant hormone 6-BA and NAA are added, 98% is reached to the inductivity of Callus of Leaf, the callus growing way induced is good,
Survival rate is high, growth is fast (see Fig. 2).
7. the multiplication of callus
The good callus of growing way is transferred in new regeneration culture medium, i.e. the organic culture medium+0.5mg/L6- of MS+B5
BA+0.1mg/L NAA, 25 DEG C, 1000~1500lx, cultivate under 14h/d illumination.Switching in time ensures culture medium nutrition supply,
Switching once every 2 weeks, it is necessary to transfer 1~2 time before evoking adventive bud, makes callus largely be proliferated (see Fig. 3).
8. evoking adventive bud
Callus after multiplication is transferred in the culture medium of evoking adventive bud, 25 DEG C, 1000~1500lx, 14h/d
It is cultivated under illumination, by many experiments, the preferable culture medium of the final definite evoking adventive bud of the present invention is：MS culture mediums+
2.0mg/LTDZ+2.0mg/L 6-BA+0.5mg/L IBA。
Technical advantage explanation：Using plant hormone TDZ, successfully induce adventitious bud and inductivity height, induction time are short by (one
Or so week), adventitious bud obtains growing way also preferably (see Fig. 4).
9. the expansion of adventitious bud is numerous
The good adventitious bud of growing way is transferred to adventitious bud and expands numerous optimum medium, i.e. MS culture mediums+2.0mg/LTDZ+
In 0.6mg/L IBA, 25 DEG C, 1000~1500lx, cultivate under 14h/d illumination, adventitious bud is made largely to expand numerous (see Fig. 5).
Technical advantage explanation：It determines optimum medium, cultivates one week or so, adventitious bud obtains fast-propagation, blade differentiation
It grows.
10. the elongation of bud
By expand it is numerous after adventitious bud be transferred to the optimum medium of bud elongation, i.e. in MS culture mediums+0.3mg/L 6-BA, 25
DEG C, 1000~1500lx, cultivate under 14h/d illumination, make bud elongation (see Fig. 6).
Technical advantage explanation：It determines optimum medium, cultivates one week or so, bud extends rapidly, and blade becomes larger in emerald green.
11. root induction
Bud after elongation is separated, is transferred to the optimum medium of root induction, i.e. 1/2MS culture mediums+0.3mg/
In LIBA, 25 DEG C, 1000~1500lx, cultivate under 14h/d illumination, root induction (see Fig. 7).
Technical advantage explanation：Hormone is few used in root media, and can effectively root induction (culture one week or so, life
100%) root rate reaches.
12. amount reproduction tissue-cultured seedling
The tissue-cultured seedling taken root is accessed into big tissue culture bottle (1/2MS culture mediums), 25 DEG C, 1000~1500lx, 14h/d light
According to lower culture, allow its amount reproduction, the quantity of tissue-cultured seedling doubles in single bottle, obtains good tissue-cultured seedling (see Fig. 8).
Technical advantage explanation：So that tissue-cultured seedling achievees the purpose that fast numerous, tissue-cultured seedling growth is vigorous, grows fine, and is the later stage
Production provides a large amount of high-quality tissue-cultured seedling.
The present invention establishes the vitro Regeneration System of " beauty " Strawberry Leaves most using the leaf of " beauty " strawberry as explant
Excellent condition includes：Blade shows sterilization method for 75% alcohol 20s+ aseptic water washings, 3+3%NaClO 10s+ sterile waters punchings
Wash 5+0.1% mercuric chloride 8min+ aseptic water washings 6 times；The size for being inoculated with blade is 3cm × 3cm；The dark treatment time is 10 days left sides
It is right；The culture medium of evoked callus is the organic culture medium+0.5mg/L 6-BA+0.1mg/L NAA of MS+B5；Callus increases
Culture medium is grown for the organic culture medium+0.5mg/L 6-BA+0.1mg/L NAA of MS+B5；The culture medium of evoking adventive bud is cultivated for MS
Base+2.0mg/L TDZ+2.0mg/L 6-BA+0.5mg/L IBA；It is MS culture mediums+2.0mg/L that adventitious bud, which expands numerous culture medium,
TDZ+0.6mg/L IBA；The elongation medium of bud is MS culture medium+0.3mg/L 6-BA；Root induction culture medium is trained for 1/2MS
Support base+0.3mg/L IBA；Condition of culture for 25 DEG C, 1000~1500lx, 14h/d illumination.
Present invention determine that strawberry leaf surfaces disinfecting times, inoculation leaf blade size, the light culture time, condition of culture, swashing
The influence to Strawberry Leaves Regeneration in Vitro such as culture medium used in element proportioning, each period, effectively solves strawberry explant materials
The problems such as difficult, surface sterilization is not thorough, easy browning, and can fast and effectively induce callus, inductivity up to 98%,
The present invention refines and specifies the optimum medium in Strawberry Leaves regenerative system needed for each stage simultaneously, shortens so as to reach
Recovery time and effectively fast numerous purpose.The present invention has the advantage of quickness and high efficiency, suitable for a large amount of acquisitions of nontoxic Strawberry Seedlings.
Claims (9)
1. a kind of Strawberry Leaves in-vitro regeneration method, it is characterised in that：Comprise the following steps：
1) using Strawberry Leaves as explant, will be seeded in after explant surface sterilization on regeneration culture medium, then in turn through dark
Culture 8~10 days is cultivated 1~2 day under illumination, obtains callus；The regeneration culture medium by the organic culture mediums of MS+B5 and
0.5~1.0mg/L 6-BA and 0.1~0.5mg/L NAA are formed, and the organic culture mediums of MS+B5 are trained for organic principle using B5
Support the MS culture mediums of the organic principle of base；
2) callus is expanded into numerous, bud elongation and root induction culture by adventitious bud inducing, adventitious bud successively, obtains strawberry group
Train seedling；
3) by Strawberry Plantlets seedling and propagating.
2. a kind of Strawberry Leaves in-vitro regeneration method according to claim 1, it is characterised in that：The explant is selected from strawberry
Nearly top blade, the explant size of inoculation is 2~3cm × 2~3cm.
3. a kind of Strawberry Leaves in-vitro regeneration method according to claim 1, it is characterised in that：The surface sterilization include with
Lower step：Prior in 75% alcohol impregnate 20~25s after aseptic water washing, then in 3~5%NaClO impregnate 10~15s after
Then aseptic water washing impregnates 8~10min, last aseptic water washing in 0.1% mercuric chloride.
4. a kind of Strawberry Leaves in-vitro regeneration method according to claim 1, it is characterised in that：It is cultivated under the illumination, with
And adventitious bud inducing, adventitious bud expand numerous, bud elongation and the condition of culture of root induction culture and tissue culture seedling and propagating is：20~25
DEG C, 1000~1500lx and 14~16h/d illumination.
5. a kind of Strawberry Leaves in-vitro regeneration method according to claim 1, it is characterised in that：The adventitious bud inducing uses
Culture medium for additionally be added with 1.5~2.0mg/L TDZ, the MS of 1.5~2.0mg/L 6-BA and 0.3~0.5mg/L IBA
Culture medium.
6. a kind of Strawberry Leaves in-vitro regeneration method according to claim 1, it is characterised in that：The adventitious bud expands numerous use
Culture medium be the additional MS culture mediums for being added with 1.5~2.0mg/L TDZ and 0.4~0.6mg/L IBA.
7. a kind of Strawberry Leaves in-vitro regeneration method according to claim 1, it is characterised in that：The training that the bud elongation uses
Base is supported as the additional MS culture mediums for being added with 0.3~0.5mg/L 6-BA.
8. a kind of Strawberry Leaves in-vitro regeneration method according to claim 1, it is characterised in that：What the root induction used
Culture medium is the additional 1/2MS culture mediums for being added with 0.3~0.5mg/L IBA.
9. a kind of Strawberry Leaves in-vitro regeneration method according to claim 1, it is characterised in that：The kind of the strawberry is selected from
Red-color strawberries.
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| CN201711287169.XA CN108094198A (en) | 2017-12-07 | 2017-12-07 | A kind of Strawberry Leaves in-vitro regeneration method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108812316A (en) * | 2018-06-27 | 2018-11-16 | 商丘师范学院 | A kind of Strawberry Leaves callus induction method |
| NL2033006B1 (en) * | 2021-11-15 | 2023-06-09 | Univ Sichuan Agricultural | High-frequency regeneration method of fragaria ananassa in vitro leaves and application method thereof in genetic transformation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1423927A (en) * | 2003-01-13 | 2003-06-18 | 贾景明 | Strawberry seedling production by tissue culture |
| CN105638465A (en) * | 2015-12-30 | 2016-06-08 | 四川禾木本业农林科技有限公司 | Strawberry tissue culture fast propagation method |
-
2017
- 2017-12-07 CN CN201711287169.XA patent/CN108094198A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1423927A (en) * | 2003-01-13 | 2003-06-18 | 贾景明 | Strawberry seedling production by tissue culture |
| CN105638465A (en) * | 2015-12-30 | 2016-06-08 | 四川禾木本业农林科技有限公司 | Strawberry tissue culture fast propagation method |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108812316A (en) * | 2018-06-27 | 2018-11-16 | 商丘师范学院 | A kind of Strawberry Leaves callus induction method |
| CN108812316B (en) * | 2018-06-27 | 2021-09-14 | 商丘师范学院 | Strawberry leaf callus induction method |
| NL2033006B1 (en) * | 2021-11-15 | 2023-06-09 | Univ Sichuan Agricultural | High-frequency regeneration method of fragaria ananassa in vitro leaves and application method thereof in genetic transformation |
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Application publication date: 20180601 |