CN106818469A - A kind of toon renovation process with blade as explant - Google Patents
A kind of toon renovation process with blade as explant Download PDFInfo
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- CN106818469A CN106818469A CN201611247958.6A CN201611247958A CN106818469A CN 106818469 A CN106818469 A CN 106818469A CN 201611247958 A CN201611247958 A CN 201611247958A CN 106818469 A CN106818469 A CN 106818469A
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- culture
- toon
- bud
- explant
- seedling
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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 invention discloses a kind of toon renovation process with blade as explant.High-efficiency tissue culture and plant regeneration method with blade as explant of the invention, can break away from the influence of external condition, and scale and factorial praluction go out healthy and strong, the toon tissue culture seedling of neat and consistent；With it has been reported that compared with using toon, big tree stem section builds regenerating system as explant, with easy to operate, materials extensively, the advantage such as repeatability is strong and efficiency is high.The method can meet demand of the market to seedling, be that toon germ plasm resource sustainable use lays the foundation.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to a kind of toon regeneration side with blade as explant
Method.
Background technology
Toon (Toona ciliata Roem.), also known as red chinaberry, are subordinate to Meliaceae (Meliaceae) Cedrela
(Toona).In China, toon is mainly distributed on south China, Central China, East China and southwest, and Natural Populations have fragmentary distribution special
Point, India, Laos, Burma, Pakistan, Australian east coast are also distributed.Toon material is good, have " the Chinese peach blossom heart
The title of wood ", is excellent furniture woodses.It has the features such as early stage fast-growing proterties is obvious, growth is fast, and increment exceedes general
The Evergreen Broad-leaved Tree Species of artificial cultivation, in the high-quality fast-growing commerical tree species that south China area has utilized as focus development.By
The poor and excessive felling destruction of change, natural regeneration ability in environment, toon has turned into endangered species at present, is 1999 years
Through national II grade of Top-rated protected wild plants of State Council approved, and list in《Chinese Main Cultivation rare tree refers to register》.
Toon material is fine and closely woven, texture is logical straight, and decorative pattern is beautiful, and core is in dark reddish brown, be widely used in furniture design, car and boat manufacture and
Interior decoration field；Its is tree-like attractive in appearance, it is also possible to planted as shade tree.
The research of current toon is primarily focused on the conventional breeding such as introduces a fine variety, breeds.Seminal propagation being used its modes of reproduction more,
But because the hold capacity of seed is limited, filial generation family can not completely preserve the excellent inhereditary feature of parental generation, therefore the skewer of toon
Insert, grafting and tissue culture technique get growing concern for.The spray requirement of cuttage and propagation by grafiting to toon is higher, adopts
The structure in fringe garden needs also exist for a large amount of manpower and materials.In a short time, traditional seminal propagation and cutting propagation can not expire in time
The sufficient seedling market demand, now, can solve the problems, such as seedling quickly and amount reproduction by tissue culture technique.Additionally, being built into
Ripe efficient regenerating system is successfully basic genetic transformation, and is established to be transferred to the toon kind of genes of interest acquisition more high-quality
Fixed basis, effectively solves the problems such as the fatal insect pest of toon treelet, efficiently the orientation genetic improvement of propulsion toon.
External toon tissue culture technique research report is less, mainly gathered from the big tree of adult explant carry out it is in vitro numerous
Grow, the axillary bud deriving rate for being obtained is relatively low, and culture of rootage and unsuccessful.Domestic toon tissue cultures it is main with seed and into
Year, big tree belt leaf stem section set up tissue culture system as explant, but proliferative conditions are unsatisfactory.
The content of the invention
It is an object of the invention to provide a kind of operation with toon blade as explant it is easier, materials widely efficiently
Toon renovation process, can quickly obtain the toon seedling of a large amount of high-quality in a short time using the method.The present invention is with toon
Blade is material, the callus for securing good health, and the regenerating system of system research toon, it is intended to solve the problems, such as leaf regeneration is carried
Toon power of regeneration high, for factorial praluction seedling, plasm resource protection and genetic engineering research provide effective reference value.
Toon renovation process with blade as explant of the invention, it is characterised in that comprise the following steps：
The acquisition of A, explant：Toon seed is removed into bilateral wing, in water soak 3~5h, it is sterilized after seed is broadcast
Plant and obtain aseptic seedling in culture on solidified MS media, cut the leaflet of aseptic seedling as explant；Condition of culture is temperature 25
± 2 DEG C, intensity of illumination 2500lx, light application time 12h/d；
The induction and elongation of B, bud：Leaflet is inoculated on bud inducement cultivation base and cultivates evoked callus, it is small during inoculation
Leaf distal shaft faces up；Condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d；Cultivate to explant
When upper differentiation is sprouted, the explant for sprouting is transferred in bud elongation medium that induction is cultivated under same culture conditions is indefinite
Bud extends；
Described bud inducement cultivation base：Every liter containing 1.8~3.2mg of 6-BA, 0.5~1.5mg of KT, NAA 0.03~
0.15mg, sucrose 30g and agar 5g, balance of MS culture mediums, pH 5.8~6.0；
Described bud elongation medium：Every liter contains 6-BA0.05~0.3mg, 0.1~0.3mg of NAA, sucrose 30g and fine jade
Fat 5g, balance of MS culture mediums, pH 5.8~6.0；
C, culture of rootage：When Elongation of adventitious bud is to 3~5cm, adventitious bud is cut, be inoculated in culture on root media and lure
Lead and take root, obtain rooted seedling；Condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d；
Described root media：Every liter contains NAA0.1~0.3mg, sucrose 15g and agar 5g, balance of MS cultures
Base, pH5.8~6.0；
D, hardening and transplanting：To be dug out from blake bottle after rooted seedling hardening, clean the culture medium on root, rooted seedling is existed
1h is soaked in running water, is transplanted on peat soil, carry out cultivation management, obtain toon plant.
It is preferred that, the sterilization of described step A is by the ethanol water of toon seed volume fraction 75% sterilizing 1min, nothing
Bacterium water is rinsed 1 time, the aqueous sodium hypochlorite solution of mass fraction 10% 15~20min of sterilizing, aseptic water washing 3~5 times.
It is preferred that, the hardening of described step D is to open culture bottle cap to adapt to 2d by rooted seedling.
It is preferred that, the peat soil of described step D is the peat soil that high-temperature sterilization is crossed.
The solidified MS media of described step A：Every liter contains sucrose 30g and agar 5g, balance of MS culture mediums, pH
5.8~6.0.
Described MS culture mediums are international culture medium, and its composition and collocation method are shown in Murashige T, Skoog
F(1962)A revised medium for rapid growth and bioassay with tobacco tissue
cultures.Physiol Plant 15:473–497。
The culture of toon high-efficiency tissue and plant regeneration method with blade as explant of the invention, can break away from external condition
Influence, scale and factorial praluction go out healthy and strong, the toon tissue culture seedling of neat and consistent；With it has been reported that with the big tree of toon
Stem section builds regenerating system and compares as explant, with easy to operate, materials extensively, that repeatability is strong and efficiency is high etc. is excellent
Gesture.The method can meet demand of the market to seedling, be that toon germ plasm resource sustainable use lays the foundation.
Brief description of the drawings
Fig. 1 is the aseptic seedling that seed culture 35d is obtained, for cutting blade.
Fig. 2 is the bud point on the leaflet explant for cultivated on bud inducement cultivation base 30d.
Fig. 3 is the adventitious bud that 30d elongations are cultivated in bud elongation medium.
Fig. 4 is the root induced after adventitious bud cultivates 30d on root media.
Fig. 5 is the toon plant of transplant survival.
Specific embodiment
Following examples are further illustrated to of the invention, rather than limitation of the present invention.
Embodiment 1
The acquisition of A, explant：Toon seed is removed into bilateral wing, 4h is soaked in water；On aseptic super-clean bench, body is used
The ethanol water of fraction 75% sterilizing 1min, aseptic water washing 1 time, the sterilizing of the aqueous sodium hypochlorite solution of mass fraction 10%
18min, sterilized water cleaning down 4 times, then by seed be seeded on solidified MS media cultivate, condition of culture be temperature 25 ±
2 DEG C, intensity of illumination 2500lx, light application time 12h/d.Seed culture 3d or so starts rudiment, and after 35d, aseptic seedling is (such as Fig. 1 institutes
Showing) compound leaf grows, and cuts individual leaflet thereon as explant.Every liter of the solidified MS media contains sucrose 30g and fine jade
Fat 5g, balance of MS culture mediums, pH5.8；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby.
The induction and elongation of B, bud：Leaflet is inoculated on bud inducement cultivation base and cultivates evoked callus, it is small during inoculation
Leaf distal shaft faces up；Condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d.Start on explant
There is callus to occur, after 30d is cultivated, be gradually divided into bud point (as shown in Figure 2), the explant for sprouting is transferred to bud elongation training
Support and cultivate evoking adventive bud elongation on base under same culture conditions.The adventitious bud extended after culture 30d is as shown in Figure 3.It is described
Bud inducement cultivation base：Every liter contains 6-BA 2.5mg, KT 1.0mg, NAA 0.1mg, sucrose 30g and agar 5g, balance of MS
Culture medium, pH5.8；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby.Described bud elongation culture
Base：Every liter contains 6-BA0.15mg, NAA 0.2mg, sucrose 30g and agar 5g, balance of MS culture mediums, pH5.8；Compound method
It is to be well mixed above-mentioned composition, adjusts pH, then sterilizes standby.
C, culture of rootage：When Elongation of adventitious bud is to 4cm, adventitious bud is cut, be inoculated in and induction is cultivated on root media
Take root, condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d.After culture of rootage 30d, adventitious bud
Grow a plurality of and obtain rooted seedling, situation of taking root is as shown in Figure 4.Described root media：Every liter contains NAA0.2mg, sucrose
15g and agar 5g, balance of MS culture mediums, pH5.8；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby
With.
D, hardening and transplanting：Open culture bottle cap and adapt to 2d by rooted seedling, then carefully rooted seedling is dug from blake bottle
Go out, clean the culture medium on root, rooted seedling is soaked into 1h in running water, be transplanted to what the high-temperature sterilization in nutrition cup was crossed
On peat soil, polybag is removed in the polybag moisturizing on set on nutrition cup at leisure after 3d, watered by growth demand.Transplant into
Toon plant living is as shown in Figure 5.
Embodiment 2
The acquisition of A, explant：Toon seed is removed into bilateral wing, 3h is soaked in water；On aseptic super-clean bench, body is used
The ethanol water of fraction 75% sterilizing 1min, aseptic water washing 1 time, the sterilizing of the aqueous sodium hypochlorite solution of mass fraction 10%
15min, sterilized water cleaning down 3 times, then by seed be seeded on solidified MS media cultivate, condition of culture be temperature 25 ±
2 DEG C, intensity of illumination 2500lx, light application time 12h/d.Seed culture 3d or so starts rudiment, and after 30d, aseptic seedling compound leaf grows,
Individual leaflet thereon is cut as explant.Every liter of the solidified MS media contains sucrose 30g and agar 5g, balance of MS
Culture medium, pH5.8；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby.
The induction and elongation of B, bud：Leaflet is inoculated on bud inducement cultivation base and cultivates evoked callus, it is small during inoculation
Leaf distal shaft faces up；Condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d.Start on explant
There is callus to occur, after 25d is cultivated, be gradually divided into bud point, the explant for sprouting is transferred in bud elongation medium in phase
Extended with evoking adventive bud is cultivated under condition of culture.Described bud inducement cultivation base：Every liter contains 6-BA 1.8mg, KT
0.5mg, NAA 0.03mg, sucrose 30g and agar 5g, balance of MS culture mediums, pH5.8；Compound method is to mix above-mentioned composition
Close uniform, adjust pH, then sterilize standby.Described bud elongation medium：Every liter contains 6-BA0.05mg, NAA 0.1mg, sucrose
30g and agar 5g, balance of MS culture mediums, pH5.8；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby
With.
C, culture of rootage：When Elongation of adventitious bud is to 3cm, adventitious bud is cut, be inoculated in and induction is cultivated on root media
Take root, condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d.After culture of rootage 30d, adventitious bud
Grow a plurality of and obtain rooted seedling.Described root media：Every liter containing NAA0.1mg, sucrose 15g and agar 5g, it is balance of
MS culture mediums, pH5.8；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby.
D, hardening and transplanting：Open culture bottle cap and adapt to 2d by rooted seedling, then carefully rooted seedling is dug from blake bottle
Go out, clean the culture medium on root, rooted seedling is soaked into 1h in running water, be transplanted to what the high-temperature sterilization in nutrition cup was crossed
On peat soil, polybag is removed in the polybag moisturizing on set on nutrition cup at leisure after 3d, watered by growth demand.Thus
To the toon plant of transplant survival.
Embodiment 3
The acquisition of A, explant：Toon seed is removed into bilateral wing, 5h is soaked in water；On aseptic super-clean bench, body is used
The ethanol water of fraction 75% sterilizing 1min, aseptic water washing 1 time, the sterilizing of the aqueous sodium hypochlorite solution of mass fraction 10%
20min, sterilized water cleaning down 5 times, then by seed be seeded on solidified MS media cultivate, condition of culture be temperature 25 ±
2 DEG C, intensity of illumination 2500lx, light application time 12h/d.Seed culture 3d or so starts rudiment, and after 40d, aseptic seedling compound leaf grows,
Individual leaflet thereon is cut as explant.Every liter of the solidified MS media contains sucrose 30g and agar 5g, balance of MS
Culture medium, pH6.0；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby.
The induction and elongation of B, bud：Leaflet is inoculated on bud inducement cultivation base and cultivates evoked callus, it is small during inoculation
Leaf distal shaft faces up；Condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d.Start on explant
There is callus to occur, after 35d is cultivated, be gradually divided into bud point, the explant for sprouting is transferred in bud elongation medium in phase
Extended with evoking adventive bud is cultivated under condition of culture.Described bud inducement cultivation base：Every liter contains 6-BA 3.2mg, KT
1.5mg, NAA 0.15mg, sucrose 30g and agar 5g, balance of MS culture mediums, pH6.0；Compound method is to mix above-mentioned composition
Close uniform, adjust pH, then sterilize standby.Described bud elongation medium：Every liter contains 6-BA 0.3mg, NAA 0.3mg, sucrose
30g and agar 5g, balance of MS culture mediums, pH6.0；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby
With.
C, culture of rootage：When Elongation of adventitious bud is to 5cm, adventitious bud is cut, be inoculated in and induction is cultivated on root media
Take root, condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d.After culture of rootage 30d, adventitious bud
Grow a plurality of and obtain rooted seedling.Described root media：Every liter containing NAA0.3mg, sucrose 15g and agar 5g, it is balance of
MS culture mediums, pH6.0；Compound method is to be well mixed above-mentioned composition, adjusts pH, is then sterilized standby.
D, hardening and transplanting：Open culture bottle cap and adapt to 2d by rooted seedling, then carefully rooted seedling is dug from blake bottle
Go out, clean the culture medium on root, rooted seedling is soaked into 1h in running water, be transplanted to what the high-temperature sterilization in nutrition cup was crossed
On peat soil, polybag is removed in the polybag moisturizing on set on nutrition cup at leisure after 3d, watered by growth demand.Thus
To the toon plant of transplant survival.
Claims (5)
1. a kind of toon renovation process with blade as explant, it is characterised in that comprise the following steps：
The acquisition of A, explant：Toon seed is removed into bilateral wing, in water soak 3~5h, it is sterilized after seed is seeded in
Culture obtains aseptic seedling on solidified MS media, cuts the leaflet of aseptic seedling as explant；Condition of culture is temperature 25 ± 2
DEG C, intensity of illumination 2500lx, light application time 12h/d；
The induction and elongation of B, bud：Leaflet is inoculated on bud inducement cultivation base and cultivates evoked callus, leaflet is remote during inoculation
Axial plane is upward；Condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d；Divide on culture to explant
When dissolving bud point, the explant for sprouting is transferred in bud elongation medium the culture evoking adventive bud under same culture conditions and is stretched
It is long；
Described bud inducement cultivation base：Every liter containing 1.8~3.2mg of 6-BA, 0.5~1.5mg of KT, NAA 0.03~
0.15mg, sucrose 30g and agar 5g, balance of MS culture mediums, pH 5.8~6.0；
Described bud elongation medium：Every liter contains 6-BA0.05~0.3mg, 0.1~0.3mg of NAA, sucrose 30g and agar
5g, balance of MS culture mediums, pH 5.8~6.0；
C, culture of rootage：When Elongation of adventitious bud is to 3~5cm, adventitious bud is cut, be inoculated on root media and cultivate induction life
Root, obtains rooted seedling；Condition of culture is 25 ± 2 DEG C of temperature, intensity of illumination 2500lx, light application time 12h/d；
Described root media：Every liter contain NAA0.1~0.3mg, sucrose 15g and agar 5g, balance of MS culture mediums,
PH5.8~6.0；
D, hardening and transplanting：To be dug out from blake bottle after rooted seedling hardening, the culture medium on root is cleaned, by rooted seedling originally
1h is soaked in water, is transplanted on peat soil, carry out cultivation management, obtain toon plant.
2. method according to claim 1, it is characterised in that the sterilization of described step A is by toon seed volume
The ethanol water of fraction 75% sterilizing 1min, aseptic water washing 1 time, the aqueous sodium hypochlorite solution of mass fraction 10% sterilizing 15~
20min, aseptic water washing 3~5 times.
3. method according to claim 1, it is characterised in that the hardening of described step D is to open culture bottle cap to allow life
Offspring adapts to 2d.
4. method according to claim 1, it is characterised in that the peat soil of described step D is the mud that high-temperature sterilization is crossed
Charcoal soil.
5. method according to claim 1, it is characterised in that the solidified MS media of described step A：Every liter contains sugarcane
Sugared 30g and agar 5g, balance of MS culture mediums, pH 5.8~6.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108782244A (en) * | 2018-06-04 | 2018-11-13 | 中国科学院华南植物园 | A kind of passion plant method for tissue culture |
| CN113215191A (en) * | 2021-04-26 | 2021-08-06 | 华南农业大学 | Agrobacterium-mediated genetic transformation method for toona sinensis |
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| CN101491215A (en) * | 2009-02-19 | 2009-07-29 | 周玉玲 | Chinese toon tissue-culture quick propagation technique and culture medium proportion |
| CN103798144A (en) * | 2014-02-28 | 2014-05-21 | 钦州市林业科学研究所 | Culture medium for tissue culture of toona ciliate roem. |
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- 2016-12-29 CN CN201611247958.6A patent/CN106818469A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101491215A (en) * | 2009-02-19 | 2009-07-29 | 周玉玲 | Chinese toon tissue-culture quick propagation technique and culture medium proportion |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108782244A (en) * | 2018-06-04 | 2018-11-13 | 中国科学院华南植物园 | A kind of passion plant method for tissue culture |
| CN113215191A (en) * | 2021-04-26 | 2021-08-06 | 华南农业大学 | Agrobacterium-mediated genetic transformation method for toona sinensis |
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