CN109006471A - A kind of rapid propagation method of teak cotyledon adventitious bud inducing - Google Patents
A kind of rapid propagation method of teak cotyledon adventitious bud inducing Download PDFInfo
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- CN109006471A CN109006471A CN201810587598.7A CN201810587598A CN109006471A CN 109006471 A CN109006471 A CN 109006471A CN 201810587598 A CN201810587598 A CN 201810587598A CN 109006471 A CN109006471 A CN 109006471A
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- adventitious bud
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Developmental Biology & Embryology (AREA)
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Abstract
The invention discloses the rapid propagation methods that a kind of teak cotyledon evoking adventive bud generates, steps are as follows: after choosing well-grown full maturity or medium well teak seed disinfection, it is inoculated on germination medium, it is transferred on adventitious bud induction culture base and cultivates after seed sprouting, the adventitious bud of acquisition is transferred in proliferated culture medium and strong seedling culture base after 30d and is cultivated, well-grown teak single plant seedling is obtained after 30d.The present invention provides a kind of method for efficiently quickly breeding seedling, has the characteristics that regeneration rate is high, budding is more, can effectively solve good seed germplasm degenerate problem, whole flow process is all easy to operate.
Description
Technical field
The present invention relates to a kind of rapid propagation methods of teak cotyledon adventitious bud inducing, belong to plant biotechnology field.
Background technique
Teak (Tectona grandis L.f.) alias Arnotto, it is tall and big for half fallen leaves property of Verenaceae Tectona fallen leaves
Arbor, natural distributed is in states such as Burma, Thailand, India and Laos.Teak growth is rapid, material is excellent, widely used, is the world
Rare commerical tree species also become main afforestation and the economic tree of China torrid areas.
Teakwood compact structure, material be tough and tensile, beautiful texture, corrosion resistant, therefore has very high utility value, has made it
As one of precious timber most short on current domestic and international market.Traditional modes of reproduction of teak is seminal propagation, by typhoon
It is lower to influence some areas seed production, and seed germination rate is low, it is difficult to meet the needs of production.From the 1980s
The country starts the teak Study on tissue culture carried out, and existing teak quick breeding by group culture method generally uses twig for explant
Disinfection treatment, is inoculated on culture medium, by its shoot proliferation culture after growing adventitious bud, subculture mode generally takes intercept
Formula, this improves the proliferative speed of teak to a certain extent.The present invention is occurred using cotyledon evoking adventive bud, can choose eight
Mature teak seed improves seed utilization rate, hides the infringement of typhoon, in addition, every cotyledon can generate largely as material
Adventitious bud substantially increases breeding efficiency.
Teak is big in China market demand, and domestic without wildwood resource, and the germ plasm resource that foreign countries introduce was being promoted
Many problems, such as low temperature freezing-disaster, pest and disease damage and typhoon influence are faced in journey, therefore on the basis of conventional breeding,
The transgenic technology of teak is developed, cold-resistant, pest-resistant, disease-resistant, fast-growing transgenosis new varieties, the popularization for China's teak are developed
Cultivation is also of great significance.Plant regeneration system is the basis of genetic plant transformations.Research team is to the teak side of regeneration indirectly
Formula has done some researchs, as a result, it has been found that can smoothly obtain callus using blade and stem section induction, but callus is difficult point
Adventitious bud is dissolved, teak not yet establishes perfect regenerating system.This research work utilizes the generation of teak cotyledon evoking adventive bud,
It lays the foundation for genetic transformation.
Summary of the invention
The present invention provides a kind of rapid propagation method of teak cotyledon adventitious bud inducing, low to solve teak regeneration rate
Problem, while laying the foundation for teak genetic transformation.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of rapid propagation method of teak cotyledon adventitious bud inducing, comprising the following steps:
(1) pericarp hard using secateurs tool removal teak, removing obtain teak seed, pay attention to keeping the complete of kind of skin, no
With seed is impregnated, directly it is ready for disinfecting.
Current year raw teak seed is chosen, the fruit of teak is divided into two with secateurs, exposes seed, then carefully shell seed
From preparation disinfection；The disinfection are as follows: on superclean bench, impregnate 30~60s, sterile water with volume fraction for 70% alcohol
It rinses 2~3 times, then sterilizes 5min~8min with mass fraction for 0.1% mercuric chloride solution, then use aseptic water washing 3~5 times.
(2) it is inoculated on MS minimal medium and cultivates after teak seed disinfection processing, shifted after cotyledon trails
It is cultivated on to adventitious bud induction culture base.The minimal medium are as follows:+30 g/L sucrose of MS+4.5 g/L agar, pH value 5.8~
6.0；Condition of culture is to cultivate 25 ± 2 DEG C of room temperature daytime in each step, and 18 ± 2 DEG C of night, intensity of illumination 1000~
1500lx, light application time 16h/d.
(3) remove kind of a skin, the cotyledon to trail is transferred on adventitious bud induction culture base, induce Multiple Buds, institute
State adventitious bud induction culture base are as follows: MS+0.1~0.2mg/L TDZ+0.5~1.0mg/L CH (caseinhydrolysate)+4.5 g/L
Agar+30g/L sucrose, pH value 5.8~6.0.
It is inoculated on MS minimal medium after teak seed disinfection processing and sprouts culture, after being inoculated with 5 ~ 7d, zygotic embryo is swollen
Greatly, it is transferred on the MS culture medium of the 6-BA or TDZ of additional various concentration and cultivates after cotyledon trails.It is inoculated with 10d
The zygotic embryo on culture medium continues to expand afterwards, and the cotyledon on partial medium starts callus, and observation discovery is additional after 20d
The cotyledon top and Surface Differentiation of 0.1mg/L TDZ and additional 0.4mg/ L 6-BA culture medium part embryo go out adventitious bud.
When TDZ concentration is 0.05 mg/L, the adventitious bud induction frequency of zygotic embryo is 43.33%, TDZ concentration when being 0.1mg/L,
The adventitious bud induction frequency of zygotic embryo be up to 66.67%, TDZ concentration be 0.05 mg/L when, the adventitious bud induction frequency of zygotic embryo
It is 40%.These results suggest that 0.1mg/L TDZ is the optimum concentration of zygote scutellum adventitious bud inducing.
6-BA concentration is 0 in 1.0 mg/L and 6.0 mg/L, the adventitious bud induction frequency of zygotic embryo.6-BA concentration is 1.0
The callus that cotyledon is formed when mg/ L expands, fine and close hard, the callus group that cotyledon is formed when 6-BA concentration is 6.0 mg/ L
It is serious to knit transparent vitrification.For 6-BA concentration in 2.0 mg/ L, merozygote scutellum and hypocotyl differentiate adventitious bud, lure
Conductance is 13.33%.For 6-BA concentration in 4.0 mg/ L, adventitious bud induction frequency highest reaches 50%.
(4) Multiple Buds for bearing cotyledon surface, which are cut into small pieces to be transferred on test tube seedling proliferated culture medium, carries out proliferation training
It supports, the test tube seedling proliferation culture medium formula: MS+0.1~0.2mg/L TDZ+4.5g/L agar+30g/L sucrose, pH value 5.8
~6.0.
(5) Multiple Buds of Multiplying culture are separated into fritter from base portion, are transferred in the elongation medium of adventitious bud and cultivate,
The Elongation of adventitious bud culture medium are as follows: 0.1 mg/L IAA+4.5g/L agar+30g/L sugarcane of MS+1.0~2.0mg/L BA+
Sugar, pH value 5.8~6.0.
The adventitious bud of zygote embryogenesis is separated into fritter from base portion, is transferred in the elongation medium of adventitious bud and cultivates,
It has been observed that not adding adventitious bud slow growth on the culture medium of plant hormone after 10 d；Additional 1.0 ~ 2.0+IAA of 6-BA
Adventitious bud obviously extends on 0.1 culture medium, and a large amount of adventitious buds are extending to 2.0 cm after 30 d；Only add the culture medium of 6-BA
Upper Elongation of adventitious bud is uneven, and the phenomena of mortality occurs in surrounding tissue, illustrate must concentration IAA stretching for adventitious bud
Length is necessary.
Advantageous effects:
The present invention provides a kind of method for efficiently quickly breeding seedling, has the characteristics that regeneration rate is high, budding is more, can effectively solve
Certainly good seed germplasm degenerate problem, whole flow process are all easy to operate.
Specific embodiment
A kind of rapid propagation method of teak cotyledon adventitious bud inducing, comprising the following steps:
(1) pericarp hard using secateurs tool removal teak, removing obtain teak seed, pay attention to keeping the complete of kind of skin, no
With seed is impregnated, directly it is ready for disinfecting.
Current year raw teak seed is chosen, the fruit of teak is divided into two with secateurs, exposes seed, then carefully shell seed
From preparation disinfection；The disinfection are as follows: on superclean bench, impregnate 30~60s, sterile water with volume fraction for 70% alcohol
It rinses 2~3 times, then sterilizes 5min~8min with mass fraction for 0.1% mercuric chloride solution, then use aseptic water washing 3~5 times.
Disinfection treatment experiment: disinfectant handles the different time, observes pollution rate situation, see the table below:
The influence that 1 disinfectant of table and disinfecting time sprout seed
| Disinfection treatment | Contamination rate | Seed germination rate |
| 0.1% mercuric chloride sterilizes 3 min | 13.33% | 80% |
| 0.1% mercuric chloride sterilizes 5 min | 3.33% | 83.33% |
| 0.1% mercuric chloride sterilizes 8 min | 0 | 76.67% |
(2) it is inoculated on MS minimal medium and cultivates after teak seed disinfection processing, be transferred into not after cotyledon trails
It is cultivated in normal bud induced medium.The minimal medium are as follows:+30 g/L sucrose of MS+4.5 g/L agar, pH value 5.8~6.0；
Condition of culture is to cultivate 25 ± 2 DEG C of room temperature daytime in each step, and 18 ± 2 DEG C of night, intensity of illumination 1000~
1500lx, light application time 16h/d.
(3) remove kind of a skin, the cotyledon to trail is transferred on adventitious bud induction culture base, induce Multiple Buds, institute
State adventitious bud induction culture base are as follows: MS+0.1~0.2mg/L TDZ+0.5~1.0mg/L CH (caseinhydrolysate)+4.5 g/L
Agar+30g/L sucrose, pH value 5.8~6.0.
Experiment: the influence that adventitious bud occurs for hormone concentration
It is inoculated on MS minimal medium after teak seed disinfection processing and sprouts culture, after being inoculated with 5 ~ 7d, zygotic embryo expands, to son
Leaf is transferred on the MS culture medium of the 6-BA or TDZ of additional various concentration after trailing and cultivates.Culture medium after inoculation 10d
On zygotic embryo continue to expand, the cotyledon on partial medium starts callus, and observation finds additional 0.1mg/L TDZ after 20d
Go out adventitious bud with the cotyledon top of additional 0.4mg/ L 6-BA culture medium part embryo and Surface Differentiation.
When TDZ concentration is 0.05 mg/L, the adventitious bud induction frequency of zygotic embryo is 43.33%, TDZ concentration when being 0.1mg/L,
The adventitious bud induction frequency of zygotic embryo be up to 66.67%, TDZ concentration be 0.05 mg/L when, the adventitious bud induction frequency of zygotic embryo
It is 40%.These results suggest that 0.1mg/L TDZ is the optimum concentration of zygote scutellum adventitious bud inducing.
6-BA concentration is 0 in 1.0 mg/L and 6.0 mg/L, the adventitious bud induction frequency of zygotic embryo.6-BA concentration is 1.0
The callus that cotyledon is formed when mg/ L expands, fine and close hard, the callus group that cotyledon is formed when 6-BA concentration is 6.0 mg/ L
It is serious to knit transparent vitrification.For 6-BA concentration in 2.0 mg/ L, merozygote scutellum and hypocotyl differentiate adventitious bud, lure
Conductance is 13.33%.For 6-BA concentration in 4.0 mg/ L, adventitious bud induction frequency highest reaches 50%.
The influence that adventitious bud occurs for 2 hormon of table
(4) Multiple Buds for bearing cotyledon surface, which are cut into small pieces to be transferred on test tube seedling proliferated culture medium, carries out Multiplying culture, institute
State test tube seedling proliferation culture medium formula: MS+0.1~0.2mg/L TDZ+4.5g/L agar+30g/L sucrose, pH value 5.8~6.0.
(5) Multiple Buds of Multiplying culture are separated into fritter from base portion, are transferred in the elongation medium of adventitious bud and cultivate,
The Elongation of adventitious bud culture medium are as follows: 0.1 mg/L IAA+4.5g/L agar+30g/L sugarcane of MS+1.0~2.0mg/L BA+
Sugar, pH value 5.8~6.0.
The elongation of 2.2 adventitious buds
The adventitious bud of zygote embryogenesis is separated into fritter from base portion, is transferred in the elongation medium of adventitious bud and cultivates, 10 d
Afterwards it has been observed that not adding adventitious bud slow growth on the culture medium of plant hormone；Additional 1.0 ~ 2.0+IAA 0.1 of 6-BA
Culture medium on adventitious bud obviously extend, a large amount of adventitious buds are extending to 2.0 cm after 30 d；Only on the culture medium of addition 6-BA
Elongation of adventitious bud is uneven, and the phenomena of mortality occurs in surrounding tissue, illustrate must concentration elongation of the IAA for adventitious bud
It is necessary.
Influence of the 3 6-BA concentration of table to Elongation of adventitious bud
It should be noted that, in this document, relational terms such as first and second and the like be used merely to an entity or
Person's operation is distinguished with another entity or operation, is appointed without necessarily requiring or implying existing between these entities or operation
What this actual relationship or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that the process, method, article or equipment for including a series of elements not only includes those elements, and
And further include other elements that are not explicitly listed, or further include for this process, method, article or equipment institute it is intrinsic
Element.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. a kind of rapid propagation method of teak cotyledon adventitious bud inducing, comprising the following steps:
(1) pericarp hard using secateurs tool removal teak, removing obtain teak seed, pay attention to keeping the complete of kind of skin, no
With seed is impregnated, directly it is ready for disinfecting.
Current year raw teak seed is chosen, the fruit of teak is divided into two with secateurs, exposes seed, then carefully remove seed,
Prepare disinfection；The disinfection are as follows: on superclean bench, impregnate 30~60s, aseptic water washing with volume fraction for 70% alcohol
2~3 times, then 5min~8min is sterilized for 0.1% mercuric chloride solution with mass fraction, then use aseptic water washing 3~5 times.
2. a kind of rapid propagation method of teak cotyledon adventitious bud inducing as described in claim 1, it is characterised in that: further include
Following steps,
(2) it is inoculated on MS minimal medium and cultivates after teak seed disinfection processing, be transferred into not after cotyledon trails
It is cultivated in normal bud induced medium.The minimal medium are as follows:+30 g/L sucrose of MS+4.5 g/L agar, pH value 5.8~6.0；
Condition of culture is to cultivate 25 ± 2 DEG C of room temperature daytime in each step, and 18 ± 2 DEG C of night, intensity of illumination 1000~
1500lx, light application time 16h/d.
(3) remove kind of a skin, the cotyledon to trail be transferred on adventitious bud induction culture base, induce Multiple Buds, it is described not
Normal bud induced medium are as follows: MS+0.1~0.2mg/L TDZ+0.5~1.0mg/L CH (caseinhydrolysate)+4.5 g/L agar+
30g/L sucrose, pH value 5.8~6.0.
3. a kind of rapid propagation method of teak cotyledon adventitious bud inducing as described in claim 1, it is characterised in that: further include
Following steps,
It is inoculated on MS minimal medium after teak seed disinfection processing and sprouts culture, after being inoculated with 5 ~ 7d, zygotic embryo expands, to son
Leaf is transferred on the MS culture medium of the 6-BA or TDZ of additional various concentration after trailing and cultivates.Culture medium after inoculation 10d
On zygotic embryo continue to expand, the cotyledon on partial medium starts callus, and observation finds additional 0.1mg/L TDZ after 20d
Go out adventitious bud with the cotyledon top of additional 0.4mg/ L 6-BA culture medium part embryo and Surface Differentiation.
When TDZ concentration is 0.05 mg/L, the adventitious bud induction frequency of zygotic embryo is 43.33%, TDZ concentration when being 0.1mg/L, zygote
The adventitious bud induction frequency of embryo be up to 66.67%, TDZ concentration be 0.05 mg/L when, the adventitious bud induction frequency of zygotic embryo is
40%.These results suggest that 0.1mg/L TDZ is the optimum concentration of zygote scutellum adventitious bud inducing.
6-BA concentration is 0 in 1.0 mg/L and 6.0 mg/L, the adventitious bud induction frequency of zygotic embryo.6-BA concentration is 1.0 mg/
The callus that cotyledon is formed when L expands, and fine and close hard, the callus that cotyledon is formed when 6-BA concentration is 6.0 mg/ L is saturating
Bright vitrifying is serious.For 6-BA concentration in 2.0 mg/ L, merozygote scutellum and hypocotyl differentiate adventitious bud, inductivity
It is 13.33%.For 6-BA concentration in 4.0 mg/ L, adventitious bud induction frequency highest reaches 50%.
4. a kind of rapid propagation method of teak cotyledon adventitious bud inducing as described in claim 1, it is characterised in that: further include
Following steps,
(4) Multiple Buds for bearing cotyledon surface, which are cut into small pieces to be transferred on test tube seedling proliferated culture medium, carries out Multiplying culture, institute
State test tube seedling proliferation culture medium formula: MS+0.1~0.2mg/L TDZ+4.5g/L agar+30g/L sucrose, pH value 5.8~6.0.
(5) Multiple Buds of Multiplying culture are separated into fritter from base portion, are transferred in the elongation medium of adventitious bud and cultivate, institute
State Elongation of adventitious bud culture medium are as follows: 0.1 mg/L IAA+4.5g/L agar+30g/L sucrose of MS+1.0~2.0mg/L BA+,
PH value 5.8~6.0.
5. a kind of rapid propagation method of teak cotyledon adventitious bud inducing as described in claim 1, it is characterised in that: further include
Following steps,
The adventitious bud of zygote embryogenesis is separated into fritter from base portion, is transferred in the elongation medium of adventitious bud and cultivates, 10 d
Afterwards it has been observed that not adding adventitious bud slow growth on the culture medium of plant hormone；Additional 1.0 ~ 2.0+IAA 0.1 of 6-BA
Culture medium on adventitious bud obviously extend, a large amount of adventitious buds are extending to 2.0 cm after 30 d.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110754361A (en) * | 2019-11-13 | 2020-02-07 | 浙江省林业科学研究院 | Quick propagation method for Zhejiang benzoin in vitro embryo induced cluster buds |
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| CN101385440A (en) * | 2008-09-26 | 2009-03-18 | 中国科学院西双版纳热带植物园 | Quick propagation method of Tectona grandis |
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2018
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| CN1471812A (en) * | 2003-06-25 | 2004-02-04 | 海南华翠棕榈园有限公司 | Method for growing small shaddock |
| CN101385440A (en) * | 2008-09-26 | 2009-03-18 | 中国科学院西双版纳热带植物园 | Quick propagation method of Tectona grandis |
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| TAMBARUSSI EV等: "Influence of antibiotics on indirect organogenesis of Teak", 《ANNALS OF FOREST RESEARCH》 * |
| 吴忠锋等: "柚木繁育技术研究进展", 《热带农业科学》 * |
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| CN110754361A (en) * | 2019-11-13 | 2020-02-07 | 浙江省林业科学研究院 | Quick propagation method for Zhejiang benzoin in vitro embryo induced cluster buds |
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Application publication date: 20181218 |