CN102301958A - Method for culturing in vitro tissues of Semiliquidambar cathayensis - Google Patents
Method for culturing in vitro tissues of Semiliquidambar cathayensis Download PDFInfo
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- CN102301958A CN102301958A CN201110211490A CN201110211490A CN102301958A CN 102301958 A CN102301958 A CN 102301958A CN 201110211490 A CN201110211490 A CN 201110211490A CN 201110211490 A CN201110211490 A CN 201110211490A CN 102301958 A CN102301958 A CN 102301958A
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Abstract
The invention relates to a method for culturing in vitro tissues of Semiliquidambar cathayensis. The method provided by the invention comprises the following steps of: taking Semiliquidambar cathayensis branches of the current year as explants; carrying out a disinfecting treatment on the explants to obtain sterile materials; culturing the sterile materials to form adventitious buds through bud induction; then carrying out a propagation culture, a rooting culture and a domestication and transplantation of tissue culture seedlings to establish a complete rapid-propagation system of the Semiliquidambar cathayensis. By utilizing the method provided by the invention, a lot of Semiliquidambar cathayensis tissue culture seedlings can be simply and efficiently cultured, so that the specie is effectively protected and propagated; the market requirements on the Semiliquidambar cathayensis are satisfied, and the endangered state of the Semiliquidambar cathayensis is eliminated, and the method lays a good foundation on further protecting and utilizing the specie in future.
Description
Technical field
The present invention relates to a kind of method for plant tissue culture, especially relate to a kind of Pterospermi Heterophylli method for tissue culture.
Background technology
Pterospermi Heterophylli
Altingia chingiiMete is the Hamamelidaceae aiphyllium, Chinese Second Class Key Protected Plant.The China endemic species have the comprehensive proterties between Liquidambar Liguidambar and Altingia Altingia two genus, and systematic growth has scientific value to the research Hamamelidaceae.Wood quality is good, and it is good to revolve plane property, can make to revolve the plane goods.Simultaneously bark is relaxed the muscles and stimulate the blood circulation, dispelled rheumatism etc., and effect is among the people is used to treat diseases such as rheumatic arthritis, chronic lumbocrural pain, hemiplegia, traumatic injury, and is evident in efficacy.Seriously reach the natural propagation difficulty because of natural stands is subjected to artificial disturbance, the distribution more and more narrow causes wild resource very rare.
The tissue culture of plant refers to isolate tissue, organ or the cell that suits the requirements from plant corpus; Protoplast etc.; Through sterile working, under the manual control condition, cultivate with whole plant that obtains regeneration or the technology of producing other products of economically valuable.Different plants right minimal medium type, hormone combination, condition of culture etc. in the cultured in vitro process require different.Utilize at present plant tissue culture technique carry out plant germplasm resource protection and the technology on the aspect such as economic nursery stock of producing to be gradually improved.Prior art all is to be the explant induction callus with the Pterospermi Heterophylli blade, through callus propagation, and callus induction differentiation and bud formation again, and do not induce adventive root.
Summary of the invention
The technical problem that the present invention will solve is: provide a kind of simple, inductivity is high, the complete method for tissue culture of the Pterospermi Heterophylli that survival rate is high.
For addressing the above problem, technical scheme provided by the invention is a kind of Pterospermi Heterophylli method for culturing in-vitro tissues, and its concrete steps are:
Draw materials: the current-year branch of getting Pterospermi Heterophylli is an explant; Getting good maternal plant current-year branch in the suitable time is explant; The said suitable time is preferably continuous sunny after one week, draws materials in noon 12 ~ 14;
The foundation of sterilizable material: the explant disinfection is obtained sterilizable material; Be specially and explant is cleaned with liquid detergent and soak, with the flowing water flushing, place on the operating desk, be cut into the stem section of 1-2 internode of band, 75% Ethanol Treatment is used 0.1% mercuric chloride immersion treatment again, during constantly shake sterile water wash;
The bud inducing culture: sterilizable material is inoculated on the bud inducing culture, and inducing culture goes out indefinite bud under the bud inducing culture condition; Said bud inducing culture is improvement MS+6-BA1.5 ~ 2.5mg/L+NAA0.2 ~ 0.4mg/L, and said bud inducing culture condition is light application time 8 ~ 12 h, intensity of illumination 3000 ~ 5000lx, 25 ~ 27 ℃ of temperature;
Enrichment culture: the indefinite bud that inducing culture is gone out is forwarded to the indefinite bud that cultivation makes new advances in the bud proliferated culture medium; Said bud proliferated culture medium is improvement MS+6-BA0.4 ~ 0.8mg/L+NAA0.05 ~ 0.15mg/L;
Culture of rootage: the indefinite bud of the 2-3cm that will newly grow is forwarded in the root media, make grow 2-3cm root; Said root media is 1/2 improvement MS+IBA0.4 ~ 0.8mg/L+NAA0.1 ~ 0.3mg/L;
Wherein modified MS medium is with NH in the MS medium
4NO
3Content is adjusted into 340 ~ 660 mg/L, KNO
3Content is adjusted into 1318 ~ 1722 mg/L, carragheen 6.7 g/L, sucrose 30g/L, pH value 5.8-6.0; 1/2 improvement MS is about to improve that macroelement reduces by half among the MS, and all the other constituent contents are constant.
Transplant: the seedling of will taking root is transplanted earlier to booth and is refined seedling, transplants the field more after treatment.Be specially the refining seedling of will taking root behind the seedling and take out, clean the root medium, 0.1% mancozeb immersion treatment; Dry in the shade; After treating that moisture on the plant limb dries in the shade, transplant in advance in the matrix of sterilizing with 0.1% potassium permanganate, matrix wherein is preferably peat soil: the peat composed of rotten mosses: perlite is 9:6:1.
The present invention provides a kind of concrete Pterospermi Heterophylli method for culturing in-vitro tissues, for:
Draw materials: continuous sunny is after one week, draw materials in noon 12 ~ 2, and be explant with good maternal plant current-year branch;
The foundation of sterilizable material: explant is cleaned with liquid detergent and immersion 10min; 30min is washed with flowing water in the back; Place on the operating desk; Be cut into the stem section of 1-2 internode of band; 75% Ethanol Treatment, 25 s, 0.1% mercuric chloride immersion treatment, 10 min, during constantly the concussion; Sterile water wash 6 times obtains sterilizable material;
The bud inducing culture: sterilizable material is inoculated on the bud inducing culture, light application time 8 ~ 12 h, intensity of illumination 3000 ~ 5000lx, 25 ~ 27 ℃ of temperature after 14 days, can induce the indefinite bud of 2 ~ 3cm;
Enrichment culture: the indefinite bud that induces is forwarded in the bud proliferated culture medium, and the place that visible indefinite bud contacts with medium after about 7 days emits the bud point that makes new advances, and bud proliferated culture medium wherein is improvement MS+6-BA0.4 ~ 0.8mg/L+NAA0.01 ~ 0.15mg/L;
Culture of rootage: the indefinite bud of the 2-3cm that will newly grow is forwarded in the root media, makes the root that grows 2-3cm; The indefinite bud that 2 ~ 3cm is long is forwarded in the root media, and the terminal incision of visible its stem section white root original hase of emerging behind the 5d grows up to the root of 2 ~ 3cm after 14 days, and root media wherein is 1/2 improvement MS+IBA0.4 ~ 0.8mg/L+NAA0.1 ~ 0.3mg/L,
Wherein modified MS medium is with NH in the MS medium
4NO
3Content is adjusted into 340 ~ 660 mg/L, KNO
3Content is adjusted into 1318 ~ 1722 mg/L, carragheen 6.7 g/L, sucrose 30g/L, pH value 5.8-6.0; 1/2 improvement MS is about to improve that macroelement reduces by half among the MS, and all the other constituent contents are constant.
Transplant: the seedling of will taking root is transplanted earlier to booth and is refined seedling; After 7 days it is carefully taken out from bottle; Clean the root medium; With 0.1% mancozeb immersion treatment, 2 ~ 3 min; Dry in the shade, treat that moisture on the plant limb dries in the shade similar the time, transplant in advance in the matrix of sterilizing with 0.1% potassium permanganate; Carry out the field temperature and humidity management after the transplanting, matrix wherein is preferably peat soil: the peat composed of rotten mosses: perlite is 9:6:1.
The technology path that present technique is complete is: with the stem section is explant; Directly induce indefinite clump bud (avoiding breaking up the situation that the clump bud is prone to variation with callus in the prior art); Propagation, culture of rootage through indefinite clump bud again; And the domestication of tissue cultivating seedling transplanting, thereby set up the complete fast traditional font of Pterospermi Heterophylli be.
Disclosed by the invention is the fast traditional font of a kind of new Pterospermi Heterophylli system, accomplishes whole group of flow process of cultivating seedling of Pterospermi Heterophylli first, sets up its fast traditional font and is.
Inductivity of the present invention is the inductivity with stem section evoking adventive bud, reaches 70%, and the propagation through indefinite clump bud, and its growth coefficient is up to 5.62.Rooting rate of the present invention and survival rate are all higher, have reached 98.7% and 94.2% respectively.Utilize the method can cultivate a large amount of Pterospermi Heterophylli tissue cultivating seedling simply, efficiently; Thereby effectively protect and breed this species; Satisfy the demand of market, remove the Critical Condition of Pterospermi Heterophylli simultaneously, lay a good foundation for further protecting and utilize these species from now on to Pterospermi Heterophylli.
Description of drawings
Fig. 1 and Fig. 2 are the figure as a result that induces of Pterospermi Heterophylli indefinite bud;
Fig. 3 and Fig. 4 are the propagation of Pterospermi Heterophylli indefinite bud figure as a result;
Fig. 5 and Fig. 6 are the figure as a result of taking root of Pterospermi Heterophylli indefinite bud;
Fig. 7 and Fig. 8 are the take root transplanting figure of seedling of Pterospermi Heterophylli.
Embodiment
To combine embodiment that embodiment of the present invention are described in detail below, but it will be understood to those of skill in the art that the following example only is used to illustrate the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person among the embodiment; According to the described technology of the document in this area or condition (for example with reference to works such as J. Sa nurse Brookers; " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be through the conventional products of commercial acquisition.
Embodiment: the tissue culture of Pterospermi Heterophylli
1, Pterospermi Heterophylli draws materials to induce and sprouts
Continuous sunny is after week; Draw materials in noon 12 ~ 14; The current-year branch of the plant that gets up with the good maternal plant seminal propagation in Longshan, sky, Quannan County, Jiangxi is an explant; Clean and immersion 10min with liquid detergent, 30min is washed with flowing water in the back, places on the operating desk; Be cut into the stem section of 1-2 internode of band; 75% Ethanol Treatment, 25 s, 0.1% mercuric chloride immersion treatment, 10 min, during constantly the concussion; Sterile water wash 6 times; Be inoculated on the bud inducing culture light application time 8 ~ 12 h, intensity of illumination 3000 ~ 5000lx; 25 ~ 27 ℃ of temperature; Behind the 14d, can induce the axillalry bud of 2 ~ 3cm, see and attach Fig. 1 and 2.Inductivity is 70%;
Bud inducing culture based component is: improvement MS (prescription is seen table 1)+6-BA1.5 ~ 2.5mg/L+NAA0.2 ~ 0.4mg/L;
2, the bud of Pterospermi Heterophylli tissue culture is bred and is taken root
The indefinite clump bud that induces is forwarded in the bud proliferated culture medium, and the place that visible indefinite bud contacts with medium behind about 7d emits the bud point that makes new advances, and its growth coefficient of statistics is 5.62% after 4 weeks, and leaf color is normal, and bottle seedling upgrowth situation is good, sees accompanying drawing 3 and 4.The indefinite bud that 2 ~ 3cm is long is forwarded in the root media light application time 8 ~ 12h, intensity of illumination 3000 ~ 5000 lx, 25 ~ 27 ℃ of temperature.The terminal incision of visible its stem section white root original hase of emerging behind the 5d grows up to the root of 2 ~ 3cm behind the 14d, see accompanying drawing 5 and 6.Rooting rate 98.7%;
Bud enrichment culture based component is improvement MS+6-BA0.4 ~ 0.8mg/L+NAA0.05 ~ 0.15mg/L)
The culture of rootage based component is 1/2 improvement MS (prescription is seen table 2)+IBA0.4 ~ 0.8mg/L+NAA0.1 ~ 0.3mg/L)
3, the transplanting in the Pterospermi Heterophylli method for tissue culture is to the land for growing field crops
Bottle seedling of will taking root goes to the transplanting booth and refines seedling, sees accompanying drawing 7 and 8.Behind the 7d it is carefully taken out from bottle; Clean the root medium; With 0.1% mancozeb immersion treatment, 2 ~ 3 min; Dry in the shade; Treat that moisture on the plant limb dries in the shade similar the time; Transplant in advance in the matrix of sterilizing with 0.1% potassium permanganate (peat soil: the peat composed of rotten mosses: perlite is 9:6:1), carry out the field temperature and humidity management after the transplanting, survival rate can reach 94.2%.
Pterospermi Heterophylli among the present invention is taken from the good maternal plant seed in Longshan, sky, Quannan County, Jiangxi, is not limited thereto, and the Pterospermi Heterophylli of other local source also can reach this goal of the invention;
Table 1 improvement MS
| Nomenclature of drug | Consumption (mg/L) |
| NH 4NO 3 | 340-660 |
| KNO 3 | 1318-1722 |
| MgSO 4·7H 2O | 370 |
| KH 2PO 4 | 170 |
| CaCl 2·2H 2O | 440 |
| MnSO 4·4H 2O | 22.3 |
| ZnSO 4·7H 2O | 8.6 |
| H 3BO 3 | 6.2 |
| KI | 0.83 |
| Na 2MoO 4·2H 2O | 0.25 |
| CuSO 4·5H 2O | 0.025 |
| CoCl 2·6H 2O | 0.025 |
| FeSO 4·7H 2O | 27.8 |
| Na 2-EDTA·2H 2O | 37.3 |
| Glycine | 2.0 |
| Thiamine hydrochloride (vitamin B1) | 0.1 |
| Puridoxine hydrochloride (vitamin B6) | 0.5 |
| IV B nicotinic acid | 0.5 |
| Inositol | 100 |
Sealing the back sterilizes according to conventional method;
Table 2 1/2 improvement MS
| Medicine | Consumption (mg/L) |
| NH 4NO 3 | 170-330 |
| KNO 3 | 659-861 |
| MgSO 4·7H 2O | 185 |
| KH 2PO 4 | 85 |
| CaCl 2·2H 2O | 220 |
| MnSO 4·4H 2O | 22.3 |
| ZnSO 4·7H 2O | 8.6 |
| H 3BO 3 | 6.2 |
| KI | 0.83 |
| Na 2MoO 4·2H 2O | 0.25 |
| CuSO 4·5H 2O | 0.025 |
| CoCl 2·6H 2O | 0.025 |
| FeSO 4·7H 2O | 27.8 |
| Na 2-EDTA·2H 2O | 37.3 |
| Glycine | 2.0 |
| Thiamine hydrochloride (vitamin B1) | 0.1 |
| Puridoxine hydrochloride (vitamin B6) | 0.5 |
| IV B nicotinic acid | 0.5 |
| Inositol | 100 |
Sealing the back sterilizes according to conventional method.
Claims (5)
1. a Pterospermi Heterophylli method for culturing in-vitro tissues is characterized in that, comprises the steps:
Draw materials: the current-year branch of getting Pterospermi Heterophylli is an explant;
The foundation of sterilizable material: the explant disinfection is obtained sterilizable material;
The bud inducing culture: sterilizable material is inoculated on the bud inducing culture, and inducing culture goes out indefinite bud under the bud inducing culture condition;
Enrichment culture: the indefinite bud that inducing culture is gone out is forwarded to the indefinite bud that cultivation makes new advances in the bud proliferated culture medium;
Culture of rootage: the indefinite bud of the 2-3cm that will newly grow is forwarded in the root media, make grow 2-3cm root;
Transplant: the seedling of will taking root is transplanted earlier to booth and is refined seedling, transplants the field more after treatment.
2. the described Pterospermi Heterophylli method for culturing in-vitro tissues of claim 1 is characterized in that,
Said draw materials into, it is explant that the suitable time is got good maternal plant current-year branch;
Being established as of said sterilizable material: explant is cleaned with liquid detergent and soak, with the flowing water flushing, place on the operating desk, be cut into the stem section of 1-2 internode of band, 75% Ethanol Treatment is used 0.1% mercuric chloride immersion treatment again, during constantly shake sterile water wash;
Said bud inducing culture is improvement MS+6-BA1.5 ~ 2.5mg/L+NAA0.2 ~ 0.4mg/L, and said bud inducing culture condition is light application time 8 ~ 12h, intensity of illumination 3000 ~ 5000 lx, 25 ~ 27 ℃ of temperature;
Said bud proliferated culture medium is improvement MS+6-BA0.4 ~ 0.8mg/L+NAA0.05 ~ 0.15mg/L;
Said root media is 1/2 improvement MS+IBA0.4 ~ 0.8mg/L+NAA0.1 ~ 0.3mg/L;
In the said transplanting, the refining seedling of will taking root behind the seedling takes out, and cleans the root medium, and 0.1% mancozeb immersion treatment is dried in the shade, treat that moisture on the plant limb dries in the shade after, transplant in advance in the matrix of sterilizing with 0.1% potassium permanganate.
3. the described Pterospermi Heterophylli method for culturing in-vitro tissues of claim 2 is characterized in that, the said suitable time is preferably continuous sunny after one week, draws materials in noon 12 ~ 14;
Said matrix is peat soil: the peat composed of rotten mosses: perlite is 9:6:1.
4. a Pterospermi Heterophylli method for culturing in-vitro tissues is characterized in that, comprises the steps:
Draw materials: continuous sunny is after one week, draw materials in noon 12 ~ 14, and be explant with good maternal plant current-year branch;
The foundation of sterilizable material: explant is cleaned with liquid detergent and immersion 10min; 30min is washed with flowing water in the back; Place on the operating desk; Be cut into the stem section of 1-2 internode of band; 75% Ethanol Treatment, 25 s, 0.1% mercuric chloride immersion treatment, 10 min, during constantly the concussion; Sterile water wash 6 times obtains sterilizable material;
The bud inducing culture: sterilizable material is inoculated on the bud inducing culture, light application time 8 ~ 12 h, intensity of illumination 3000 ~ 5000 lx, 25 ~ 27 ℃ of temperature after 14 days, can induce the indefinite bud of 2 ~ 3cm;
Enrichment culture: the indefinite bud that induces is forwarded in the bud proliferated culture medium, and the place that visible indefinite bud contacts with medium after about 7 days emits the bud point that makes new advances, and bud proliferated culture medium wherein is improvement MS+6-BA0.4 ~ 0.8mg/L+NAA0.05 ~ 0.15mg/L;
Culture of rootage: the indefinite bud of the 2-3cm that will newly grow is forwarded in the root media, makes the root that grows 2-3cm; The indefinite bud that 2 ~ 3cm is long is forwarded in the root media, and the terminal incision of visible its stem section white root original hase of emerging behind the 5d grows up to the root of 2 ~ 3cm after 14 days, and root media wherein is 1/2 improvement MS+IBA0.4 ~ 0.8mg/L+NAA0.1 ~ 0.3mg/L;
Transplant: the seedling of will taking root is transplanted earlier to booth and is refined seedling; After 7 days it is carefully taken out from bottle; Clean the root medium; With 0.1% mancozeb immersion treatment, 2 ~ 3 min; Dry in the shade; Treat that moisture on the plant limb dries in the shade similar the time, transplant in advance in the matrix of sterilizing with 0.1% potassium permanganate, carry out the field temperature and humidity management after the transplanting.
5. the described Pterospermi Heterophylli method for culturing in-vitro tissues of claim 4 is characterized in that,
Said matrix is peat soil: the peat composed of rotten mosses: perlite is 9:6:1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726295A (en) * | 2012-06-30 | 2012-10-17 | 江苏汤氏园林有限公司 | Tissue culture method for acer truncatum |
| CN104206186A (en) * | 2014-08-25 | 2014-12-17 | 从江神瑶保健品有限公司 | Method for grafting semiliquidambar cathayensis |
| CN106171986A (en) * | 2016-07-12 | 2016-12-07 | 玉林师范学院 | A kind of tissue culture and rapid propagation method of Pterospermi Heterophylli seed germination approach |
| CN107494264A (en) * | 2017-08-31 | 2017-12-22 | 地缘（厦门）生物科技有限公司 | A kind of method of Pterospermi Heterophylli tissue culture outside sprout-cultivating-bottle radication |
-
2011
- 2011-07-27 CN CN 201110211490 patent/CN102301958B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| 《南京林业大学研究生硕士学位论文》 20100215 李亮 枫香、枫香"变异体"、细柄阿丁枫、半枫荷的组培研究 正文第35-39页 1 , * |
| 李亮: "枫香、枫香"变异体"、细柄阿丁枫、半枫荷的组培研究", 《南京林业大学研究生硕士学位论文》 * |
| 陈世红等: "半枫荷组织培养研究", 《中药材》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726295A (en) * | 2012-06-30 | 2012-10-17 | 江苏汤氏园林有限公司 | Tissue culture method for acer truncatum |
| CN104206186A (en) * | 2014-08-25 | 2014-12-17 | 从江神瑶保健品有限公司 | Method for grafting semiliquidambar cathayensis |
| CN106171986A (en) * | 2016-07-12 | 2016-12-07 | 玉林师范学院 | A kind of tissue culture and rapid propagation method of Pterospermi Heterophylli seed germination approach |
| CN106171986B (en) * | 2016-07-12 | 2018-03-30 | 玉林师范学院 | A kind of Pterospermi Heterophylli seed sprouts the tissue culture and rapid propagation method of approach |
| CN107494264A (en) * | 2017-08-31 | 2017-12-22 | 地缘（厦门）生物科技有限公司 | A kind of method of Pterospermi Heterophylli tissue culture outside sprout-cultivating-bottle radication |
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