CN102301958B - 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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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 Traits between Liquidambar Liguidambar and Altingia Altingia two genus, and systematic growth has scientific value to the research Hamamelidaceae.Wood quality is good, revolves plane property good, 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 for the treatment of the diseases such as rheumatic arthritis, chronic lumbocrural pain, hemiplegia, traumatic injury, and is evident in efficacy.Seriously reach the natural propagation difficulty because natural stands is subjected to artificial disturbance, distribution is more and more narrow, causes wild resource very rare.
The tissue of plant is cultivated and is referred to isolate from plant corpus tissue, organ or the cell that suits the requirements, protoplast etc., by sterile working, cultivate the technology of other products that the whole plant that obtains to regenerate or production has economic worth under the manual control condition.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 carrying out technology on the aspects such as plant germplasm resource protection and production economy nursery stock to be gradually improved.Prior art is all take the Pterospermi Heterophylli blade as the explant induction callus, breeds by callus, then the callus induction differentiation and bud formation, and do not induce adventive root.
Summary of the invention
The technical problem to be solved in the present invention 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 method for culturing in vitro tissues of Semiliquidambar cathayensis, and its concrete steps are:
Draw materials: the current-year branch of getting Pterospermi Heterophylli is explant; Getting good maternal plant current-year branch in the suitable time is explant; The described 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 explant cleaned with liquid detergent and soaks, then rinse with flowing water, be placed on operating desk, be cut into the stem section with 1-2 internode, 75% Ethanol Treatment, then use 0.1% mercuric chloride immersion treatment, during constantly shake, sterile water wash;
Bud is induced cultivation: sterilizable material is inoculated on the bud inducing culture, and bud is induced to induce under condition of culture and is turned out indefinite bud; Described bud inducing culture is improvement MS+6-BA1.5 ~ 2.5mg/L+NAA0.2 ~ 0.4mg/L, and it is light application time 8 ~ 12 h that described bud is induced condition of culture, intensity of illumination 3000 ~ 5000lx, 25 ~ 27 ℃ of temperature;
Propagation is cultivated: will induce the indefinite bud of turning out to be forwarded to the indefinite bud that in the bud proliferated culture medium, cultivation makes new advances; Described 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 root media, make grow 2-3cm root; Described 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 in MS, macroelement reduces by half, and all the other constituent contents are constant.
Transplant: the seedling of taking root is first transplanted to booth and is carried out hardening, then is transplanted into after treatment the field.Be specially the seedling of to take root after hardening and take out, clean the root medium, 0.1% mancozeb immersion treatment, dry in the shade, after moisture on plant to be planted limb dries in the shade, be transplanted in prior matrix with 0.1% potassium permanganate sterilization, matrix wherein is preferably peat soil: the peat composed of rotten mosses: perlite is 9:6:1.
The invention provides a kind of concrete method for culturing in vitro tissues of Semiliquidambar cathayensis, for:
Draw materials: continuous sunny is after one week, draws materials in noon 12 ~ 2, take good maternal plant current-year branch as explant;
The foundation of sterilizable material: explant is cleaned with liquid detergent and soak 10min, rinse 30min with flowing water afterwards, be placed on operating desk, be cut into the stem section with 1-2 internode, 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;
Bud is induced cultivation: sterilizable material is inoculated on the bud inducing culture, and 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;
Propagation is cultivated: the indefinite bud that induces is forwarded in the bud proliferated culture medium, approximately 7 days afterwards the place that contact with medium of visible indefinite bud emit the bud point that makes new advances, bud proliferated culture medium wherein is to improve 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 root media, makes the root that grows 2-3cm; The indefinite bud that 2 ~ 3cm is long is forwarded in root media, and visible its stem section end incision former base of white root of emerging after 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 in MS, macroelement reduces by half, and all the other constituent contents are constant.
Transplant: the seedling of taking root is first transplanted to booth and is carried out hardening, 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, the moisture on plant to be planted limb dries in the shade when similar, is transplanted in advance in the matrix of sterilizing with 0.1% potassium permanganate, carry out the field temperature and humidity management after 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: take the stem section as explant, directly induce indefinite clump bud (avoiding being prone to callus differentiation clump bud in prior art the situation of variation), propagation, culture of rootage by indefinite clump bud again, and the rooting culture of group training seedling, thereby set up Pterospermi Heterophylli complete fast traditional font system.
Disclosed by the invention is a kind of new Pterospermi Heterophylli fast traditional font system, completes first the flow process of the whole tissue culture of Pterospermi Heterophylli, sets up its fast traditional font system.
Inductivity of the present invention is with the inductivity of stem section evoking adventive bud, reaches 70%, and the propagation by indefinite clump of bud, and its growth coefficient is up to 5.62.Rooting rate of the present invention and survival rate are all higher, have reached respectively 98.7% and 94.2%.Utilize the method can cultivate simply, efficiently a large amount of Pterospermi Heterophylli group training seedlings; thereby effectively protect and breed this species; the demand of satisfying the market to Pterospermi Heterophylli removed the Critical Condition of Pterospermi Heterophylli simultaneously, lays a good foundation for further protecting from now on and utilize these species.
Description of drawings
Fig. 1 and Fig. 2 are the indefinite spore induction of Pterospermi Heterophylli figure as a result;
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
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition (such as works such as reference J. Pehanorm 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 by the conventional products of commercial acquisition.
Embodiment: the tissue of Pterospermi Heterophylli is cultivated
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 take the good maternal plant seminal propagation in Longshan, sky, Quannan County, Jiangxi is as explant, clean and soak 10min with liquid detergent, rinse 30min with flowing water afterwards, be placed on operating desk, be cut into the stem section with 1-2 internode, 75% Ethanol Treatment 25 s, 0.1% mercuric chloride immersion treatment 10 min, constantly concussion during this time, 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, after 14d, can induce the axillalry bud of 2 ~ 3cm, see attached Fig. 1 and 2.Inductivity is 70%;
Bud inducing culture composition is: improvement MS(formula sees Table 1)+6-BA1.5 ~ 2.5mg/L+NAA0.2 ~ 0.4mg/L;
2, the bud of Pterospermi Heterophylli tissue cultivation is bred and is taken root
The indefinite clump bud that induces is forwarded in the bud proliferated culture medium, and the place that approximately after 7d, visible indefinite bud contacts with medium emits the bud point that makes new advances, and after 4 weeks, its growth coefficient of statistics is 5.62%, 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 root media, light application time 8 ~ 12h, intensity of illumination 3000 ~ 5000 lx, 25 ~ 27 ℃ of temperature.Visible its stem section end incision former base of white root of emerging after 5d grows up to the root of 2 ~ 3cm after 14d, see accompanying drawing 5 and 6.Rooting rate 98.7%;
Bud proliferated culture medium composition is improvement MS+6-BA0.4 ~ 0.8mg/L+NAA0.05 ~ 0.15mg/L)
The culture of rootage based component is that 1/2 improvement MS(formula sees 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 taking root goes to the transplanting booth and carries out hardening, sees accompanying drawing 7 and 8.After 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, moisture on plant to be planted limb dries in the shade when similar, be transplanted 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 transplanting, survival rate can reach 94.2%.
Pterospermi Heterophylli in the present invention is taken from the good maternal plant seed in Longshan, sky, Quannan County, Jiangxi, is not limited to this, 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 |
Sterilize according to conventional method after sealing;
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 |
Sterilize according to conventional method after sealing.
Claims (4)
1. a method for culturing in vitro tissues of Semiliquidambar cathayensis, is characterized in that, comprises the steps:
Draw materials: the current-year branch of getting Pterospermi Heterophylli is explant;
The foundation of sterilizable material: the explant disinfection is obtained sterilizable material;
Bud is induced cultivation: sterilizable material is inoculated on the bud inducing culture, and bud is induced to induce under condition of culture and is turned out indefinite bud;
Propagation is cultivated: will induce the indefinite bud of turning out to be forwarded to the indefinite bud that in the bud proliferated culture medium, cultivation makes new advances;
Culture of rootage: the indefinite bud of the 2-3cm that will newly grow is forwarded in root media, makes the root that grows 2-3cm;
Transplant: the seedling of taking root is first transplanted to booth and is carried out hardening, then is transplanted into after treatment the field;
Described draw materials into, it is explant that the suitable time is got good maternal plant current-year branch; The described suitable time is that fine day is after one week, in noon 12 ~ 14 points;
Being established as of described sterilizable material: explant cleaned with liquid detergent and soak, then rinse with flowing water, being placed on operating desk, being cut into the stem section with 1-2 internode, 75% Ethanol Treatment, then use 0.1% mercuric chloride immersion treatment, during constantly shake, sterile water wash;
Described bud inducing culture is improvement MS+6-BA1.5 ~ 2.5mg/L+NAA0.2 ~ 0.4mg/L, and it is light application time 8 ~ 12 h that described bud is induced condition of culture, intensity of illumination 3000 ~ 5000 lx, 25 ~ 27 ℃ of temperature;
Described bud proliferated culture medium is improvement MS+6-BA0.4 ~ 0.8mg/L+NAA0.01 ~ 0.15mg/L;
Described root media is 1/2 improvement MS+IBA0.4 ~ 0.8mg/L+NAA0.1 ~ 0.3mg/L;
Described modified MS medium is with NH in MS
4NO
3Content is adjusted into 340 ~ 660 mg/L, KNO
3Content is adjusted into 1318 ~ 1722 mg/L, and medium adds carragheen 6.7 g/L, sucrose 30g/L, pH value 5.8-6.0;
In MS, macroelement reduces by half described 1/2 improvement MS in order improveing, and all the other constituent contents are constant;
In described transplanting, the seedling of taking root after hardening takes out, and cleans the root medium, and 0.1% mancozeb immersion treatment is dried in the shade, and after the moisture on plant to be planted limb dries in the shade, is transplanted in prior matrix with 0.1% potassium permanganate sterilization.
2. method for culturing in vitro tissues of Semiliquidambar cathayensis claimed in claim 1, is characterized in that, described matrix is peat soil: the peat composed of rotten mosses: perlite is 9:6:1.
3. a method for culturing in vitro tissues of Semiliquidambar cathayensis, is characterized in that, comprises the steps:
Draw materials: continuous sunny is after one week, draws materials in noon 12 ~ 14, take good maternal plant current-year branch as explant;
The foundation of sterilizable material: explant is cleaned with liquid detergent and soak 10min, rinse 30min with flowing water afterwards, be placed on operating desk, be cut into the stem section with 1-2 internode, 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;
Bud is induced cultivation: sterilizable material is inoculated on the bud inducing culture, and 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; Described bud inducing culture is improvement MS+6-BA1.5 ~ 2.5mg/L+NAA0.2 ~ 0.4mg/L;
Propagation is cultivated: the indefinite bud that induces is forwarded in the bud proliferated culture medium, approximately 7 days afterwards the place that contact with medium of visible indefinite bud emit the bud point that makes new advances, wherein the bud proliferated culture medium is to improve MS+6-BA0.4 ~ 0.8mg/L+NAA0.01 ~ 0.15mg/L;
Described modified MS medium is with NH in MS
4NO
3Content is adjusted into 340 ~ 660mg/L, KNO
3Content is adjusted into 1318 ~ 1722 mg/L, and medium adds carragheen 6.7 g/L, sucrose 30g/L, pH value 5.8-6.0;
Culture of rootage: the indefinite bud of the 2-3cm that will newly grow is forwarded in root media, makes the root that grows 2-3cm; The indefinite bud that 2 ~ 3cm is long is forwarded in root media, visible its stem section end incision former base of white root of emerging after 5d, the root that grows up to 2 ~ 3cm after 14 days, wherein root media is 1/2 improvement MS+IBA0.4-0.8mg/L+NAA0.1 ~ 0.3mg/L, in MS, macroelement reduces by half described 1/2 improvement MS in order improveing, and all the other constituent contents are constant;
Transplant: the seedling of taking root is first transplanted to booth and is carried out hardening, 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, when the moisture on plant to be planted limb dries in the shade almost, be transplanted in prior matrix with 0.1% potassium permanganate sterilization, carry out the field temperature and humidity management after transplanting.
4. method for culturing in vitro tissues of Semiliquidambar cathayensis claimed in claim 3, is characterized in that, described matrix is peat soil: the peat composed of rotten mosses: perlite is 9:6:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110211490 CN102301958B (en) | 2011-07-27 | 2011-07-27 | Method for culturing in vitro tissues of Semiliquidambar cathayensis |
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| CN 201110211490 CN102301958B (en) | 2011-07-27 | 2011-07-27 | Method for culturing in vitro tissues of Semiliquidambar cathayensis |
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| CN102726295A (en) * | 2012-06-30 | 2012-10-17 | 江苏汤氏园林有限公司 | Tissue culture method for acer truncatum |
| CN104206186B (en) * | 2014-08-25 | 2016-05-04 | 从江神瑶保健品有限公司 | A kind of engrafting method of Sassafras tsumu Hemsl |
| 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 |
| CN107494264B (en) * | 2017-08-31 | 2020-01-14 | 地缘（厦门）生物科技有限公司 | Method for rooting semiliquidambar cathayensis tissue culture seedling outside bottle |
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