CN104604688A - In-vitro culture method capable of promoting efficient propagation of dendrobium officinale by nano materials having antibacterial effect - Google Patents
In-vitro culture method capable of promoting efficient propagation of dendrobium officinale by nano materials having antibacterial effect Download PDFInfo
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- CN104604688A CN104604688A CN201510061294.3A CN201510061294A CN104604688A CN 104604688 A CN104604688 A CN 104604688A CN 201510061294 A CN201510061294 A CN 201510061294A CN 104604688 A CN104604688 A CN 104604688A
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Abstract
The invention discloses an in-vitro culture method capable of promoting efficient propagation of dendrobium officinale by nano materials having an antibacterial effect. The method comprises the following steps that nano materials promote wild dendrobium officinale protocorm to be induced and propagated, and the antibacterial effect of the nano materials in in-vitro culture is researched. According to the invention, the problems of slow propagation, inconsistent growth vigor and pollution easiness in in-vitro culture and the like for the growth of the wild dendrobium officinale protocorm are overcome; the used nano materials on living plants can induce photobiology effect, such as the improvement of the photosynthetic function, and the growth and development of plants, and have high capability of eliminating bacteria, fungi, viruses and cancer cells. The technical process is easy to operate, few in flow, and high in yield; the obtained dendrobium officinale protocorm is high in quality, rapid in propagation speed, and is consistent in growth vigor; moreover, the nano materials can promote the growth of plants and eliminate microorganism pollution in plant tissue culture and the like, and provide technical support for the practical and industrial development for dendrobium officinale tissue culture seedlings.
Description
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, be specifically related to a kind of nano material and promote that dendrobium candidum is efficiently bred and the in-vitro culture method of tool antibacterial action.
Background technology
Dendrobium candidum (Dendrobium officinal Kimura et Migo) is grown nonparasitically upon another plant for the orchid family Dendrobium is perennial type herbaceous plant, because its eustipes part is pitchy, and also name ribbed hedyotis herb.Dendrobium candidum is the traditional rare Chinese herbal medicine of China, and " Chinese Pharmacopoeia " includes 3 kinds of former plants of the stem of noble dendrobium, and wherein dendrobium candidum is most precious kind, has nourishing the stomach to improve the production of body fluid, nourishing Yin and clearing heat, the effect such as moisten the lung and relieve the cough.Modern pharmacological research shows, dendrobium candidum contains the compositions such as polysaccharide, alkaloid, amino acid, have antitumor, anti-ageing, strengthen the effect such as immunity of organisms, hemangiectasis and anti-platelet aggregation, be therefore widely used in clinical and Chinese medicine compound prescription.Dendrobium candidum is mainly distributed in the ground such as Yunnan, Guizhou, Guangdong, Guangxi, Zhejiang, Sichuan in China, the orchid family epiphyte that it is a kind of poor growth, self-reproduction ability is extremely low, add that people excessively excavate the continuous destruction of natural crude drugs and overall ecotope in recent years, dendrobium officinale resource is close to exhausted.From 20 century 70s, scientific research personnel just starts to be studied dendrobium candidum artificial cultivation technique, fundamentally solves resource problem, and therefore development dendrobium candidum stock breeding and artificial cultivation become the road of the certainty of conservation and utilization dendrobium candidum resource.
Nano material and the nano particle whole world that is applied in recent years emerges rapidly, and becomes a most active modern scientific research.The kind of nano material is a lot, respectively has different function and application scopes, wherein nano titanium oxide (TiO
2) apply comparatively extensive in life science and agriculture field.A few studies is had to point out nano-TiO at present
2photosemiconductor material can bring out photobiology effect on live plant, as improved photosynthetic function, Promoting plant growth growth etc.In addition, nano-TiO
2be a new non-toxic material, it shows the ability of very strong elimination bacterium, fungi, virus and cancer cell.In view of nano-TiO
2the growth of plant can be promoted and to eliminate in Plant Tissue Breeding the advantages such as microbial contamination, be expected to for candidum tissue culturing seedling is practical and industrialized development provides technical support.
Summary of the invention
Primary and foremost purpose of the present invention is to provide a kind of nano material to promote dendrobium candidum efficiently to breed and the in-vitro culture method of tool antibacterial action, the method is cultivated from dendrobium officinale aseptic seed, obtain the dendrobium candidum protocorm of genetic stability, acellular variation occurs in this process; One of feature of the method adds nano material in the medium.
Another object of the present invention is to provide the dendrobium candidum protocorm polluted obtained by said method, reduce through nanomaterial solution cleaning after stain situation; Above-mentioned nanomaterial solution has the effect of removing medium microorganism.
Object of the present invention is achieved through the following technical solutions:
In this article, after protocorm (Protocorm) refers to seed germination after the expanding of embryo, seed coat break, naked eyes are light yellow in spherical proembryo as seen, its be bead, cripetura, the organ of cells,primordial, similar young stem, can breed and form protocorm clump.
Nano material promotes that dendrobium candidum is efficiently bred and the in-vitro culture method of tool antibacterial action, comprises the following steps:
(1) dendrobium officinale protocorm is induced
Get dendrobium officinale capsule, after surface cleaning, on superclean bench, the capsule scalpel of sterilizing half-and-half is cut, shakes gently with one end of tweezers clamping capsule, seed is dispersed evenly in protocorm induction medium, cover bottle cap, be placed in culturing room and cultivate.
Containing 1 ~ 10g/L nano-TiO in described inducing culture
2;
The preferred N6 medium of described inducing culture, MS medium, 1/2MS medium, B5 medium, LS medium or KM medium;
During tissue cultures, observe the situation of being induced protocorm by Seeds of Dendrobium Candidum.
(2) Multiplying culture of dendrobium candidum protocorm
The dendrobium candidum protocorm of above-mentioned induction is moved in proliferated culture medium in superclean bench, covers bottle cap, be placed in culturing room and cultivate;
Containing 1 ~ 10g/L nano-TiO in medium used
2, preferred N6 medium, MS medium, 1/2MS medium, B5 medium, LS medium or KM medium;
(3) antibacterial action of nano material
The dendrobium candidum protocorm by microbial contamination of above-mentioned acquisition is taken out from tissue culture bottle, inserts nano-TiO
2soaking and washing in solution, be inoculated in new medium and cultivate, result shows nano-TiO
2solution has the effect eliminating microorganism.
Described use nano-TiO
21 ~ 10g/L.
The present invention has following advantage and effect relative to prior art:
1, the present invention can not induced environment problem to the inducation and proliferation cultural method of dendrobium candidum protocorm, and Absorbable organic halogens provides the utility deriving from dendrobium candidum (as antioxidant, cancer-resisting substance and functional health product additive etc.).
2, the quality that instant invention overcomes the growth of dendrobium officinale protocorm is low, propagation is slow, growing way is inconsistent and the problem of cultured in vitro vulnerable to pollution, thus improves culture efficiency and reduce production cost.The present invention possesses skills the advantages such as technique is easy to operate, flow process is few, quantum of output is large.
Accompanying drawing explanation
Fig. 1 is dendrobium officinale capsule and drawing of seeds thereof.
Fig. 2 is the protocorm result of the test figure obtained through dendrobium officinale Seed inducement.
Fig. 3 is dendrobium candidum protocorm contaminated situation minimizing figure after nano material process.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
Nano material promotes the induction of dendrobium officinale protocorm
1.1 material
The material preservation that this experiment adopts, in the plant tissue culture room of medicinal plant teaching and research room of Traditional Chinese Medicine University Of Guangzhou, is accredited as the orchid family Dendrobium Sw dendrobium candidum Dendrobium officinaleKimura et Migo through Chinese traditional medicine identification teaching and research room of Traditional Chinese Medicine University Of Guangzhou professor Lai little Ping.
1.2 experimental technique
First with liquid detergent cleaning 3 ~ 5min, after tap water 5 ~ 8min, on dislocation superclean bench, with after 75% alcohol disinfecting with sterile water wash, then with disinfectant soak shake sterilize, sterile water wash, aseptic paper handkerchief blots rear for subsequent use.On superclean bench, the capsule scalpel of sterilizing half-and-half is cut, shake gently with one end of tweezers clamping capsule, seed is interspersed among on inducing culture equably, covers bottle cap, be placed in culturing room and cultivate.
Using MS as minimal medium, containing agar, sucrose in medium, add 1 ~ 10g/L nano-TiO
2, adjust pH for 5.8, at 121 DEG C of high-pressure sterilizing pot sterilizing 20min with 1.0mol/LNaOH or 0.1mol/L HCl.Condition of culture: temperature (25 ± 1) DEG C, illumination 12h/d, intensity of illumination is 1500 ~ 2000lx.
1.3 experimental results and conclusion
Dendrobium candidum protocorm induction situation the results are shown in Figure 1 ~ 2.Result shows, and cultivates seed after 1 week and develops into yellow green granule by yellow powder, induce the protocorm of green, smooth surface, roundlet shape after 4 weeks.Compare with control group, in medium, add nano-TiO
2material can improve inductivity and the growth conditions (P<0.05) of dendrobium candidum protocorm significantly.
Above experimental result shows, nano-TiO
2material can improve dendrobium candidum protocorm induction situation.
Embodiment 2
Nano material promotes dendrobium candidum Protocorm Multiplication
2.1 material
Get the dendrobium candidum protocorm obtained in " embodiment 1 ".
2.2 experimental technique
Select the protocorm that size is basically identical, be inoculated in Protocorm Multiplication medium.Using MS as minimal medium, containing agar, sucrose in medium, add 1 ~ 10g/L nano-TiO
2, adjust pH for 5.8, at 121 DEG C of high-pressure sterilizing pot sterilizing 20min with 1.0mol/LNaOH or 0.1mol/LHCl.Condition of culture: temperature (25 ± 1) DEG C, illumination 12h/d, intensity of illumination is 1500 ~ 2000lx.
2.3 experimental results and conclusion
Result shows, and compares, add nano-TiO in medium with control group
2material can improve proliferation times and growth size (P<0.05) of dendrobium candidum protocorm significantly.
Above experimental result shows, nano-TiO
2material can promote dendrobium candidum Protocorm Multiplication.
Embodiment 3
The antibacterial action test of nano material
2.1 material
Get the contaminated dendrobium candidum protocorm obtained in " embodiment 2 ".
2.2 experimental technique
Select the protocorm by microbial contamination that size is basically identical, adopt nano-TiO
2solution carries out soaking and cleans, and be then inoculated in new medium by the protocorm after cleaning, condition of culture: temperature (25 ± 1) DEG C, illumination 12h/d, intensity of illumination is 1500 ~ 2000lx.
2.3 experimental results and conclusion
Nano-TiO
2the antibacterial action of material the results are shown in Figure 3.Result shows, through nano-TiO
2after solution cleaning, the beginning microbiological contamination time by the dendrobium candidum protocorm of microbial contamination obviously postpones, and obviously reduces through 3 ~ 6 Secondary Culture after stain situations.
Above experimental result shows, nano-TiO
2the antibacterial action that material has in tissue cultures.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (8)
1. promote that dendrobium candidum is efficiently bred and the in-vitro culture method of tool antibacterial action by nano material, it is characterized in that comprising the following steps:
(1) nano material promotes the induction of dendrobium officinale protocorm
Get dendrobium candidum capsule, after surface cleaning, the capsule scalpel of sterilizing half-and-half cuts by superclean bench, shake gently with one end of tweezers clamping capsule, seed is dispersed evenly in protocorm induction medium;
(2) nano material promotes dendrobium candidum Protocorm Multiplication
The dendrobium candidum protocorm of above-mentioned induction is moved in proliferated culture medium in superclean bench, covers bottle cap, be placed in culturing room and cultivate;
Containing 1 ~ 10 g/L nano-TiO in medium used
2, preferred N6 medium, MS medium, 1/2MS medium, B5 medium, LS medium or KM medium;
(3) antibacterial action of nano material
The dendrobium candidum protocorm by microbial contamination of above-mentioned acquisition is taken out from tissue culture bottle, inserts nano-TiO
2soaking and washing in solution, be inoculated in new medium and cultivate, result shows nano-TiO
2solution has the effect eliminating microorganism.
2. a kind of nano material according to claim 1 promotes dendrobium candidum efficiently to breed and the in-vitro culture method of tool antibacterial action, it is characterized in that: containing 1 ~ 10 g/L nano-TiO in the inducing culture described in step (1)
2.
3. a kind of nano material according to claim 1 promotes dendrobium candidum efficiently to breed and the in-vitro culture method of tool antibacterial action, it is characterized in that: the inducing culture described in step (1) is N6 medium, MS medium, 1/2MS medium, B5 medium, LS medium or KM medium.
4. a kind of nano material according to claim 1 promotes dendrobium candidum efficiently to breed and the in-vitro culture method of tool antibacterial action, it is characterized in that: in step (2), containing 1 ~ 10 g/L nano-TiO in described inducing culture
2.
5. a kind of nano material according to claim 1 promotes dendrobium candidum efficiently to breed and the in-vitro culture method of tool antibacterial action, it is characterized in that: in step (2), described inducing culture is N6 medium, MS medium, 1/2MS medium, B5 medium, LS medium or KM medium.
6. a kind of nano material according to claim 1 promotes dendrobium candidum efficiently to breed and the in-vitro culture method of tool antibacterial action, it is characterized in that: in step (3), described use nano-TiO
21 ~ 10 g/L.
7. promote that dendrobium candidum is efficiently bred and the in-vitro culture method of tool antibacterial action by nano material, it is characterized in that: be obtained by the method described in any one of claim 1-6.
8. nano material tool antibacterial action according to claim 7, is characterised in that: its action mode is suspension.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110972935A (en) * | 2019-10-16 | 2020-04-10 | 中国农业科学院麻类研究所 | Application of iron-based nanoparticles in promoting growth of plant stem tips |
| CN112237140A (en) * | 2020-10-16 | 2021-01-19 | 云南省农业科学院花卉研究所 | Method for blocking dendrobium officinale tissue culture seedling from being polluted by bacteria |
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| CN101548644B (en) * | 2009-03-25 | 2012-04-04 | 刘星华 | Tissue culturing method for applying nanotechnology to promote fast proliferation of dendrobium officinale |
| CN103864538A (en) * | 2014-04-04 | 2014-06-18 | 广东国方医药科技有限公司 | Production method for moisturizing antibacterial dendrobium candidum culture medium containing nano-material |
| CN104067940A (en) * | 2014-06-30 | 2014-10-01 | 广州花都先锋园艺有限公司 | Hormone-free tissue culture method for dendrobium officinale |
| CN104082135A (en) * | 2014-06-11 | 2014-10-08 | 陕西恒富源林业有限公司 | Method for rapidly breeding dendrobium officinale seedlings |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548644B (en) * | 2009-03-25 | 2012-04-04 | 刘星华 | Tissue culturing method for applying nanotechnology to promote fast proliferation of dendrobium officinale |
| CN103864538A (en) * | 2014-04-04 | 2014-06-18 | 广东国方医药科技有限公司 | Production method for moisturizing antibacterial dendrobium candidum culture medium containing nano-material |
| CN104082135A (en) * | 2014-06-11 | 2014-10-08 | 陕西恒富源林业有限公司 | Method for rapidly breeding dendrobium officinale seedlings |
| CN104067940A (en) * | 2014-06-30 | 2014-10-01 | 广州花都先锋园艺有限公司 | Hormone-free tissue culture method for dendrobium officinale |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110972935A (en) * | 2019-10-16 | 2020-04-10 | 中国农业科学院麻类研究所 | Application of iron-based nanoparticles in promoting growth of plant stem tips |
| CN110972935B (en) * | 2019-10-16 | 2021-07-16 | 中国农业科学院麻类研究所 | Application of iron-based nanoparticles in promoting growth of plant stem tips |
| CN112237140A (en) * | 2020-10-16 | 2021-01-19 | 云南省农业科学院花卉研究所 | Method for blocking dendrobium officinale tissue culture seedling from being polluted by bacteria |
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Application publication date: 20150513 |