CN104813936B - Method for inducing Chinese pholidota herb plant to regenerate and propagate by using pseudobulbs - Google Patents
Method for inducing Chinese pholidota herb plant to regenerate and propagate by using pseudobulbs Download PDFInfo
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- CN104813936B CN104813936B CN201510234195.0A CN201510234195A CN104813936B CN 104813936 B CN104813936 B CN 104813936B CN 201510234195 A CN201510234195 A CN 201510234195A CN 104813936 B CN104813936 B CN 104813936B
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Abstract
The invention belongs to a rapid propagation method for an important plant material in endangered wild plants and particularly relates to a method for inducing a Chinese pholidota herb plant to regenerate and propagate by using pseudobulbs. The method comprises the steps of material selection and sterilization; inducing culture; multiplication culture; inducing rooting; and transplanting. The method aims at solving the problems of seed germination difficulty in a natural state, poor natural propagation capacity, poor diffusion propagation capacity, natural regeneration difficulty, low division propagation speed and the like in the prior art, and provides the technical support for large-scale industrial production. By using the method, the propagation coefficient and the rooting rate are high, the plant growth time is short, the adaptability after culture is strong, a nursery stock is robust and straight, good in growth vigor, neat and consistent, the culture processes of the nursery stock are greatly shortened, and the cost is greatly reduced. The technical support is provided for protecting Chinese pholidota herbs and large-scale industrial production, popularization and application.
Description
Technical field
The invention belongs to important vegetable material method for quickly breeding in wild plant in imminent danger, more particularly to a kind of using false
The method that bulb induces Pseudobulbus Pholidotae Chinensis plant regeneration and breeding.
Background technology
Pseudobulbus Pholidotae Chinensis（Pholidota chinensis Lindl.）It is orchid family (orchidaceae) Pholidota
(pholidota) plant, Prunus persica f. compressa and Semen Nelumbiniss on another name stone Fructus Canarii albi, stone.Perennial draft of growing nonparasitically upon another plant, meat is plump are in ampuliform or avette
Root stock it is horizontal walk, leaf two panels, basidixed on pseudobulb, papery and it is tenderer.Pseudobulbus Pholidotae Chinensis polysaccharide has preferable antioxidation
Effect.Particularly superoxide anion resisitance effect.In Pseudobulbus Pholidotae Chinensis, not only content is higher for polysaccharide, and has yin nourishing, lung heat clearing, dampness removing,
The functions such as repercussive, for dizziness, headache, cough, spit blood, nocturnal emission, dysentery, leucorrhea, infantile malnutrition, while also having：Anesthesia, stomach reinforcing,
Cough-relieving, anticancer and raising immunization.Pholidota plant the whole world about 30 kinds, be distributed in Tropical Asia to Australia and too
Flat some island of ocean.China be distributed mainly on Yunnan, Guangdong, Guangxi, Guizhou, Zhejiang, Fujian, Jiangxi etc. province, wherein especially with
Yunnan southwest is common.The Pholidota plant majority for commercially occurring at present is from wild resource, and Pseudobulbus Pholidotae Chinensis seed
The extremely difficult sprouting under natural situation, mainly by division propagation, speed is slow.If excessively excavating without plan, without purpose, blindness, will
Heavy damage its natural resources, rare species would be at the state for gradually endangering or gradually facing extinction.Therefore it is necessary to the protection that takes measures
Its natural resources.Induction Pseudobulbus Pholidotae Chinensis bulb plant regeneration has no report at present.
The content of the invention
In order to solve seed extremely difficult sprouting, natural procreation ability, Spreading and diffusion ability under natural situation in prior art
Not by force, the problems such as natural renovation is difficult, division propagation speed is slow, the invention provides a kind of using pseudobulb induction Pseudobulbus Pholidotae Chinensis plant
Strain regeneration and the method bred, provide technical support for scale, industrialization production.
The method of the present invention is realized as follows：
A kind of method of employing pseudobulb induction Pseudobulbus Pholidotae Chinensis plant regeneration and breeding of the present invention, comprises the following steps that
1）Material selection and sterilization：It is explant to choose fresh full Pseudobulbus Pholidotae Chinensis pseudobulb, goes 2 of disleaf, 3 parts and vacation
Bulb surrounding impurities, use detergent rinsed clean, are placed in punching drop 0.5-1h in flowing water, and then disinfection, sterilization are completed
After be inoculated with；
2）Inducing culture：By sterile-processed material, Jing high temperature, high pressure are inoculated in Jing after filter paper blots surface moisture
In the bulb bud inducement cultivation base of sterilizing；Condition of culture：Culture room temperature is 23 ± 2 DEG C, light application time 8h/d, intensity of illumination
500～1000lx；
3）Enrichment culture：Jing above-mentioned inducing culture, resulting well-grown diameter are grown into into 0.2-0.5 cm's
Pseudobulb, is seeded in proliferated culture medium, light application time 8h/d, 1000～1500lx of intensity of illumination；
4）Root induction：By the enrichment culture pseudobulb unrooted seedling individual plant with vanelets out, it is transferred to and takes root
In culture medium；Light application time 10h/d, 1500～1800lx of intensity of illumination；
5）Transplant：When root induction culture test tube Seedling grows to root 1～3, during young leaves 1-2 pieces, test tube seedling is placed on certainly
So light lower refining seedling 5～7 days, open bottle cap seedling exercising 1-2 days；Cultivating seedling is then taken out, the culture medium being attached on root system is washed away, is moved
It is 1 to enter pasture and water and fertile soil mass ratio:In 2 mixed-matrix, moisturizing is sheltered from heat or light, and at 15～25 DEG C, humidity should keep temperature control
75%～85%, it is to avoid direct sunlight, the complete regenerated plant of robust growth is obtained.
Above-mentioned involved culture medium：
（1）Bulb bud inducement cultivation base：VW+2.0 mg/L 6-BA +0.01 mg/L TDZ+0.3mg/LNAA+
1.5g·L-1Peptone+25g/L Su+6.5g/L Ag+1.5 gL-1Ac+30 g·L-1 + 60 gL of Fructus Musae-1Rhizoma Solani tuber osi, pH
It is worth for 5.4-5.6；
（2）Proliferated culture medium：VW+3.0mg/L 6-BA +0.01 mg/L TDZ+ 0.5mg/LNAA + 2g·L-1Albumen
+ 30 gL of peptone-1 + 70 gL of Fructus Musae-1Rhizoma Solani tuber osi+25g/L Su+6.5g/L-1 Ag+1.5 g·L-1Ac, pH value are 5.4-
5.6；
（3）Root media：1/2VW+1.0 mg/L IBA+0.3 mg/L NAA+25g/L Su+6.5g/LAg+1.5
g·L-1Ac, pH value are 5.4-5.6；
Above is referred to：1st, Ag is（The agent of Agar culture medium solidifyings and the abbreviation of holder agar powder）.
2nd, Su is（Sugar provides plant carbon source and maintains the abbreviation of osmotic pressure sucrose）.
3rd, Ac is activated carbon.
4th, Maturity is 80% Fructus Musae（Without various process of accelerating the ripening）.
5th, Rhizoma Solani tuber osi（For commercially available fresh food Rhizoma Solani tuber osi, the poor kind of preferred starch）.
Present invention is disclosed a kind of Pseudobulbus Pholidotae Chinensis plant regeneration and quick breeding technology, through 25-35d inducing culture, 60-
70d enrichment cultures, 30-40d root inductions, moisturizing shelter from heat or light, it is to avoid direct sunlight, and transplanting survival rate is up to more than 95%.2 Zhou Houzhi
Strain adaptability gradually strengthens, and obtains the complete regenerated plant of robust growth.Applied Biotechnology method obtains Pseudobulbus Pholidotae Chinensis regeneration
Plant contributes to solving scarcity of resources, alleviates the pressure of wild resource subject to severe risks of damage, effectively to protect limited wild money
Source, provides technical support for scale, industrialization production.
The remarkable advantage of the present invention：
One：Existing resource can be applied to greatest extent by the inventive method, by animal nutrition in the short time
The interior application demand for solving scarce resource；
Two：Contribute to solving scarcity of resources, alleviate the pressure of wild resource subject to severe risks of damage, effectively protect limited
Wild resource, provides technical support for scale, industrialization.
Three：Pseudobulbus Pholidotae Chinensis bulb division propagation speed is slow, and relevant Pseudobulbus Pholidotae Chinensis bulb efficient propagation method has no report.
Four：Pseudobulbus Pholidotae Chinensis clove cultivate on the bulb bud inducement cultivation base after gradually greening, grow up, growth coefficient is high, raw
Root rate is high, and the demand of plant growth time is short, and strong adaptability after cultivation, nursery stock stalwartness are tall and straight, grow fine, neat and consistent, significantly
Nursery stock incubation is shortened, great amount of cost has been saved.Promote and answer for the protection of Pseudobulbus Pholidotae Chinensis, and scale, industrialization production
With offer technical support.
Five：Sum up it is a set of be available for the efficient micropropagation technology of production unit application Pseudobulbus Pholidotae Chinensis bulb, by using the skill
Art sets up Pseudobulbus Pholidotae Chinensis production base.Therefore, the extensive application that Pseudobulbus Pholidotae Chinensis bulb is bred can directly improve rare kind artificial forest land
Yield and quality and afforestation level, realize that resource high-efficiency is cultivated, with very wide Developmental Prospect of Industrialization.The present invention
Strong innovation, with high content of technology, public welfare is strong, for promoting the research of rare cymbidium variety, utilizing, have good prospect and and
Its significance, provides theoretical foundation and technical support to develop this kind of resource.
Specific embodiment
Embodiment 1
A kind of induction Pseudobulbus Pholidotae Chinensis bulb plant regeneration and breeding application technology, comprise the following steps that
1）Material selection and sterilization：1st, it is explant to choose full Pseudobulbus Pholidotae Chinensis pseudobulb, and freshness protection package packaging, band are used after harvesting
2 of disleaf, 3 parts and pseudobulb surrounding impurities are removed after returning laboratory, detergent rinsed clean is used, punching drop 0.5h in flowing water is placed in；
2nd, plant is disinfected：Band leaf pseudobulb plant distilled water after flushing is dashed 2 times, is put in superclean bench and is used 75% ethanol
Ethanol is removed after sterilization 30s, 0.1% mercuric chloride (HgCl is added2) 8min is processed, pour mercury solution into useless hydrargyrum bottle, sterilized water is dashed 3 times,
Can inoculation；
2）Inducing culture：Material will be disinfected, Jing after filter paper blots surface moisture band 13 leaf portions point and pseudobulb plant
Strain is entirely inoculated in Jing high temperature, autoclaved bulb bud inducement cultivation base；Condition of culture：Culture room temperature is 23 DEG C, light
According to time 8h/d, intensity of illumination 800lx, incubation time 25d；Pseudobulb gradually adapts to culture medium the here that special design is prepared
In culture medium cultivate after gradually greening, grow up；
3）Enrichment culture：Jing above-mentioned inducing culture, resulting well-grown diameter are grown into into the pseudobulb of 0.4cm, point
It is not seeded in proliferated culture medium, light application time 8h/d, intensity of illumination 1200lx, enrichment culture time 60d；
4）Root induction：By the subculture inducing culture tap of the pseudobulb unrooted seedling individual plant with vanelets out, turn
Move on in root media；Light application time 10h/d, intensity of illumination 1600lx, root induction time 30d；
5）Test tube seedling culture is completed：When root induction culture test tube Seedling grows to root 1, complete to induce stone during young leaves 1
Prunus persica f. compressa plant regeneration simultaneously can carry out Periodical Reproduction culture；
6）Transplant：Root culture test tube seedling will be completed and be placed on natural light lower refining seedling 6 days, open bottle cap seedling exercising 1 day, strengthen examination
Adaptability of the Guan Miao to outdoor environment；Then with take the photograph son cultivating seedling is taken out from culture bottle, wash away the training being attached on root system
Foster base, moves into pasture and water and fertile soil (1:2), in the substrate for mixing, moisturizing is sheltered from heat or light（Temperature control is at 20 DEG C）, humidity is maintained at
80%, it is to avoid direct sunlight, survival rate is up to more than 95%.After 1 week, plant adaptability gradually strengthens, and obtains robust growth
Complete regenerated plant.
Culture medium is prepared：Culture medium thickness are 1.5cm or so, and pH value is 5.5；
（1）+ 0.01 mg/L TDZ+0.3mg/LNAA+ of bulb bud inducement cultivation base VW+2.0 mg/L 6-BA
1.5g·L-1Peptone+25g/L Su+6.5g/L Ag+1.5 gL-1Ac+30 g·L-1 + 60 gL of Fructus Musae-1Rhizoma Solani tuber osi；
（2）+ 0.01 mg/L TDZ+ 0.5mg/LNAA+2gL of proliferated culture medium VW+3.0mg/L 6-BA-1Albumen
+ 30 gL of peptone-1 + 70 gL of Fructus Musae-1Rhizoma Solani tuber osi+25g/L Su+6.5g/L-1 Ag+1.5 g·L-1Ac；
（3）Root media 1/2VW+1.0 mg/L IBA+0.3 mg/L NAA+25g/L Su+6.5g/L Ag+1.5
g·L-1Ac；
Above is referred to：1st, Ag is（The agent of Agar culture medium solidifyings and the abbreviation of holder agar powder）
2nd, Su is（Sugar provides plant carbon source and maintains the abbreviation of osmotic pressure sucrose）
3rd, Ac is activated carbon
4th, Maturity is 80% Fructus Musae（Without various process of accelerating the ripening）
5th, Rhizoma Solani tuber osi（For commercially available fresh food Rhizoma Solani tuber osi）.
Claims (1)
1. a kind of method that employing pseudobulb induces Pseudobulbus Pholidotae Chinensis plant regeneration and breeding, it is characterised in that：The method includes following
Step：
1）Material selection and sterilization：It is explant to choose fresh full Pseudobulbus Pholidotae Chinensis pseudobulb, removes 2 of disleaf, 3 parts and pseudobulb
Surrounding impurities, use detergent rinsed clean, are placed in punching drop 0.5-1h in flowing water, and then disinfection, sterilization complete laggard
Row inoculation；
2）Inducing culture：By sterile-processed material, Jing high temperature, autoclaving are inoculated in Jing after filter paper blots surface moisture
Bulb bud inducement cultivation base in；Condition of culture：Culture room temperature is 23 ± 2 DEG C, light application time 8h/d, intensity of illumination 500～
1000lx；
3）Enrichment culture：Jing above-mentioned inducing culture, resulting well-grown diameter are grown into into the false squama of 0.2-0.5 cm
Stem, is seeded in proliferated culture medium, light application time 8h/d, 1000～1500lx of intensity of illumination；
4）Root induction：By the enrichment culture pseudobulb unrooted seedling individual plant with vanelets out, root culture is transferred to
In base；Light application time 10h/d, 1500～1800lx of intensity of illumination；
5）Transplant：When root induction culture test tube Seedling grows to root 1～3, during young leaves 1-2 pieces, test tube seedling is placed on into natural light
Lower refining seedling 5～7 days, opens bottle cap seedling exercising 1-2 days；Cultivating seedling is then taken out, the culture medium being attached on root system is washed away, water is moved into
Grass and fertile soil mass ratio are 1:In 2 mixed-matrix, moisturizing is sheltered from heat or light, and at 15～25 DEG C, humidity should be maintained at temperature control
75%～85%, it is to avoid direct sunlight, obtain the complete regenerated plant of robust growth；
The bulb bud inducement cultivation base：VW+2.0 mg/L 6-BA +0.01 mg/L TDZ+0.3mg/L NAA+ 1.5g·L-1Peptone+25g/L Su+6.5g/L Ag+1.5 gL-1 Ac+30 g·L-1 + 60 gL of Fructus Musae-1Rhizoma Solani tuber osi, pH value is
5.4-5.6；Ag is the agent of Agar culture medium solidifyings and the abbreviation of holder agar powder；
The proliferated culture medium：VW+3.0mg/L 6-BA +0.01 mg/L TDZ+ 0.5mg/L NAA + 2g·L-1Peptone
+30 g·L-1 + 70 gL of Fructus Musae-1Rhizoma Solani tuber osi+25g/L Su+6.5g/L-1 Ag+1.5 g·L-1Ac, pH value are 5.4-5.6；
Ag is the agent of Agar culture medium solidifyings and the abbreviation of holder agar powder；
The root media：1/2VW+1.0 mg/L IBA+0.3 mg/L NAA+25g/L Su+6.5g/L Ag+1.5 g·
L-1Ac, pH value are 5.4-5.6；Ag is the agent of Agar culture medium solidifyings and the abbreviation of holder agar powder.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510234195.0A CN104813936B (en) | 2015-05-11 | 2015-05-11 | Method for inducing Chinese pholidota herb plant to regenerate and propagate by using pseudobulbs |
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| CN201510234195.0A CN104813936B (en) | 2015-05-11 | 2015-05-11 | Method for inducing Chinese pholidota herb plant to regenerate and propagate by using pseudobulbs |
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| CN102422811B (en) * | 2011-10-06 | 2012-12-26 | 红河州巨丰生物科技有限公司 | Dendrobium officinale tissue culture reproduction method |
| CN103299903B (en) * | 2013-05-23 | 2014-08-06 | 广西壮族自治区中国科学院广西植物研究所 | Efficient bletilla propagation method using pseudobulbs to induce pseudobulbs |
| CN104255497B (en) * | 2014-09-23 | 2016-11-09 | 福建省农业科学院农业生物资源研究所 | With the method that Pseudobulbus Pholidotae Chinensis root stock is outer implant Fast-propagation Pseudobulbus Pholidotae Chinensis |
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