CN101138322A - Cremastra appendiculata tissue culturing rhizome fungus root seedling raising technique - Google Patents
Cremastra appendiculata tissue culturing rhizome fungus root seedling raising technique Download PDFInfo
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Abstract
The present invention belongs to the technical field of plant tissue culture seedling fungus breeding; the present invention mainly provides the crucial technology method of Cremastra appendiculata tissue culture rhizomorphous fungus breeding. The present invention relates to the methods of separation of mycorrhizal fungi, culture of mycorrhizal fungi, preparation of mixed fungus agent, rhizomorphous stem fungus breeding and tissue culture seedling fungus cultivation. The whole technology simplifies the variety and complexity of arethusa mycorrhizal fungi which closely grasps the nature of the synergistic action of a plurality of fungi and with the method, the phase of rhizomorphous stem is chosen for breeding which greatly shortens the tissue culture time and promotes the survival rate and reduces the cost; the present invention provides a referential novel pattern for the application of arethusa mycorrhizal fungi.
Description
One, technical field
The invention belongs to plant tissue culture seedling mycorrhizal seedling growing process field
Two, background technology
Cremastra appendiculata (Cremastra appendiculata (D.Don) Makino), have another name called PSEUDOBULBUS CREMASTRAE APPENDICULATAE, twisted-stalk, for the orchid family PSEUDOBULBUS CREMASTRAE APPENDICULATAE belongs to herbaceos perennial, be China's rare and endangered medicinal plant, also be the genuine traditional Chinese medicine of the formed uniqueness of the three-dimensional climatic environment in Guizhou.Cremastra appendiculata beginning sees and supplement to the Herbal is used as medicine with the soccer fraud stem, and the mucous and glucomannan of its rhizome has detumescence, dissipating bind, reduces phlegm, detoxifies, controls that the ulcer malignant boil is swollen, scrofula, larynx numbness swell and ache scrofula, effects such as snake, worm, mad dog wound.Cremastra appendiculata requires tighter to ecotope, happiness is shady and cool moistening, the main at present division propagation that relies on, and reproduction coefficient is extremely low, a general protocorm new bulb of only growing in 1 year; The Cremastra appendiculata seed is very tiny, has only embryo simple in structure, and endosperm is degenerated, and does not have enough nutrition to make it to sprout under nature in the seed and forms protocorm and seedling, utilizes the seed natural propagation can't be successful; Utilizing seed to carry out tissue-culturing rapid propagation becomes the important channel that addresses this problem, but its transplanting survival rate is low, and poor growth.
Big quantity research report shows that orchid and its mycorhiza bacterium exist confidential relation both at home and abroad, and no chloroplast and chlorophyllous orchid rely on the mycorhiza bacterium throughout one's life nutrition is provided; Adult plant has chloroplast and chlorophyllous orchid, in root-like stock (protocorm) stage of seed and seed formation, also do not form chloroplast and chlorophyll, rely on the mycorhiza bacterium fully nutrition is provided, battalion's bacterium parasitism, the back has formed chloroplast and chlorophyll although grow up, but its root system is undeveloped, also rely on bacterium and obtain moisture content and mineral salt, some also obtains auxin substance from the mycorhiza bacterium, the mycorhiza bacterium can also be transitted to orchid with the nutriment of soil and other plant generation, promotes the plant corpus g and D, has the research report to show that the mycorhiza bacterium can also promote the raising of active ingredients of plants.
The important function of orchid being grown in view of the mycorhiza bacterium, Chinese scholars should be used as number of research projects to orchid mycorhiza bacterium, especially on the research of giving birth to blue mycorhiza bacterium saprophyticly and using, obtained important breakthrough, having broken rhizoma Gastrodiae as the application of rhizoma Gastrodiae mycorhiza bacterium can not tame mythology.But all the time, the application of the mycorhiza bacterium that can carry out photosynthetic orchid to growing up does not obtain breakthrough, its main cause is fundamentally not understand kind and the dynamic change of this class plant mycorhiza bacterium, return its reason, the one, mainly adopt processes for disinfecting surfaces isolate root fungus, the slow bacterial strain of some growths can't be separated, the 2nd, the suitableeest infection period and the many bacterium to the mycorhiza bacterium do not do deep research, can't set up suitable co-culture system, make a large amount of mycorhiza bacterium be used as pathogenic bacteria and get rid of.This bacterium that finally causes Many researchers to filter out but is not real mycorhiza bacterium, and the application of mycorhiza bacterium is walked more difficult and more difficult.
The present invention adopts single hypha body isolation technics to separate from root and obtains real mycorhiza bacterium, according to the kind and the ratio of separating the mycorhiza bacterium that obtains, the preparation composite bacteria agent capable, composite bacteria agent capable is applied to root-like stock grows seedlings, save group training root-like stock and become the seedling time (40～50 days), greatly reduce energy consumption, also improved planting percent, survival rate and growth rate simultaneously; The cultivation of the tissue cultivating seedling that composite bacteria agent capable is applied to break up has improved the survival rate and the growth rate of transplanted seedling greatly.The present invention has solved the application problem of Cremastra appendiculata mycorhiza bacterium first, has enriched the application study of orchid mycorhiza bacterium in theory, has promoted the industrialization of Cremastra appendiculata on using.
Three, summary of the invention
The subject matter that the present invention solves is: the mycorhiza bacterium is applied to the industrialization of Cremastra appendiculata, has promptly realized Cremastra appendiculata tissue culturing rhizome fungus root seedling raising and the cultivation of differentiation group cultivation nursery garden rooting.The present invention relates to the preparation, root-like stock mycorrhizal seedling raising of cultivation, the composite bacteria agent capable of separation, the mycorhiza bacterium of mycorhiza bacterium, break up the group cultivation nursery garden rooting cultivation.Whole technique is oversimplified the diversity of orchid mycorhiza bacterium and complexity, holds on to the synergistic essence of many bacterium, for the application of orchid mycorhiza bacterium provides a new pattern.
Among the present invention, the moment of blue tissue culture given birth in term " root-like stock (Rhizome) " with being, and culture is the root shape, and this stage can be cut cultivation, realizes propagation in a large number, similar to protocorm (Protocorm) stage of epiphytic orchid.All living orchids in ground of occurring in nature all form this structure, and this stage mainly in soil, does not form chloroplast and chlorophyll, and the mycorhiza bacterium that places one's entire reliance upon provides nutrition, this in stage plant and the mycorhiza bacterium in close relations.
(1) scope of application of the present invention
The present invention is applicable to all orchid that forms root-like stock stage group training root-like stock mycorrhizal seedling raising and cultivations.
(2) technical scheme of the present invention
1, the separation of mycorhiza bacterium
Adopting single hypha body separating method to carry out the mycorhiza bacterium separates, be first key, can separate the slow bacterial strain of various growths to greatest extent, and strain separated can be the endophyte root fungus certainly, need not to carry out the screening of bacterial strain, can directly carry out the preparation of composite bacteria agent capable.
2, the cultivation of mycorhiza bacterium
Each isolated strains is an endogenetic fungus, and itself is little to the demand of oxygen, and all strain growths are slow, incubation time is long, easily pollutes, and preventing to pollute is the key in this step, can adopt to leave standstill to cultivate to address this problem, this also is to cultivate method inequality with other fungi liquids.
3, the preparation of compound bacteria root fungus liquid bacterial agent
This is second key of present technique, studies show that in a large number that single mycorhiza bacterium and group training thing are cultivated altogether often causes that plant causes a disease dead, present technique is not considered single bacterium effect, and consider the synergy of many bacterium composite bacteria agent capable, make full use of the interaction of each bacterium, each bacteria growing gesture of active balance promotes the formation of mycorhiza bacterium and plant symbiosis balance, greatly reduce lethality rate, this is first core of present technique.
4, root-like stock mycorrhizal seedling raising
This is the 3rd key of present technique, adopt the direct Mycorrhizal of root-like stock, directly plant into Mycorrhizal matrix by tissue culture bottle, on the one hand can fine maintenance humidity, need not pass through seedling differentiation and hardening stage on the other hand, improve survival rate greatly, reduce group training cost, this is the maximum bright spot of present technique, is second core of present technique.
5, group cultivation nursery garden rooting cultivation
This is the 4th key of present technique, pine soil has pine needle and the detritus soil that becomes thoroughly decomposed, pine needle can strengthen gas permeability, this to the Cremastra appendiculata root grow and the mycorhiza bacterium to play a role be necessary, and the existence of detritus soil makes matrix have good moisture capacity, can guarantee humidity suitable in the soil, this is undoubtedly suitable cultivation matrix cheap and easy to get.
Four, description of drawings
The tissue cultivating seedling of Fig. 1 group training root-like stock and differentiation
Fig. 2 root-like stock mycorrhizal seedling raising
The cultivation of Fig. 3 group cultivation nursery garden rooting
Five, embodiment
1, the separation of mycorhiza bacterium
Adopt single hypha body separating method to carry out the mycorhiza bacterium and separate, see patent " separation technology of orchidaceal plant mycorrhizal fungi monobyssisede ", application number: 00610051196.2, publication number: CN1912101.Isolate 7 kinds of mycorhiza bacterium altogether, it all is rhizoctonia, its code name is Rhizoctonia R1, Rhizoctonia R2, Rhizoctonia R3, Rhizoctonia R4, Rhizoctonia R5, RhizoctoniaR6, RhizoctoniaR7, and its segregation ratio is 1: 3: 1: 1: 2: 1: 5.
2, the cultivation of mycorhiza bacterium
Picking test tube strains grain of rice size switching 60cmPDA plate is cultivated activation in 15 days for 25 ℃; Utilize the card punch of diameter 0.5cm to beat from plate and get 3 bacterium cakes, adopt line-of-sight course to be inoculated in the PDA plate of diameter 90cm, 25 ℃ are cultured to bacterium colony and cover with plate soon, obtain the solid original seed; Utilize card punch to beat and get 5～10 bacterium cakes, transfer in the 500ml triangular flask that the 100mlPDA liquid nutrient medium is housed, 25 ℃ leave standstill cultivation and obtained in 1 month to produce to plant.
3, the preparation of compound bacteria root fungus liquid bacterial agent
Utilize vavuum pump that the liquid production kind of cultivating is carried out suction filtration and obtain mycelia, the ratio of each bacterial classification that obtains according to single hypha body partition method takes by weighing each bacterium mycelia of respective quality, put into refiner and smash mixing, then bacterium liquid is added the sterile water dilution, make the liquid bacterial agent that mycelial concentration is 14g/L.
4, root-like stock mycorrhizal seedling raising
The preparation of Mycorrhizal matrix (volume ratio): hardwood sawdust: vermiculite: perlite=4: 2: 1 or detritus soil: hardwood sawdust: perlite=2: 2: 1, the Hou Jiashui that is mixed reaches to the water content of matrix and can extrude 2～3 with hand and drip and be advisable.
Cultivate according to the stem Mycorrhizal: choose length 1～2cm and begin to be differentiated to form the root-like stock of bud, the medium of clean surface dries.The matrix for preparing is packed in the nutritive cube or seedbed of diameter 10cm, and bottom is the thick aseptic substrate of 3cm; With sprayer at this laminar surface spraying liquid composite bacteria agent capable (75ml/m
2), or cover thick liquid bacterial agent of one deck 1cm and substrate mixture (75ml microbial inoculum/10L matrix), or each is planted the seedling point and executes 1ml bacterium liquid.The root-like stock base portion is inserted in stromal surface, or in the matrix of carrying disease germs, or execute bacterium and plant the seedling point, bud upwards covers the thick matrix of 2cm then and covers root-like stock.
The mycorrhizal seedling raising management: temperature is controlled at 20～25 ℃, and the highest above 30 ℃, relative air humidity is controlled at 75～80% shade densities and reaches 70～80% of full sun.
5, group cultivation nursery garden rooting cultivation
The preparation of Mycorrhizal matrix: the pine soil of having become thoroughly decomposed, the water content of matrix is dripped and is advisable can extrude 2～3 with hand.
Mycorrhizal cultivation: the tissue cultivating seedling of selecting for use the tissue cultivating seedling that broken up or root-like stock Mycorrhizal to obtain, clean and remove medium (root-like stock Mycorrhizal breed seedling need not to clean), dry.Pine soil is covered in the seedbed, and bottom 3cm is thick; With sprayer at this laminar surface spraying liquid composite bacteria agent capable (75ml/m
2), or cover thick liquid bacterial agent of one deck 1cm and pine soil mixture (75ml microbial inoculum/10L matrix), or each is planted the seedling point and executes 1ml bacterium liquid.To break up shoot root and be tiled in pine soil surface or execute bacterium and plant the seedling point, or root plants in the pine soil of carrying disease germs, seedling upwards covers the thick matrix of 2cm then and covers shoot root portion anchor root.
The Mycorrhizal management: temperature is controlled at 20～25 ℃, and the highest above 30 ℃, relative air humidity is controlled at 75～80%.
Claims (6)
1. utilize single hypha body separating method, from the Cremastra appendiculata root, isolate 7 kinds of mycorhiza bacterium altogether, it all is rhizoctonia, its code name is Rhizoctonia R1, Rhizoctonia R2, Rhizoctonia R3, Rhizoctonia R4, Rhizoctonia R5, Rhizoctonia R6, Rhizoctonia R7, its segregation ratio is 1: 3: 1: 1: 2: 1: 5, these rhizoctonias are carried out single bacterium to be cultivated, according to each strain separating ratio, take by weighing each bacterial classification mycelia preparation composite fluid microbial inoculum of different amounts, liquid bacterial agent is applied to Cremastra appendiculata group training root-like stock and has broken up the tissue cultivating seedling cultivation that produces root and bud, and this technology mainly may further comprise the steps:
1) cultivation of mycorhiza bacterium
2) preparation of compound bacteria root fungus liquid bacterial agent
3) root-like stock mycorrhizal seedling raising
4) broken up the group cultivation nursery garden rooting cultivation
2. require described method according to right 1, it is characterized in that, step 1) test tube strains switching PDA plate, cultivate activation in 15 days for 25 ℃, utilize the card punch of diameter 0.5cm to beat from plate and get 3 bacterium cakes, line-of-sight course is inoculated in the PDA plate of diameter 90cm, 25 ℃ are cultured to bacterium colony and cover with plate soon, utilize card punch to beat and get 5～10 bacterium cakes, transfer in the 500ml triangular flask that the 100mlPDA liquid nutrient medium is housed, 25 ℃ leave standstill cultivate 1 month standby.
3. require described method according to right 1, it is characterized in that, step 2) utilizes vavuum pump that the liquid spawn of cultivating is carried out suction filtration and obtain mycelia, the ratio of each bacterial classification that obtains according to single hypha body partition method takes by weighing each bacterial classification mycelia of respective quality, put into refiner and smash mixing, then bacterium liquid is added the sterile water dilution, make the liquid bacterial agent that mycelial concentration is 14g/L.
4. require described method according to right 1, it is characterized in that, the used Mycorrhizal matrix of step 3) is (volume ratio): hardwood sawdust: vermiculite: perlite=4: 2: 1 or detritus soil: hardwood sawdust: perlite=2: 2: 1, the water content of matrix is dripped and is advisable can extrude 2～3 with hand.
5. require described method according to right 1, it is characterized in that, the step 3) specific practice is to select long 1～2cm and broken up the group training root-like stock that sprouts, cleans up the medium on surface; The matrix for preparing is packed in the nutrient cup or seedbed of diameter 10cm, and bottom is the thick aseptic substrate of 3cm; With sprayer at this laminar surface spraying liquid composite bacteria agent capable (75ml/m
2), or cover thick liquid bacterial agent of one deck 1cm and substrate mixture (75ml microbial inoculum/10L matrix), or each is planted the seedling point and executes 1ml bacterium liquid.The root-like stock base portion is inserted in stromal surface, or in the matrix of carrying disease germs, or execute bacterium and plant the seedling point, bud upwards covers the thick matrix of 2cm then and covers root-like stock.
6. require described method according to right 1, it is characterized in that, step 4) Mycorrhizal cultivation matrix is the pine soil of having become thoroughly decomposed under the pine forest, and specific practice is identical with the root-like stock Mycorrhizal, but the tissue cultivating seedling leaf will expose matrix.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103039366A (en) * | 2013-01-18 | 2013-04-17 | 通化师范学院 | Industrial production method of mycorrhizal seedlings of Changbai Mountain rhododendron plants |
| CN103589648A (en) * | 2013-10-30 | 2014-02-19 | 中国热带农业科学院热带作物品种资源研究所 | Method and fungal elicitor for rapid breeding of dendrobium tissue culture seedling |
| CN105075632A (en) * | 2015-09-01 | 2015-11-25 | 大理苍山植物园生物科技有限公司 | Rhododendron decorum aseptic seedling mycorrhization technology |
| CN107148886A (en) * | 2017-05-18 | 2017-09-12 | 广州华苑园林股份有限公司 | A kind of Mycorrhizal technology of azalea tree seedling |
| CN109105264A (en) * | 2018-11-02 | 2019-01-01 | 成都大学 | A kind of fast breeding method of Cremastra appendiculata regeneration plant |
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2007
- 2007-08-09 CN CNA2007100778785A patent/CN101138322A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103039366A (en) * | 2013-01-18 | 2013-04-17 | 通化师范学院 | Industrial production method of mycorrhizal seedlings of Changbai Mountain rhododendron plants |
| CN103039366B (en) * | 2013-01-18 | 2014-02-26 | 通化师范学院 | Industrial production method of mycorrhizal seedlings of Changbai Mountain rhododendron plants |
| CN103589648A (en) * | 2013-10-30 | 2014-02-19 | 中国热带农业科学院热带作物品种资源研究所 | Method and fungal elicitor for rapid breeding of dendrobium tissue culture seedling |
| CN103589648B (en) * | 2013-10-30 | 2016-04-20 | 中国热带农业科学院热带作物品种资源研究所 | A kind of method of dendrobium tissue cultured seedling fast breeding and fungal elicitor |
| CN105075632A (en) * | 2015-09-01 | 2015-11-25 | 大理苍山植物园生物科技有限公司 | Rhododendron decorum aseptic seedling mycorrhization technology |
| CN107148886A (en) * | 2017-05-18 | 2017-09-12 | 广州华苑园林股份有限公司 | A kind of Mycorrhizal technology of azalea tree seedling |
| CN109105264A (en) * | 2018-11-02 | 2019-01-01 | 成都大学 | A kind of fast breeding method of Cremastra appendiculata regeneration plant |
| CN109105264B (en) * | 2018-11-02 | 2022-01-11 | 成都大学 | Rapid cultivation method of rhododendron regeneration plant |
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