CN101543184A - Method for culturing open type tissue of konjak - Google Patents
Method for culturing open type tissue of konjak Download PDFInfo
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- CN101543184A CN101543184A CN200810047258A CN200810047258A CN101543184A CN 101543184 A CN101543184 A CN 101543184A CN 200810047258 A CN200810047258 A CN 200810047258A CN 200810047258 A CN200810047258 A CN 200810047258A CN 101543184 A CN101543184 A CN 101543184A
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Abstract
The invention belongs to the field of quick reproduction of plants, and in particular relates to a method for culturing an open type tissue of konjak. The invention provides the method for culturing the open type tissue of konjak aiming at the defects existing in the prior tissue culture quick reproduction method. The culture process comprises the following steps: inoculation: a tissue of the konjak is used as an explant, inoculated to a konjak culture medium and picked up until the tissue is expanded to a certain number; differentiated culture: the inoculated konjak tissue is put in a differentiated culture medium to be differentiated into a large number of spores, the auxiliary buds are differentiated from the spores, and simultaneously roots grow; re-differentiated culture: the konjak tissue with differentiated auxiliary buds is put in a re-differentiated nutrient solution for culture, so that the konjak tissue is changed from the heterotrophic type to the autotrophic type; and transplant transplanting: the complete konjak plant is transplanted to a seed bed. The method has the advantages that a novel process is adopted; and the production of test-tube plantlet does not need to be performed in a strictly aseptic environment, so that the operational difficulty is reduced, and the production efficiency is improved.
Description
Technical field
The invention belongs to the plant fast propagation field, particularly a kind of open method for tissue culture of konjaku.
Background technology
Konjaku belongs to Araeceae Amorphophallus herbaceos perennial, because of it is that unique in the world high plant that contains Glucomannan causes the extensive concern in the world.About 1,200,000 mu of present konjakus in the cultivated area in the whole nation, mainly be distributed in the outlying mountain area that economize in Hubei, Hunan, Yunnan, Guizhou, Sichuan, Shaanxi etc., the plantation konjaku is the important channel that the local peasant programme of guiding is striven for a relatively comfortable life, and also is the main economic crops that a lot of local governments adjust the structure of rural undertaking.
The tissue culture technique research of konjaku originates in 1980, but it is just progressively developed later on by 2000, the application of konjaku tissue culture technique, the one, the problem of solution present stage konjaku kind taro wretched insufficiency, the 2nd, solve slow, the slow problem of new varieties promotion rate of konjaku kind taro reproduction speed, the 3rd, solve konjaku kind taro because virus accumulates the serious problem of degenerating, this has played very important effect in the development that promotes the konjaku industry.
Plant Tissue Breeding develops so far, and its theoretical research is quite profound, but also there are many problems in sport technique segment.Traditional group training process is numerous and diverse, formulates around preventing to pollute, and control has been polluted into the primary technology in the group training, can not be reduced to the acceptable scope to pollution rate, just means that also this technology can not go on.And the factor that influence is polluted is varied, as kind, concentration, disinfecting time and sterilization method, medium and the vessel of the kind of explant, the season of drawing materials, time, preprocess method, disinfectant go out the requirement of mattress, operating personnel, working environment, the work quality of superclean bench etc. all with pollute closely related, therefore, make pollution problem very complicated, also be difficult to control, become one of major reason of restriction group training industrialization.
Pollution in the group training process mainly shows as the pollution of medium, and nutritious medium provides the suitable place that grows for bacterium, fungi.Therefore, both guarantee the plant tissue normal growth as long as medium is transformed into, have the medium of sterilization, antibacterial functions again, in a single day mushroom loses and grows the place, just can not work the mischief to group training process.The contaminated solution problem makes group training process need not strict gnotobasis, fundamentally simplification group training link, and it is fully possible carrying out open tissue culture.And the key of transforming medium is to locate a kind of broad-spectrum germicide that can add medium to.Since 2000, related scientific research unit trains cost around development and use, simplification group training link, the reduction group of bactericide, set up the complete feasible open tissue culture pattern of a cover (hereinafter to be referred as " open group training ") and done number of research projects, and in the training of tens plant species groups such as potato, banana, having obtained success, this technology will promote the use of other plant.
In the application of group culturation rapid propagating technology, the technical difficulty that Different Crop is cultivated is made a world of difference, the relative potato of konjaku is more complex, the one, potato makes explant with stem apex, there is not endophyte pollution problems in successive transfer culture, the initial cultivation of konjaku adopts bulb or root-like stock to make explant, contains a large amount of endophytes, can show in successive transfer culture; The 2nd, potato test-tube plantlet forms the back by cutting section propagation, expands numerously not pass through the callus stage, and fast growth, general 20 days is a breeding cycle, can repeatedly breed; Konjaku expand numerous must be by the callus stage, growth rate is slower, subculture cycle is longer, stripping and slicing is expanded numerously once generally needs 30-45 days, and 1 can only be cut the 3-4 piece, after subculture 5-6 time, differentiation capability weakens gradually, need carry out initial cultivation again; The 3rd, it is simple than the konjaku medium to be used for the numerous medium component of potato expansion, does not generally add hormone, even need not add organic matter, the probability of living contaminants is greatly reduced, therefore, at different crops, need the corresponding open tissue culture technique of research, could practical application.
Summary of the invention
The objective of the invention is to improve, a kind of open method for tissue culture of konjaku is provided at existing tissue culture and rapid propagation method deficiency.
The object of the present invention is achieved like this: a kind of open method for tissue culture of konjaku, and it is waited to levy and is to include in the fast culture process following steps:
A. inoculation: adopt the tissue of konjaku to make explant, be inoculated in the konjaku medium, expand and numerously after some, take out;
B. differentiation culture: postvaccinal konjaku tissue is put into differential medium, make tissue differentiate the brood cell in a large number, the differentiation bud of growing thickly from the brood cell is with the duration root;
C. differentiation culture again: the konjaku tissue that will differentiate the bud of growing thickly is put into and is broken up nutrient solution again and cultivate, and makes it change autotrophic type into from heterotroph;
D. transplant: the complete konjak plantlet of transplant is arrived the seedbed.
The present invention is the open method for tissue culture of konjaku further, wherein includes step down:
A. inoculation: adopt the tissue with vitality of konjaku to make explant, be inoculated in the konjaku medium, make the basic stitch volume expand 2 times-3 times.Contain in the composition of konjaku medium: MS+6-BA1.5mg/L-3.0mg/L+NAA 0.1mg/L-0.5mg/L+IBA 0.5mg/L-1.0mg/L+30% sucrose.
B. differentiation culture: postvaccinal konjaku tissue is put into differential medium, make tissue differentiate the brood cell in a large number, under suitable temperature and illumination condition, the differentiation bud of growing thickly from the brood cell is with the duration root.Contain in the composition of konjaku differential medium: MS+6-BA 1.5mg/L-3.0mg/L+NAA 0.1mg/L-0.5mg/L+IBA0.5mg/L-1.0mg/L+KT1mg/L-5mg/L+30% sucrose.
C. differentiation culture again: the konjaku tissue that will differentiate the bud of growing thickly is cut into the 3-4 piece, and 3-5 bud of growing thickly of every band put into and broken up nutrient solution again and cultivate, and adds certain density bacteriostatic agent in the nutrient solution, makes it change autotrophic type into from heterotroph.Konjaku breaks up in the composition of nutrient solution again and contains: macroelement and microelement concentration are 1/5-1/10MS medium concentration in the nutrient solution, and hormone concentration is 6-BA 1.0mg/L-10.0mg/L+NAA0.1mg/L-0.5mg/L+IBA 0.5mg/L-1.0mg/L+KT 2mg/L-10mg/L.
D. transplant: the differentiation complete konjak plant of the long root of petiole base is downcut with blade, be with a small amount of callus, be transplanted to the seedbed, band brood cell's callus continues nutrient solution evoked callus with the band hormone and expands differentiation with the brood cell, successive transfer culture under non-sterile condition.
The open method for tissue culture of a kind of konjaku provided by the present invention, owing to adopt new technology, shortened the production time of test-tube plantlet, improved labor productivity, test-tube plantlet production need not carried out under the gnotobasis of strictness, significantly reduce the difficulty of technology operation, improved production efficiency.
Embodiment
The present invention is further described below by embodiment.
The fast culture process of konjaku of the present invention.At first obtain the propagating materials of bud of growing thickly of band callus as open tissue culture by traditional tissue culture technique, the bud of will growing thickly then cuts the back and cultivates as fixture with the sponge of can repeated multiple times using in containing the nutrient solution of hormone, and it is numerous to adopt the mode that differentiates a large amount of buds of growing thickly under wide-open condition when callus is expanded to carry out the expansion of konjaku test-tube plantlet.The present invention makes the tissue culture of konjaku just break away from blake bottle and gnotobasis after breeding certain basic seedling, under wide-open condition, expand numerous, the agar that adopts sponge to replace price constantly to rise, do not add sucrose and organic matter, adopt natural lighting to replace fluorescent lamp, adopt the cheap special-purpose blake bottle of container replacement group training, saved investment in fixed assets, reduced the test-tube plantlet production cost more than 80%; Test-tube plantlet production need not carried out under the gnotobasis of strictness, has significantly reduced the difficulty of technology operation, has improved production efficiency.
The invention process is as follows:
(1) become pink annual 3-6 month elephant-foot yam main bud, the physiology that begins to sprout enlivens period, get health, intact bottom set, taro whip or arrow fruit are as propagating materials, after running water is cleaned and is dried, in closed container, smoked gas 20-30 minute with formaldehyde-potassium permanganate, take out, after treating smell volatilization to the greatest extent, place on the superclean bench, with 75% alcoholic solution sterilization 1 minute, used 0.1% mercuric chloride solution sterilization again 25 minutes, take out and clean, in the culture dish that fills 0.05%PVP+0.5%Vc solution, be cut into the fritter of 1cm * 1cm size, put into the blake bottle that MS+6-BA1.5-3.0mg/L+NAA0.1-0.5mg/L+IBA0.5-1.0mg/L+30% sucrose (pH=5.8-6.5) medium is housed, place 1, under 25-28 ℃ of condition, secretly cultivate for every bottle.About about 15 days, visible tissue was obviously expanded, and about 30 days, the volume that expands is about 2-3 times of basic stitch volume.On superclean bench, callus is taken out, be cut into the 4-6 piece, put into identical cultivation, place the 2-3 piece, carry out successive transfer culture for every bottle.About successive transfer culture 30 days, take out, be cut into the 3-6 piece, put into the differential medium of MS+6-BA1.5-3.0mg/L+NAA0.1-0.5mg/L+IBA0.5-1.0mg/L+KT1-5mg/L+30% sucrose, the dark cultivation 20-30 days under uniform temp, differentiate the brood cell on the callus in a large number, blake bottle moved to the window limit or open fluorescent lamp on the culturing rack, shone every day 12 hours, after 5-10 days, from the brood cell, break up the bud of growing thickly, with the duration root; Bud grows to 1-2cm, as the material of open tissue culture;
(2) callus lines that will differentiate the bud of growing thickly takes out from blake bottle, be cut into the 3-4 piece with blade, 3-5 bud of growing thickly of every band, with behind a small amount of sponge wrapping sponge being soaked with nutrient solution, put into the inorganic salt concentration of 1/2-1/10MS medium, hormone is that the 0.5-5 of differentiation culture based formulas is doubly and in the nutrient solution of finite concentration bacteriostatic agent, do not add sugar in the nutrient solution, agar and organic matter, temperature 20-30 ℃, adopt the irradiation of natural lighting or fluorescent lamp, the bud of growing thickly of band callus is placed in the greenhouse of uniform temperature and illumination to be cultivated, change autotrophic type into from heterotroph, the bud of growing thickly utilizes luminous energy to make nutrient, nutritive element in callus and the root absorption nutrient solution is for plant strain growth; Under the effect of exogenous hormone, callus continues to expand, and mounted blade is taken root from petiole base, continues to differentiate the bud of growing thickly simultaneously.
(3) differentiation of the long root of petiole base is complete plant is downcut with blade, be with a small amount of callus, 1000 times of liquid of agricultural streptomycin wetting powder with 72% soaked after 5-8 minute is transplanted to the seedbed, band brood cell's callus continues nutrient solution evoked callus with the band hormone and expands differentiation with the brood cell, can carry out repeatedly successive transfer culture.
The medium of inducing the initial differentiation of konjaku tissue culture is a MS+6-BA1.5-3.0mg/L+NAA0.1-0.5mg/L+IBA0.5-1.0mg/L+30% sucrose; Inducing the medium of a large amount of brood cells' formation and differentiation and seedling emergence is MS+6-BA1.5-3.0mg/L+NAA0.1-0.5mg/L+IBA0.5-1.0mg/L+KT1-5mg/L+30% sucrose, and condition of culture is temperature 23-28 ℃, secretly cultivates 30-50 days.
The callus that differentiates the bud of growing thickly continue to expand and the nutrient solution that breaks up in inorganic salt concentration be the 1/5-1/20 of medium, hormone is that hormone dosage 0.5-5 does not doubly add sugar and organic matter in the medium; Bacteriostatic agent is for to have inhibiting Chinese herbal medicine extract to multiple fungi, bacterium and algal grown; But replace agar with the fixture that other repeated multiple times are used.
The production of test-tube plantlet need not to operate under strict aseptic environments, and culture vessel only needs general cheap vessel, need not special-purpose blake bottle.
Macroelement and microelement concentration are 1/5-1/10MS medium concentration in the nutrient solution, and hormone concentration is 6-BA5.0-10.0mg/L+NAA0.1-0.5mg/L+IBA0.5-1.0mg/L+KT2-10mg/ L.
The 100-200 that is numbered HUST-RF198, the HUST-RF214 times dilution that bacteriostatic agent concentration provides for the Central China University of Science and Technology; The fixture of explant replaces agar with sponge, degreasing silk floss etc.
The cutting and the inoculation of bud of growing thickly do not carried out on superclean bench, and carry out on plane bench, but the laboratory will keep clean relatively, and inoculating instruments such as used blade and tweezers will carry out strict sterilization; Culture vessel can be used disposal plastic cup, dish, seals with preservative film.
Claims (8)
1. the open method for tissue culture of a konjaku, it is waited to levy and is to include in the incubation following steps:
A. inoculation: adopt the tissue of konjaku to make explant, be inoculated in the konjaku medium, expand and numerously after some, take out;
B. differentiation culture: postvaccinal konjaku tissue is put into differential medium, make tissue differentiate the brood cell in a large number, the differentiation bud of growing thickly from the brood cell is with the duration root;
C. differentiation culture again: the konjaku tissue that will differentiate the bud of growing thickly is put into and is broken up nutrient solution again and cultivate, and makes it change autotrophic type into from heterotroph;
D. transplant: the complete konjak plantlet of transplant is arrived the seedbed.
2. according to the open method for tissue culture of the described a kind of konjaku of claim 1, it is waited to levy in the composition that is konjaku medium among the described step a and contains: MS+6-BA 1.5mg/L-3.0mg/L+NAA 0.1mg/L-0.5mg/L+IBA 0.5mg/L-1.0mg/L+30% sucrose.
3. according to the open method for tissue culture of the described a kind of konjaku of claim 1, it is waited to levy in the composition that is konjaku differential medium among the described step b and contains: MS+6-BA 1.5mg/L-3.0mg/L+NAA 0.1mg/L-0.5mg/L+IBA 0.5mg/L-1.0mg/L+KT1mg/L-5mg/L+30% sucrose.
4. according to the open method for tissue culture of the described a kind of konjaku of claim 1, it is waited to levy and is that konjaku among the described step c breaks up in the composition of nutrient solution again and contains: macroelement and microelement concentration are 1/5MS-1/10MS medium concentration in the nutrient solution, and hormone concentration is 6-BA 1.0mg/L-10.0mg/L+NAA 0.1mg/L-0.5mg/L+IBA 0.5mg/L-1.0mg/L+KT 2mg/L-10mg/L.
5. according to the open method for tissue culture of the described a kind of konjaku of claim 1, it is waited to levy and is that described step a is: a. inoculation: adopt the tissue with vitality of konjaku to make explant, be inoculated in the konjaku medium, make the basic stitch volume expand 2 times-3 times.
6. according to the open method for tissue culture of the described a kind of konjaku of claim 1, it is waited to levy and is that described step b is: the b. differentiation culture: postvaccinal konjaku tissue is put into differential medium, make tissue differentiate the brood cell in a large number, under 23 ℃ of-28 ℃ of temperature and 1200Lux-2000Lux illumination condition, from the brood cell, break up the bud of growing thickly, with the duration root.
7. according to the open method for tissue culture of the described a kind of konjaku of claim 1, it is waited to levy and is that described step c is: c. is differentiation culture again: the konjaku tissue that will differentiate the bud of growing thickly is cut into 3-4,3-5 bud of growing thickly of every band, put into and break up nutrient solution again and under non-sterile condition, cultivate, add certain density bacteriostatic agent in the nutrient solution, do not add sugar and organic matter,, make it change autotrophic type into from heterotroph with sponge or the continuous agar that replaces of degreasing.
8. according to the open method for tissue culture of the described a kind of konjaku of claim 1, it is waited to levy and is that described steps d is: d. transplants: the differentiation complete konjak plant of the long root of petiole base is downcut with blade, be with a small amount of callus, be transplanted to the seedbed, band brood cell's callus continues nutrient solution evoked callus with the band hormone and expands differentiation with the brood cell, successive transfer culture under non-sterile condition.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106993532A (en) * | 2017-03-31 | 2017-08-01 | 中国林业科学研究院热带林业研究所 | A kind of open tissue culture method of yearning between lovers |
| CN107211891A (en) * | 2017-05-27 | 2017-09-29 | 安康市农业科学研究所 | A kind of live fast breeding technique of konjaku callus |
| CN107318305A (en) * | 2017-08-18 | 2017-11-07 | 安康学院 | A kind of konjaku true seed fast breeding method |
| CN111602596A (en) * | 2020-07-17 | 2020-09-01 | 中国热带农业科学院橡胶研究所 | Method for obtaining regeneration plant by tissue culture of bulbil konjak flower device |
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2008
- 2008-03-29 CN CN200810047258A patent/CN101543184A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106993532A (en) * | 2017-03-31 | 2017-08-01 | 中国林业科学研究院热带林业研究所 | A kind of open tissue culture method of yearning between lovers |
| CN106993532B (en) * | 2017-03-31 | 2019-10-22 | 中国林业科学研究院热带林业研究所 | A kind of open tissue culture method of yearning between lovers |
| CN107211891A (en) * | 2017-05-27 | 2017-09-29 | 安康市农业科学研究所 | A kind of live fast breeding technique of konjaku callus |
| CN107318305A (en) * | 2017-08-18 | 2017-11-07 | 安康学院 | A kind of konjaku true seed fast breeding method |
| CN107318305B (en) * | 2017-08-18 | 2020-12-25 | 安康学院 | Rapid breeding method for konjac seedling seeds |
| CN111602596A (en) * | 2020-07-17 | 2020-09-01 | 中国热带农业科学院橡胶研究所 | Method for obtaining regeneration plant by tissue culture of bulbil konjak flower device |
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