CN102577973A - Tissue culture method for Cymbidium floribundum - Google Patents
Tissue culture method for Cymbidium floribundum Download PDFInfo
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- CN102577973A CN102577973A CN2012100636713A CN201210063671A CN102577973A CN 102577973 A CN102577973 A CN 102577973A CN 2012100636713 A CN2012100636713 A CN 2012100636713A CN 201210063671 A CN201210063671 A CN 201210063671A CN 102577973 A CN102577973 A CN 102577973A
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- 241000320571 Cymbidium floribundum Species 0.000 title abstract 5
- 238000012136 culture method Methods 0.000 title abstract 5
- 241000196324 Embryophyta Species 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 18
- 238000009331 sowing Methods 0.000 claims abstract description 13
- 230000001954 sterilising effect Effects 0.000 claims abstract description 13
- 230000035755 proliferation Effects 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims abstract description 7
- 229910052956 cinnabar Inorganic materials 0.000 claims description 81
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 37
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 36
- 229930006000 Sucrose Natural products 0.000 claims description 36
- 239000005720 sucrose Substances 0.000 claims description 36
- 239000002609 medium Substances 0.000 claims description 25
- 239000002775 capsule Substances 0.000 claims description 24
- 241000234295 Musa Species 0.000 claims description 18
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 18
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 18
- 238000005286 illumination Methods 0.000 claims description 15
- 229960002523 mercuric chloride Drugs 0.000 claims description 12
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000000306 component Substances 0.000 claims description 9
- 230000004069 differentiation Effects 0.000 claims description 9
- 230000007226 seed germination Effects 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 240000007509 Phytolacca dioica Species 0.000 claims description 6
- 235000013399 edible fruits Nutrition 0.000 claims description 6
- 230000013020 embryo development Effects 0.000 claims description 6
- 238000011010 flushing procedure Methods 0.000 claims description 6
- 230000035784 germination Effects 0.000 claims description 6
- 239000012869 germination medium Substances 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 235000013372 meat Nutrition 0.000 claims description 6
- 230000000877 morphologic effect Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 239000012533 medium component Substances 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 235000013619 trace mineral Nutrition 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
- 239000005972 6-Benzyladenine Substances 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract 2
- 239000003086 colorant Substances 0.000 abstract 1
- 230000000249 desinfective effect Effects 0.000 abstract 1
- 230000006698 induction Effects 0.000 abstract 1
- 230000002062 proliferating effect Effects 0.000 abstract 1
- 230000000644 propagated effect Effects 0.000 abstract 1
- 238000005728 strengthening Methods 0.000 abstract 1
- 238000002054 transplantation Methods 0.000 abstract 1
- 241000233855 Orchidaceae Species 0.000 description 12
- 239000005556 hormone Substances 0.000 description 8
- 229940088597 hormone Drugs 0.000 description 8
- 239000006870 ms-medium Substances 0.000 description 7
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000035040 seed growth Effects 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 241000732800 Cymbidium Species 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- 206010043949 Tongue discolouration Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
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- 239000004744 fabric Substances 0.000 description 1
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- 230000002068 genetic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a tissue culture method for a plant, in particular to a tissue culture method for Cymbidium floribundum, and belongs to the technical field of agricultural plantation. The tissue culture method for the Cymbidium floribundum comprises the following six steps of: collecting an explant, disinfecting and sterilizing, sowing, proliferating, strengthening seedlings, and performing rooting culture. When the Cymbidium floribundum is propagated through plant tissue culture by a tissue culture technology, the advantage of rapid propagation can be exerted, and a large number of seedlings are obtained in a short time; compared with cottage, the tissue culture method provided by the invention is characterized in that flower colors of plants are seldomly mutated in later culture; and the plant tissue culture serving as a vegetative propagation mode can ensure that the seedlings are regular, facilitates late unified transplantation and plantation management, and can also ensure that various characters of grown product plants and bonsai are consistent. Compared with the conventional propagation mode, the invention has the advantages that a used culture medium is short in induction time, quick in seedling emergence and high in proliferation frequency; and particularly, the seedlings are regular, the method is easy to operate, and the Cymbidium floribundum can be produced on a large scale.
Description
Technical field
The present invention relates to the method for tissue culture of a plant species, especially relate to the blue method for tissue culture of cinnabar.Belong to the agricultural planting technique field.
Background technology
The cinnabar orchid is the plant of the orchid family Cymbidium, gains the name like cinnabar because of pattern is red; Ming Dynasty cinnabar in period orchid once was taken as tribute, and " tribute is blue " and " superb work is blue " therefore is otherwise known as.The blue plant of cinnabar is robust, and root is slightly grown, bloom the annual 9-10 month (generally be to hold around the Double Ninth Festival, minority is extended down to the end of the year, even around the Spring Festival), the florescence is January approximately; The high 30-40cm of Roripa, the line rod rust red, every Roripa is blossomed 3-5,6-8 centimetre in flower footpath, the willow leaf lobe, sepal drapes over one's shoulders needle-like, is triangular arranged, and the look Vermilion is seen purple, and fragrance is pure, flower tongue white background, the scarlet fleck of cloth, but spot is diffusing, shapeless.
The blue existing over thousands of year domestic history of cinnabar is, slight snow plain equally celebrated for their achievements Yunnan traditional orchid plain with heavy snow, belongs to one of blue four big famous-objects in Yunnan.Yunnan is among the people thinks that this flower of breeding can increase the celebrating atmosphere of family, and its leaf is strong luxuriant and spend bright and beautifully, is possessed of dignity very much, and big expensive, the in the limelight implied meaning of Da Fu is arranged, and is loved by the people.The cinnabar orchid once obtained bonus time and again in the blue rich meeting of all types, obtain two pieces of gold medals, also obtain gold medal Kunming blue exhibition in autumn on National Day in 2003 at China Lan Bohui in the tenth autumn.
The blue traditional modes of reproduction of cinnabar is to utilize plant division to breed, and reproduction coefficient is low, and pattern can produce variation in late growing stage.The relevant report that does not have the blue tissue culture of cinnabar aspect at present in the documents and materials.
Summary of the invention
The objective of the invention is to overcome the defective that above-mentioned prior art exists; A kind of fast, propagation frequency height that obtains in a short time to emerge is provided; Especially it is neat to emerge, and produces the consistent seedling of proterties, guarantees the uniform commodity performance of subsequent product; Easy operating, the blue method for tissue culture of cinnabar that can carry out large-scale production.
The object of the invention can be realized through following technical scheme: the blue method for tissue culture of cinnabar is characterized in that this method may further comprise the steps:
(1) explant collection
The 10-11 month is the blue capsule mature period of cinnabar, when the capsule appearance transfers brown to, reaches morphological maturity and gathers when not ftractureing as yet; Time is too early, and seed embryonic development not umbra rings germination rate; Time is too late, and capsule breaks, and can cause unnecessary loss;
(2) sterilization
Get the blue uncracked capsule of cinnabar; Flowing water flushing 25-35min; After handling 25-35s with 70-75% alcohol on the superclean bench,, remove mercuric chloride solution, with aseptic water washing 4-5 time again with 0.1-0.2% mercuric chloride liquid immersion 10-15min; Be no less than 4-5min, fruit surface moisture is blotted with aseptic paper in the back at every turn;
(3) sowing
Under aseptic condition, with the blue planting seed of the cinnabar of sterilizing on the sowing medium of manual work preparation; Cultivated 30-40 days, and treated that culture expands when forming former spheroid, promptly began next stage;
(4) propagation
A last step gained culture is being transferred under the aseptic condition on the artificial proliferated culture medium of preparing; Through cultivating about 30-40 days; The former spheroid that expands begins to occur the blue seedling of the tender cinnabar of children, can seedling be transferred on the strong seedling culture base when treating seedling length to routine program desired height in strong sprout;
(5) strong sprout
The blue seedling of the cinnabar of a last step gained is being transferred under the aseptic condition on the artificial strong seedling culture base of preparing; Seedling through cultivation in 25-35 days begins to occur 2-3 sheet leaf, forms independently plant, treats that plant length can be transferred to it on root media when routine is taken root the program desired height;
(6) take root
The cinnabar of a last step gained is transferred on the root media under aseptic condition blue strong sprout, begins to occur growing tall and 5-6 sheet leaf, grow many meats, green aerial root, form complete plant at last at the base portion of seedling through cultivating 30-50 days plant; Final rooting rate is up to 80-95%;
Described seed germination medium comprises: MS+ benzyladenine 6-BA0.1-0.5mg/L+1-3% sucrose;
Described proliferation and differentiation medium comprises: MS+ methyl NAA 0.1-0.3mg/L+6 benzyladenine 6-BA0.8-1.2mg/L+1-3% sucrose;
Described strong seedling culture base comprises: MS+10-15% banana+methyl NAA 1-2mg/L+1-3% sucrose;
Described root media comprises: MS+10-15% banana+methyl NAA 1.5-2.5mg/L+ indolebutyric acid IBA 0.8-1.2mg/L+1-3% sucrose.
Described MS minimal medium is made up of macroelement, trace element and organic principle.
Said seed germination medium component is preferably MS+ benzyladenine 6-BA0.1mg/L+2% sucrose.
Said proliferation and differentiation medium component is preferably MS+ methyl NAA0.2mg/L+ benzyladenine 6-BA 1mg/L+2% sucrose.
Said strong seedling culture based component is preferably MS+10% banana+methyl NAA1.5mg/L+2% sucrose.
Said culture of rootage based component is preferably MS+10% banana+methyl NAA 2mg/L+ indolebutyric acid IBA 1mg/L+2% sucrose.
Each medium pH value is 5.5-6.0, and cultivation temperature is 23-25 ℃, intensity of illumination 1600-2000LX, light application time 9-12h/d.
Compared with prior art, the present invention when utilizing Plant Tissue Breeding that the cinnabar orchid is bred, can bring into play fast numerous advantage through tissue culture technology, obtains a large amount of seedlings in a short time; Cuttage relatively, the also less generation variation of plant pattern in the later stage cultivation; And Plant Tissue Breeding can guarantee that as a kind of vegetative propagation mode seedling is neat, both has been convenient to unified transplanting of later stage and has planted planting management, product plant that also can guarantee to be grown and potted plant consistent on each proterties.With respect to modes of reproduction in the past, used medium culture is brought out short, fast, the propagation frequency height of emerging of time, and it is neat especially to emerge, and easy operating can carry out large-scale production.
Description of drawings
Accompanying drawing 1 is the flow chart of the blue method for tissue culture of cinnabar of the present invention.
Embodiment
Below in conjunction with specific embodiment the present invention is elaborated.
(1) explant collection
The 10-11 month is the blue capsule mature period of cinnabar, when the capsule appearance transfers brown to, reaches morphological maturity and gathers when not ftractureing as yet.Time is too early, and seed embryonic development not umbra rings germination rate; Time is too late, and capsule breaks, and can cause unnecessary loss;
(2) sterilization
Get the blue uncracked capsule of cinnabar; Flowing water flushing 25-35min (minute); After handling 25-35s (second) with 70-75% alcohol on the superclean bench again with 0.1-0.2% mercuric chloride liquid immersion 10-15min (minute), remove mercuric chloride solution, with aseptic water washing 4-5 time; Be no less than at every turn 4-5min (minute), the back blot fruit surface moisture with aseptic paper;
(3) sowing
Under aseptic condition, the blue planting seed of the cinnabar of sterilizing on the sowing medium of manual work preparation, was cultivated 30-40 days, when treating that culture expands the former spheroid of formation, promptly begin next stage;
(4) propagation
A last step gained culture is being transferred under the aseptic condition on the artificial proliferated culture medium of preparing; Through cultivating about 30-40 days; The former spheroid that expands begins to occur the blue seedling of the tender cinnabar of children, can seedling be transferred on the strong seedling culture base when treating seedling length to routine program desired height in strong sprout;
(5) strong sprout
The blue seedling of the cinnabar of a last step gained is being transferred under the aseptic condition on the artificial strong seedling culture base of preparing; Seedling through cultivation in 25-35 days begins to occur 2-3 sheet leaf; Form independently plant, treat that plant length can be transferred to it on root media when routine is taken root the program desired height;
(6) take root
The cinnabar of a last step gained is transferred on the root media under aseptic condition blue strong sprout; Begin to occur growing tall and 5-6 sheet leaf through cultivating 30-50 days plant; Base portion at seedling grows many meats, green aerial root, forms complete plant at last, and final rooting rate is up to 80-95%.
Described seed germination medium comprises MS+ benzyladenine 6-BA 0.1-0.5mg/L+1-3% sucrose.
Described proliferation and differentiation medium comprises MS+ methyl NAA 0.1-0.3mg/L+ benzyladenine 6-BA0.8-1.2mg/L+1-3% sucrose.
Described strong seedling culture base comprises MS+10-15% banana+methyl NAA 1-2mg/L+1-3% sucrose.
Described root media comprises MS+10-15% banana+methyl NAA 1.5-2.5mg/L+ indolebutyric acid IBA 0.8-1.2mg/L+1-3% sucrose.
Described MS minimal medium is made up of macroelement, trace element and organic principle.
Said seed germination medium component preferably includes MS+ benzyladenine 6-BA 0.1mg/L+2% sucrose.
Said proliferation and differentiation medium component preferably includes MS+ methyl NAA 0.2mg/L+ benzyladenine 6-BA 1mg/L+2% sucrose.
Said strong seedling culture based component preferably includes MS+10% banana+methyl NAA 1.5mg/L+2% sucrose.
Said culture of rootage based component preferably includes MS+10% banana+methyl NAA 2mg/L+ indolebutyric acid IBA 1mg/L+2% sucrose.
Each blue medium pH value of described cinnabar is 5.5-6.0, and cultivation temperature is 23-25 ℃ (degree), intensity of illumination 1600-2000LX (lux), light application time 9-12h/d (hour/day).
Embodiment 1
(1) explant collection
The 10-11 month is the blue capsule mature period of cinnabar, when the capsule appearance transfers brown to, reaches morphological maturity and gathers when not ftractureing as yet.Time is too early, and seed embryonic development not umbra rings germination rate; Time is too late, and capsule breaks, and can cause unnecessary loss;
(2) sterilization
Get the blue uncracked capsule of cinnabar, flowing water flushing 25min, after handling 25s with 75% alcohol on the superclean bench again with 0.1% mercuric chloride liquid immersion 10min; Remove mercuric chloride solution; With aseptic water washing 4 times, be no less than 4min at every turn, fruit surface moisture is blotted with aseptic paper in the back;
(3) sowing
The MS medium is to the sprouting of seed and grow preferably, and sucrose concentration 1%, pH value are 5.5, and 6-BA (benzyladenine) concentration 0.1mg/L (mg/litre) is fit to seed germination and growth.Under aseptic condition, with the blue planting seed of the cinnabar of sterilizing on the sowing medium of manual work preparation.Cultivation temperature is 23 ℃, intensity of illumination 1600LX (lux), and light application time 9h/d (hour/day) cultivated 30 days, treated that culture expands when forming former spheroid, promptly begins next stage;
(4) propagation
The optimum medium of the blue former spheroid proliferation and differentiation cultivation stage of cinnabar is MS+NAA 0.1mg/L+6-BA (6 a benzyladenine) 0.8mg/L+1% sucrose, and pH value is 5.5.A last step gained culture is being transferred under the aseptic condition on the artificial proliferated culture medium of preparing.Cultivation temperature is 23 ℃, intensity of illumination 1600LX, and light application time 9h/d, through cultivating about 30 days, the former spheroid that expands begins to occur the blue seedling of the tender cinnabar of children, can seedling be transferred on the strong seedling culture base when treating seedling length to routine program desired height in strong sprout;
(5) strong sprout
The optimum medium in blue stage in strong sprout of cinnabar is MS+10% banana+NAA 1mg/L+1% sucrose, and pH value is 5.5; The blue seedling of the cinnabar of a last step gained is being transferred under the aseptic condition on the artificial strong seedling culture base of preparing.Cultivation temperature is 23 ℃, intensity of illumination 1800LX, and light application time 9h/d begins to occur 2-3 sheet leaf through the seedling of cultivating in 25 days, forms independently plant, treats that plant is long when routine is taken root the program desired height, can it be transferred on the root media.
(6) take root
The take root medium in stage of cinnabar orchid is MS+10% banana+NAA 1.5mg/L+IBA (indolebutyric acid) 0.8mg/L+1% sucrose, and pH value is 5.5.The cinnabar of a last step gained is transferred on the root media under aseptic condition blue strong sprout; Cultivation temperature is 23 ℃, intensity of illumination 1800LX, and 10h/d begins to occur growing tall and 5-6 sheet leaf through cultivating 30 days plant, grows many meats, green aerial root at the base portion of seedling, forms complete plant at last.Final rooting rate is 86.3%.
Embodiment 2
(1) explant collection
The 10-11 month is the blue capsule mature period of cinnabar, when the capsule appearance transfers brown to, reaches morphological maturity and gathers when not ftractureing as yet.Time is too early, and seed embryonic development not umbra rings germination rate; Time is too late, and capsule breaks, and can cause unnecessary loss;
(2) sterilization
Get the blue uncracked capsule of cinnabar, flowing water flushing 30min, after handling 30s with 75% alcohol on the superclean bench again with 0.1% mercuric chloride liquid immersion 15min; Remove mercuric chloride solution; With aseptic water washing 5 times, be no less than 5min at every turn, fruit surface moisture is blotted with aseptic paper in the back.
(3) sowing
The MS medium is to the sprouting of seed and grow preferably, and sucrose concentration 2%, pH value are 6.0, and 6-BA (benzyladenine) concentration 0.1mg/L (mg/litre) is fit to seed germination and growth.Under aseptic condition, with the blue planting seed of the cinnabar of sterilizing on the sowing medium of manual work preparation.Cultivation temperature is 25 ℃, intensity of illumination 2000LX (lux), and light application time 10h/d (hour/day) cultivated 40 days, treated that culture expands when forming former spheroid, promptly begins next stage.
(4) propagation
The optimum medium of the blue former spheroid proliferation and differentiation cultivation stage of cinnabar is MS+NAA 0.2mg/L+6-BA (6 a benzyladenine) 1mg/L+2% sucrose, and pH value is 6.0.A last step gained culture is being transferred under the aseptic condition on the artificial proliferated culture medium of preparing.Cultivation temperature is 25 ℃, intensity of illumination 2000LX, and light application time 10h/d, through cultivating about 40 days, the former spheroid that expands begins to occur the blue seedling of the tender cinnabar of children, can seedling be transferred on the strong seedling culture base when treating seedling length to routine program desired height in strong sprout.
(5) strong sprout
The optimum medium in blue stage in strong sprout of cinnabar is MS+10% banana+NAA 1.5mg/L+2% sucrose, and pH value is 6.0.The blue seedling of the cinnabar of a last step gained is being transferred under the aseptic condition on the artificial strong seedling culture base of preparing.Cultivation temperature is 25 ℃, intensity of illumination 2000LX, and light application time 10h/d begins to occur 2-3 sheet leaf through the seedling of cultivating in 30 days, forms independently plant, treats that plant is long when routine is taken root the program desired height, can it be transferred on the root media.
(6) take root
The take root medium in stage of cinnabar orchid is MS+10% banana+NAA 2mg/L+IBA (indolebutyric acid) 1mg/L+2% sucrose, and pH value is 6.0.The cinnabar of a last step gained is transferred on the root media under aseptic condition blue strong sprout.Cultivation temperature is 25 ℃, intensity of illumination 2000LX, and light application time 10h/d begins to occur growing tall and 5-6 sheet leaf through cultivating 50 days plant, grows many meats, green aerial root at the base portion of seedling, forms complete plant at last.Final rooting rate is 95%.
Embodiment 3
(1) explant collection
The 10-11 month is the blue capsule mature period of cinnabar, when the capsule appearance transfers brown to, reaches morphological maturity and gathers when not ftractureing as yet.Time is too early, and seed embryonic development not umbra rings germination rate; Time is too late, and capsule breaks, and can cause unnecessary loss;
(2) sterilization
Get the blue uncracked capsule of cinnabar, flowing water flushing 35min, after handling 35s with 75% alcohol on the superclean bench again with 0.2% mercuric chloride liquid immersion 15min; Remove mercuric chloride solution; With aseptic water washing 5 times, be no less than 5min at every turn, fruit surface moisture is blotted with aseptic paper in the back.
(3) sowing
The MS medium is to the sprouting of seed and grow preferably, and sucrose concentration 3%, pH value are 6.0, and 6-BA (benzyladenine) concentration 0.5mg/L (mg/litre) is fit to seed germination and growth.Under aseptic condition, with the blue planting seed of the cinnabar of sterilizing on the sowing medium of manual work preparation.Cultivation temperature is 25 ℃, intensity of illumination 2000LX (lux), and light application time 11h/d (hour/day) cultivated 40 days, treated that culture expands when forming former spheroid, promptly begins next stage.
(4) propagation
The optimum medium of the blue former spheroid proliferation and differentiation cultivation stage of cinnabar is MS+NAA 0.3mg/L+6-BA (6 a benzyladenine) 1.2mg/L+3% sucrose, and pH value is 6.0.A last step gained culture is being transferred under the aseptic condition on the artificial proliferated culture medium of preparing.Cultivation temperature is 25 ℃, intensity of illumination 2000LX, and light application time 11h/d, through cultivating about 40 days, the former spheroid that expands begins to occur the blue seedling of the tender cinnabar of children, can seedling be transferred on the strong seedling culture base when treating seedling length to routine program desired height in strong sprout.
(5) strong sprout
The optimum medium in blue stage in strong sprout of cinnabar is MS+15% banana+NAA 2mg/L+3% sucrose, and pH value is 6.0.The blue seedling of the cinnabar of a last step gained is being transferred under the aseptic condition on the artificial strong seedling culture base of preparing.Cultivation temperature is 25 ℃, intensity of illumination 2000LX, and light application time 11h/d begins to occur 2-3 sheet leaf through the seedling of cultivating in 30 days, forms independently plant, treats that plant is long when routine is taken root the program desired height, can it be transferred on the root media.
(6) take root
The take root medium in stage of cinnabar orchid is MS+15% banana+NAA 2.5mg/L+IBA (indolebutyric acid) 1.2mg/L+3% sucrose, and pH value is 6.0.The cinnabar of a last step gained is transferred on the root media under aseptic condition blue strong sprout.Cultivation temperature is 25 ℃, intensity of illumination 2000LX, and light application time 11h/d begins to occur growing tall and 5-6 sheet leaf through cultivating 50 days plant, grows many meats, green aerial root at the base portion of seedling, forms complete plant at last.Final rooting rate is 83.5%.Result of the test and analysis
The 6-BA of table 1 variable concentrations induces the influence of formation to the cinnabar orchid
Observe in the MS medium, add that concentration is respectively 0.1,0.3, the cinnabar orchid is induced performance during the hormone 6-BA of 0.5mg/L.The blue seed of cinnabar changes green at first on the level of 0.1mg/L, the time that forms protocorm is the shortest, and appreciation rate is also a lot of than exceeding of all the other two levels.So it is 0.1mg/L that the cinnabar orchid is induced optimal 6-BA level.
The 6-BA of table 2 variable concentrations is to the influence of the blue propagation of cinnabar
Observe in the MS medium, add that concentration is respectively 0.8,1, protocorm propagation performance during the hormone 6-BA of 1.2mg/L.The blue protocorm of cinnabar average plant height on the level of 0.1mg/L is the highest, and average leaf is long the longest, and the sturdy degree of stem is good than all the other two levels also.So the optimal 6-BA level of the blue propagation of cinnabar is 1mg/L.
The NAA of table 3 variable concentrations is to the cinnabar influence in blue strong sprout
Observe in the MS medium, add that concentration is respectively 1,1.5, the blue performance in strong sprout of cinnabar during the hormone NAA of 2mg/L.1.5mg/L root is long the longest on the level, radical is maximum, and leaf is long the longest, and rooting rate is also a lot of than exceeding of all the other two levels.Though the NAA rooting rate of hormone 1mg/L than the height of 2mg/L a bit, the quality of its plant is not fine, and blade has the situation of jaundice the cultivation in later stage to occur being unfavorable for.The NAA of 2mg/L is on rooting rate, and it is all much lower to compare other level of aggregation of two.
The influence that the NAA of table 4 variable concentrations is taken root to the cinnabar orchid
Observe in the MS medium, add that concentration is respectively 1.5,2, the tissue cultivating seedling performance of taking root during the hormone NAA of 2.5mg/L.Root is long the longest on the 2mg/L level, and radical is maximum, and plant height is the highest, and survival rate is also a lot of than exceeding of all the other two levels.Though being the NAA survival rate of 1.5mg/L, hormone reached 82.3%, relatively poor, the tiny and poor growth of its root system.Hormone be the NAA survival rate of 2.5mg/L than other two groups low, to reach radical slowly very few because of the too high plant strain growth of hormone.
Adopt method for tissue culture of the present invention, carry out the blue in-vitro propagate work of cinnabar, be the blue germ plasm resource of protection cinnabar, carry out the blue genetic improvement of cinnabar, promote the blue factorial seedling growth of cinnabar, effective way is provided.
Claims (7)
1. the blue method for tissue culture of cinnabar is characterized in that this method may further comprise the steps:
(1) explant collection
The 10-11 month is the blue capsule mature period of cinnabar, when the capsule appearance transfers brown to, reaches morphological maturity and gathers when not ftractureing as yet; Time is too early, and seed embryonic development not umbra rings germination rate; Time is too late, and capsule breaks, and can cause unnecessary loss;
(2) sterilization
Get the blue uncracked capsule of cinnabar; Flowing water flushing 25-35min; After handling 25-35s with 70-75% alcohol on the superclean bench,, remove mercuric chloride solution, with aseptic water washing 4-5 time again with 0.1-0.2% mercuric chloride liquid immersion 10-15min; Be no less than 4-5min, fruit surface moisture is blotted with aseptic paper in the back at every turn;
(3) sowing
Under aseptic condition, with the blue planting seed of the cinnabar of sterilizing on the sowing medium of manual work preparation; Cultivated 30-40 days, and treated that culture expands when forming former spheroid, promptly began next stage;
(4) propagation
A last step gained culture is being transferred under the aseptic condition on the artificial proliferated culture medium of preparing; Through cultivating about 30-40 days; The former spheroid that expands begins to occur the blue seedling of the tender cinnabar of children, can seedling be transferred on the strong seedling culture base when treating seedling length to routine program desired height in strong sprout;
(5) strong sprout
The blue seedling of the cinnabar of a last step gained is being transferred under the aseptic condition on the artificial strong seedling culture base of preparing; Seedling through cultivation in 25-35 days begins to occur 2-3 sheet leaf, forms independently plant, treats that plant length can be transferred to it on root media when routine is taken root the program desired height;
(6) take root
The cinnabar of a last step gained is transferred on the root media under aseptic condition blue strong sprout, begins to occur growing tall and 5-6 sheet leaf, grow many meats, green aerial root, form complete plant at last at the base portion of seedling through cultivating 30-50 days plant; Final rooting rate is up to 80-95%;
Described seed germination medium comprises: MS+ benzyladenine 6-BA 0.1-0.5mg/L+1-3% sucrose;
Described proliferation and differentiation medium comprises: MS+ methyl NAA 0.1-0.3mg/L+6 benzyladenine 6-BA0.8-1.2mg/L+1-3% sucrose;
Described strong seedling culture base comprises: MS+10-15% banana+methyl NAA 1-2mg/L+1-3% sucrose;
Described root media comprises: MS+10-15% banana+methyl NAA 1.5-2.5mg/L+ indolebutyric acid IBA 0.8-1.2mg/L+1-3% sucrose.
2. the blue method for tissue culture of cinnabar according to claim 1 is characterized in that described MS minimal medium is made up of macroelement, trace element and organic principle.
3. the blue method for tissue culture of cinnabar according to claim 1 is characterized in that said seed germination medium component is preferably MS+ benzyladenine 6-BA 0.1mg/L+2% sucrose.
4. the blue method for tissue culture of cinnabar according to claim 1 is characterized in that said proliferation and differentiation medium component is preferably MS+ methyl NAA0.2mg/L+ benzyladenine 6-BA 1mg/L+2% sucrose.
5. the method for tissue culture that cinnabar according to claim 1 is blue is characterized in that said strong seedling culture based component is preferably MS+10% banana+methyl NAA 1.5mg/L+2% sucrose.
6. the blue method for tissue culture of cinnabar according to claim 1 is characterized in that said culture of rootage based component is preferably MS+10% banana+methyl NAA 2mg/L+ indolebutyric acid IBA 1mg/L+2% sucrose.
7. the method for tissue culture that cinnabar according to claim 1 is blue is characterized in that each medium pH value is 5.5-6.0, and cultivation temperature is 23-25 ℃, intensity of illumination 1600-2000LX, light application time 9-12h/d.
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| CN113796315A (en) * | 2021-10-13 | 2021-12-17 | 湖南文理学院 | Quick propagation method of endangered wild plant cymbidium floribundum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113796315A (en) * | 2021-10-13 | 2021-12-17 | 湖南文理学院 | Quick propagation method of endangered wild plant cymbidium floribundum |
| CN113796315B (en) * | 2021-10-13 | 2022-05-17 | 湖南文理学院 | Quick propagation method of endangered wild plant cymbidium floribundum |
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