CN101491213B - Freshwater reeds non-callus culture method - Google Patents
Freshwater reeds non-callus culture method Download PDFInfo
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- CN101491213B CN101491213B CN2009100256642A CN200910025664A CN101491213B CN 101491213 B CN101491213 B CN 101491213B CN 2009100256642 A CN2009100256642 A CN 2009100256642A CN 200910025664 A CN200910025664 A CN 200910025664A CN 101491213 B CN101491213 B CN 101491213B
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- 235000014676 Phragmites communis Nutrition 0.000 title claims abstract description 37
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 14
- 238000012136 culture method Methods 0.000 title claims description 5
- 239000013505 freshwater Substances 0.000 title claims description 4
- 244000089486 Phragmites australis subsp australis Species 0.000 title 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 230000001954 sterilising effect Effects 0.000 claims description 15
- 229920001817 Agar Polymers 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- 239000002689 soil Substances 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 10
- 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 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 230000003203 everyday effect Effects 0.000 claims description 8
- 238000005286 illumination Methods 0.000 claims description 8
- 230000001939 inductive effect Effects 0.000 claims description 6
- 239000006870 ms-medium Substances 0.000 claims description 6
- 239000012879 subculture medium Substances 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 244000273256 Phragmites communis Species 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 3
- 235000019362 perlite Nutrition 0.000 claims description 3
- 239000010451 perlite Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 230000007704 transition Effects 0.000 claims description 3
- 230000000763 evoking effect Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 238000010276 construction Methods 0.000 abstract 1
- 238000002054 transplantation Methods 0.000 abstract 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 10
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 10
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 9
- 239000005972 6-Benzyladenine Substances 0.000 description 6
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229920002955 Art silk Polymers 0.000 description 1
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 241000283986 Lepus Species 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 230000011681 asexual reproduction Effects 0.000 description 1
- 238000013465 asexual reproduction Methods 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention belongs to the technical field of plant tissue culture. A reed stem is used to establish a rapid in-vitro propagation method through the non-callus culture and obtain a complete high quality reed plant. The method has a simple process, a short propagation cycle, the inductivity as high as 92.3 percent, and the transplantation survival rate as high as 90.2 percent. The method can be used to supply high quality reed germchits rapidly, meet the increasingly improved market demand, and give full play to the actions of the reed in the ecological environment construction, water environment comprehensive treatment and wetland restoration projects.
Description
One, technical field
The invention belongs to field of plant tissue culture technique.
Two, background technology
The natural population of reed (Phragmites australis) is mostly occurred at the shallow water wetland, is common in pond, river alongshore, beach, many water area, He Xi limit.Reed is as economic crops, and the reed stalk can be used for papermaking and artificial silk, establishment reed mat, straw mattress; The reed in vegetative period contains amounts of protein and sugar, is good feed; The leaf of reed, flower, stem, root, bamboo shoot all can be used as medicine.Simultaneously, reed has important ecological functions, protects native dyke strengthening, the wave unhurried current that disappears, short silt corrosion protection etc.; In recent years, reed resistant, decontamination, the ability of improving eutrophication water quality enjoys attention, and in lakeside band, river sloping bank and marsh wetland recovered and rebuild, reed became essential plant, by establishing in large scale.Along with a large amount of enforcements that China's improvement of the ecological environment, the water environment comprehensive regulation and wetland recover engineering, seedling of reed and provenance demand can be more and more urgent, and current technology will be difficult to satisfy the market demand that grows to even greater heights; Therefore, a kind of method that high-quality reed seedling can be provided fast of invention needs that are inevitable.
Through Chinese patent net and relevant paper retrieved web, find at present more existing reports about the reed tissue culture, cultivate by callus with seed in " formation of reed embryo callus and the regeneration of plant " as Wu Guoliang etc. and to obtain whole plant, but the cycle of callus is longer, and process is complicated; Wang Xiyou has only studied callus Growth in " reed embryo callus Study on Growth ", do not grow whole plant; Guo Rui also is to obtain plant by callus culture in " research of blast generation of salt tolerant reed stipes organizer and plant regeneration ", has long problem of cycle equally; In addition, in the patent " asexual reproduction method of salt tolerant reed " of Yang Yingen etc., also describe the process that obtains whole plant by callus culture, had long problem of cycle equally.
Three, summary of the invention
The problem that the present invention need solve is: invent the method for a kind of quick in-vitro propagate reed, make to obtain a large amount of high-quality same specification reed seedlings in a short time, satisfy the needs in wetland recovery and the reconstruction engineering, promote the recovery of environment.
Technical scheme of the present invention is:
(1) the tall and big reed of Luoma Lake growing way is selected in open-air seed selection;
(2) sterilization, the reed stem that will fetch from the field is cut into segment, every section 2-3cm is long, under distilled water, clean, then on super-clean bench with 70% alcohol immersion 30s, rinse with distilled water and to wash 2-3 time, use 0.1%HgCl thereafter
2Aqueous solution sterilization 5min is again with sterile distilled water washing 4-5 time;
(3) the stem section of the band bud original hase of the reed after will sterilizing is inoculated into evoking adventive bud on the inducing culture, has added polyvinylpyrrolidone (PVP), 6-benzyladenine (BA) and methyl (NAA) in the inducing culture; Temperature is that 25 2 ℃ in soil, light intensity are 2000lx, illumination every day 14h; Cultivate 7-23d, the bud of most stem sections can be grown 3cm about two weeks;
(4) indefinite bud that produces is transferred to carried out successive transfer culture on the subculture medium, added PVP, BA and NAA in the subculture medium; Temperature is that 25 2 ℃ in soil, light intensity are to cultivate 11-24d under 2000lx, the illumination every day 14h condition;
(5) lure root on the root media when bud length moves on to during to 4-5cm, lure and added PVP, IBA and NAA in the root medium; Temperature is that 25 2 ℃ in soil, light intensity are 2000lx, illumination every day 14h;
(6) when the test tube shoot root grows to about 2cm, open bottleneck and add a small amount of distilled water hardening 2-3d, then root agar is cleaned, move into transition in the perlite, move into soil behind the 3-5d.
The culture medium prescription of each cultivation stage (hormone combination) is as following table:
The culture medium prescription in each stage
| The group training stage | The medium constituent |
| Adventitious bud inducing | MS medium+3% sucrose+0.43% agar+BA 6mg/L+NAA 0.2mg/L+PVP 50mg/L |
| The indefinite bud subculture | MS medium+3% sucrose+0.43% agar+BA 5mg/L+NAA 0.2mg/L+PVP 50mg/L |
| Plant takes root | 1/2MS medium+3% sucrose+0.43% agar+IBA 1.0mg/L+NAA 1.0mg/L+PVP 50mg/L |
Annotate: because pH can slightly descend behind the medium sterilization, so the pH of medium transfers to 6.1 when preparing; All medium are 121 ℃ of sterilization 20min in high-pressure sterilizing pot, and culture medium after sterilization is placed on the super-clean bench behind the 1d usefulness again, to dry moisture film.
The present invention has set up the integral framework of a quick in-vitro propagate reed, compared with prior art its beneficial effect is: (1) obtains complete reed tissue cultivating seedling by non-callus culture method, its process is simple, the cycle is short, and growing complete test-tube plantlet by explant only need about 35-65d; (2) inductivity is up to 92.3%, and rooting rate is also up to 95.8%; (3) transplanting survival rate height is 90.2%; (4) eliminated browning by adding 50mg/L PVP, stoped the influence of brownization incubation; (5) material therefor is taken at Luoma Lake among the present invention, and plant is tall and big, and on average about 6m can be widely used in shore, river course bank and lakeside band and recover.
Four, embodiment
1. explant sterilization
The reed stem that to fetch from the field is cut into segment, and every section 2-3cm is long, under distilled water, clean, then on super-clean bench with 70% alcohol immersion 30s, rinse with distilled water and to wash 2-3 time, use 0.1%HgCl thereafter
2Aqueous solution sterilization 5min is again with sterile distilled water washing 4-5 time;
2. indefinite bud induces
On super-clean bench, stem section after the sterilization is inoculated on the inducing culture, being MS medium+3% sucrose+0.43% agar+BA6mg/L+NAA0.2mg/L+PVP 50mg/L, is that 25 2 ℃ in soil, light intensity are to cultivate under the condition of 2000lx, illumination every day 14h in temperature.Cultivate 7-23d, the bud of most stem sections can be grown 3cm about two weeks; Best when BA is 6mg/l, inductivity is up to 92.3%.
3. successive transfer culture
The bud that induces is transferred on the subculture medium, i.e. MS medium+3% sucrose+0.43% agar+BA5mg/L+NAA0.2mg/L+PVP 50mg/L is that 25 2 ℃ in soil, light intensity are to cultivate 11-24d under the condition of 2000lx, illumination every day 14h in temperature.
4. culture of rootage
Clump shape indefinite bud is separated into single bud, move on on the root media, be 1/2MS medium+3% sucrose+0.43% agar+IBA1.0mg/L+NAA1.0mg/L+PVP50mg/L, temperature is that 25 2 ℃ in soil, light intensity are that 2000lx, every sight are according under the condition of 14h, turn out complete regeneration plant, the rooting of vitro seedling rate reaches 95.8%.
5, transplant
When the test tube shoot root grows to the 2cm left and right sides, open bottleneck and add a small amount of distilled water hardening 2-3d, then root agar is cleaned, move into transition in the perlite, move into soil behind the 3-5d, survival rate reaches 90.2%.
Claims (2)
1. a freshwater reeds non-callus culture method is characterized in that carrying out non-callus with the stem section of tall growing reed cultivates, and breeds complete reed seedling fast, is made of following steps:
(1) will pick up from open-air reed stem section and be cut into segment, every section 2-3cm is long, under distilled water, clean, then on super-clean bench with 70% alcohol immersion 30s, rinse with distilled water and to use 0.1%HgCl after washing 2-3 time
2Aqueous solution sterilization 5min is again with sterile distilled water washing 4-5 time;
(2) the reed stem section after will sterilizing is inoculated into evoking adventive bud on the inducing culture; Inducing culture is MS medium+3% sucrose+0.43% agar+BA6mg/L+NAA0.2mg/L+PVP 50mg/L; Temperature is that 25 2 ℃ in soil, light intensity are 2000lx, illumination every day 14h;
(3) indefinite bud that produces is transferred to carried out successive transfer culture on the subculture medium; Subculture medium is MS medium+3% sucrose+0.43% agar+BA5mg/L+NAA0.2mg/L+PVP 50mg/L; Temperature is that 25 2 ℃ in soil, light intensity are 2000lx, illumination every day 14h;
(4) on root media, lure root, produce complete test-tube plantlet; Root media is 1/2MS medium+3% sucrose+0.43% agar+IBA1.0mg/L+NAA1.0mg/L+PVP50mg/L; Temperature is that 25 2 ℃ in soil, light intensity are 2000lx, illumination every day 14h;
(5) when the long 2cm of test tube shoot root, open bottleneck and add a small amount of distilled water hardening 2-3d, then root agar is cleaned, move into transition in the perlite, move into soil behind the 3-5d.
2. according to the described freshwater reeds non-callus culture method of claim 1, transfer to 6.1 when it is characterized in that the pH preparation of all medium; All medium are 121 ℃ of sterilization 20min in high-pressure sterilizing pot, and culture medium after sterilization is placed on the super-clean bench behind the 1d usefulness again, to dry moisture film.
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| CN2009100256642A CN101491213B (en) | 2009-03-05 | 2009-03-05 | Freshwater reeds non-callus culture method |
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| CN2009100256642A CN101491213B (en) | 2009-03-05 | 2009-03-05 | Freshwater reeds non-callus culture method |
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| CN101491213B true CN101491213B (en) | 2011-03-30 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102150620B (en) * | 2011-03-18 | 2012-10-10 | 广西壮族自治区药用植物园 | Tissue culture and propagation method and induction medium of tinospora capillipes gagnep |
| CN105638464B (en) * | 2015-12-30 | 2018-01-09 | 四川七彩林业开发有限公司 | A kind of tissue culture and rapid propagation method of silk ribbon grass |
| CN106171982A (en) * | 2016-07-12 | 2016-12-07 | 广西壮族自治区中国科学院广西植物研究所 | The breeding method of Arundo donax seedling |
| CN110902834A (en) * | 2019-11-28 | 2020-03-24 | 东华大学 | Device and method for inducing manganese film to cover root surface of emergent aquatic plant |
| CN112293260B (en) * | 2020-11-24 | 2022-08-23 | 福建农林大学 | Method for obtaining tissue culture seedlings of reed in Africa |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100407901C (en) * | 2004-12-13 | 2008-08-06 | 中国科学院植物研究所 | Asexual generation method for salt tolerant reed |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100407901C (en) * | 2004-12-13 | 2008-08-06 | 中国科学院植物研究所 | Asexual generation method for salt tolerant reed |
Non-Patent Citations (3)
| Title |
|---|
| 吴国良，等.芦苇胚性愈伤组织的形成及植株的再生.《植物学报（英文版）》.1987,第29卷(第4期),361-366. |
| 吴国良等.芦苇胚性愈伤组织的形成及植株的再生.《植物学报(英文版)》.1987,第29卷(第4期),361-366. * |
| 王希友.芦苇胚性愈伤组织生长的研究.《现代农业》.2008,(第4期),87-88. * |
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| CN101491213A (en) | 2009-07-29 |
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