CN102239808B - Tissue culture medium and tissue culture method of silvergrass - Google Patents
Tissue culture medium and tissue culture method of silvergrass Download PDFInfo
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- CN102239808B CN102239808B CN 201110207383 CN201110207383A CN102239808B CN 102239808 B CN102239808 B CN 102239808B CN 201110207383 CN201110207383 CN 201110207383 CN 201110207383 A CN201110207383 A CN 201110207383A CN 102239808 B CN102239808 B CN 102239808B
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- 239000003104 tissue culture media Substances 0.000 title claims description 11
- 238000012136 culture method Methods 0.000 title abstract description 6
- 241000878007 Miscanthus Species 0.000 title abstract 4
- 230000006698 induction Effects 0.000 claims abstract description 25
- 229920001817 Agar Polymers 0.000 claims abstract description 15
- 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 abstract description 15
- 229930006000 Sucrose Natural products 0.000 claims abstract description 15
- 239000008272 agar Substances 0.000 claims abstract description 15
- 230000035755 proliferation Effects 0.000 claims abstract description 14
- 229960004793 sucrose Drugs 0.000 claims abstract description 14
- 230000001939 inductive effect Effects 0.000 claims abstract description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 10
- 235000014676 Phragmites communis Nutrition 0.000 claims description 33
- 239000002609 medium Substances 0.000 claims description 17
- 238000005286 illumination Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 235000013681 dietary sucrose Nutrition 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 230000003203 everyday effect Effects 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 abstract description 7
- 239000001963 growth medium Substances 0.000 abstract 5
- 230000001850 reproductive effect Effects 0.000 abstract 2
- 239000012883 rooting culture medium Substances 0.000 abstract 2
- 230000002062 proliferating effect Effects 0.000 abstract 1
- 239000007787 solid Substances 0.000 abstract 1
- 238000009395 breeding Methods 0.000 description 8
- 230000001488 breeding effect Effects 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 1
- 241001074119 Miscanthus sacchariflorus Species 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 1
- 229960001669 kinetin Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- WHFQORJQTYYOBK-UHFFFAOYSA-N purine-6,6-diamine Chemical compound NC1(N)N=CN=C2N=CN=C12 WHFQORJQTYYOBK-UHFFFAOYSA-N 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to adventitious bud induction, proliferation and rooting culture mediums of silvergrass, which comprise four culture mediums at different culture stages: an initial culture medium(1), an adventitious bud inducing culture medium (2), an adventitious bud proliferating culture medium (3) and an inducing and rooting culture medium (4); in the four culture mediums, an MS(Murashig and Skoog) culture medium is used as a basic culture medium, matters such as cane sugar, agar and the like are added, and the pH is 5. A tissue culture method for adventitious bud induction, proliferation and rooting of silvergrass by adopting the four culture mediums comprises the following five steps of: 1. collection and disinfection of explantes;2. initial culture; 3. adventitious bud induction; 4. proliferation and culture of the adventitious buds; and 5. rooting and induction. The cultivation of the five steps lays a solid foundation for rapidly cultivating silvergrass seedlings with high quality. By adopting the tissue culture of the culture mediums, regenerated plants can keep excellent characters of a female parent, the reproductive speed of seedlings is increased, the reproductive output is increased, seeds can be effectively used, the seedling cultivation period is shortened and the society requirement is satisfied.
Description
Technical field
The present invention relates to take reed seed as explant, carry out substratum and the tissue culture method of tissue culture.
Background technology
Reed (Miscanthus sacchariflorus (Maxim.) Benth) is that the Gramineae awns is drafted a document this land and water two living plants.Under physical environment, the sturdy paleate rhizome of reed tool, stalk is upright, without hair, tool more piece, tool whisker on the joint, high 100～400cm.It is in all parts of the country to distribute, and grows on mountain valley, hillside and the beach more.Reed happiness light, cold-resistant, waterlogging, strong stress resistance, the biomass of over-ground part is large.In environment protection, Landscape construction, biomass energy, pulping and paper-making, sand prevention dyke strengthening, huge potentiality are arranged, also can replace the raw material of timber and plastics do manufacturing wood-based plate etc.Therefore, reed is that exploitation is worth very high important energy source plant and material plant.The tissue culture of awns platymiscium begins to have successively report from twentieth century the nineties, but focuses mostly in the research of the aspects such as awns and Caulis Miscanthis floriduli callus and plant regeneration.Through retrieval, find so far by reed tissue culture, set up the fast traditional font of indefinite bud system, and breeding takes root, cultivate into the relevant report of whole plant.
Summary of the invention
Group culturation rapid propagating technology is used as economic, efficient propagation method on a lot of plants, and the cycle is short, breeding coefficient is high, cost is low, is not subjected to the restriction in time and space.In the existing breeding technology, the cost of artificial breeding was high when reed was developed as items in commerce, and its germinative seed amount is few, is difficult to the big area artificial growth.For satisfying the needs of the artificial fast breeding of reed, big area high-yield cultivating, the technical problem to be solved in the present invention be seed take reed as explant, set up substratum and the tissue culture breeding method of adventitious bud inducing, propagation and the root induction of a kind of reed.
Technical problem of the present invention solves by following technical solution:
The tissue culture medium (TCM) of reed comprises four kinds of substratum of different Induction periods, and raw material and the additional content thereof of various substratum are respectively:
(1) first culture base: take MS as minimum medium, additional saccharose 20～30g/L, agar 6.5～8.5g/L, the pH value of substratum is 5.7;
(2) adventitious bud induction culture base: take MS as minimum medium, additional 6-BA0.5～2.0mg/L, NAA 0.05～0.1mg/L, sucrose 20～30g/L, agar 6.5～8.5g/L, the pH value of substratum is 5.7;
(3) adventitious bud proliferation substratum: take MS as minimum medium, additional 6-BA0.3～1.5mg/L, NAA 0.1～0.2mg/L, sucrose 20～30g/L, agar 6.5～8.5g/L, the pH value of substratum is 5.7;
(4) root induction substratum: take MS as minimum medium, additional saccharose 20～30g/L, agar 6.5～8.0g/L, NAA 0.2～1.0mg/L, the pH value of substratum is 5.7.
Carry out the method for reed tissue culture with above-mentioned substratum, carry out as follows:
(1) explant collection and sterilization: choose healthy and strong reed seed, carrying out successively washing composition, to clean on 1min, tap water flushing 2h, the Bechtop with sterilize 30s, aseptic water washing 3～4 times, concentration of 75% ethanol be that 0.525% NaClO vacuum-sterilisation 20min, aseptic water washing blot seed-coat moisture with sterilization filter paper 5～6 times afterwards;
(2) first culture: in the seed access first culture base that will reed gets under step (1) aseptic condition, wherein placed dark cabinet to cultivate in front 7 days, placing light dark period in rear 7 days is to cultivate under the condition of the dark 8h of light 16h/, and culture temperature is 25 ± 2 ℃, and reed gets seedling;
(3) adventitious bud inducing: cut radicle with growing healthy and strong seedling behind the first culture, in the access adventitious bud induction culture base, through the 15d inducing culture, reed gets indefinite bud, and every day, light dark period was the dark 8h of light 16h/, and intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 2 ℃;
(4) adventitious bud proliferation is cultivated: in the indefinite bud access adventitious bud proliferation substratum that reed is got, through 28 days multiplication culture, growth coefficient was 10～20,28 days subcultures 1 time, and subculture is 3 times altogether; Illumination cultivation, light dark period are the dark 8h of light 16h/, and intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 2 ℃, turns out indefinite bud;
(5) root induction: choose in the healthy and strong indefinite bud access root media, carry out root induction, cultivated 21 days, rooting rate reaches 99%; Illumination cultivation, light dark period are the dark 8h of light 16h/, and intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 1 ℃, turns out seedling, the whole process of group training finishes.
The invention has the beneficial effects as follows:
Using reed seed is explant, carries out adventitious bud inducing and carries out root culture with the seedling after sprouting, and effectively improves the breeding coefficient of reed, commercially produces the seedling source that provides sufficient for reed.Compare with seed propagation, tissue culture method can effectively utilize seed, improves germination rate, shortens growing-seedling period, seed only needs can to sprout in 2 weeks under the aseptic condition, is 100～150 times of seed propagation at the breeding coefficient of a growth cycle inner tissue culture method of 6 months by a definite date.
Embodiment
The present invention is described in further detail below in conjunction with embodiment: in the substratum of this reed adventitious bud inducing and adventitious bud proliferation, select MS to make minimum medium, this is through all additional saccharose 20～30g/L, agar 6.5～8.5g/L, 6-BA are 6-aminoadenine 0～2.0mg/L separately with MS, 1/2MS, four kinds of minimum mediums of B5, N6, KT is that kinetin 0～2.0mg/L, NAA are α-naphthaleneacetic acid 0～0.1mg/L, pH value 5.7, test, every group is repeated 3 times, and every kind of minimum medium has the simultaneous test of 20 explants to draw.Because do to process 28 days in the minimum medium with MS after, uncertain buds growth is best, and the indefinite bud number of inducing is maximum, best in quality, determines to select MS to make minimum medium.
In a word, its proportioning value of substratum disclosed in this invention, temperature, peptizer etc. are also available to be substituted such as white sugar, quartzy agar etc., and the proportioning value of each raw material of substratum also can adopt respectively replacing near value of preferred plan definite value of the present invention.
Given below is embodiments of the invention.
The tissue culture medium (TCM) of reed comprises four kinds of substratum of different Induction periods.Now raw material and the additional content thereof of four kinds of substratum are listed in table 1-4 by seven embodiment respectively:
First culture base (table 1):
Adventitious bud induction culture base (table 2):
Adventitious bud proliferation substratum (table 3)
Root induction substratum (table 4)
Embodiment 1 (raw material of corresponding embodiment and additional content among the synopsis 1-4):
(1) first culture base: take MS as minimum medium, additional saccharose 20g/L, agar 6.5g/L, the pH value of substratum is 5.7;
(2) adventitious bud induction culture base: additional 6-BA0.5mg/L, NAA 0.05mg/L, sucrose 20g/L, agar 6.5g/L take MS as minimum medium, the pH value of substratum is 5.7;
(3) adventitious bud proliferation substratum: the MS minimum medium adds 6-BA 0.3mg/L, NAA 0.1mg/L, sucrose 20g/L, agar 6.5g/L, and the pH value of substratum is 5.7;
(4) root induction substratum: take MS as minimum medium, additional saccharose 20g/L, agar 6.5g/L, NAA 0.2mg/L, the pH value of substratum is 5.7.
Carry out the method for reed tissue culture carries out as follows with above-mentioned substratum:
(1) explant collection and sterilization: choose healthy and strong reed seed, carrying out successively washing composition, to clean on 1min, tap water flushing 2h, the Bechtop with sterilize 30s, aseptic water washing 3～4 times, concentration of 75% ethanol be that 0.525% NaClO vacuum-sterilisation 20min, aseptic water washing blot seed-coat moisture with sterilization filter paper 5～6 times afterwards;
(2) first culture: in reed seed access first culture base that will reed gets under step (1) aseptic condition, wherein placed dark cabinet to cultivate in front 7 days, placing light dark period in rear 7 days is to cultivate under the condition of the dark 8h of light 16h/, and culture temperature is 25 ± 2 ℃, and reed gets seedling;
(3) adventitious bud inducing: cut radicle with growing healthy and strong seedling behind the first culture, in the access adventitious bud induction culture base, through the 15d inducing culture, reed gets indefinite bud, and every day, light dark period was the dark 8h of light 16h/, and intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 2 ℃;
(4) adventitious bud proliferation is cultivated: the indefinite bud that reed is got accesses in the adventitious bud proliferation substratum, and through 28 days multiplication culture, growth coefficient was 10～20, subculture was 1 time in 28 days, and subculture is 3 times altogether, illumination cultivation, light dark period is the dark 8h of light 16h/, and intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 2 ℃, turns out indefinite bud;
(5) root induction: choose in the healthy and strong indefinite bud access root media, carry out root induction, cultivated 21 days, rooting rate reaches 99%, illumination cultivation, and light dark period is the dark 8h of light 16h/, intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 1 ℃, turns out seedling, all processes of group training finishes.
MS minimum medium and other related chemicals can get by commercially available reed in the above-mentioned substratum, press the catalogue adapted, and be known for going together.
All the other implement 2-7, and all corresponding embodiment raw material and additional content among the synopsis 1-4 get tissue culture medium (TCM) and group training seedling with embodiment 1 same procedure reed.
Among seven embodiment, preferred with embodiment 1-3.
Claims (2)
1. the tissue culture medium (TCM) of reed is characterized in that comprising four kinds of substratum of different Induction periods, and raw material and the additional content thereof of various substratum are respectively:
(1) first culture base: take MS as minimum medium, additional saccharose 20～30g/L, agar 6.5～8.5g/L, the pH value of substratum is 5.7;
(2) adventitious bud induction culture base: take MS as minimum medium, additional 6-BA0.5～2.0mg/L, NAA 0.05～0.1mg/L, sucrose 20～30g/L, agar 6.5～8.5g/L, the pH value of substratum is 5.7;
(3) adventitious bud proliferation substratum: take MS as minimum medium, additional 6-BA0.3～1.5mg/L, NAA 0.1～0.2mg/L, sucrose 20～30g/L, agar 6.5～8.5g/L, the pH value of substratum is 5.7;
(4) root induction substratum: take MS as minimum medium, additional saccharose 20～30g/L, agar 6.5～8.0g/L, NAA 0.2～1.0mg/L, the pH value of substratum is 5.7.
2. one kind is obtained the method for tissue culture with substratum claimed in claim 1, it is characterized in that through following steps:
(1) explant collection and sterilization: choose the healthy and strong seed that obtains, carrying out successively washing composition, to clean on 1min, tap water flushing 2h, the Bechtop with sterilize 30s, aseptic water washing 3～4 times, concentration of 75% ethanol be 0.525% NaClO vacuum-sterilisation 20min, and aseptic water washing blots seed-coat moisture with sterilization filter paper 5～6 times afterwards;
(2) first culture: what will obtain under step (1) aseptic condition obtains in the seed access first culture base, wherein placed dark cabinet to cultivate in front 7 days, placing light dark period in rear 7 days is to cultivate under the condition of the dark 8h of light 16h/, and culture temperature is 25 ± 2 ℃, obtains seedling;
(3) adventitious bud inducing: cut radicle with growing healthy and strong seedling behind the first culture, in the access adventitious bud induction culture base, through the 15d inducing culture, obtain indefinite bud, every day, light dark period was the dark 8h of light 16h/, and intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 2 ℃;
(4) adventitious bud proliferation is cultivated: in the indefinite bud access adventitious bud proliferation substratum that obtains, through 28 days multiplication culture, growth coefficient was 10～20,28 days subcultures 1 time, and subculture is 3 times altogether; Illumination cultivation, light dark period are the dark 8h of light 16h/, and intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 2 ℃, turns out indefinite bud;
(5) root induction: choose in the healthy and strong indefinite bud access root media, carry out root induction, cultivated 21 days, rooting rate reaches 99%; Illumination cultivation, light dark period are the dark 8h of light 16h/, and intensity of illumination is 40～50 μ molm
-2S
-1, culture temperature is 25 ± 1 ℃, turns out seedling, the whole process of group training finishes.
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| CN 201110207383 CN102239808B (en) | 2011-07-22 | 2011-07-22 | Tissue culture medium and tissue culture method of silvergrass |
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| CN 201110207383 CN102239808B (en) | 2011-07-22 | 2011-07-22 | Tissue culture medium and tissue culture method of silvergrass |
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| CN102239808A CN102239808A (en) | 2011-11-16 |
| CN102239808B true CN102239808B (en) | 2013-04-24 |
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| CN102524068B (en) * | 2011-12-29 | 2014-01-01 | 湖北光芒能源植物有限公司 | Method for quickly reproducing miscanthus plant miscanthus and triarrhena hybrid NO.9 somatic embryo by tissue culture |
| CN105039396A (en) * | 2015-07-08 | 2015-11-11 | 浙江大学 | Silvergrass seed callus transforming method mediated by agrobacterium and application of method |
| CN113179950B (en) * | 2021-05-18 | 2022-06-07 | 广西壮族自治区农业科学院 | Method for sterilizing explants by using reduced pressure |
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| CN101711504B (en) * | 2009-12-14 | 2012-04-25 | 湖南农业大学 | Rapid propagation method of triarrhena sacchariflora |
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