CN109588316A - A kind of method for tissue culture of cyperue esculentus - Google Patents
A kind of method for tissue culture of cyperue esculentus Download PDFInfo
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- CN109588316A CN109588316A CN201910067381.8A CN201910067381A CN109588316A CN 109588316 A CN109588316 A CN 109588316A CN 201910067381 A CN201910067381 A CN 201910067381A CN 109588316 A CN109588316 A CN 109588316A
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- culture
- cyperue esculentus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a kind of method for tissue culture of cyperue esculentus.The method for tissue culture of cyperue esculentus disclosed by the invention includes: that cyperue esculentus stem tuber is carried out Fiber differentiation to obtain stem tuber bud in germination medium；Stem tuber bud is subjected to Multiplying culture in proliferated culture medium, obtains proliferation bud；Proliferation bud is subjected to culture of rootage in root media, cyperue esculentus plant is obtained, completes the tissue cultures of cyperue esculentus.The present invention realizes the tissue cultures and Vitro Quick Reproduction of cyperue esculentus using method for tissue culture and germination medium, the proliferated culture medium and root media of cyperue esculentus, optimizes based theoretical for efficiently breeding cyperue esculentus seedling, germ plasm resource and provides technical support.
Description
Technical field
The present invention relates to the method for tissue culture of cyperue esculentus a kind of in field of biotechnology.
Background technique
Cyperue esculentus also known as chufa, underground walnut and ginseng beans, some places are called oily beans.Common English is entitled
Yellow nutsedge, tiger nut, Latin are Cyperus esculentus.Cyperue esculentus category Angiospermae, list
Leaf plant guiding principle, Poales, Cyperaceae, Cyperus.Nutgrass flatsedge platymiscium about 5,500 kinds, be distributed in China shares more than 30 kinds,
But it is a kind of that edible nutgrass flatsedge platymiscium is currently known only cyperue esculentus.
Cyperue esculentus is to be currently known the extraordinary industrial crops that a large amount of greases are uniquely accumulated in stem tuber nutrition organs.Cyperue esculentus
It is raw-eaten or it is ripe eat, can also be beaten and be made into soymilk, can also be milled as edible flour.Cyperue esculentus can be used to extract oil, refine sugar and
Wine brewing, and surplus slag can be used to make feed.Cauline leaf is the good of papermaking, packaging filling and braiding in addition to it can make green manure and greenfeed
Material.Therefore cyperue esculentus integrates grain, oil, feeding, comprehensive utilization value with higher.
But, the biomass of cyperue esculentus and oil content are unstable, no according to growth place, environmental condition and soil regime etc.
Together, it is changed between strain very big.But the excellent variety of existing cyperue esculentus is seldom, the especially cyperue esculentus genetic diversity in China
It is very low, it is relatively difficult that fine quality is cultivated using conventional hybridization.
Tissue culture and it is fast it is numerous be simple, the efficiently effective ways for realizing micropropagation of plants, excellent variety may be implemented
The large-scale production of germplasm innovation, seedling, is used widely in agricultural production.Currently, tissue cultures of cyperue esculentus and again
Raw and genetic conversion system is not yet mature to be established, so that carrying out genetic improvement by very to cyperue esculentus character by molecular breeding
Big limitation.
Summary of the invention
The technical problem to be solved by the present invention is to how carry out the tissue cultures of cyperue esculentus.
In order to solve the above technical problems, present invention firstly provides the method for tissue culture of cyperue esculentus, the method includes
1) and 2):
1) the stem tuber bud of cyperue esculentus is subjected in proliferated culture medium Multiplying culture, obtains proliferation bud；The Multiplying culture
Base is made of solvent and solute, and the solvent is MS culture medium, the solute and its concentration point in the proliferated culture medium
Not Wei 1mg/L 6-BA, 1.5~2% (mass percent concentration) sucrose, pH be 5.8~6；
2) the proliferation bud is subjected in root media culture of rootage, obtains cyperue esculentus plant, completes cyperue esculentus
Tissue cultures.
The stem tuber bud is the bud grown by stem tuber.
Concentration of the sucrose in the proliferated culture medium concretely 2% (mass percent concentration).
The pH of the proliferated culture medium concretely 6.
The root media can be made of solvent and solute, and the solvent is MS culture medium, the solute and its in institute
Stating the concentration in root media is respectively 0.2mg/L IBA, 1.5~2% (mass percent concentration) sucrose, and pH is 5.8~
6。
Concentration of the sucrose in the root media concretely 2% (mass percent concentration).
The pH of the root media concretely 6.
When the stem tuber bud of cyperue esculentus to be carried out to Multiplying culture in proliferated culture medium, subculture can be carried out to obtained proliferation bud
Culture.The number of the squamous subculture is no more than 2 times.The squamous subculture can carry out in the proliferated culture medium.
In the above method, the stem tuber bud can be obtained by the way that cyperue esculentus stem tuber to be carried out to Fiber differentiation in germination medium
It arrives.
The germination medium can be made of solvent and solute, and the solvent is MS culture medium, the solute and its in institute
Stating the concentration in germination medium is respectively 1mg/L NAA, 1.5~2% (mass percent concentration) sucrose, and pH is 5.8~6.
Concentration of the sucrose in the germination medium concretely 2% (mass percent concentration).
The pH of the germination medium concretely 6.
In the above method, the temperature of the Multiplying culture, the culture of rootage and/or the Fiber differentiation can be 24-26
℃。
The humidity of the Multiplying culture, the culture of rootage and/or the Fiber differentiation can be 60-70%.
In the above method, the periodicity of illumination of the Multiplying culture, the culture of rootage and/or the Fiber differentiation can be 14
~16h illumination and 8~10h are dark.
The light intensity of the Multiplying culture, the culture of rootage and/or the Fiber differentiation can be 130~150 μm of olm-2·s-1。
The Multiplying culture, the culture of rootage and/or the Fiber differentiation can be carried out aseptically.
The present invention also provides a kind of complete set of culture medium, the complete set of culture medium includes the proliferated culture medium and the life
Root culture medium.
The complete set of culture medium may also include the germination medium.
Specifically, the complete set of culture medium can be made of the proliferated culture medium and the root media, it can also be by institute
Proliferated culture medium, the root media and the germination medium is stated to form.
The complete set of culture medium has following any purposes:
X1) cyperue esculentus tissue cultures；
X2 cyperue esculentus tissue culture products) are prepared.
Following any applications of the complete set of culture medium, also belong to protection scope of the present invention:
Y1) cyperue esculentus tissue cultures；
Y2) preparation is used for cyperue esculentus tissue culture products；
Y3) cyperue esculentus genetic transformation；
Y4) preparation is used for cyperue esculentus genetic transformation product；
Y5) cyperue esculentus molecular breeding；
Y6) preparation is used for cyperue esculentus molecular breeding product.
It is demonstrated experimentally that the method for tissue culture and complete set of culture medium using cyperue esculentus of the invention can be realized to cyperue esculentus
Tissue cultures and Vitro Quick Reproduction can optimize based theoretical and provide for efficiently breeding cyperue esculentus seedling, germ plasm resource
Technical support.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
The tissue cultures of embodiment 1, cyperue esculentus
1. vegetable material
Cyperue esculentus used be cultivar Cyperus esculentus L.var.sativus Boeck (Yang Z, Ji H,
Liu D.Oil biosynthesis in underground oil-rich storage vegetative tissue:
Comparison of Cyperus esculentus tuber with oil seeds and fruits.Plant Cell
Physiol.2016,57:2519-2540.)。
2. culture medium
(1) liquid germination medium: liquid germination medium is made of solvent and solute, and solvent is MS culture medium, solute
And its concentration in liquid germination medium is respectively 1mg/L NAA (α-naphthaleneacetic acid, α-naphthalene second
Acid), 2% (mass percent concentration) sucrose, pH 6.0.Solid germination medium is from adding agar into liquid germination medium
It obtains, concentration of the agar in solid germination medium is 0.8% (mass percent concentration).
(2) liquid proliferated culture medium: liquid proliferated culture medium is made of solvent and solute, and solvent is MS culture medium, solute
And its concentration in liquid proliferated culture medium is respectively that (6-benzylaminopurine, 6- benzamido group are fast by 1.0mg/L 6-BA
Purine), 2% (mass percent concentration) sucrose, pH 6.0.Solid multiplication culture medium is from adding agar into liquid proliferated culture medium
It obtains, concentration of the agar in solid multiplication culture medium is 0.8% (mass percent concentration).
(3) liquid root media: liquid root media is made of solvent and solute, and solvent is MS culture medium, solute
And its concentration in liquid root media is respectively 0.2mg/L IBA (indole-3-butyric acid, indoles -3- fourth
Acid), 2% (mass percent concentration) sucrose, pH 6.0.Solid root media is from adding agar into liquid root media
It obtains, concentration of the agar in solid root media is 0.8% (mass percent concentration).
3. condition of culture
Culture carries out in growth climate box, and growth climate box condition is as follows: 25.0 ± 1.0 DEG C of temperature, humidity 60-
70%, periodicity of illumination is 16h illumination/8h dark, and light intensity is 150 μm of olm-2·s-1。
4. tissue cultures
(1) sterilizing of cyperue esculentus
The full cyperue esculentus stem tuber chosen fanout free region, have complete bud eye, tap water repeated flushing are clean；Then by stem tuber elder generation
Impregnated 1 minute in 75% (volume ratio) ethanol water, then with sterilized solution (sterilized solution be 10% (matter to available chlorine content
Amount ratio) NaClO aqueous solution in the obtained solution of addition Triton X-100, quality hundred of the Triton X-100 in sterilized solution
Dividing specific concentration is 0.1%) to impregnate 30min to sterilize, and during which constantly shakes, stem tuber is made to come into full contact with sterilized solution；Sterilizing terminates
It uses sterile water wash stem tuber 5~6 times afterwards, 2~3min every time；Stem tuber is blotted into block with sterilized filter paper in gnotobasis after cleaning
Surface moisture on stem, the stem tuber after being sterilized.
(2) stem eye induces
After the completion of step (1), cultivated in the stem tuber access solid germination medium after being sterilized, about one week
Stem tuber starts to sprout afterwards.After sprouting 1 day, white bud sharp (i.e. initial bud) is completely cut with sterilized blade, is transferred into solid
On body proliferated culture medium.
(3) clump bud is proliferated
White bud point is cultivated 10 days or so on solid multiplication culture medium, induces sprouting (i.e. proliferation bud).Hereafter every 15
Sprouting is transferred and carries out 2 squamous subcultures into new solid multiplication culture medium by it, continues to obtain proliferation bud, the quantity for being proliferated bud can
Up to 5 times of initial bud.
(4) seedling takes root
After the completion of step (3), after the proliferation bud (for Multiple Buds) of 2~3cm high is opened from bottom cutting, it is respectively connected to solid
It is cultivated in root media, 4 days or so bastem portions of culture start to take root, and when culture was to 10 days or so, rooting rate is reachable
80% or more, average out to 82% obtains cyperue esculentus seedling after taking root.
Step (2)-(4) carry out in growth climate box.
(5) plant culture
The cyperue esculentus seedling that step (4) obtains is uncapped experienced seedling 2 days or so, seedling is then moved into the culture by disinfection
Continue to cultivate in soil (compost is made of Nutrition Soil and vermiculite, Nutrition Soil: the volume ratio of vermiculite is 1:1).Survival rate of seedling can
Up to 80% or more, average out to 86%.
Claims (10)
1. the method for tissue culture of cyperue esculentus, including 1) and 2):
1) the stem tuber bud of cyperue esculentus is subjected in proliferated culture medium Multiplying culture, obtains proliferation bud；The proliferated culture medium by
Solvent and solute composition, the solvent are MS culture medium, and the solute and its concentration in the proliferated culture medium are respectively
1mg/L 6-BA, 1.5~2% (mass percent concentration) sucrose, pH are 5.8~6；
2) the proliferation bud is subjected in root media culture of rootage, obtains cyperue esculentus plant, completes the tissue of cyperue esculentus
Culture.
2. described according to the method described in claim 1, it is characterized by: the root media is made of solvent and solute
Solvent is MS culture medium, and the solute and its concentration in the root media are respectively 0.2mg/L IBA, 1.5~2%
(mass percent concentration) sucrose, pH are 5.8~6.
3. method according to claim 1 or 2, it is characterised in that: the stem tuber bud is by sprouting cyperue esculentus stem tuber
Fiber differentiation is carried out in culture medium to obtain.
4. described according to the method described in claim 3, it is characterized by: the germination medium is made of solvent and solute
Solvent is MS culture medium, and the solute and its concentration in the germination medium are respectively 1mg/L NAA, 1.5~2%
(mass percent concentration) sucrose, pH are 5.8~6.
5. method according to any one of claims 1-4, it is characterised in that: the Multiplying culture, the culture of rootage and/
Or the temperature of the Fiber differentiation is 24-26 DEG C；
And/or the humidity of the Multiplying culture, the culture of rootage and/or the Fiber differentiation is 60-70%.
6. any method in -5 according to claim 1, it is characterised in that: the Multiplying culture, the culture of rootage and/
Or the periodicity of illumination of the Fiber differentiation is that 14~16h illumination and 8~10h are dark；
And/or the light intensity of the Multiplying culture, the culture of rootage and/or the Fiber differentiation is 130~150 μm of olm-2·s-1。
7. complete set of culture medium, including proliferated culture medium described in claims 1 or 2 and the root media.
8. complete set of culture medium according to claim 7, it is characterised in that: the complete set of culture medium further includes claim 4
Described in germination medium.
9. complete set of culture medium according to claim 7 or 8, it is characterised in that: the complete set of culture medium has following any
Purposes:
X1) cyperue esculentus tissue cultures；
X2 cyperue esculentus tissue culture products) are prepared.
10. following any applications of any complete set of culture medium in claim 7-9:
Y1) cyperue esculentus tissue cultures；
Y2) preparation is used for cyperue esculentus tissue culture products；
Y3) cyperue esculentus genetic transformation；
Y4) preparation is used for cyperue esculentus genetic transformation product；
Y5) cyperue esculentus molecular breeding；
Y6) preparation is used for cyperue esculentus molecular breeding product.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910067381.8A CN109588316A (en) | 2019-01-24 | 2019-01-24 | A kind of method for tissue culture of cyperue esculentus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111837505A (en) * | 2020-07-27 | 2020-10-30 | 黑龙江省农业科学院农村能源与环保研究所 | Method for efficiently germinating cyperus esculentus seeds |
| CN115413576A (en) * | 2022-08-22 | 2022-12-02 | 呼伦贝尔市农牧科学研究所 | Culture medium formula for cyperus esculentus tissue culture technology and preparation process |
-
2019
- 2019-01-24 CN CN201910067381.8A patent/CN109588316A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111837505A (en) * | 2020-07-27 | 2020-10-30 | 黑龙江省农业科学院农村能源与环保研究所 | Method for efficiently germinating cyperus esculentus seeds |
| CN111837505B (en) * | 2020-07-27 | 2021-11-23 | 黑龙江省农业科学院农村能源与环保研究所 | Method for germinating cyperus esculentus seeds |
| CN115413576A (en) * | 2022-08-22 | 2022-12-02 | 呼伦贝尔市农牧科学研究所 | Culture medium formula for cyperus esculentus tissue culture technology and preparation process |
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Application publication date: 20190409 |