CN109220801A - A kind of method for plant tissue culture - Google Patents
A kind of method for plant tissue culture Download PDFInfo
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- CN109220801A CN109220801A CN201811234674.2A CN201811234674A CN109220801A CN 109220801 A CN109220801 A CN 109220801A CN 201811234674 A CN201811234674 A CN 201811234674A CN 109220801 A CN109220801 A CN 109220801A
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000004161 plant tissue culture Methods 0.000 title claims abstract description 17
- 239000003375 plant hormone Substances 0.000 claims abstract description 133
- 239000001963 growth medium Substances 0.000 claims abstract description 105
- 238000002360 preparation method Methods 0.000 claims abstract description 83
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 60
- 230000006698 induction Effects 0.000 claims abstract description 53
- 239000002609 medium Substances 0.000 claims abstract description 36
- 229920001817 Agar Polymers 0.000 claims abstract description 35
- 239000008272 agar Substances 0.000 claims abstract description 35
- 239000007787 solid Substances 0.000 claims description 112
- 230000001954 sterilising effect Effects 0.000 claims description 62
- 238000004659 sterilization and disinfection Methods 0.000 claims description 44
- 239000012528 membrane Substances 0.000 claims description 31
- 238000001914 filtration Methods 0.000 claims description 26
- 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 24
- 229930006000 Sucrose Natural products 0.000 claims description 24
- 239000005720 sucrose Substances 0.000 claims description 24
- 230000001939 inductive effect Effects 0.000 claims description 23
- 238000007711 solidification Methods 0.000 claims description 20
- 230000008023 solidification Effects 0.000 claims description 20
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 19
- 239000000843 powder Substances 0.000 claims description 11
- 238000004321 preservation Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 2
- 229940088597 hormone Drugs 0.000 claims description 2
- 238000003306 harvesting Methods 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 238000011017 operating method Methods 0.000 abstract 2
- 238000011177 media preparation Methods 0.000 abstract 1
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 22
- 210000001519 tissue Anatomy 0.000 description 22
- VGKONPUVOVVNSU-UHFFFAOYSA-N naphthalen-1-yl acetate Chemical compound C1=CC=C2C(OC(=O)C)=CC=CC2=C1 VGKONPUVOVVNSU-UHFFFAOYSA-N 0.000 description 21
- 241000196324 Embryophyta Species 0.000 description 16
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 11
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 11
- 229960001669 kinetin Drugs 0.000 description 11
- 240000005373 Panax quinquefolius Species 0.000 description 10
- 235000003140 Panax quinquefolius Nutrition 0.000 description 10
- 244000309465 heifer Species 0.000 description 9
- 238000007789 sealing Methods 0.000 description 9
- 238000003501 co-culture Methods 0.000 description 8
- 238000007654 immersion Methods 0.000 description 7
- 241000244987 Daiswa polyphylla Species 0.000 description 5
- 239000009636 Huang Qi Substances 0.000 description 5
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 5
- 235000008434 ginseng Nutrition 0.000 description 5
- 241001533947 Psammosilene Species 0.000 description 4
- 241000202807 Glycyrrhiza Species 0.000 description 3
- 244000132619 red sage Species 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 description 2
- 229940107666 astragalus root Drugs 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- LTINPJMVDKPJJI-UHFFFAOYSA-N iodinated glycerol Chemical compound CC(I)C1OCC(CO)O1 LTINPJMVDKPJJI-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- WTPPRJKFRFIQKT-UHFFFAOYSA-N 1,6-dimethyl-8,9-dihydronaphtho[1,2-g][1]benzofuran-10,11-dione;1-methyl-6-methylidene-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCCC(=C)C3=CC=C2C2=C1C(C)=CO2.O=C1C(=O)C2=C3CCC=C(C)C3=CC=C2C2=C1C(C)=CO2 WTPPRJKFRFIQKT-UHFFFAOYSA-N 0.000 description 1
- OVSKIKFHRZPJSS-UHFFFAOYSA-N 2,4-D Chemical compound OC(=O)COC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-UHFFFAOYSA-N 0.000 description 1
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 description 1
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 description 1
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 1
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 description 1
- 240000008917 Glycyrrhiza uralensis Species 0.000 description 1
- 235000000554 Glycyrrhiza uralensis Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229940010454 licorice Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of method for plant tissue culture, the preparation of (1) combination culture medium is specifically included that, the circular filter paper piece for being moistened with plant hormone is replaced into laying with the layering of MS agar medium；(2) the induction of callus, takes aseptic explant to be placed on filter paper, by dark culture, obtains callus；(3) callus absorbs bottom induction plant hormone and starts to grow, and forms adventitious bud and adventitious root.The present invention also protects a kind of culture medium preparation method and operating procedure, is the combination culture medium and operating procedure.Inductivity height may be implemented in the present invention, and without transferring, growth cycle is short, and harvest is convenient, without carrying out culture medium separation, meets the market demand, has great promotional value.
Description
Technical field
The invention belongs to plant organ culture technique field, in particular to a kind of method for plant tissue culture.
Background technique
Plant Tissue Breeding is also known as in vitro culture, is to have totipotency theoretical using plant cell, sterile and be suitable for
Under artificial medium and external factor, the isolated organ of plant, tissue, cell and protoplast, induced synthesis callus are utilized
Tissue, adventitious bud, adventitious root, finally development are intact plant.
In recent years under the overall situation that Chinese Medicine Industry rapidly develops, due to the further investigation to medicinal plant, make it not
Disconnected to be developed, demand also constantly increases, tissue cultures regeneration with seminal propagation mode compared with the period it is short, not by four seasons gas
The advantages of waiting the harmful substances pollutions, small investment, high financial profit such as limitation, heavy metal free, pesticide, therefore by the extensive of people
Concern, it is considered to be a kind of to be hopeful to alleviate land resource shortage and the different important channel of Chinese medicine quality standard.
Summary of the invention
The purpose of the present invention is to provide a kind of method for plant tissue culture.Compared with traditional planting patterns, weight of the present invention
Renaturation is good, inductivity is high, growth is rapid, active constituent content is high, harvest is convenient.
A kind of method for plant tissue culture is superimposed the combination of preparation using the filter paper layer containing plant hormone with solid culture
Culture medium cultivates plant tissue, includes the following steps:
The induction of S1, callus: aseptic explant root, tender stem segments 2cm of plant or so or young leaflet tablet is taken to be placed in
In the top layer filter paper layer of combination culture medium, 10~15d of dark sterile culture, obtains fresh callus at 24~28 DEG C；
Wherein, the combination culture medium is alternately formed by stacking from the bottom to top by filter paper layer and solid layer；The filter paper layer is
1 or multi-disc contain the filter paper of plant hormone, and the solid layer is solid medium, the bottom and top layer of the combination culture medium
It is filter paper layer；The filter paper layer plays the role of being sustained plant hormone in plant tissue culture course；
The induction of S2, plant tissue: after obtaining callus, aseptic liquid nutrient medium, liquid are added into combination culture medium
Body did not had top layer filter paper layer 1mm or so, continued to induce adventitious root or adventitious root and adventitious bud；Wherein, adventitious root lures item
Part are as follows: 24~28 DEG C, 15~20d of dark sterile culture obtain the adventitious root with callus；Adventitious root and adventitious bud inducing
Condition are as follows: 24~28 DEG C, illumination 12h/d 15~20d of sterile culture obtain the intact plant with adventitious root and adventitious bud.
Under preferred embodiment, plant hormone described in step S1 is 2,4- dichlorphenoxyacetic acid (2,4-D), 6- benzyl aminoadenine
One of (6-BA), indolebutyric acid (IBA), methyl α-naphthyl acetate (NAA), kinetin (KT) are a variety of.
Under preferred embodiment, solid medium described in step S1 with a thickness of 0.5~1cm.
Under preferred embodiment, solid medium described in step S1 is MS solid medium.
Under preferred embodiment, combination culture medium described in step S1 is alternately folded from the bottom to top by four layers of filter paper layer and three layers of solid layer
Add.
Under preferred embodiment, combination culture medium described in step S1 is alternately folded from the bottom to top by three layers of filter paper layer and two layers of solid layer
Add.
Under preferred embodiment, combination culture medium described in step S1 is alternately folded from the bottom to top by two layers of filter paper layer and one layer of solid layer
Add.
Under preferred embodiment, culture medium described in step S2 is MS fluid nutrient medium.
Under preferred embodiment, combination culture medium described in step S1 from the bottom to top be respectively the 1st layer of filter paper layer, the 1st layer of solid layer,
2nd layer of filter paper layer, the 2nd layer of solid layer and top layer filter paper layer；The 1st layer of filter paper layer contain 2.5~5.0mg/L IBA, 0.2~
1.0mg/L NAA, 0.5~4.0mg/L 6-BA, 0.5~1.0mg/L KT；The 2nd layer of filter paper layer contains 1.0~2.0mg/L
IBA, 0.5~1.0mg/L 2,4-D, 0.5~2.0mg/L 6-BA and 0.2mg/L NAA；The top layer filter paper layer contains 0.5~
1.0mg/L 2,4-D and 0.2~1.0mg/L 6-BA；The 1st layer of solid layer is that the concentration of the sucrose with a thickness of 0.5cm is 30
~50g/L, the MS solid medium that agar content is 8g/L, the 2nd layer of solid layer be the concentration of the sucrose with a thickness of 1cm be 30~
50g/L, the MS solid medium that agar content is 8g/L；
Under preferred embodiment, combination culture medium described in step S1 the preparation method comprises the following steps:
S11, cut out filter paper: taking qualitative filter paper to be cut into sterile tissue culture bottle bottom diameter of the same size is 8cm
Circular filter paper piece, put it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
Be blended in after filtration sterilization in a sterile small beaker, obtain mixed plant hormone 1, in the mixed plant hormone 1 containing 2.5~
5.0mg/L IBA, 0.2~1.0mg/L NAA, 0.5~4.0mg/L 6-BA, 0.5~1.0mg/L KT, by 4~8 steps
Filter paper after sterilizing obtained by S11 is stacked together, and impregnates 20s in mixed plant hormone 1, obtains the 1st layer of filter paper layer, for pair
Adventitious root is induced；The preparation of 2nd layer of filter paper layer: aperture is used to swash for 0.22 μm of filter membrane to plant in superclean bench
Element is blended in a sterile small beaker after being filtered degerming, is obtained mixed plant hormone 2, is contained 1.0 in the mixed plant hormone 2
~2.0mg/L IBA, 0.5~1.0mg/L 2,4-D, 0.5~2.0mg/L 6-BA and 0.2mg/L NAA, by 3~8 steps
Filter paper after sterilizing obtained by S11 is stacked together, and is placed in the mixed plant hormone 2 and impregnates 20s, obtains the 2nd layer of induction filter
Paper layer, for inducing adventitious root；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
Be blended in after row filtration sterilization in a sterile small beaker, obtain mixed plant hormone 3, in the mixed plant hormone 3 containing 0.5~
1.0mg/L 2,4-D and 0.2~1.0mg/L 6-BA, the filter paper after 3~5 step S11 are sterilized are stacked together, are planting
20s is impregnated in object hormone 3, top layer induction filter paper layer is obtained, for inducing callus；
S13, prepare culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=5.8~
6.0,121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 30~50g/L, agar
Content is the MS agar medium of 8g/L；
S14, preparation combination culture medium: the 1st layer of filter paper layer obtained by step S12 is laid in sterile tissue culture described in step S11
The culture medium of step S13 preparation is poured into bottom of bottle portion in the 1st layer of filter paper layer, and the 1st layer of solid with a thickness of 1cm is formed after solidification
2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer by layer, and step S13 preparation is poured into the 2nd layer of filter paper layer
Culture medium forms the 2nd layer of solid layer with a thickness of 0.5 after solidification, top layer filter paper layer obtained by step S12 is laid in the 2nd layer admittedly
On body layer, combination culture medium is obtained；The combination culture medium is respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid layer, the 2nd from top to bottom
Layer filter paper layer, the 2nd layer of solid layer and top layer filter paper layer.
The beneficial effects of the present invention are:
High inductivity may be implemented in the present invention, and required raw material are few, and contamination rate is low, and callus is not necessarily to transfer,
Growth cycle is short, and harvest is convenient, without carrying out culture medium separation, meets the market demand, has great promotional value.
Detailed description of the invention
Fig. 1 is combination culture medium schematic diagram prepared by the embodiment of the present invention 1,
Wherein 1. the 1st layers of filter paper layer；2. the 1st layer of solid layer；3. the 2nd layer of filter paper layer；4. the 2nd layer of solid layer；5. top layer is filtered
Paper layer；6. tissue culture bottle；7. bottle cap；8. filter membrane.
Specific embodiment
Below by specific implementation example, the present invention will be further described.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material as used in the following examples, reagent etc., unless otherwise specified, commercially obtain.MS is solid
Body powder is purchased from: chembase, article No.: 27-11-2016.
Embodiment 1
A kind of cultural method of plant tissue:
The induction of S1, ginseng callus: the root segment 2cm or so for taking sterile Ginseng Growth vigor high is placed in combination culture medium
Top layer filter paper layer on, dark sterile culture 10d, obtains fresh ginseng callus at 24 DEG C；
The induction of S2, ginseng adventitious root: after obtaining callus, sterile MS Liquid Culture is added into combination culture medium
Base, liquid height did not had top layer filter paper layer 1mm or so, induced plant hormone since callus absorbs bottom, at 24 DEG C and black
Sterile culture 15d under dark condition, adventitious root start to grow, and obtain the ginseng adventitious root with callus, co-culture 35 days, no
Determine radical amount 6, length 3.2cm, inductivity 95.1%.
Wherein, combination culture medium described in step S1 is alternately folded from the bottom to top by three layers of filter paper layer and two layers of MS solid medium
Add, the preparation method comprises the following steps:
S11, cut out filter paper: taking qualitative filter paper to be cut into sterile tissue culture bottle bottom diameter of the same size is 8cm
Circular filter paper piece, put it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 1, contain in the mixed plant hormone 1
2.5mg/L IBA, 0.2mg/L NAA, 0.5mg/L 6-BA, 0.5mg/L KT, by the filter paper after sterilizing obtained by 8 step S11
Piece is stacked together, and impregnates 20s in mixed plant hormone 1, the 1st layer of filter paper layer is obtained, for inducing adventitious root；2nd
The preparation of layer filter paper layer: aperture is used to be filtered after degerming for 0.22 μm of filter membrane to plant hormone etc. in superclean bench
Volume mixture obtains mixed plant hormone 2 in a sterile small beaker, IBA containing 1.0mg/L in the mixed plant hormone 2,
Filter paper after sterilizing obtained by 3 step S11 is overlayed one by 0.5mg/L2,4-D, 0.5mg/L 6-BA and 0.2mg/L NAA
It rises, is placed in the mixed plant hormone 2 and impregnates 20s, the 2nd layer of induction filter paper layer is obtained, for inducing adventitious root；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
It is blended in equal volume after row filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 3
There are 0.5mg/L 2,4-D and 0.5mg/L6-BA, the filter paper after 5 step S11 are sterilized is stacked together, in plant hormone 3
Middle immersion 20s obtains top layer induction filter paper layer, for inducing callus；
S13, it prepares culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=5.8,
121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 30g/L, agar content 8g/
The MS agar medium of L；
S14, preparation combination culture medium: the 1st layer of filter paper layer obtained by step S12 is laid in culture bottle bottom described in step S11
The culture medium of step S13 preparation is poured into portion in the 1st layer of filter paper layer, and the 1st layer of solid layer with a thickness of 1cm is formed after solidification, will
2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, and the culture of step S13 preparation is poured into the 2nd layer of filter paper layer
Base forms the 2nd layer of solid layer with a thickness of 0.5 after solidification；Top layer filter paper layer obtained by step S12 is laid in the 2nd layer of solid layer
On, obtain combination culture medium；The combination culture medium is respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid layer, the 2nd layer of filter from top to bottom
Paper layer, the 2nd layer of solid layer and top layer filter paper layer.
Embodiment 2
A kind of cultural method of plant tissue:
The induction of S1, Callus Tissue of Danshen: the root segment 2cm or so for taking sterile Salvia miltiorrhiza Growth vigor high is placed in combination culture medium
Top layer filter paper layer on, at 28 DEG C after dark sterile culture 15d, obtain fresh salvia miltiorrhiza callus；
The induction of S2, Radix Salviae Miltiorrhizae adventitious root: after obtaining callus, sterile MS Liquid Culture is added into combination culture medium
Base, liquid height did not had top layer filter paper layer 1mm or so, induced plant hormone since callus absorbs bottom, at 28 DEG C and black
Sterile culture 20d under dark condition, adventitious root start to grow, and obtain the Radix Salviae Miltiorrhizae adventitious root with callus, co-culture 35d, no
Determine radical amount 5, length 2.9cm, inductivity 97.3%.
Wherein, combination culture medium described in step S1 is alternately folded from the bottom to top by three layers of filter paper layer and two layers of MS solid medium
Add, the preparation method comprises the following steps:
The preparation of S11, filter paper layer: it takes qualitative filter paper to be cut into be with sterile tissue culture bottle bottom diameter of the same size
The circular filter paper piece of 8cm puts it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 1, contain in the mixed plant hormone 1
5.0mg/L IBA, 0.4mg/L NAA, 1.0mg/L 6-BA, 0.5mg/L KT, by the filter paper after sterilizing obtained by 4 step S11
Piece is stacked together, and impregnates 20s in mixed plant hormone 1, the 1st layer of filter paper layer is obtained, for inducing adventitious root；2nd
The preparation of layer filter paper layer: it is mixed after using aperture to be filtered degerming to plant hormone for 0.22 μm of filter membrane in superclean bench
It closes in a sterile small beaker, obtains mixed plant hormone 2, IBA containing 1.2mg/L, 0.5mg/L in the mixed plant hormone 2
2,4-D, the filter paper after sterilizing obtained by 3 step S11 is stacked together, is placed in by 0.6mg/L 6-BA and 0.2mg/L NAA
20s is impregnated in the mixed plant hormone 2, the 2nd layer of induction filter paper layer is obtained, for inducing adventitious root；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
It is blended in equal volume after row filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 3
There are 0.5mg/L 2,4-D and 0.5mg/L6-BA, the filter paper after 3 step S11 are sterilized is stacked together, in plant hormone 3
Middle immersion 20s obtains top layer induction filter paper layer, for inducing callus；
S13, it prepares culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=5.8,
121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 30g/L, agar content 8g/
The M agar medium of L；
S14, preparation combination culture medium: the 1st layer of filter paper layer obtained by step S12 is laid in culture bottle bottom described in step S11
The culture medium of step S13 preparation is poured into portion in the 1st layer of filter paper layer, and the 1st layer of solid layer with a thickness of 1cm is formed after solidification；It will
2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, and the culture of step S13 preparation is poured into the 2nd layer of filter paper layer
Base forms the 2nd layer of solid layer with a thickness of 0.5cm after solidification；Top layer filter paper layer obtained by step S12 is laid in the 2nd layer of solid
On layer, combination culture medium is obtained；The combination culture medium is respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid layer, the 2nd layer from top to bottom
Filter paper layer, the 2nd layer of solid layer and top layer filter paper layer.
Embodiment 3
A kind of cultural method of plant tissue:
The induction of S1, Callus Tissue of Glycyrrhiza: the root segment 2cm or so for taking sterile growth of Glycyrrhiza uralensis vigor high is placed in combination culture medium
Top layer filter paper layer on, at 26 DEG C after dark sterile culture 12d, obtain fresh Callus Tissue of Glycyrrhiza；
The induction of S2, licorice adventitious root and adventitious bud: it after obtaining callus, is added into combination culture medium a small amount of sterile
MS fluid nutrient medium did not had top layer filter paper layer 1mm or so, plant hormone was induced since callus absorbs bottom, in 26 DEG C, light
According to sterile culture 18d under the conditions of 12h/d, adventitious root and adventitious bud start to grow, and form complete Radix Glycyrrhizae plant, co-culture 35 days,
Adventitious root quantity 6, adventitious bud quantity 11, adventitious root length 3.0cm, adventitious bud height 5.5cm, inductivity 96.9%.
Wherein, combination culture medium described in step S1 is alternately folded from the bottom to top by three layers of filter paper layer and two layers of MS solid medium
Add, the preparation method comprises the following steps:
S11, cut out filter paper: taking qualitative filter paper to be cut into sterile tissue culture bottle bottom diameter of the same size is 8cm
Circular filter paper piece, put it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 1, contain in the mixed plant hormone 1
4.0mg/L IBA, 0.5mg/L NAA, 4.0mg/L 6-BA, 0.8mg/L KT, by the filter paper after sterilizing obtained by 4 step S11
Piece is stacked together, and impregnates 20s in mixed plant hormone 1, the 1st layer of filter paper layer is obtained, for inducing adventitious root；
The preparation of 2nd layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 2, contain in the mixed plant hormone 2
1.5mg/L IBA, 2 0.8mg/L, 4-D, 1.5mg/L 6-BA and 0.2mg/L NAA, after sterilizing obtained by 5 step S11
Filter paper is stacked together, and is placed in the mixed plant hormone 2 and impregnates 20s, obtains the 2nd layer of induction filter paper layer, for indefinite
Root is induced；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
It is blended in equal volume after row filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 3
There are 0.8mg/L 2,4-D and 0.8mg/L6-BA, the filter paper after 3 step S11 are sterilized is stacked together, in plant hormone 3
Middle immersion 20s obtains top layer induction filter paper layer, for inducing callus；
S13, it prepares culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=5.9,
121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 50g/L, agar content 8g/
The MS agar medium of L；
S14, preparation combination culture medium: the 1st layer of filter paper layer obtained by step S12 is laid in culture bottle bottom described in step S11
The culture medium of step S13 preparation is poured into portion in the 1st layer of filter paper layer, and the 1st layer of solid layer with a thickness of 1cm is formed after solidification；It will
2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, and the culture of step S13 preparation is poured into the 2nd layer of filter paper layer
Base forms the 2nd layer of solid layer with a thickness of 0.5cm after solidification, the 2nd layer of combination culture medium is by the 2nd layer of filter paper layer and the 2nd layer
Solid layer composition；Top layer filter paper layer obtained by step S12 is laid on the 2nd layer of solid layer, combination culture medium is obtained；The combination training
Feeding base is respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid layer, the 2nd layer of filter paper layer, the 2nd layer of solid layer and top layer filter paper from top to bottom
Layer.
Embodiment 4
The induction of S1, Radix Astragali callus: taking sterile Radix Astragali young leaflet tablet to be placed in the top layer filter paper layer of combination culture medium,
At 25 DEG C after dark sterile culture 10d, fresh Radix Astragali callus is obtained；
The induction of S2, astragalus root of Radix Astragali: after obtaining callus, sterile MS Liquid Culture is added into combination culture medium
Base did not had top layer filter paper layer 1mm or so, induced plant hormone, the nothing under 25 DEG C, dark condition since callus absorbs bottom
Bacterium cultivates 16d, and adventitious root starts to grow, and obtains the astragalus root of Radix Astragali with callus, co-cultures 35 days, adventitious root quantity 7,
Adventitious root length 3.2cm, inductivity 98.3%.
Wherein, combination culture medium described in step S1 is alternately folded from the bottom to top by three layers of filter paper layer and two layers of MS solid medium
Add, the preparation method comprises the following steps:
S11, cut out filter paper: taking qualitative filter paper to be cut into sterile tissue culture bottle bottom diameter of the same size is 8cm
Circular filter paper piece, put it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 1, contain in the mixed plant hormone 1
3.5mg/L IBA, 0.5mg/L NAA, 1.0mg/L 6-BA, 0.5mg/L KT, by the filter paper after sterilizing obtained by 6 step S11
Piece is stacked together, and impregnates 20s in mixed plant hormone 1, the 1st layer of filter paper layer is obtained, for inducing adventitious root；
The preparation of 2nd layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 2, contain in the mixed plant hormone 2
2.0mg/L IBA, 2 0.5mg/L, 4-D, 0.8mg/L 6-BA and 0.2mg/L NAA, after sterilizing obtained by 3 step S11
Filter paper is stacked together, and is placed in the mixed plant hormone 2 and impregnates 20s, obtains the 2nd layer of induction filter paper layer, for indefinite
Root is induced；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
It is blended in equal volume after row filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 3
There are 0.8mg/L 2,4-D and 0.5mg/L6-BA, the filter paper after 4 step S11 are sterilized is stacked together, in plant hormone 3
Middle immersion 20s obtains top layer induction filter paper layer, for inducing callus；
S13 prepares culture medium: it accurately weighs MS solid powder and is dissolved in water, add sucrose and agar, adjust pH=5.8,121
DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 30g/L, and agar content is 8g/L's
MS agar medium；
The 1st layer of S14, preparation combination culture medium layer: the 1st layer of filter paper layer obtained by step S12 is laid in training described in step S11
Bottom of bottle portion is supported, the culture medium of step S13 preparation is poured into the 1st layer of filter paper layer, is formed after solidification with a thickness of the 1st of 1cm or so
Layer solid layer；2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, pours into step S13 in the 2nd layer of filter paper layer
The culture medium of preparation forms the 2nd layer of solid layer with a thickness of 0.5cm or so after solidification；Top layer filter paper layer obtained by step S12 is put down
It is laid on the 2nd layer of solid layer, obtains combination culture medium；The combination culture medium is respectively as follows: the 1st layer of filter paper layer, the 1st layer from top to bottom
Solid layer, the 2nd layer of filter paper layer, the 2nd layer of solid layer and top layer filter paper layer.
Embodiment 5
The induction of S1, tuniclike psammosilene root callus: the top layer filter paper for taking sterile tuniclike psammosilene root to there is leaflet tablet to be placed in combination culture medium
On layer, at 27 DEG C after dark sterile culture 12d, fresh tuniclike psammosilene root callus is obtained；
The induction of S2, tuniclike psammosilene root adventitious root and adventitious bud: after obtaining callus, sterile MS is added into combination culture medium
Fluid nutrient medium did not had top layer filter paper layer 1mm or so, plant hormone was induced since callus absorbs bottom, in 27 DEG C, illumination
Sterile culture 15d under the conditions of 12h/d, adventitious root and adventitious bud start to grow, and form complete golden iron lock plant, co-culture 35 days,
Adventitious root quantity 7, adventitious bud quantity 9, adventitious root length 3.5cm, adventitious bud height 5.1cm, inductivity 95.9%.
Wherein, combination culture medium described in step S1 is alternately folded from the bottom to top by three layers of filter paper layer and two layers of MS solid medium
Add, the preparation method comprises the following steps:
S11, cut out filter paper: taking qualitative filter paper to be cut into sterile tissue culture bottle bottom diameter of the same size is 8cm
Circular filter paper piece, put it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 1, contain in the mixed plant hormone 1
3.5mg/L IBA, 0.5mg/L NAA, 4.0mg/L 6-BA, 1.0mg/L KT, by the filter paper after sterilizing obtained by 7 step S11
Piece is stacked together, and impregnates 20s in mixed plant hormone 1, the 1st layer of filter paper layer is obtained, for inducing adventitious root；
The preparation of 2nd layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 2, contain in the mixed plant hormone 2
1.5mg/L IBA, 2 1.0mg/L, 4-D, 2.0mg/L 6-BA and 0.2mg/L NAA, after sterilizing obtained by 4 step S11
Filter paper is stacked together, and is placed in the mixed plant hormone 2 and impregnates 20s, obtains the 2nd layer of induction filter paper layer, for indefinite
Root is induced；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
It is blended in after row filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 3
1.0mg/L 2,4-D and 1.0mg/L6-BA, the filter paper after 3 step S11 are sterilized is stacked together, in plant hormone 3
20s is impregnated, top layer induction filter paper layer is obtained, for inducing callus；
S13, it prepares culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=5.9,
121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 45g/L, agar content 8g/
The MS agar medium of L；
S14, preparation combination culture medium layer: the 1st layer of filter paper layer obtained by step S12 is laid in culture bottle described in step S11
The culture medium of step S13 preparation is poured into bottom in the 1st layer of filter paper layer, is formed after solidification solid with a thickness of the 1st layer of 1cm or so
Body layer；2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, step S13 preparation is poured into the 2nd layer of filter paper layer
Culture medium, after solidification formed with a thickness of 0.5cm or so the 2nd layer of solid layer；Top layer filter paper layer obtained by step S12 is laid in
On 2nd layer of solid layer, combination culture medium is obtained；The combination culture medium is respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid from top to bottom
Layer, the 2nd layer of filter paper layer, the 2nd layer of solid layer and top layer filter paper layer.
Embodiment 6
The induction of S1, Radix Angelicae Sinensis callus: sterile Radix Angelicae Sinensis tender stem segments 2cm or so is taken to be placed in the top layer filter of combination culture medium
On paper layer, at 25 DEG C after dark sterile culture 11d, fresh Radix Angelicae Sinensis callus is obtained；
The induction of S2, Radix Angelicae Sinensis adventitious root: after obtaining callus, sterile MS Liquid Culture is added into combination culture medium
Base did not had top layer filter paper layer 1mm or so, induced plant hormone, the nothing under 25 DEG C of dark conditions since callus absorbs bottom
Bacterium cultivates 18d, and adventitious root starts to grow, and obtains the Radix Angelicae Sinensis adventitious root with callus, co-cultures 35 days, adventitious root quantity 5,
Adventitious root length 2.7cm, inductivity 97.6%.
Wherein, combination culture medium described in step S1 is alternately folded from the bottom to top by three layers of filter paper layer and two layers of MS solid medium
Add, the preparation method comprises the following steps:
S11, cut out filter paper: taking qualitative filter paper to be cut into sterile tissue culture bottle bottom diameter of the same size is 8cm
Circular filter paper piece, put it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 1, contain in the mixed plant hormone 1
4.0mg/L IBA, 1.0mg/L NAA, 0.5mg/L 6-BA, 1.0mg/L KT, by the filter paper after sterilizing obtained by 6 step S11
Piece is stacked together, and impregnates 20s in mixed plant hormone 1, the 1st layer of filter paper layer is obtained, for inducing adventitious root；
The preparation of 2nd layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 2, contain in the mixed plant hormone 2
1.5mg/L IBA, 2 1.0mg/L, 4-D, 0.5mg/L 6-BA and 0.2mg/L NAA, after sterilizing obtained by 2 step S11
Filter paper is stacked together, and is placed in the mixed plant hormone 2 and impregnates 20s, obtains the 2nd layer of induction filter paper layer, for indefinite
Root is induced；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
It is blended in equal volume after row filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 3
There are 1.0mg/L 2,4-D and 0.8mg/L6-BA, the filter paper after 4 step S11 are sterilized is stacked together, in plant hormone 3
Middle immersion 20s obtains top layer induction filter paper layer, for inducing callus；
S13, it prepares culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=6.0,
121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 30g/L, agar content 8g/
The MS agar medium of L；
S14, preparation combination culture medium: the 1st layer of filter paper layer obtained by step S12 is laid in culture bottle bottom described in step S11
The culture medium of step S13 preparation is poured into portion in the 1st layer of filter paper layer, and the 1st layer of solid with a thickness of 1cm or so is formed after solidification
Layer；2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, step S13 preparation is poured into the 2nd layer of filter paper layer
Culture medium forms the 2nd layer of solid layer with a thickness of 0.5cm or so after solidification；Top layer filter paper layer obtained by step S12 is laid in the
On 2 layers of solid layer, combination culture medium is obtained；The combination culture medium is respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid from top to bottom
Layer, the 2nd layer of filter paper layer, the 2nd layer of solid layer and top layer filter paper layer.
Embodiment 7
The induction of S1, paris polyphylla callus: paris polyphylla tender stem segments 2cm or so is taken to be placed in the top layer filter of combination culture medium
On paper layer, at 26 DEG C after dark sterile culture 10d, new paris polyphylla callus is obtained；
The induction of S2, paris polyphylla adventitious root: after obtaining callus, sterile MS Liquid Culture is added into combination culture medium
Base, liquid height did not had top layer filter paper layer 1mm or so, plant hormone were induced since callus absorbs bottom, in 26 DEG C of dark
Under the conditions of sterile culture 15d, adventitious root starts to grow, and obtains the paris polyphylla adventitious root with callus, co-cultures 35 days, no
Determine radical amount 8, adventitious root length 3.1cm, inductivity 96.9%.
Wherein, combination culture medium described in step S1 is alternately folded from the bottom to top by two layers of filter paper layer and one layer of MS solid medium
Add, the preparation method comprises the following steps:
S11, cut out filter paper: taking qualitative filter paper to be cut into sterile tissue culture bottle bottom diameter of the same size is 8cm
Circular filter paper piece, put it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 1, contain in the mixed plant hormone 1
3.5mg/L IBA, 2 1.0mg/L, 4-D, 0.5mg/L 6-BA and 1.0mg/L NAA, after sterilizing obtained by 8 step S11
Filter paper is stacked together, and impregnates 20s in mixed plant hormone 1, the 1st layer of filter paper layer is obtained, for inducing adventitious root；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
It is blended in equal volume after row filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 3
There are 1.0mg/L 2,4-D and 1.0mg/L6-BA, the filter paper after 4 step S11 are sterilized is stacked together, in plant hormone 3
Middle immersion 20s obtains top layer induction filter paper layer, for inducing callus；
S13, it prepares culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=5.8,
121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 30g/L, agar content 8g/
The MS agar medium of L；
S14, preparation combination culture medium: the 1st layer of filter paper layer obtained by step S12 is laid in culture bottle bottom described in step S11
The culture medium of step S13 preparation is poured into portion in the 1st layer of filter paper layer, and the 1st layer of solid with a thickness of 1cm or so is formed after solidification
Layer；Top layer filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, combination culture medium is obtained；The combination culture medium is under
To being above respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid layer and top layer filter paper layer.
Embodiment 8
The induction of S1, American Ginseng callus: vigorous root segment 2cm of sterile American Ginseng growth vigor or so is taken to be placed in combination training
In the top layer filter paper layer for supporting base, at 25 DEG C after dark sterile culture 10d, fresh American Ginseng callus is obtained；
The induction of S2, American Ginseng adventitious root: after obtaining callus, a small amount of sterile MS liquid is added into combination culture medium
Culture medium, it is 1mm or so that liquid, which did not had top layer filter paper layer height, plant hormone is induced since callus absorbs bottom, 25
Sterile culture 17d under DEG C dark condition, adventitious root start to grow, and obtain the American Ginseng adventitious root with callus, co-culture
35 days, adventitious root quantity 8, adventitious root length 2.9cm, inductivity 98.4%.
Wherein, combination culture medium described in step S1 is alternately folded from the bottom to top by four layers of filter paper layer and three layers of MS solid medium
Add, the preparation method comprises the following steps:
S11, cut out filter paper: taking qualitative filter paper to be cut into sterile tissue culture bottle bottom diameter of the same size is 8cm
Circular filter paper piece, put it into clean culture dish heifer paper sealing, 121 DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 1, contain in the mixed plant hormone 1
4.0mg/L IBA, 0.5mg/L NAA, 0.5mg/L 6-BA, 0.5mg/L KT, by the filter paper after sterilizing obtained by 4 step S11
Piece is stacked together, and impregnates 20s in mixed plant hormone 1, the 1st layer of filter paper layer is obtained, for inducing adventitious root；
The preparation of 2nd layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 2, contain in the mixed plant hormone 2
1.0mg/L IBA, 2 1.0mg/L, 4-D, 1.0mg/L 6-BA and 0.2mg/L NAA, after sterilizing obtained by 3 step S11
Filter paper is stacked together, and is placed in the mixed plant hormone 2 and impregnates 20s, obtains the 2nd layer of induction filter paper layer；
The preparation of 3rd layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in equal volume after filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 2
2.0mg/L IBA, 2 1.5mg/L, 4-D, 4.0mg/L 6-BA and 0.8mg/L NAA, after sterilizing obtained by 3 step S11
Filter paper is stacked together, and is placed in the mixed plant hormone 3 and impregnates 20s, obtains the 2nd layer of induction filter paper layer；
Top layer induction filter paper layer preparation: used in superclean bench aperture for 0.22 μm filter membrane to plant hormone into
It is blended in equal volume after row filtration sterilization in a sterile small beaker, obtains mixed plant hormone 3, contain in the mixed plant hormone 3
There are 0.8mg/L 2,4-D and 0.5mg/L6-BA, the filter paper after 4 step S11 are sterilized is stacked together, in plant hormone 3
Middle immersion 20s obtains top layer induction filter paper layer, for inducing callus；
S13, it prepares culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=5.8,
121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 30g/L, agar content 8g/
The MS agar medium of L；
S14, preparation combination culture medium: the 1st layer of filter paper layer obtained by step S12 is laid in culture bottle bottom described in step S11
The culture medium of step S13 preparation is poured into portion in the 1st layer of filter paper layer, is formed after solidification solid with a thickness of the 1st layer of 0.5cm or so
Body layer；2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, step S13 preparation is poured into the 2nd layer of filter paper layer
Culture medium, after solidification formed with a thickness of 0.5cm or so the 2nd layer of solid layer；3rd filter paper layer obtained by step S12 is laid in
On 2nd layer of solid layer, the culture medium of step S13 preparation is poured into the 3rd layer of filter paper layer, is formed after solidification with a thickness of the left side 0.5cm
The 3rd layer of right solid layer；Top layer filter paper layer obtained by step S12 is laid on the 3rd layer of solid layer, combination culture medium is obtained；It is described
Combination culture medium is respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid layer, the 2nd layer of filter paper layer, the 2nd layer of solid layer, the 3rd from top to bottom
Layer filter paper layer, the 3rd layer of solid layer and top layer filter paper layer.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art within the technical scope of the present disclosure, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of method for plant tissue culture, which comprises the steps of:
The induction of S1, callus: the aseptic explant root, tender stem segments 2cm or young leaflet tablet of plant is taken to be placed in combination culture
In the top layer filter paper layer of base, 10~15d of dark sterile culture at 24~28 DEG C obtains callus；
Wherein, the combination culture medium is alternately formed by stacking from the bottom to top by filter paper layer and solid layer；The filter paper layer be containing
The filter paper of plant hormone, the solid layer are solid medium, and the bottom and top layer of the combination culture medium are filter paper layer；
The induction of S2, plant tissue: fluid nutrient medium being added into combination culture medium, and liquid did not had top layer filter paper layer 1mm or so,
Adventitious root or adventitious root and adventitious bud are induced；
Wherein, adventitious root lures condition are as follows: 24~28 DEG C, 15~20d of dark sterile culture, it is indefinite with callus to obtain
Root；Adventitious root and adventitious bud inducing condition are as follows: 24~28 DEG C, illumination 12h/d 15~20d of sterile culture are obtained with adventitious root
With the intact plant of adventitious bud.
2. method for plant tissue culture according to claim 1, which is characterized in that plant hormone described in step S1 be 2,4-D,
One of 6-BA, IBA, NAA, KT or a variety of.
3. method for plant tissue culture according to claim 1, which is characterized in that combination culture medium described in step S1 is by four layers
Filter paper layer and three layers of solid layer are alternately formed by stacking from the bottom to top.
4. method for plant tissue culture according to claim 1, which is characterized in that combination culture medium described in step S1 is by three layers
Filter paper layer and two layers of solid layer are alternately formed by stacking from the bottom to top.
5. method for plant tissue culture according to claim 1, which is characterized in that combination culture medium described in step S1 is by two layers
Filter paper layer and one layer of solid layer are alternately formed by stacking from the bottom to top.
6. according to claim 1 to 5 any method for plant tissue culture, which is characterized in that solid layer described in step S1
With a thickness of 0.5~1cm.
7. according to claim 1 to 5 any method for plant tissue culture, which is characterized in that solid layer described in step S1 is
MS solid medium.
8. according to claim 1 to 5 any method for plant tissue culture, which is characterized in that Liquid Culture described in step S2
Base is MS fluid nutrient medium.
9. method for plant tissue culture according to claim 1, which is characterized in that combination culture medium described in step S1 by down toward
Upper is respectively the 1st layer of filter paper layer, the 1st layer of solid layer, the 2nd layer of filter paper layer, the 2nd layer of solid layer and top layer filter paper layer；Described 1st layer
Filter paper layer by 4~8 containing 2.5~5.0mg/L IBA, 0.2~1.0mg/L NAA, 0.5~4.0mg/L 6-BA, 0.5~
The filter paper of 1.0mg/L KT stacks together；The 2nd layer of filter paper layer by 3~8 containing 1.0~2.0mg/L IBA, 0.5~
1.0mg/L 2,4-D, 0.5~2.0mg/L 6-BA and 0.2mg/L NAA filter paper stack together；The top layer filter paper layer is by 3
~5 filter paper containing 0.5~1.0mg/L 2,4-D and 0.2~1.0mg/L 6-BA stack together；The 1st layer of solid layer
The MS solid medium that concentration for the sucrose with a thickness of 0.5cm is 30~50g/L, agar content is 8g/L, the 2nd layer of solid layer
The MS solid medium that concentration for the sucrose with a thickness of 1cm is 30~50g/L, agar content is 8g/L.
10. method for plant tissue culture according to claim 9, which is characterized in that the system of combination culture medium described in step S1
Preparation Method are as follows:
S11, cut out filter paper: take qualitative filter paper be cut into the sterile consistent circular filter paper piece of tissue culture bottle bottom shape, 121
DEG C, 0.1MPa high-temperature sterilization 25min；
S12, preparation filter paper layer:
The preparation of 1st layer of filter paper layer: aperture is used to be filtered for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in after degerming in a sterile small beaker, obtains mixed plant hormone 1, contain 2.5~5.0mg/L in the mixed plant hormone 1
IBA, 0.2~1.0mg/L NAA, 0.5~4.0mg/L 6-BA, 0.5~1.0mg/L KT will go out obtained by 4~8 step S11
Filter paper after bacterium is stacked together, and impregnates 20s in mixed plant hormone 1, obtains the 1st layer of filter paper layer, for adventitious root into
Row induction；The preparation of 2nd layer of filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
It is blended in a sterile small beaker after filtering out bacterium, obtains mixed plant hormone 2, contain 1.0~2.0mg/ in the mixed plant hormone 2
L IBA, 0.5~1.0mg/L 2,4-D, 0.5~2.0mg/L 6-BA and 0.2mg/L NAA will go out obtained by 3~8 step S11
Filter paper after bacterium is stacked together, and is placed in the mixed plant hormone 2 and impregnates 20s, obtains the 2nd layer of induction filter paper layer, is used for
Adventitious root is induced；
The preparation of top layer induction filter paper layer: aperture is used to carry out for 0.22 μm of filter membrane to plant hormone in superclean bench
Be blended in a sterile small beaker after filtering out bacterium, obtain mixed plant hormone 3, in the mixed plant hormone 3 containing 0.5~
1.0mg/L 2,4-D and 0.2~1.0mg/L 6-BA, the filter paper after 3~5 step S11 are sterilized are stacked together, are planting
20s is impregnated in object hormone 3, top layer induction filter paper layer is obtained, for inducing callus；
S13, it prepares culture medium: accurately weighing MS solid powder and be dissolved in water, add sucrose and agar, adjust pH=5.8~6.0,
121 DEG C, heat preservation prevents from solidifying after 0.1MPa high-temperature sterilization 25min, the concentration for being configured to sucrose is 30~50g/L, agar content
For the MS agar medium of 8g/L；
S14, preparation combination culture medium: the 1st layer of filter paper layer obtained by step S12 is laid in sterile tissue culture bottle bottom described in step S11
The culture medium of step S13 preparation is poured into portion in the 1st layer of filter paper layer, and the 1st layer of solid layer with a thickness of 1cm is formed after solidification, will
2nd filter paper layer obtained by step S12 is laid on the 1st layer of solid layer, and the culture of step S13 preparation is poured into the 2nd layer of filter paper layer
Base, forms the 2nd layer of solid layer with a thickness of 0.5 after solidification, top layer filter paper layer obtained by step S12 is laid in the 2nd layer of solid layer
On, obtain combination culture medium；The combination culture medium is respectively as follows: the 1st layer of filter paper layer, the 1st layer of solid layer, the 2nd layer of filter from top to bottom
Paper layer, the 2nd layer of solid layer and top layer filter paper layer.
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