CN106258974A - A kind of propagation method of the little rhizome of Chinese podophyllum root - Google Patents
A kind of propagation method of the little rhizome of Chinese podophyllum root Download PDFInfo
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- CN106258974A CN106258974A CN201610699256.5A CN201610699256A CN106258974A CN 106258974 A CN106258974 A CN 106258974A CN 201610699256 A CN201610699256 A CN 201610699256A CN 106258974 A CN106258974 A CN 106258974A
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- chinese podophyllum
- podophyllum root
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- 238000000034 method Methods 0.000 title claims abstract description 41
- YJGVMLPVUAXIQN-XVVDYKMHSA-N podophyllotoxin Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YJGVMLPVUAXIQN-XVVDYKMHSA-N 0.000 title claims abstract description 37
- 241001495452 Podophyllum Species 0.000 title claims abstract description 31
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 25
- 230000006698 induction Effects 0.000 claims abstract description 10
- 230000011218 segmentation Effects 0.000 claims abstract description 7
- 230000035800 maturation Effects 0.000 claims abstract description 6
- 238000003860 storage Methods 0.000 claims abstract description 6
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 4
- HFCYZXMHUIHAQI-UHFFFAOYSA-N Thidiazuron Chemical compound C=1C=CC=CC=1NC(=O)NC1=CN=NS1 HFCYZXMHUIHAQI-UHFFFAOYSA-N 0.000 claims description 32
- 239000007943 implant Substances 0.000 claims description 32
- 230000001939 inductive effect Effects 0.000 claims description 24
- 229920001817 Agar Polymers 0.000 claims description 23
- 229930006000 Sucrose Natural products 0.000 claims description 23
- 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 23
- 239000008272 agar Substances 0.000 claims description 23
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- 238000005286 illumination Methods 0.000 claims description 21
- 239000005972 6-Benzyladenine Substances 0.000 claims description 20
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 20
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 claims description 20
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 claims description 20
- 229940023877 zeatin Drugs 0.000 claims description 20
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 claims description 16
- 239000002253 acid Substances 0.000 claims description 16
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 16
- 235000021240 caseins Nutrition 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 14
- IXVMHGVQKLDRKH-YEJCTVDLSA-N (22s,23s)-epibrassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@H](O)[C@@H](O)[C@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-YEJCTVDLSA-N 0.000 claims description 13
- IXVMHGVQKLDRKH-VRESXRICSA-N Brassinolide Natural products O=C1OC[C@@H]2[C@@H]3[C@@](C)([C@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC3)CC[C@@H]2[C@]2(C)[C@@H]1C[C@H](O)[C@H](O)C2 IXVMHGVQKLDRKH-VRESXRICSA-N 0.000 claims description 13
- GPXLRLUVLMHHIK-UHFFFAOYSA-N forchlorfenuron Chemical compound C1=NC(Cl)=CC(NC(=O)NC=2C=CC=CC=2)=C1 GPXLRLUVLMHHIK-UHFFFAOYSA-N 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- YNWVFADWVLCOPU-MAUPQMMJSA-N uniconazole P Chemical compound C1=NC=NN1/C([C@@H](O)C(C)(C)C)=C/C1=CC=C(Cl)C=C1 YNWVFADWVLCOPU-MAUPQMMJSA-N 0.000 claims description 12
- -1 Nitrophenols Ketone Chemical class 0.000 claims description 11
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 11
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 8
- 229930191978 Gibberellin Natural products 0.000 claims description 8
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 claims description 8
- 239000003448 gibberellin Substances 0.000 claims description 8
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 claims description 8
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims description 6
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- 229960001237 podophyllotoxin Drugs 0.000 description 6
- YVCVYCSAAZQOJI-UHFFFAOYSA-N podophyllotoxin Natural products COC1=C(O)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YVCVYCSAAZQOJI-UHFFFAOYSA-N 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 5
- 230000004792 oxidative damage Effects 0.000 description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
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- JLIDBLDQVAYHNE-LXGGSRJLSA-N 2-cis-abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\C1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-LXGGSRJLSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
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- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
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- 238000005457 optimization Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000004576 sand Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 239000001117 sulphuric acid Substances 0.000 description 2
- 235000011149 sulphuric acid Nutrition 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 229940124321 AIDS medicine Drugs 0.000 description 1
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- 206010006002 Bone pain Diseases 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
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- 208000025747 Rheumatic disease Diseases 0.000 description 1
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- 229930003935 flavonoid Natural products 0.000 description 1
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- 231100000614 poison Toxicity 0.000 description 1
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- 239000005017 polysaccharide Substances 0.000 description 1
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- 230000001902 propagating effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- KXXXUIKPSVVSAW-UHFFFAOYSA-K pyranine Chemical compound [Na+].[Na+].[Na+].C1=C2C(O)=CC(S([O-])(=O)=O)=C(C=C3)C2=C2C3=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C2=C1 KXXXUIKPSVVSAW-UHFFFAOYSA-K 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
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- 239000003053 toxin Substances 0.000 description 1
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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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides the propagation method of the little rhizome of a kind of Chinese podophyllum root, by obtaining the steps such as aseptic embryo, the induction of Multiple Buds, the maturation of Multiple Buds and segmentation, the growth of seedling, the formation of little rhizome and storage, establish the tissue culture method of the little rhizome of Chinese podophyllum root, the method has that reproductive efficiency is high, breeding coefficient is big, cultivation cycle is short, convenient cultivation storage, convenient transport, easy and simple to handle, the advantage of low cost, improving Chinese podophyllum root tissue culture technology, the plantation that expands further for Chinese podophyllum root provides guarantee.
Description
Technical field
The present invention relates to agricultural biological technical field, a kind of directly breed Fructus Persicae youngster by the means of cell engineering
The method of the seven little rhizomes of test tube.
Background technology
Chinese podophyllum root (Sinopodophyllum hexandrum Royle) it is Berberidaceae Sinopodophyllum herbaceous plant, state
Family three-level protective plant, is mainly distributed on China western.Face due to natural resources deficient and in imminent danger, be included into " China
Rare or endangered species register " and " Chinese Plants Red Data Book ".
Chinese podophyllum root is mainly used in the diseases such as pain furuncle and phyma poison, rheumatic ostealgia, tracheitis among the people, and modern pharmacy research shows,
Mainly containing the compositions such as lignanoids, flavonoid, saponin, polysaccharide in its root and rhizome, wherein the podophyllotoxin of lignanoids contains
Measure the highest.Podophyllotoxin has pharmaceutical active widely, as 0.5% podophyllotoxin tincture has onset in terms for the treatment of condyloma acuminatum
The feature such as hurry up, cure rate is high, safety is good, nineteen ninety world health organisation recommendations its be first-line drug, within 1994, China is " national
Essential drugs " skin antiviral is uniquely selected in medicine.Podophyllotoxin still synthesize cancer therapy drug GP7, VP-16, VM-26,
NK611 etc. and the precursor substance of anti-AIDS drug.
Under natural conditions, Chinese podophyllum root based on seminal propagation, seed dormancy phase length (6～8 months), growing pullets after germination
For slowly, within 5～6 years, just can complete sexual maturity, and the requirement to growing environment is the harshest.In order to alleviate Chinese podophyllum root resource danger
Machine, produces main active component podophyllotoxin, and Chinese scholars utilizes callus culture, cell suspension cultures to carry out Rhizoma Dysosmae Versipellis
The production of toxin, but there is easy brownization of callus, breed slow and that podophyllotoxin content is low problem；In vitro about Chinese podophyllum root
Embryo culture, Shoot propagation are cultivated, the acclimatization and transplants technology of test tube Seedling has been reported, although to some extent solve the numerous of resource
Grow problem, but still suffer from the problems such as breeding coefficient low, test tube Seedling white silk Seedling complex operation, time-consuming long, production cost height.
Chinese podophyllum root, in growth course, can form the abnormal organ of similar flattened spherical at petiole base with root upper area
Rhizome, rhizome, as the raw organ in a kind of place, has the former base of bud of different development stage, can germinate, take root, and convenient transport,
Transplant, resistance.During group training, petiole base also has similar little rhizome formation.Group currently for Chinese podophyllum root trains work
Making, the technology still carrying out breeding not over little rhizome occurs.
Summary of the invention
It is an object of the invention to for above technical problem, it is provided that a kind of Chinese podophyllum root test tube little Propagation of Rhizomes method.We
Method breeding coefficient is high, with low cost, can be effectively improved the reproductive efficiency from test tube to land for growing field crops, to Chinese podophyllum root protection of resources and kind
Seedling produces significant.
Present disclosure, is to provide a kind of efficient Chinese podophyllum root little Propagation of Rhizomes method, comprises the following steps:
(1) aseptic embryo is obtained
By the Chinese podophyllum root seed of fresh collection, remove and damage by worms and the seed that goes rotten, clean up rear wet concentration, with a little concentrated sulphuric acid surface
Sterilization, corrosion seed coat, the sodium carbonate soaking at room temperature of 0.5% removes the hydrophobic ingredients such as oil, waxiness in 20 hours, and 80mg/L's is red mould
Element (GA4+7) 4 DEG C soaks 28 hours, the mercuric chloride surface sterilization of 0.2% 6 minutes.Repeatedly rinse with sterilized water, with sterilized filter
Paper blots the surface of the seed moisture, is cut by seed, and the half granule seed taking band embryo is stand-by.
As preferably, with sterilized needle-nose pliers, embryo is extruded stand-by from the germinal aperature of kind of umbilical part.
(2) induction of Multiple Buds
Above-mentioned embryo is inoculated in inducing clumping bud culture medium, at proportion of red blue light (3:1), intensity of illumination 600-1200lux, light
Cycle is that 14 hours illumination/10 hour are dark, and corresponding cultivation temperature 23/18 DEG C is cultivated about 35 days, indefinite to being clearly visible
Bud bud point, it is thus achieved that outer implant.
Inducing culture based formulas is as follows: MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L,
Heteroauxing (IAA) 0.1-0.5mg/L, 6-benzyladenine (6-BA) 0.5mg/L, zeatin (ZT) 0.5-2mg/L.
As preferably, heteroauxing (IAA) concentration is 0.3mg/L, and zeatin (ZT) concentration is 1.5mg/L.
As preferably, available Thidiazuron (TDZ) 0.05-0.2mg/L of the zeatin (ZT) in inducing culture substitutes.
As preferably, Thidiazuron (TDZ) concentration is 0.1mg/L.
(3) maturation of Multiple Buds and segmentation
Above-mentioned outer implant is transferred into subculture medium, in proportion of red blue light (3:1), intensity of illumination 600-1200lux, photoperiod
It is that 14 hours illumination/10 hour are dark, corresponding cultivation temperature 23/18 DEG C, cultivate 10-20 days, grow 4-6 to every outer implant
Individual leaf bud, splits with 1-2 leaf bud for cutting unit.
Successive transfer culture based formulas is as follows: MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L,
Naphthalene acetic acid (NAA) 0.6mg/L, Thidiazuron (TDZ) 0.01-0.06mg/L, abscisic acid (ABA) 0.5-2mg/L.
As preferably, Thidiazuron (TDZ) concentration is 0.03mg/L, abscisic acid concentration is (ABA) 1mg/L.
(4) growth of seedling
Leaf bud after segmentation is proceeded to growth of seedling culture medium, at proportion of red blue light (3:1), intensity of illumination 600-1200lux, light
Cycle is that 14 hours illumination/10 hour are dark, and corresponding cultivation temperature 23/18 DEG C is cultivated and grown tall to seedling, become strong for 40 days, and
And have root restriction to be formed.
Growth of seedling culture medium prescription is as follows: MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, poly-second
Alkene pyrrole irons alkanone 1g/L, agar 5g/L, gibberellins (GA4+7) 0.2mg/L, brassinosteroid (BR) 0.01-0.1mg/L, excitement
Element (KT) 0.5-2mg/L.
As preferably, brassinosteroid (BR) concentration is 0.03mg/L, kinetins (KT) concentration is 1.5mg/L.
(5) formation of little rhizome
Seedling is proceeded in little rhizome inducing culture, at 26 DEG C, cultivate 60 days under dark condition, receive after seedling is gradually withered
Obtain little rhizome.
Little rhizome inducing culture formula is as follows: MS basic culture solution, sucrose 80g/L, yeast extract 1g/L, activity
Charcoal 1g/L, agar 5g/L, indolebutyric acid (IBA) 0.5mg/L, uniconazole P (S-3307) 2-6mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate (MEJA)
1-4mg/L, 6-benzyladenine (6-BA) 1.5mg/L.
As preferably, in little rhizome inducing culture, uniconazole P concentration is (S-3307) 4mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate
(MEJA) concentration is 2mg/L.
As preferably, in little rhizome inducing culture, 6-benzyladenine (6-BA) can use forchlorfenuron (CPPU) 0.5-
3mg/L substitutes.
As preferably, forchlorfenuron (CPPU) concentration is 1mg/L.
(6) storage of little rhizome
After being cleaned up by little rhizome, sand is hidden in 4 DEG C, and after about two months take out, it is little that 100mg/L gibberellins (GA4+7) soaks 1
Time, dry surface moisture and can sow.
The invention has the beneficial effects as follows: the mode that embryo is extruded from seed coat is reduced cut kind cause a loss of seeds,
Embryo injury, embryo brownization, ensure that the integrity of embryo, improve the utilization rate of seed, and reduce in endosperm raw
Bacterium is to the germination inhibitor suppression to embryo germination in the pollution of incubation and endosperm；The foundation of little Propagation of Rhizomes method is by Fructus Persicae
The induction of ERQI Multiple Buds and the induction of test tube little rhizome combine, and the little rhizome of test tube of this method induction can be as propagating materials land for growing field crops
Cultivation, compared with traditional tissue culture method, this method has reproductive efficiency height, breeding coefficient is big, cultivation cycle is short, convenient cultivation storage
Hide, convenient transport, easy and simple to handle, the advantage of low cost, improve Chinese podophyllum root tissue culture technology, expand cultivation further for Chinese podophyllum root
Plant and provide guarantee.
Detailed description of the invention
The following examples can further illustrate the present invention, but should not be construed as limitation of the present invention, without departing substantially from
In the case of present invention spirit and essence, the amendment that the inventive method, step or condition are made or replacement, belong to the present invention
Scope.
Hereinafter test the Chinese podophyllum root seed used, all pick up from Xinglong Mountain, Lanzhou height above sea level more than 2300 meters, be identified as
Chinese podophyllum root seed, is fresh seeds.
Embodiment 1: the acquisition of aseptic embryo.
By fresh peaches ERQI seed, remove and damage by worms and the seed that goes rotten, with a little concentrated sulphuric acid corrosion seed coat to color after washing
Blackout, the sodium carbonate soaking at room temperature with 0.5% removes the hydrophobic ingredients such as oil, waxiness, the gibberellins (GA4+ of 80mg/L in 20 hours
7) 4 DEG C are soaked 28 hours, the mercuric chloride surface sterilization of 0.2% 6 minutes.
Experimental group, by the seed of above-mentioned pretreatment, blots the surface of the seed moisture with sterilized filter paper, with sterilized point
Embryo is extruded from the germinal aperature of kind of umbilical part by mouth pincers, is inoculated in MS culture medium；Matched group Seeds preprocess mode and aforementioned phase
With, only go embryo to adopt with the following method: to be cut by seed with dissecting knife, the half granule seed with embryo is inoculated in MS culture medium.
Two groups of co-cultivation, observe sprouting, pollution, brownization situation.
Experimental result is as follows, experimental group: the germination rate of seed can reach more than 80%, does not find brownization and pollutes existing
As, simple to operate, loss of seeds is few, and speed of operation is fast；Matched group: the germination percentage of seed reaches about 70%, in operating process often
The problem occurring embryo to cut off, causes loss of seeds more serious, due to damage and the endosperm endophyte of embryo in incubation
Problem cause a certain degree of brownization and pollution, in endosperm, the existence of germination inhibitor there is also certain suppression to germination percentage
Effect.
Test result indicate that, the method that experimental group is used, will the method extruded from germinal aperature of embryo be preferred side
Method.
Embodiment 2: the hormon impact on inducing clumping bud.
Being outer implant with embryo, in proportion of red blue light (3:1), intensity of illumination 800lux, the photoperiod is 14 hours illumination/10
Hour dark, under conditions of corresponding cultivation temperature 23/18 DEG C, it is inoculated in basal medium (MS, sucrose 50g/L, agar 5g/
L, acid hydrolyzed casein 1000mg/L) and add hormon and cultivate, the proportioning of hormon sees following table, culture medium
PH value is 6.5.
| Hormone | 6-BA(mg/L) | IAA(mg/L) | ZT(mg/L) | Experimental result |
| 1# | 0.5 | 0.1 | 1 | Within about 20 days, outer implant is substantially expanded, inductivity 92% |
| 2# | 0.5 | 0.3 | 1 | Within about 13 days, outer implant is substantially expanded, inductivity 92%, IAA optium concentration |
| 3# | 0.5 | 0.5 | 1 | Within about 20 days, outer implant is substantially expanded, inductivity 92% |
| 4# | 0.5 | 0.3 | 0.5 | Within about 13 days, outer implant is substantially expanded, inductivity 90% |
| 5# | 0.5 | 0.3 | 1.5 | Within about 13 days, outer implant is substantially expanded, inductivity 96%, ZT optium concentration |
| 6# | 0.5 | 0.3 | 2 | Within about 13 days, outer implant is substantially expanded, inductivity 94%, and quality is loose frangible |
Above-mentioned process can induce Multiple Buds, and along with the change of IAA concentration 0.1-0.5mg/L, the induction time of Multiple Buds is not
With, 2# induced velocity is the fastest, 1# and 3# induced velocity does not has notable difference, and 2# is preferred concentration.ZT can improve inductivity, along with
The change of ZT concentration 0.5-2mg/L, inductivity increases, but the ZT(6# of high concentration) Multiple Buds induced, quality is loose frangible, no
Preferably carry out subsequent process steps.5# is ZT preferred concentration.By above experiment, obtain inducing culture based formulas I formula: MS is basic
Culture fluid, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L, heteroauxing (IAA) 0.1-0.5mg/L, 6-benzyl gland
Purine (6-BA) 0.5mg/L, zeatin (ZT) 0.5-2mg/L.Preferably inducing culture based formulas I formula: MS basic culture solution,
Sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L, heteroauxing (IAA) 0.3mg/L, 6-benzyladenine (6-BA)
0.5mg/L, zeatin (ZT) 1.5mg/L.
Experimental technique ibid, changes different hormones and processes, and obtains following result:
| Hormone | 6-BA(mg/L) | IAA(mg/L) | TDZ(mg/L) | Experimental result |
| 1# | 0.5 | 0.1 | 0.15 | Within about 20 days, outer implant is substantially expanded, inductivity 96% |
| 2# | 0.5 | 0.3 | 0.15 | Within about 13 days, outer implant is substantially expanded, inductivity 96%, IAA optium concentration |
| 3# | 0.5 | 0.5 | 0.15 | Within about 20 days, outer implant is substantially expanded, inductivity 96% |
| 4# | 0.5 | 0.3 | 0.05 | Within about 13 days, outer implant is substantially expanded, and inductivity 91%, quality is hard |
| 5# | 0.5 | 0.3 | 0.1 | Within about 13 days, outer implant is substantially expanded, inductivity 98%, and quality is hard, TDZ optium concentration |
| 6# | 0.5 | 0.3 | 0.2 | Within about 13 days, outer implant is substantially expanded, and inductivity 96% has wound healing trend |
Above-mentioned process can induce Multiple Buds, and along with the change of IAA concentration 0.1-0.5mg/L, the induction time of Multiple Buds is not
With, 2# induced velocity is the fastest, 1# and 3# induced velocity does not has notable difference, and 2# is preferred concentration.TDZ can improve inductivity, with
The change of TDZ concentration 0.05-0.2, inductivity increases, but the TDZ(6# of high concentration) there is the trend of callus induction, unsuitable
Carrying out subsequent process steps, 5# is TDZ preferred concentration.By above experiment, obtain inducing culture based formulas II formula: MS is basic
Culture fluid, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L, heteroauxing (IAA) 0.1-0.5mg/L, 6-benzyl gland
Purine (6-BA) 0.5mg/L, Thidiazuron (TDZ) 0.05-0.2mg/L.Preferably inducing culture based formulas II formula: MS cultivates substantially
Liquid, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L, heteroauxing (IAA) 0.3mg/L, 6-benzyladenine (6-
BA) 0.5mg/L, Thidiazuron (TDZ) 0.1mg/L.
Embodiment 3: the illumination impact on inducing clumping bud.
Use the preferred inducing culture determined in embodiment 2 that embryo is cultivated, conversion proportion of red blue light and light
According to intensity, remaining condition of culture is constant, carries out inducing clumping bud experiment, and result is as follows:
| Hormone | 6-BA(mg/L) | IAA(mg/L) | ZT(mg/L) | TDZ(mg/L) | Proportion of red blue light | Light intensity (lux) | Experimental result |
| 1# | 0.5 | 0.3 | 1.5 | 0 | 3:1 | 600 | Late stage of culture has excessive growth phenomenon |
| 2# | 0.5 | 0.3 | 1.5 | 0 | 3:1 | 800 | Late stage of culture robust growth, leaf color is dark green |
| 3# | 0.5 | 0.3 | 1.5 | 0 | 3:1 | 1000 | Late stage of culture robust growth, leaf color is dark green |
| 4# | 0.5 | 0.3 | 1.5 | 0 | 3:1 | 1200 | The outer implant of late stage of culture has withered, the oxidative damage sign of browning |
| 5# | 0.5 | 0.3 | 0 | 0.1 | 3:1 | 600 | Late stage of culture has excessive growth phenomenon |
| 6# | 0.5 | 0.3 | 0 | 0.1 | 3:1 | 800 | Late stage of culture robust growth, leaf color is dark green |
| 7# | 0.5 | 0.3 | 0 | 0.1 | 3:1 | 1000 | Late stage of culture robust growth, leaf color is dark green |
| 8# | 0.5 | 0.3 | 0 | 0.1 | 3:1 | 1200 | The outer implant of late stage of culture has withered, the oxidative damage sign of browning |
| 9# | 0.5 | 0.3 | 1.5 | 0 | 1:1 | 800 | The outer implant of late stage of culture has withered, the oxidative damage sign of browning |
| 10# | 0.5 | 0.3 | 0 | 0.1 | 1:1 | 800 | The outer implant of late stage of culture has withered, the oxidative damage sign of browning |
Under the conditions of the light quality of proportion of red blue light (3:1), along with the increase (600-1200lux) of intensity of illumination, bud point more enriching
Green, growth effect more rapid, 800lux and 1000lux is best, it is contemplated that cost, 800lux is optimization process, along with light
The continuation of illumination increases, and the outer implant of 1200lux has withered, the trend of color browning.At 9# and 10# proportion of red blue light (1:1)
Under the conditions of light quality, along with the prolongation of incubation time, outer planting soma is withered, black, show the sign of oxidative damage.So really
Determining illumination cultivation condition is proportion of red blue light (3:1), intensity of illumination 800lux.
Embodiment 4: the maturation of Multiple Buds and segmentation.
The outer implant that above-mentioned induced bundle is sprouted, in proportion of red blue light (3:1), intensity of illumination 800lux, the photoperiod is 14
Hour illumination/10 hour are dark, under conditions of corresponding cultivation temperature 23/18 DEG C, are inoculated in basal medium (MS, sucrose
50g/L, agar 5g/L, acid hydrolyzed casein 1000mg/L) and add hormon and cultivate, the proportioning of hormon sees
Following table, the pH value of culture medium is 6.5.After being clearly visible leaf bud, split with 1-2 leaf bud for cutting unit.
| Hormone | NAA(mg/L) | TDZ(mg/L) | ABA(mg/L) | Experimental result |
| 1# | 0.6 | 0.01 | 1.5 | The subculture of Multiple Buds and breeding are relatively slow, every average 4 leaf buds of outer implant |
| 2# | 0.6 | 0.03 | 1.5 | The subculture of Multiple Buds and breeding are very fast, every average 5 leaf buds of outer implant, TDZ optium concentration |
| 3# | 0.6 | 0.06 | 1.5 | The subculture of Multiple Buds and breeding are very fast, every average 6 leaf buds of outer implant, but deformity bud increases |
| 4# | 0.6 | 0.03 | 0.5 | The subculture of Multiple Buds and breeding are very fast, every average 5 leaf buds of outer implant, but have a small amount of deformity bud |
| 5# | 0.6 | 0.03 | 1 | The subculture of Multiple Buds and breeding are the fastest, every average 6 leaf buds of outer implant, and deformity bud is few, ABA optium concentration |
| 6# | 0.6 | 0.03 | 2 | The subculture of Multiple Buds and breeding are very fast, every average 5 leaf buds of outer implant, and deformity bud is few, but browning increases |
More than process and can promote the maturation of Multiple Buds, along with the increase (0.01-0.06mg/L) of TDZ concentration, Multiple Buds growth,
Breeding accelerate, but the TDZ(0.06mg/L of high concentration) deformity bud increase, consider TDZ(0.03mg/L) be optium concentration.With
The increase (0.5-2mg/L) of ABA concentration, the maturation of bud can be promoted, suppress the generation of deformity, but high concentration (2mg/L), bud
The speed of growth slows down, and has the trend of brownization, considers, ABA(1mg/L) it is optimization process.According to above experiment, obtain
Successive transfer culture based formulas is as follows: MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L, naphthalene acetic acid
(NAA) 0.6mg/L, Thidiazuron (TDZ) 0.01-0.06mg/L, abscisic acid (ABA) 0.5-2mg/L.Preferably successive transfer culture basigamy
Side: MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L, naphthalene acetic acid (NAA) 0.6mg/L, Thidiazuron
(TDZ) 0.03mg/L, abscisic acid (ABA) 1mg/L.
Embodiment 5: the growth of seedling.
The outer implant of above-mentioned segmentation, at proportion of red blue light (3:1), intensity of illumination 800lux, the photoperiod be illumination in 14 hours/
10 hours dark, under conditions of corresponding cultivation temperature 23/18 DEG C, is inoculated in basal medium (MS, sucrose 50g/L, agar
5g/L, acid hydrolyzed casein 1000mg/L, Nitrophenols ketone 1g/L) and add hormon and cultivate, hormon
Proportioning see following table, the pH value of culture medium is 6.5.When the height of seedling grows to about about 5cm, and root length 1cm, turn
Enter little rhizome inducing culture.
| Hormone | GA4+7(mg/L) | BR(mg/L) | KT(mg/L) | Experimental result |
| 1# | 0.2 | 0.01 | 1 | Seedling elongation speed, takes root preferably |
| 2# | 0.2 | 0.03 | 1 | Seedling elongation is fastest, takes root preferably, BR optium concentration |
| 3# | 0.2 | 0.06 | 1 | Seedling elongation is fastest, takes root preferably, and blade, petiole have increase, increase thick trend |
| 4# | 0.2 | 0.1 | 1 | Seedling elongation speed, takes root preferably, and blade, petiole have increase, increase thick trend |
| 5# | 0.2 | 0.03 | 0.5 | Seedling elongation is fastest, leaf color is dark green, fresh, has hestening rooting sign |
| 6# | 0.2 | 0.03 | 1.2 | Seedling elongation is fastest, leaf color is dark green, fresh, takes root preferably |
| 7# | 0.2 | 0.03 | 1.5 | Seedling elongation is fastest, leaf color is dark green, fresh, and rooting efficiency is best, KT optium concentration |
| 8# | 0.2 | 0.03 | 2 | Seedling elongation is fastest, leaf color is dark green, fresh, and rooting efficiency is best |
Above-mentioned process can promote the growth of seedling, along with the increase (0.01-0.1mg/L) of BR concentration, seedling elongation speedometer
Reveal trend in a slight decrease after first increasing, but along with concentration increases, blade, petiole have increase, increase thick trend.BR
(0.03mg/L) it is optium concentration.Along with the increase (0.5-2mg/L) of KT concentration, seedling leaf color is more dark green, fresh, and has
The trend of hestening rooting, 7# and 8# process does not has notable difference, it is contemplated that cost, KT(1.5mg/L) it is optium concentration.By upper
State experiment, obtain growth of seedling culture medium prescription as follows: be MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, poly-
Ethylene pyrrole irons alkanone 1g/L, agar 5g/L, gibberellins (GA4+7) 0.2mg/L, brassinosteroid (BR) 0.01-0.1mg/L, swashs
Therbligs (KT) 0.5-2mg/L.Preferably growth of seedling culture medium prescription is as follows: MS basic culture solution, sucrose 50g/L, acid hydrolysis cheese
Albumen 1g/L, Nitrophenols ketone 1g/L, agar 5g/L, gibberellins (GA4+7) 0.2mg/L, brassinosteroid (BR)
0.03mg/L, kinetins (KT) 1.5mg/L.
Embodiment 6: the induction of little rhizome.
Above-mentioned seedling, under the dark condition of 26 DEG C, is inoculated in basal medium (MS, sucrose 80g/L, agar 5g/L, ferment
Female extract 1000mg/L, activated carbon 1g/L) and add hormon and cultivate, the proportioning of hormon sees following table, training
The pH value supporting base is 6.5.
Above-mentioned process can be induced the generation of little rhizome and be expanded, along with the increase (1-4mg/L) of MEJA concentration, induced velocity and
Little rhizome number is gradually increased, but high concentration MEJA(4mg/L) browning increases, 2# and 3# does not has obvious difference, it is considered to
To cost MEJA(2mg/L) it is optium concentration.Along with the increase (2-6mg/L) of S-3307 concentration, little rhizome is gradually increased, but high
The S-3307(6mg/L of concentration) abnormal phenomenon increase, consider, optimal S-3307 concentration is 4mg/L.By above experiment,
Obtain little rhizome inducing culture formula I as follows: MS basic culture solution, sucrose 80g/L, yeast extract 1g/L, activated carbon
1g/L, agar 5g/L, indolebutyric acid (IBA) 0.5mg/L, uniconazole P (S-3307) 2-6mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate (MEJA) 1-
4mg/L, 6-benzyladenine (6-BA) 1.5mg/L.The least rhizome inducing culture formula I is as follows: S basic culture solution, sugarcane
Sugar 80g/L, yeast extract 1g/L, activated carbon 1g/L, agar 5g/L, indolebutyric acid (IBA) 0.5mg/L, uniconazole P (S-
3307) 4mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate (MEJA) 2mg/L, 6-benzyladenine (6-BA) 1.5mg/L.
Experimental technique ibid, changes different hormones and processes, and obtains following result:
Above-mentioned process can be induced the generation of little rhizome and be expanded, along with the increase (0.5-3mg/L) of CPPU concentration, little rhizome
Induced efficiency and size are gradually increased, but the CPPU(3mg/L of high concentration) little rhizome deformity can be increased, 2# and 3# is the poorest
Different, it is contemplated that cost, optimal CPPU concentration is 1mg/L.By above experiment, obtain little rhizome inducing culture formula II such as
Under: MS basic culture solution, sucrose 80g/L, yeast extract 1g/L, activated carbon 1g/L, agar 5g/L, indolebutyric acid (IBA)
0.5mg/L, uniconazole P (S-3307) 2-6mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate (MEJA) 1-4mg/L, forchlorfenuron (CPPU) 0.5-
3mg/L.The least rhizome inducing culture formula II is as follows: MS basic culture solution, sucrose 80g/L, yeast extract 1g/L,
Activated carbon 1g/L, agar 5g/L, indolebutyric acid (IBA) 0.5mg/L, uniconazole P (S-3307) 4mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate
(MEJA) 2mg/L, forchlorfenuron (CPPU) 1mg/L.
Embodiment 7: the storage of little rhizome.
Above-mentioned little rhizome falls after Seedling, removes residual leaf, cleans up culture medium, and sand is hidden in 4 DEG C, taking-up in about two months,
100mg/L gibberellins (GA4+7) soaks 1 hour, dries surface moisture and can sow.Sha Zangzhong has part rootlet stem rot rotten, just
Normal little rhizome is seeded in turfy soil: perlite: the mixed-matrix of Vermiculitum (3:1:1), cultivates, about 40 days under the conditions of 18 DEG C
Grow seedling.
Claims (9)
1. a Chinese podophyllum root little Propagation of Rhizomes method, it is characterised in that comprise the steps:
(1) aseptic embryo is obtained: by fresh Chinese podophyllum root seed disinfection, take out aseptic embryo；
(2) induction of Multiple Buds: aseptic embryo is inoculated in inducing clumping bud culture medium, in proportion of red blue light 3:1, intensity of illumination
600-1200lux, the photoperiod is that 14 hours illumination/10 hour are dark, and corresponding cultivation temperature 23/18 DEG C is cultivated 35 days, to seeing
To adventitious bud bud point, it is thus achieved that outer implant；
Inducing culture based formulas: MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L, heteroauxing
0.1-0.5mg/L, 6-benzyladenine 0.5mg/L, zeatin 0.5-2mg/L；
(3) maturation of Multiple Buds and segmentation: outer implant is transferred into subculture medium, illumination and temperature same step (2), cultivate
10-20 days, grow 4-6 leaf bud to every outer implant, split for cutting unit with 1-2 leaf bud；
Successive transfer culture based formulas: MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, agar 5g/L, naphthalene acetic acid
0.6mg/L, Thidiazuron 0.01-0.06mg/L, abscisic acid 0.5-2mg/L；
(4) growth of seedling: the leaf bud after segmentation is proceeded to growth of seedling culture medium, illumination and temperature same step (2), cultivates
Within 40 days, grow tall to seedling, become strong, and have root restriction to be formed；
Growth of seedling culture medium prescription: MS basic culture solution, sucrose 50g/L, acid hydrolyzed casein 1g/L, Nitrophenols
Ketone 1g/L, agar 5g/L, gibberellins 0.2mg/L, brassinosteroid 0.01-0.1mg/L, kinetins 0.5-2mg/L；
(5) formation of little rhizome: proceeded to by seedling in little rhizome inducing culture, at 26 DEG C, cultivates 60 days under dark condition,
After seedling is gradually withered, gathers in the crops little rhizome and stores；
Little rhizome inducing culture formula: MS basic culture solution, sucrose 80g/L, yeast extract 1g/L, activated carbon 1g/L, fine jade
Fat 5g/L, indolebutyric acid 0.5mg/L, uniconazole P 2-6mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate 1-4mg/L, 6-benzyladenine 1.5mg/L.
Chinese podophyllum root the most according to claim 1 little Propagation of Rhizomes method, it is characterised in that described step (1) aseptic embryo
Removing method is to be extruded from the germinal aperature of kind of umbilical part by embryo.
Chinese podophyllum root the most according to claim 1 little Propagation of Rhizomes method, it is characterised in that heteroauxing in described step (2)
Concentration is 0.3mg/L, and zeatin concentration is 1.5mg/L.
Chinese podophyllum root the most according to claim 1 little Propagation of Rhizomes method, it is characterised in that in described step (2), zeatin can
Substituting with Thidiazuron 0.05-0.2mg/L, Thidiazuron concentration is 0.1mg/L.
Chinese podophyllum root the most according to claim 1 little Propagation of Rhizomes method, it is characterised in that in described step (3), Thidiazuron is dense
Degree is 1mg/L for 0.03mg/L, abscisic acid concentration.
Chinese podophyllum root the most according to claim 1 little Propagation of Rhizomes method, it is characterised in that in described step (4) in Brassica campestris L element
Ester concentration is 0.03mg/L, kinetins concentration is 1.5mg/L.
Chinese podophyllum root the most according to claim 1 little Propagation of Rhizomes method, it is characterised in that in described step (5), uniconazole P is dense
Degree is 2mg/L for 4mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate concentration.
Chinese podophyllum root the most according to claim 1 little Propagation of Rhizomes method, it is characterised in that 6-benzyl gland in described step (5)
Purine can substitute with forchlorfenuron 0.5-3mg/L, and forchlorfenuron concentration is 1mg/L.
Chinese podophyllum root the most according to claim 1 little Propagation of Rhizomes method, it is characterised in that described step (5) medium and small rhizome
Storage method is: after being cleaned up by little rhizome, and after 4 DEG C of husky Tibetan are taken out for two months, 100mg/L gibberellins soaks 1 hour, dries
Surface moisture can be sowed.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1849882A (en) * | 2005-04-22 | 2006-10-25 | 甘肃省科学院生物研究所 | Method for producing podophyllotoxin induced from sino podophyllum hexandrum callus |
| CN102763599A (en) * | 2012-08-20 | 2012-11-07 | 甘肃省科学院生物研究所 | Podophyllum hexandrum tissue culture seedling and exercising seedling transplanting acclimatization method |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1849882A (en) * | 2005-04-22 | 2006-10-25 | 甘肃省科学院生物研究所 | Method for producing podophyllotoxin induced from sino podophyllum hexandrum callus |
| CN102763599A (en) * | 2012-08-20 | 2012-11-07 | 甘肃省科学院生物研究所 | Podophyllum hexandrum tissue culture seedling and exercising seedling transplanting acclimatization method |
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| QI GUO ET AL.: "In vitro Rooting of Podophyllum Hexandrum and Transplanting Technique", 《ENGINEERING》 * |
| 叶耀辉等: "珍稀藏药桃儿七组织培养研究", 《江西农业大学学报》 * |
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