CN106973795A - A kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo - Google Patents
A kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo Download PDFInfo
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- CN106973795A CN106973795A CN201710328848.0A CN201710328848A CN106973795A CN 106973795 A CN106973795 A CN 106973795A CN 201710328848 A CN201710328848 A CN 201710328848A CN 106973795 A CN106973795 A CN 106973795A
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- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 39
- 230000000392 somatic effect Effects 0.000 title claims abstract description 37
- 230000001939 inductive effect Effects 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 13
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 46
- 238000012258 culturing Methods 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 238000009966 trimming Methods 0.000 claims abstract description 4
- 230000000763 evoking effect Effects 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 6
- 230000006698 induction Effects 0.000 claims description 4
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- 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 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 238000009940 knitting Methods 0.000 claims 1
- 238000005286 illumination Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 230000004069 differentiation Effects 0.000 abstract description 2
- 230000036512 infertility Effects 0.000 abstract 1
- 241001092371 Bergenia Species 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- YWJXCIXBAKGUKZ-HJJNZUOJSA-N Bergenin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@H]2C3=C(O)C(OC)=C(O)C=C3C(=O)O[C@@H]21 YWJXCIXBAKGUKZ-HJJNZUOJSA-N 0.000 description 2
- XULPLJSODQQHPH-UHFFFAOYSA-N Bergenin Natural products OCC1OC2C(OC(=O)c3cc(O)c(CO)c(O)c23)C(O)C1O XULPLJSODQQHPH-UHFFFAOYSA-N 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002932 luster Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 240000004972 Bergenia crassifolia Species 0.000 description 1
- 235000014785 Bergenia crassifolia Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000625 blastula Anatomy 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000025366 tissue development Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 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
- A01H4/001—Culture apparatus for tissue culture
-
- 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)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo, takes Rhizoma Seu Herba Bergeniae tests for sterility, wound occurs in trimming blade edge, and blade is positioned over into the mg/L+V of the mg/L+KT0.01 of culture medium MS+2,4 D0.1~1.0~0.1COn 0.05~1.0 mg/L, dark culturing；The callus that induces is peeled off, is transferred into the mg/L+V of the mg/L+NAA0.01 of proliferated culture medium MS+ZT0.5~2.0~0.1C0.05~1.0 mg/L, dark culturing；Callus is transferred into the mg/L+V of the mg/L+IBA0.01 of inducing culture MS+ZT0.1~2.0~0.1C0.05~1.0 mg/L, dark culturing is induced after somatic embryo, is transferred under illumination and is cultivated.The differentiation rate of somatic embryo is up to 26.67% in the present invention.
Description
Technical field
The invention belongs to field of biology, it is related to the Rhizoma Seu Herba Bergeniae of Bergenia, is a kind of induction Rhizoma Seu Herba Bergeniae body
The method of cell stage.
Background technology
Rhizoma Seu Herba Bergeniae (Bergenia crassifolia (L.) Fritsch) is that Saxifragaceae Bergenia is perennial often
Greenweed sheet, is the larger species of body in the category, and domestic wild species are rarely seen to be distributed in Altai in Xinjiang Province, and pole early spring blooms, winter
Blade is in aubergine, and floral leaf is all beautiful.Herb contains Bergenin, is the specific drug for treating respiratory disease.Under nature,
Rhizoma Seu Herba Bergeniae broadcasts breeding, low reproduction rate mainly by plant division or seed.Wild resource is utilized by excessive excavation, and resource is on the point of increasingly
Danger.
The content of the invention
It is an object of the invention to provide a kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo.Described this induction
The method of Rhizoma Seu Herba Bergeniae somatic embryo will be solved in the prior art, the technical problem of Rhizoma Seu Herba Bergeniae low reproduction rate, and be
Genetic transformation lays the foundation.
The invention provides a kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo, comprise the following steps：
1) evoked callus：Second pair of in vitro cuttings morphology upper end, the 3rd pair of blade are taken, trimming blade edge goes out
Existing wound, blade distal shaft is put in MS+2,0.1~1.0mg/L+KT of 4-D, 0.01~0.1mg/L+V downC0.05~
On 1.0mg/L culture mediums, dark culturing is cultivated 25~35 days；
2) callus is bred：The callus induced is peeled off, is transferred into 0.5~2.0mg/ of proliferated culture medium MS+ZT
0.01~0.1mg/L+V of L+NAAC0.05~1.0mg/L, dark culturing is cultivated 25~35 days；
3) inducing somatic embryo：Callus is transferred into 0.1~2.0mg/L+IBA0.01 of inducing culture MS+ZT
~0.1mg/L+VC0.05~1.0mg/L, dark culturing is cultivated 55~65 days, induces after somatic embryo, transfer to
Cultivated under illumination.
Further, in the culture medium of evoked callus, 2,4-D concentration can be any interval 0.1~1.0mg/L
Concentration；KT concentration can be any concentration interval 0.01~0.1mg/L；VCConcentration can be 0.05~1.0mg/L intervals
Any concentration.Wherein optimal callus induction culture medium is MS+2,4-D 1.0mg/L+KT 0.1mg/L+VC0.5mg/L, it is dark
Culture 30 days.
Further, in callus proliferation medium, ZT concentration can be any concentration interval 0.5~2.0mg/L；
NAA concentration can be any concentration interval 0.01~0.1mg/L；VCConcentration can be any interval 0.05~1.0mg/L
Concentration.Its optimal callus proliferation medium is MS+ZT 1.0mg/L+NAA 0.1mg/L+VC0.5mg/L, dark culturing
30 days.
Further, in the culture medium of inducing somatic embryo, ZT concentration can be any interval 0.1~2.0mg/L
Concentration；IBA concentration can be any concentration interval 0.01~0.1mg/L；VCConcentration can be 0.05~1.0mg/L intervals
Any concentration.Its optimal inducing somatic embryo culture medium is MS+ZT 1.0mg/L+IBA 0.1mg/L+VC0.5mg/L is black
Light culture 60 days.
Further, above-mentioned various culture mediums, its pH value is 5.6~5.9 interval any values；The quality percentage of sucrose
Specific concentration is 2.5%~3.5% interval any value；The mass percent concentration of agar is 0.55%~0.75% interval
Any value.
Rhizoma Seu Herba Bergeniae is as the wider Chinese medicine of purposes, and demand constantly rises, the size of body, fast growth
Slowly, can annidation and Determination of Bergenin height be to determine its several key factor that promote.The present invention is established more
Injured tissue development ways, have induced Bergenia somatic embryo first, are to expand numerous popularization, genetic transformation and ploidy from now on to educate
Cultivation new varieties are planted to lay the foundation.
The present invention is compared with prior art, is had the technical effect that actively and it will be evident that and unique, novelty and innovation
Property.The present invention induces callus, and then induce somatic embryo using the tender leaf of Rhizoma Seu Herba Bergeniae aseptic seedling as parent material
Tire, differentiation rate is up to 26.67%, is that genetic transformation and ploidy breeding are laid a good foundation.
Brief description of the drawings
Fig. 1 is the photo of Rhizoma Seu Herba Bergeniae callus.
Fig. 2 is the photo of Rhizoma Seu Herba Bergeniae somatic embryo.
Fig. 3 is the photo of Rhizoma Seu Herba Bergeniae cotyledonary embryos.
Fig. 4 is the section of Rhizoma Seu Herba Bergeniae cotyledonary embryos.
Embodiment
Initialism bilingual table in following embodiments
Every kind of processing investigation 30 in following embodiments, statistics evoked callus rate, callus weightening multiple, body are thin
Blastula tire inductivity.Unless otherwise indicated, every kind of processing is repeated 3 times.
Evoked callus rate=callus number/inoculation number, callus weightening multiple=(heavy after callus tissue culture
Weight before amount-callus tissue culture) weight, somatic embryo inductivity=somatic embryo number/inoculation before/callus tissue culture
Number.
Statistical analysis is carried out to data using Excel 2003 and the softwares of SPSS 18.0.Using single factor test (one-way
) and Duncan methods carry out variance analysis and Multiple range test (α=0.05) ANOVA
The pH value of various culture mediums is 5.6~5.9 in following embodiments, the mass percent concentration of sucrose for 2.5%~
3.5%, the mass percent concentration of agar is 0.55%~0.75%.
The evoked callus of embodiment 1
Take second pair of in vitro cuttings morphology upper end, the 3rd pair of blade.
There is wound in trimming blade edge, so that nutrient absorption with breaking up again, and blade distal shaft is positioned over culture down
Base MS+2,4-D0.1~1.0mg/L+KT0.01~0.1mg/L+VC0.05~1.0mg/L, dark culturing.
Blade starts at edge notches to produce the callus group of milk yellow in the 10d of evoked callus medium culture
Knit, with the extension of incubation time, callus increases, when cultivating to 30d, can peel, carry out Multiplying culture.
Embodiment 2 breeds callus
Induce obtained callus to be divided into 5mm sizes primary, squamous subculture on proliferated culture medium of transferring, using MS as
Basal medium, ZT, BA concentration is arranged between 0.5~2.0mg/L, and NAA concentration is arranged between 0.01~0.1mg/L, VC
Concentration is arranged between 0.05~1.0mg/L, dark culturing, and 30d is a cycle.
The influence that table 1 BA, ZT and various concentrations level are bred to callus
Note：Data are mean+SD in table, and letter different after numeral represents that 0.05 level difference is notable in table
Property (P ﹤ 0.05)
Culture medium designed by the present invention, callus can carry out propagation growth, but be very easy to brown stain, Suo Youpei
Support in base and all with the addition of VC0.05~1.0mg/L, and dark culturing is taken, also brown stain journey is simply alleviated to a certain extent
Degree, extends the brown stain time, but can not fundamentally solve the brown stain of callus.Cultivated through 30d, callus enlarged volume,
Weight gain is original 1.1~2.5 times (tables 1), wherein in MS+ZT1.0mg/L+NAA0.1mg/L+VC0.5mg/L culture mediums
On, growth rate is most fast, and compared with other processing, significant difference, part callus is creamy white (Fig. 1), later stage culture card
It is bright to be easier to break up.
Callus is in MS+ZT1.0mg/L+NAA0.1mg/L+VCCultivated on 0.5mg/L culture mediums after 30d, select color and luster
Milky white, compact structure, size is similar, consistent callus of originating, and transfers on the MS minimal mediums without any hormone, no
How long is pipe incubation time, can not all differentiate adventitious bud, can not induce somatic embryo.Its callus further divides
Change or the derived need addition basic element of cell division, cultivation results depend on the hormone kind of addition, and addition BA culture medium is differentiated
Adventitious bud, and the culture medium for adding ZT can then induce somatic embryo.
The inducing somatic embryo of embodiment 3
The culture medium of inducing somatic embryo culture medium based on MS, ZT concentration is arranged between 0.1~2.0mg/L,
IBA concentration is arranged between 0.01~0.1mg/L, VCConcentration is arranged between 0.05~1.0mg/L, dark culturing, is induced
After somatic embryo, transfer under illumination and cultivate.
On addition ZT culture medium, Rhizoma Seu Herba Bergeniae callus can induce somatic embryo, when its concentration is super
When crossing 1.0mg/L, compared with less than other groups of this concentration, treatment effect difference is significantly (table 2).Found through screening in MS+
ZT1.0mg/L+IBA0.1mg/L+VCWhen being cultivated on 0.5mg/L culture mediums to 60d, inductivity is up to 26.67%, goes out embryo and is cured
Injured tissue can be with the individual cells embryo (Fig. 2) of induced synthesis 2~3.The somatic embryo form induced is normal, and color and luster is tender white,
Plumule end radiculodium substantially, easily departs from from callus, and individually development (Fig. 3), is observed, its cotyledonary embryos by histotomy
With independent vascular structure, and with obvious bipolarity (Fig. 4).But callus needs to be constantly in dark and trained
Support, cultivated if callus is gone under light, when culture is to 10d, start gradually brown stain, it is completely dead when cultivating to 30d
Die, can not break up somatic embryo.
The influence that the hormon of table 2 and hormon level are induced somatic embryo
Note：Data are mean+SD in table, and letter different after numeral represents that 0.05 level difference is notable in table
Property (P ﹤ 0.05)
Conclusion：
Chinese Academy Of Sciences Xinjiang physics ＆ chemistry Technology Research Institute differentiates adventitious bud by callus approach, but has no and induce
The report of somatic embryo, the present invention induces somatic embryo, is that the platymiscium is being lured finally by the exploration up to 2 years
Breakthrough first in terms of conductor cell stage.But in the culture of the present invention, equally occur in that the feelings of the serious brown stain of callus
Condition, adds anti-browning agent VC, PVP, sodium thiosulfate when, browning rate decreases, but still can not fundamentally solve brown stain
Problem, the brown stain of callus is also to cause current somatic embryo inductivity than relatively low major reason.The present invention is mainly adopted
With the pattern of light culture, induce after somatic embryo, transfer under light training mode to reduce brown stain.
Claims (5)
1. a kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo, it is characterised in that：Comprise the following steps：
1) evoked callus：Second pair of in vitro cuttings morphology upper end, the 3rd pair of blade are taken, trimming blade edge is hindered
Mouthful, blade distal shaft is put in MS+2, the mg/L+V of 0.1~1.0 mg/L+KT of 4-D 0.01~0.1 downC 0.05~1.0 mg/
On L culture mediums, dark culturing is cultivated 25 ~ 35 days；
2) callus is bred：The callus induced is peeled off, is transferred into the mg/L+ of proliferated culture medium MS+ZT 0.5~2.0
The mg/L+V of NAA 0.01~0.1C 0.05~1.0 mg/L, dark culturing is cultivated 25 ~ 35 days；
3) inducing somatic embryo：By callus transfer into the mg/L+IBA 0.01 of inducing culture MS+ZT 0.1~2.0~
0.1 mg/L+VC 0.05~1.0 mg/L, dark culturing is cultivated 55 ~ 65 days, induces after somatic embryo, transfer to light
According to lower culture.
2. a kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo according to claim 1, it is characterised in that：Induction is cured
The optimal medium of injured tissue is the mg/L+KT0.1 mg/L+V of MS+2,4-D 1.0C 0.5 mg/L, dark culturing 30 days is optimal.
3. a kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo according to claim 1, it is characterised in that：Callus group
The optimal medium for knitting propagation is the mg/L+V of 1.0 mg/L+NAA of MS+ZT 0.1C 0.5 mg/L, dark culturing 30 days is optimal.
4. a kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo according to claim 1, it is characterised in that：Inductor
The optimal medium of cell stage is the mg/L+V of 1.0 mg/L+IBA of MS+ZT 0.1C 0.5 mg/L, dark culturing 60 days is most
It is good.
5. a kind of method for inducing Rhizoma Seu Herba Bergeniae somatic embryo according to claim 1, it is characterised in that：It is above-mentioned each
The pH value planted in culture medium is 5.6~5.9, and the mass percent concentration of sucrose is 2.5%~3.5%, the quality hundred of agar
It is 0.55%~0.75% to divide specific concentration.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101116425A (en) * | 2007-09-19 | 2008-02-06 | 中国科学院新疆理化技术研究所 | A method for preparing rock cabbage with biological tissue culture method |
| CN102948367A (en) * | 2012-03-16 | 2013-03-06 | 上海市园林科学研究所 | Method for performing in-vitro culturing and rapid propagating on bergenia crassifolia |
| CN103392599A (en) * | 2013-07-26 | 2013-11-20 | 云南农业大学 | Bergenia purpurascens(hook.f.et Thoms.)Engl. tender leaf tissue culture rapid propagation method |
-
2017
- 2017-05-11 CN CN201710328848.0A patent/CN106973795A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101116425A (en) * | 2007-09-19 | 2008-02-06 | 中国科学院新疆理化技术研究所 | A method for preparing rock cabbage with biological tissue culture method |
| CN102948367A (en) * | 2012-03-16 | 2013-03-06 | 上海市园林科学研究所 | Method for performing in-vitro culturing and rapid propagating on bergenia crassifolia |
| CN103392599A (en) * | 2013-07-26 | 2013-11-20 | 云南农业大学 | Bergenia purpurascens(hook.f.et Thoms.)Engl. tender leaf tissue culture rapid propagation method |
Non-Patent Citations (1)
| Title |
|---|
| 吕秀立等: "厚叶岩白菜叶片离体培养研究", 《上海交通大学学报（农业科学版）》 * |
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Application publication date: 20170725 |