CN103718962B - Culture mediums for tissue culturing of maiden pink - Google Patents
Culture mediums for tissue culturing of maiden pink Download PDFInfo
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- CN103718962B CN103718962B CN201310716702.5A CN201310716702A CN103718962B CN 103718962 B CN103718962 B CN 103718962B CN 201310716702 A CN201310716702 A CN 201310716702A CN 103718962 B CN103718962 B CN 103718962B
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- 241001166698 Dianthus deltoides Species 0.000 title abstract description 6
- 238000012258 culturing Methods 0.000 title abstract description 5
- 239000001963 growth medium Substances 0.000 claims abstract description 37
- 240000003421 Dianthus chinensis Species 0.000 claims description 49
- 238000000034 method Methods 0.000 claims description 15
- 230000001939 inductive effect Effects 0.000 claims description 11
- 238000012549 training Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims 1
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- 230000006698 induction Effects 0.000 abstract description 5
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- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 abstract description 3
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 abstract description 3
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- 239000012883 rooting culture medium Substances 0.000 abstract 1
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- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 238000004017 vitrification Methods 0.000 description 4
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 4
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 3
- 235000017491 Bambusa tulda Nutrition 0.000 description 3
- 241001330002 Bambuseae Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 3
- 239000011425 bamboo Substances 0.000 description 3
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- 241000219321 Caryophyllaceae Species 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
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- 229940064880 inositol 100 mg Drugs 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 229960003512 nicotinic acid Drugs 0.000 description 2
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Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to culture mediums for tissue culturing of a maiden pink. The optimal culture mediums for different stages in the tissue culturing of the maiden pink are screened; and the matching proportions of various hormones in the culture mediums are determined, so that the culture mediums prepared according to a formula provided by the invention can meet the nutritional requirement and the growing development requirement of the maiden pink in all stages. The culture media respectively comprise: an initial induction culture medium formed by MS, 0.01mg/L of 6-BA and 0.05mg/L of NAA (Naphthalene Acetic Acid), a mulitiplication culture medium formed by the MS, 0.01mg/L of 6-BA and 0.05mg/L of NAA, and a rooting culture medium formed by 1/2 of MS and 0.05mg/L of IBA (Indole Butyric Acid). The culture mediums are particularly suitable for the tissue culturing of the maiden pink.
Description
Technical field
The invention belongs to plant biotechnology field, specifically, it is related in a kind of tissue culture of maiden's Dianthus chinensis each
The culture medium in stage.The present invention filters out the optimal medium for different times in maiden's Dianthus chinensis tissue culture it is determined that training
The proportioning of each hormone in foster base, the culture medium that the formula according to the present invention is prepared can meet the nutrition in maiden's each period of Dianthus chinensis
Need and growth promoter.
Background technology
Dianthus chinensis are perennial work one, the showy flowers of herbaceous plants of biennial cultivation, and because its pattern enriches, the florescence is long, and cultivation management is extensive, is
Important ornamental flower.The whole world there are about more than 600 kinds, is widely distributed in north temperate zone, is distributed mainly on Eurasia, especially Mediterranean ground
Area, minority produces North America and north African.Record according to " Chinese Plants will ", China Carnation has 17 kinds, 1 subspecies, 9 mutation, many
It is distributed in Northern grassland and hill country, is mostly born in and area without shade is dried, some are planted and are born in border or sylvan life, desert and half famine
Unconcerned.Inner Mongol water resource famine, landscape planting will use water by multiplex sunlight less, should popularize drought resisting green plants in an all-round way, and give birth to
The long Dianthus chinensis in Inner Mongol arid, semiarid zone, have the vitality of tanacity, can smoothly survive the winter in the wild, few, no in rainwater
In the case of pouring also can normal growth, its root system is huge, can prevent erosion and can anti-harmful gas and pest and disease damage, have weight
The hygienic environment-protecting function of wanting, can be as the important component part in gardens it can also be used to the roadbed such as highway, sky way, road
The the afforesting and beautifying of multiple place such as slope.But due to from mankind's activity or naturally threaten so as to population quantity of both transition
Exist in retrogression of nature problem and tissue culture expanding propagation kind and sparkling phenomenon occurs, lead to industrial seedling rearing limited amount.Solve
Method be exactly to improve the existing method for tissue culture to Dianthus chinensis, this is Fast-propagation, detoxification rejuvenation and reduces the having of sparkling
Efficacious prescriptions method.
Under isolated culture condition, the histiocyte of different plants and kindred plant different parts is to nutrition for tissue culture
Require different, only meet the particular/special requirement of each of which, could preferably growth promoter.Grasp preparation and the sieve of culture medium
Choosing method is to obtain one of successful key link of tissue culture.
Plant tissue culture is critically important animal nutrition.The cell of plant, stem section, blade etc. all can be cultivated.
Take cell, organ and the tissue of plant, insert in the container of appropriate culture medium, under certain condition of culture, can be differentiated to form
Whole plant.As the tissue culture of biotechnology powerful measure, it is increasingly subject to pay attention to, in the detoxifying fast breeding, prominent of farm-forestry crop
Become the aspects such as induction, cell engineering and genetic engineering all to play an important role.
So far, the successful Caryophyllaceae kind of domestic and international tissue culture is a lot, but be more common in report is for fragrant stone
The production of bamboo and cultivation, less to maiden's Dianthus chinensis Study on tissue culture.Summarize the data in literature over nearly 20 years, maiden's Caryophyllaceae product
Kind of tissue culture majority is that stem section, blade are more successful, and being even more of relevant maiden's Dianthus chinensis Shoot Tip Culture is very few.Maiden's Dianthus chinensis
Artificial propagation still mainly adopt seeding method and cuttage, reproductive number is limited, time length and easily lead to adventitious bud sparkling,
Disease infection and quality deterioration.It is presently available for solution to shorten seedling raise period, factorial praluction, seedling Vitrification Occurred, update again
Strong tissue culture method study on reproduction is less, and therefore carrying out maiden's Dianthus chinensis Study on tissue culture in a deep going way has become researcher urgently
One of pending work.Carry out varieties distribution with the wild resource of China's abundant, apply diversified breeding technique, paying attention to
On the basis of conventional cross-breeding, carry out breeding of new variety in conjunction with modern biotechnology means, made with detoxication and tissue culture seedling simultaneously
To carry out the batch production exploitation of bulb for productivity original seed, to be the developing direction of maiden's Dianthus chinensis tissue culture.Tissue culture method is numerous
Grow and draw materials conveniently, do not limited by natural conditions, constantly can carry out amount reproduction, be maiden's Dianthus chinensis Fast-propagation, reduce glass
The most effectual way of glass, detoxification rejuvenation and rearing new variety, can obtain a large amount of seedlings in a short time, solve maiden's stone
Bamboo vitrification, shorten seedling raise period, the less problem of introduces a collection in cultivation, be the research from now on of maiden's Dianthus chinensis and scale, product
Industryization develops and provides theoretical foundation.
Content of the invention
The complete procedure of tissue culture is generally divided into formulation culture scheme, explant selects and processes, inoculates, is just commissioned to train
Support, successive transfer culture, strong sprout and several sport technique segments such as root culture, rooting culture of test tube seedling.Tissue culture is in isolated culture
Under the conditions of, the histiocyte of different plants and kindred plant different parts is different to nutritional requirement, only meets them each
From particular/special requirement, could preferably growth promoter.And grasping the preparation of culture medium and screening technique is that acquirement tissue culture is successful
One of key link.
The purpose of the present invention be filter out optimal medium for different times in maiden's Dianthus chinensis tissue culture it is determined that
The proportioning of each hormone in culture medium, the culture medium that the formula according to the present invention is prepared can meet the battalion in maiden's each period of Dianthus chinensis
Support and need and growth promoter.
The present invention is carrying out information search, collecting and is formulating embodiment on the basis of analyzing, comprehensive, multi-faceted
Carry out tissue culture experiment, the present invention has screened the culture medium from different times such as first generation induction → subculture increment → root culture.Make
Just generation, breed, the culture in each period of taking root reaches optimum efficiency.
Embodiment of the present invention comprises the optimal medium of each period of duration in the whole tissue culture procedures of Dianthus chinensis, and they are
Draw through careful, repeated tests, be on the basis of minimal medium according to each period of duration of Dianthus chinensis need add
Different types of plant growth agent and different proportionings.
In embodiments of the invention, there is provided the culture needed for a kind of maiden's Dianthus chinensis tissue culture procedures each stage
Base is it is characterised in that this culture medium is by just forming for inducing culture, proliferated culture medium, root media.These Dianthus chinensis tissue cultures
Each stage optimal medium filters out through comparative test.
The culture medium that the present invention provides is applied to the tissue culture of maiden's Dianthus chinensis, specifically it is adaptable to maiden's Dianthus chinensis
Shoot Tip Culture.
One embodiment of the present invention there is provided a kind of maiden's Dianthus chinensis first for inducing culture: ms+6-
Ba0.01mg/l+naa0.05mg/l.Research finds, all inducible maiden's Dianthus chinensis differentiation Multiple Buds of hormone combinations not of the same race, but respectively
The differentiation rate of individual combination is different, and ms+6-ba0.01mg/l+naa0.05mg/l is maiden's Dianthus chinensis Shoot Tip Culture base is optimal luring
Lead culture medium, not only inductivity is high, and the newly-increased bud number of each explant is many, differentiation rate highest, its differentiation rate is up to 200%.
One embodiment of the present invention there is provided a kind of subculture medium of maiden's Dianthus chinensis: ms+6-ba0.01mg/l+
Naa0.05mg/l.Research finds, the 6-ba of high concentration has inhibitory action, excessive concentration to Dianthus chinensis propagation, can suppress the life of bud
Long, with increasing of 6-ba concentration, when 6-ba concentration is more than 0.2mg/l, grows thickly dwarfing substantially, be also easy to produce vitrification Seedling.Sieve
The optimum multiplication medium formula selected is ms+6-ba0.01mg/l+naa0.05mg/l.
One embodiment of the present invention there is provided a kind of root media of maiden's Dianthus chinensis: 1/2ms+iba0.05mg/
L, research finds, low salt concn is favourable to the growth of maiden's Radix et Rhizoma Dispori Cantoniensis, and the culture medium suitably taken root is 1/2ms+iba0.05mg/
l.
In the present embodiment, in scope as well known to those skilled in the art, naa, 6-ba, iba are naphthalene second respectively
Acid, 6-benzyl aminopurine, the abbreviation of indolebutyric acid.
In the whole tissue culture procedures of maiden's Dianthus chinensis that the present invention selects, the optimal medium of each period of duration can fully meet
The nutritional need in maiden's each period of Dianthus chinensis and growth promoter, are to ensure that the successful implementation of maiden's Dianthus chinensis tissue culture, reach stream
Key and core that journeyization produces, are that maiden's Dianthus chinensis large-scale production provides strong support.
Specific embodiment
The material of embodiment 1. maiden's Dianthus chinensis explant and screening statistical method
1st, material
Maiden's Dianthus chinensis of experiment are adopted in grinding from Inner Mongolia Hexinyuan Monsod Drought-Resistance Greening Co., Ltd.'s drought-resistant plant
Study carefully the test nursery of institute's Experimental Base, supplemented according to test progress, different times sampling sampling at any time.Adopt maiden Dianthus chinensis children tender
The stem apex of 0.5-1.0cm is about on branch, after rinsing through flowing water, with 70% alcohol-pickled 10sec, then with 0.1% liter
Hydrargyrum sterilization 5min, aseptic water washing 2 times, each 5min, it is inoculated in the Initial culture base (ms) of improvement, set up test tube seedling asexual
Line of breeding.Choose subculture 10d about Multiple Buds, Multiple Buds cut off base portion；Take the stem apex of Multiple Buds, be about 0.5-1.0cm, make
Explant for regeneration.
2nd, the statistical computation formula of screening effect
Survival rate=(the explant sum of viable explant number/inoculation) × 100%
Just for bud induction rate=(the explant sum of the explant number/inoculation sprouted) × 100%
Subculture multiplication multiple=(sum of the Multiple Buds number inducing/access individual plant bud) × 100%
Rooting rate=(the explant sum of the explant number/inoculation of differentiation root) × 100%
The screening of embodiment 2. each stage optimal medium of maiden's Dianthus chinensis tissue culture
With ms as minimal medium, make firming agent with the carrageenan of 3.5g/l, 3% edible sugar replaces sucrose as carbon
Source, replaces distilled water with tap water, adds the basic element of cell division of different ratio respectively and auxin carries out the screening of culture medium.
1st, the selection of inductive differentiation medium
The concentration of 6-ba is set five Concentraton gradient of 0.01mg/l, 0.05mg/l, 0.1mg/l, 0.2mg/l, 0.5mg/l,
The concentration of naa sets tetra- Concentraton gradient of 0.01mg/l, 0.05mg/l, 0.1mg/l, 0.2mg/l, carries out difference and assembles, during inoculation
Every bottle connects 2-3 maiden's Dianthus chinensis stem apex (stem apex is about 0.5-1cm), observes the generation feelings of stem apex adventitious bud on different culture media
Condition.
As seen from Table 1,7 kinds of hormone combinations all can induce maiden's Dianthus chinensis differentiation Multiple Buds, but differentiation rate of each combination is not
With No. 1 ms+6-ba0.01mg/l+naa0.05mg/l is maiden's Dianthus chinensis Shoot Tip Culture base is optimal inducing culture, not only
Inductivity is high, and the newly-increased bud number of each explant is many, differentiation rate highest, and its differentiation rate is up to 200%.
The impact to maiden's Dianthus chinensis stem apex inducing clumping bud for table 1 hormone concentration and combinations thereof
2nd, the selection of subculture multiplication medium
The Multiple Buds being about 0.5-1.0cm differentiating are taken out, is inoculated in ms+6-ba0.01mg/l+naa0.05mg/l
Subculture multiplication medium on, add edible white sugar 40g/l, carrageenan 3.5g/l, in successive transfer culture 3 generation, breed and maiden's stone
Bamboo Seedling；Then the just above-mentioned Multiple Buds being about 0.5-1.0cm differentiating take out, and are inoculated in subculture in the ms culture medium of improvement
Enrichment culture, adds edible white sugar 40g/l, carrageenan 3.5g/l, in successive transfer culture 2 generation, breeds and maiden's Dianthus chinensis Seedling, can reduce
The sparkling phenomenon of maiden's Dianthus chinensis；Finally, the above-mentioned Multiple Buds being about 0.5-1.0cm differentiating are taken out, be inoculated in ms+6-
In the subculture multiplication medium of ba0.01mg/l+naa0.05mg/l, add edible white sugar 40g/l, carrageenan 3.5g/l, continue
In culture 1 generation, breed and maiden's Dianthus chinensis Seedling.
The adventitious bud that above-mentioned all inductions differentiate is inoculated on proliferated culture medium, 6-ba set 0.01mg/l, 0.05mg/l,
Tetra- Concentraton gradient of 0.1mg/l, 0.2mg/l, naa sets tetra- concentration ladders of 0.01mg/l, 0.05mg/l, 0.1mg/l, 0.2mg/l
Degree totally eight kinds of culture medium, every bottle connects 4 individual plants, the differential growth situation of observed and recorded regenerated adventitious bud, counts different after 15 days
The rate of increase of bud under the conditions of hormone combinations.
After inducing culture, the 0.5-1.0cm budlet producing in inducing culture is moved in proliferated culture medium respectively
(being shown in Table 2), after 2-3 days, the bastem portion of portion of material occurs starting observed and recorded during bud clump, statistical results after 15 days.Observe
Show, the 6-ba of high concentration has inhibitory action, excessive concentration to maiden's Dianthus chinensis propagation, can suppress the growth of bud, dense with 6-ba
Increasing of degree, when 6-ba concentration is more than 0.2mg/l, grows thickly dwarfing substantially, is also easy to produce vitrification Seedling.The optimal propagation filtering out
Culture medium prescription is ms+6-ba0.01mg/l+naa0.05mg/l.
The impact to maiden's Dianthus chinensis adventitious bud proliferation for table 2 hormone concentration and combinations thereof
Test shows, maiden's Dianthus chinensis no matter in initial culture or in successive transfer culture, in ms+6-ba0.01mg/l+
In naa0.05mg/l culture, the growth to Multiple Buds has facilitation, and it is even more ideal to compare other combinations, Multiple Buds quantity
Many, seedling is larger, transplants emergence rate best results.
3rd, the selection of root media
By preferable for growing way in subculture medium Seedling, it is divided into individual plant and proceeds in root media, every bottle is accessed 4 plants, observes
The situation of taking root of Seedling on record different culture media, counts rooting rate after 10 days.
From the point of view of the root culture result of the test of 10 days, two culture medium all have an inducing action to the formation of root, wherein No. 2
Preferably, rooting rate reaches 88.89% to root media rooting efficiency.Base portion of taking root can increase the Multiple Buds with root newly, averagely up to 3-4
Individual.It can be seen that low salt concn is favourable to the growth of maiden's Radix et Rhizoma Dispori Cantoniensis, the culture medium suitably taken root is 1/2ms+iba0.05mg/l (table
3).
The impact that table 3 inorganic salt is taken root to seedling
In the embodiment of the present invention 1, the first of improvement is to change part inorganic salt and ion in ms culture medium for ms culture medium
The ionic equilibrium solution of concentration, for the minimal medium of maiden's Dianthus chinensis initial culture, it specifically comprises:
A great number of elements: kno3950mg/l；nh4no3825mg/l；cacl2·2h2O220mg/l；mgso4·7h2o185mg/
l；kh2po485mg/l
Trace element: ki0.83mg/l；h3bo36.2mg/l；mnso4·4h2O22.3mg/l；znso4·7h2O8.6mg/
l；na2moo4·2h2O0.25mg/l；cuso4·5h2o0.25mg/l；cocl2·6h2O0.025mg/l
Iron salt: feso4·7h2o27.8mg/l；na2-edta·2h2o37.3mg/l
Organic substance: inositol 100mg/l；Nicotinic acid 0.5mg/l；Pyridoxine hydrochloride (vitamin b6) 0.5mg/l；Thiamine hydrochloride
Plain (vitamin b1) 0.1mg/l；Glycine 2.0mg/l
6-ba2.5mg/l；naa1.0mg/l；Activated carbon 1g/l；Hydrolysis network albumen 0.5g/l；Antibiotic 50mg/l.
In the embodiment of the present invention 2, the ms culture medium of improvement is to change part inorganic salt and ion concentration in ms culture medium
Ionic equilibrium solution, for maiden's Dianthus chinensis subculture multiplication culture minimal medium, it specifically comprises:
A great number of elements: kno31900mg/l；nh4no3413mg/l；cacl2·2h2o440mg/l；mgso4·
7h2o370mg/l；kh2po4310mg/l
Trace element: ki0.83mg/l；h3bo36.2mg/l；mnso4·4h2O22.3mg/l；znso4·7h2o8.6mg/
l；na2moo4·2h2o0.25mg/l；cuso4·5h2O0.25mg/l；cocl2·6h2o0.025mg/l
Iron salt: feso4·7h2O27.8mg/l；na2-edta·2h2o37.3mg/l
Organic substance: inositol 100mg/l；Nicotinic acid 0.5mg/l；Pyridoxine hydrochloride (vitamin b6) 0.5mg/l；Thiamine hydrochloride
Plain (vitamin b1) 0.1mg/l；Glycine 2.0mg/l
6-ba1.0mg/l；naa0.5mg/l.
Above example purpose is for illustrating present disclosure, but should not be construed as limitation of the present invention.?
In the case of present invention spirit and essence, the modification that the inventive method, step or condition are made or replacement, belong to
The scope of the present invention.
Claims (1)
1. the culture medium needed for maiden's Dianthus chinensis tissue culture procedures each stage is it is characterised in that this culture medium induces training by first generation
Foster base, proliferated culture medium, root media forms: just consists of 0.01mg/l 6-ba, 0.05mg/l for inducing culture
The ms culture medium of naa；Proliferated culture medium consist of 0.01mg/l 6-ba, the ms culture medium of 0.05mg/l naa；Root culture
The 1/2ms culture medium consisting of containing 0.05mg/l iba of base；In incubation using the stem apex taking from maiden's Dianthus chinensis twig as
Explant is cultivated.
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| CN110810244B (en) * | 2019-11-29 | 2022-08-26 | 蒙草生态环境(集团)股份有限公司 | Culture medium for tissue culture of floral rod |
| CN112400695B (en) * | 2020-12-28 | 2022-04-08 | 内蒙古蒙草生态环境（集团）股份有限公司 | Culture medium for culturing evergreen common summer pink |
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