CN103828716B - The method for tissue culture of maiden China pink - Google Patents
The method for tissue culture of maiden China pink Download PDFInfo
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The method for tissue culture of a kind of maiden China pink, adopt the stem apex of maiden China pink spray as explant, after sterilization treatment, cultivation temperature 25 ± 1 DEG C, intensity of illumination 2500Lx, except natural lighting on daytime, under light filling at the night condition of culture of 4 hours/day, be inoculated into successively by MS, 0.01mg/L6-BA, 0.05mg/L? it is first for inducing culture that NAA forms, by MS, 0.01mg/L6-BA, 0.05mg/L? the subculture medium of NAA composition, by 1/2MS, 0.05mg/L? the root media of IBA composition is cultivated, this cultural method can obtain the maiden China pink training seedling of indoor large-scale breeding in the short time, productivity ratio is high, there is potential ecological benefits and social benefit.
Description
Technical field
The invention belongs to plant biotechnology field, specifically, relate to the method for tissue culture of a kind of maiden China pink, this cultural method is specially adapted to maiden China pink, be much better than the method for tissue culture of this medium for other China pinks, comprise the selection of China pink explant, stem-tip tissue cultivate in subculture medium and cultivate the selection of algebraically, the content such as selection of condition of culture in tissue culture procedures.
Background technology
China pink is the showy flowers of herbaceous plants of perennial work one, biennial cultivation, and because its pattern enriches, the florescence is long, and cultivation management is extensive, is important ornamental flower.The whole world about has more than 600 to plant, and is widely distributed in north temperate zone, is mainly distributed in Eurasia, especially Mediterranean Region, and minority produces North America and north African.Record according to " Chinese Plants will ", China Carnation has 17 kinds, 1 subspecies, 9 mutation, is distributed in Northern grassland and hill country more, is mostly born in dry area without shade, and some kind is born in border or sylvan life, desert and half-desert.Inner Mongol water resource famine, landscape planting wants multiplex sunlight to use water less, drought resisting green plants should be popularized in an all-round way, and growth is the China pink of Inner Mongol arid, semiarid zone, there is indomitable vitality, can survive the winter smoothly in the wild, few at rainwater, without also can normal growth when pouring, its root system is huge, can prevent erosion and energy anti-harmful gas and damage by disease and insect, there is important hygienic environment-protecting function, can be used as the important component part in gardens, also can be used for afforesting and beautifying of the multiple places such as the roadbed such as highway, sky way, Lu Po.But due to from mankind's activity or the threat naturally changing two aspects, make its population quantity there is retrogression of nature problem and in tissue culture expanding propagation kind, occur that sparkling phenomenon causes factorial seedling growth limited amount.Solution is exactly the existing method for tissue culture to China pink of improvement, and this is Fast-propagation, detoxification rejuvenation and reduce the effective ways of sparkling.In laboratory conditions, reducing its sparkling is a new problem need putting into practice to grope and inquire into, when formulating technical scheme can the data of reference few, all must be settleed one by one by test, key will solve explant and draw materials position, period; The selection of each cultivation period medium; Fast breeding technique etc. after condition of culture and seedling.The present invention, to develop, reduces costs as target, to be formed for the purpose of batch production, industrialization production, formulates corresponding work plan and implementing method.
Tissue cultures is under isolated culture condition, and the histocyte of different plant and kindred plant different parts is different to nutritional requirement, only meets their respective particular/special requirements, could grow better.Preparation and the screening technique of grasping medium obtain one of successful key link of group training.
Plant Tissue Breeding is very important animal nutrition.The cell, stem section, blade etc. of plant all can be cultivated.Get the cell of plant, Organ and tissue, insert in the container of appropriate culture medium, under certain condition of culture, can whole plant be differentiated to form.As the tissue cultures of biotechnology powerful measure, day by day come into one's own, bring out in the detoxifying fast breeding of farm-forestry crop, sudden change, all play an important role in cell engineering and gene engineering etc.
In the tissue cultures of pinkwort, normal stem section, blade, petal and the stem apex etc. selected obtains regeneration plant as explant.Stem section is also one of common used material in pinkwort Fast-propagation, because it has the advantage and being widely adopted such as to draw materials conveniently, quantity is many.Watad etc. have studied and make explant with stem section, adopt the cultivation of gel entrapment culture base, liquid shake is cultivated and on liquid nutrient medium, floating interfacial film assists 3 kinds of different training methods such as cultivation on the impact of carnation regeneration frequency, result shows, the regeneration frequency of the third training method can improve two to three times than other two kinds of modes, and this can be greatly cost-saving in plant fast propagation process.Godo etc. are studied LychnisFlos-Cuculi stem section culture, and the regeneration frequency of explant on 1/2MS+0.2mg/LBA and 1/2MS medium is respectively 30% and 70%.On MS+10mg/LBA medium, each stem section node on average can generate 8 and can utilize bud, and the genetic stability quite stable of bud.
The leaf culture of pinkwort obtains regeneration plant by direct or indirect Regeneration Ways.Jethwani etc. add the callus of growth hormone PAA to Chinese phyllostachys nuda leaf sheet and break up research again in medium.Found that, the suitableeest Calli Differentiation medium is MS+BA (2.0,5.0mg/L)+PAA (0.5,1.0mg/L).No matter be used alone or in combination BA, NAA, 2,4-D, callus all can not break up generation bud again.In addition, they also compare the 1st leaf (from top) of Chinese China pink and the callus induction situation of the 2nd leaf, research shows that the 1st leaf and the 2nd callus that basil portion induces have regeneration capacity, and the callus of the 2nd leaf remainder induction does not have regeneration capacity.Kantia etc. study report: Chinese China pink leaf explant is substantially cultivated on tomb at the MS of 3mg/LBA+0.5mg/LNAA and 3mg/LBA+1mg/LNAA and cultivated, and all directly can produce indefinite bud; And at 0.5mg/LBA+1mg/L2, though the MS medium of 4-D also can produce indefinite bud, being attended by the generation of callus, the indefinite bud of generation could will grow containing on the medium of BA and NAA.Paresk etc. with the blade of carnation, Chinese China pink and sweetwilliam for explant, adopt MS+1mg/L2,4-D liquid nutrient medium has induced somatic embryo, somatic embryo develops into seedling on MS+1mg/LGA3 solid culture medium, establishes the direct somatic embryo inducement regenerating system of pinkwort first.
Nakano etc. with Carnation Petal, blade and stem section for explant induction indefinite bud.Result only has petal to have higher regeneration frequency, and suitable medium is MS+1.1-2.2mg/LBA and MS+0.9mg/LNAA.Fzsiter etc. are that explant have studied two kinds of training methods to the impact of China pink shoot regeneration frequency with Carnation Petal.Result shows, China pink petal liquid medium within higher than the regeneration rate on solid culture medium (the former can reach 100%, and the latter only has 50%); Each position of petal all can form regeneration bud, and each explant can produce at most 7 indefinite buds.Casanova etc. also induce regeneration bud by Carnation Petal, and inductivity reaches 84.4%, and the regeneration bud inductivity of ro1C Transgenic carnation petal is up to 90.4%.
Stem apex is a position of drawing materials conventional in pinkwort tissue cultures, except for except Fast-propagation, also for virus-free culture, Germ-plasma resources protection etc.From early 1990s, report the achievement in research of this respect both at home and abroad.Carnation due to produce in adopt cottage propagation cause quality degradation, therefore, its Shoot Tip Culture has important production practices meaning.Can etc. have studied different growth hormone, the basic element of cell division and the gibberellin combination impact numerous soon on fringed pink stem apex, and result shows: shoot apical meristem in MS+5.0mg/LBA+1.0mg/LIAA medium than to contain the Multiple Buds produced in the medium of other several combination of regulators many.Onamu etc. report the dosage of TDZ and the impact of processing time on carnation stem apex in-vitro propagate efficiency very large.Their research shows, the concentration of TDZ is 1-5mg/L, and the processing time is 3-10d, and when carrying out in-vitro propagate to carnation stem apex, efficiency can reach 100%.
Up to now, the successful Caryophyllaceae kind of domestic and international tissue cultures is a lot, but what be more common in report is production for carnation and cultivation, less to maiden China pink Study on tissue culture.Sum up the data in literature over nearly 20 years, maiden's Caryophyllaceae kind tissue cultures majority is that stem section, blade are relatively more successful, caning be counted on one's fingers especially of relevant maiden China pink Shoot Tip Culture.The artificial propagation of maiden China pink still mainly adopts seeding method and cuttage, and reproductive number is limited, the time is long and very easily cause indefinite bud sparkling, disease infection and quality deterioration.Can be used at present separating shorten seedling raise period, factorial praluction, seedling Vitrification Occurred, rejuvenating tissue culture method study on reproduction less, therefore carrying out maiden China pink Study on tissue culture in a deep going way has become one of work that researcher urgently carries out.Varieties distribution is carried out with the wild resource of China's abundant, apply diversified breeding technique, on the basis paying attention to conventional cross-breeding, breeding of new variety is carried out in conjunction with modern biotechnology means, carry out the batch production exploitation of kind of ball using detoxication and tissue culture seedling as yielding ability original seed, be the developing direction of maiden China pink tissue cultures simultaneously.Tissue culture method breeding is drawn materials conveniently; not by the restriction of natural conditions; constantly can carry out amount reproduction; maiden China pink Fast-propagation, the most effectual way reducing vitrifying, detoxification rejuvenation and rearing new variety; a large amount of seedling can be obtained in a short time; solve the vitrifying of maiden China pink, shorten seedling raise period, the less problem of provenance in cultivation, for the research from now on of maiden China pink and scale, industrialization development utilize and provide theoretical foundation.
The complete procedure that stem-tip tissue is cultivated generally be divided into formulate culture scheme, explant select with process, inoculate, several sport technique segment such as Initial culture, shoot proliferation expand numerous cultivation, the rooting culture of strong sprout and culture of rootage, test-tube plantlet, front mentioned grasp medium preparation and screening technique be obtain to organize to train successful key link.The present invention filters out the optimal medium for different times in maiden China pink tissue cultures, the proportioning of adjustment to hormone in different times medium, meet the nutritional need in maiden China pink each period according to the medium of different formulations preparation and grow, solving a large amount of vitrification phenomenon of maiden China pink seedling and the long problem of growing-seedling period simultaneously.
Before mentioned the tissue cultures of domestic and international many scholars to China pink and be also studied, be applied in the production of carnation China pink at present, but what be more common in report is that cultivar produces for fresh cut-flowers, medium and method are not also suitable for maiden China pink, have studied the selection of the explant of maiden China pink, optimal culture condition, the optimal medium of different phase and Multiplying culture algebraically present system.
The present invention is easy and simple to handle, with low cost, has a good application prospect.Have studied the optimal medium of different phase in the group training of maiden China pink present system, the suitableeest condition of culture and training method, solve a difficult problem for the problem that provenance is less in the vitrifying of maiden China pink, shortening seedling raise period, cultivation, add kind for realizing drought resisting landscape planting new concept and there is stronger practicality, for the factorial praluction realizing China pink has established reliable basis.
Summary of the invention
In embodiment of the present invention, comprise the method for tissue culture of a kind of maiden China pink, it is characterized in that it is made up of following steps:
Step 1: explant takes from the stem apex of maiden China pink spray;
Step 2: explant is inoculated in just on inducing culture MS+6-BA0.01mg/L+NAA0.05mg/L, differentiation-inducing go out Multiple Buds;
Step 3: the Multiple Buds of just generation induction was cultivated for 3 generations on subculture medium MS+6-BA0.01mg/L+NAA0.05mg/L, then its Multiple Buds was cultivated for 2 generations on the MS medium of improvement, after finally above-mentioned Multiple Buds being cultivated for 1 generation on subculture medium, transferred on root media 1/2MS+IBA0.05mg/L and cultivated;
Step 4: seedling of being taken root by the maiden China pink after above-mentioned root induction is transplanted.
The condition of culture of each step is: cultivation temperature 25 ± 1 DEG C, intensity of illumination 2500Lx, except natural lighting on daytime, night light filling 4 hours/day.
Wherein, the MS medium of improvement is the ionic equilibrium solution changing part mineral salt and ion concentration in MS medium, and for the minimal medium that maiden China pink shoot proliferation is cultivated, it specifically consists of:
Macroelement: KNO
31900mg/L; NH
4nO
3413mg/L; CaCl
22H
2o440mg/L; MgSO
47H
2o370mg/L; KH
2pO
4310mg/L
Trace element: KI0.83mg/L; H
3bO
36.2mg/L; MnSO
44H
2o22.3mg/L; ZnSO
47H
2o8.6mg/L; Na
2moO
42H
2o0.25mg/L; CuSO
45H
2o0.25mg/L; CoCl
26H
2o0.025mg/L
Molysite: FeSO
47H
2o27.8mg/L; Na
2-EDTA2H2O37.3mg/L
Organic substance: inositol 100mg/L; Nicotinic acid 0.5mg/L; Puridoxine hydrochloride (vitamin B6) 0.5mg/L; Thiamine hydrochloride (vitamin B1) 0.1mg/L; Glycine 2.0mg/L
6-BA1.0mg/L；NAA0.5mg/L。
In the present embodiment, in the tissue culture procedures 1 of maiden China pink, the sterilisation process of maiden China pink explant is: on super-clean bench, put into sterile chamber, drip in the alcohol of 75% of " Tween " at often liter of dropping 2-3 and soak 10 seconds, use the mercuric chloride solution process 5 minutes of 0.15% again, aseptic washing 2 times, suck dry moisture on last aseptic filter paper.
In the present embodiment, in tissue culture procedures 2 seeded process of maiden China pink, maiden China pink spray stem apex leaf primordium otch inserts medium.
The present invention, also through comparative test, filters out each stage optimal medium of maiden China pink group training:
A. just for inducing culture: MS+6-BA0.01mg/L+NAA0.05mg/L is the first for inducing culture of maiden China pink the best, and not only inductivity is high, and each explant to increase bud number newly many, differentiation rate is the highest, and its differentiation rate can reach 200%.
B. subculture multiplication medium: MS+6-BA0.01mg/L+NAA0.05mg/L, the 6-BA of high concentration has inhibitory action to maiden China pink propagation, excessive concentration, the growth of bud can be suppressed, along with increasing of 6-BA concentration, when 6-BA concentration is more than 0.2mg/L, grows thickly and downgrade obviously, easily produce vitrifying seedling.
C. root media: 1/2MS+IBA0.05mg/L, low salt concn is favourable to taking root of maiden China pink.
Maiden China pink, no matter at Initial culture or in squamous subculture, in MS+6-BA0.01mg/L+NAA0.05mg/L cultivates, has facilitation to the growth of Multiple Buds, compare other combinations even more ideal, Multiple Buds quantity is many, and seedling is comparatively large, transplants emergence rate best results.
Also comprise in specific embodiments of the present invention maiden China pink take root after the acclimatization and transplants stage, result shows, vermiculite+sheep excrement+diammonium phosphate, as cultivation matrix best results, is very applicable to the growth of seedling root system.
Embodiment
Choosing and the impact of method of operating on adventitious bud inducing of embodiment 1. maiden China pink explant
The maiden China pink of experiment is adopted in the test nursery from drought-resistant plant research institute of Inner Mongolia Hexinyuan Monsod Drought-Resistance Greening Co., Ltd. Experimental Base, and according to test progress, different times samples and sampling supplements at any time.Adopt stem apex maiden China pink edible tender branch being about 0.5-1.0cm, after running water, with the alcohol-pickled 10sec of 70%, then with 0.1% mercuric chloride sterilization 5min, aseptic water washing 2 times, each 5min, is inoculated on the Initial culture base (MS) of improvement, sets up test-tube plantlet clone.Choose the Multiple Buds of subculture about 10d, Multiple Buds cuts off base portion; Get the stem apex of Multiple Buds, be about 0.5-1.0cm, as the explant of regeneration.
Wherein, the first of improvement is the ionic equilibrium solution changing part mineral salt and ion concentration in MS medium for MS medium, and for the minimal medium of maiden China pink Initial culture, it specifically consists of:
Macroelement: KNO
3950mg/L; NH
4nO
3825mg/L; CaCl
22H
2o220mg/L; MgSO
47H
2o185mg/L; KH
2pO
485mg/L
Trace element: KI0.83mg/L; H
3bO
36.2mg/L; MnSO
44H
2o22.3mg/L; ZnSO
47H
2o8.6mg/L; Na
2moO
42H
2o0.25mg/L; CuSO
45H
2o0.25mg/L; CoCl
26H
2o0.025mg/L
Molysite: FeSO
47H
2o27.8mg/L; Na
2-EDTA2H2O37.3mg/L
Organic substance: inositol 100mg/L; Nicotinic acid 0.5mg/L; Puridoxine hydrochloride (vitamin B6) 0.5mg/L; Thiamine hydrochloride (vitamin B1) 0.1mg/L; Glycine 2.0mg/L
6-BA2.5mg/L; NAA1.0mg/L; Active carbon 1g/L; Hydrolysis network albumen 0.5g/L; Antibiotic 50mg/L.The screening of each stage optimal medium of embodiment 2. maiden China pink tissue cultures
1, 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 of sprouting) × 100%
Shoot proliferation multiple=(sum of the Multiple Buds number induced/access individual plant bud) × 100%
Rooting rate=(the explant sum of the explant number/inoculation of differentiation root) × 100%
2, the screening of each stage optimal medium of China pink tissue cultures
Take MS as minimal medium, make curing agent with the carragheen of 3.5g/L, the edible sugar of 3% replaces sucrose as carbon source, replaces distilled water with running water, adds the screening that the basic element of cell division of different ratio and growth hormone carry out medium respectively.
2.1, the selection of inductive differentiation medium
The concentration of 6-BA is established 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L, 0.5mg/L five concentration gradients, the concentration of NAA establishes 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L tetra-concentration gradients, carry out different assembly, every bottle graft 2-3 maiden China pink stem apex (stem apex is about 0.5-1cm) during inoculation, observe stem apex on different culture media indefinite bud a situation arises.
As seen from Table 1,7 kinds of hormone combinations all can induce maiden China pink to break up Multiple Buds, but the differentiation rate of each combination is different, No. 1 MS+6-BA0.01mg/L+NAA0.05mg/L is maiden China pink Shoot Tip Culture base is best inducing culture, not only inductivity is high, and each explant to increase bud number newly many, differentiation rate is the highest, and its differentiation rate can reach 200%.
Table 1 hormone concentration and the impact of combination on maiden China pink stem apex inducing clumping bud thereof
2.2, the selection of subculture multiplication medium
Taken out by the Multiple Buds being about 0.5-1.0cm differentiated, be inoculated in the subculture multiplication medium of MS+6-BA0.01mg/L+NAA0.05mg/L, additional edible white granulated sugar 40g/L, carragheen 3.5g/L, in squamous subculture 3 generation, breed and maiden China pink seedling; Then the just above-mentioned Multiple Buds being about 0.5-1.0cm differentiated takes out, and on the MS medium being inoculated in improvement, shoot proliferation is cultivated, additional edible white granulated sugar 40g/L, carragheen 3.5g/L, in squamous subculture 2 generation, breed and maiden China pink seedling, the sparkling phenomenon of maiden China pink can be reduced; Finally, the above-mentioned Multiple Buds being about 0.5-1.0cm differentiated is taken out, is inoculated in the subculture multiplication medium of MS+6-BA0.01mg/L+NAA0.05mg/L, additional edible white granulated sugar 40g/L, carragheen 3.5g/L, in squamous subculture 1 generation, breeds and maiden China pink seedling.
Wherein, the MS medium of improvement is the ionic equilibrium solution changing part mineral salt and ion concentration in MS medium, and for the minimal medium that maiden China pink shoot proliferation is cultivated, it specifically consists of:
Macroelement: KNO
31900mg/L; NH
4nO
3413mg/L; CaCl
22H
2o440mg/L; MgSO
47H
2o370mg/L; KH
2pO
4310mg/L
Trace element: KI0.83mg/L; H
3bO
36.2mg/L; MnSO
44H
2o22.3mg/L; ZnSO
47H
2o8.6mg/L; Na
2moO
42H
2o0.25mg/L; CuSO
45H
2o0.25mg/L; CoCl
26H
2o0.025mg/L
Molysite: FeSO
47H
2o27.8mg/L; Na
2-EDTA2H2O37.3mg/L
Organic substance: inositol 100mg/L; Nicotinic acid 0.5mg/L; Puridoxine hydrochloride (vitamin B6) 0.5mg/L; Thiamine hydrochloride (vitamin B1) 0.1mg/L; Glycine 2.0mg/L
6-BA1.0mg/L；NAA0.5mg/L。
Above-mentioned all differentiation-inducing go out indefinite bud be inoculated on proliferated culture medium, 6-BA establishes 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L tetra-concentration gradients, NAA establishes 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L tetra-concentration gradients totally eight kinds of medium, every bottle graft 4 individual plants, the differential growth situation of observed and recorded regenerated adventitious bud, adds up the rate of increase of bud under different hormone combinations condition for 15 days afterwards.
After Fiber differentiation, the 0.5-1.0cm budlet produced in inducing culture is moved into (see table 2) in proliferated culture medium respectively, start observed and recorded when bud clump appears in the bastem portion of 2-3 days rear section materials, statistical results after 15 days.Observation shows, the 6-BA of high concentration has inhibitory action, excessive concentration to maiden China pink propagation, can suppress the growth of bud, along with increasing of 6-BA concentration, when 6-BA concentration is more than 0.2mg/L, grows thickly and downgrades obviously, easily produce vitrifying seedling.The optimum multiplication medium formula filtered out is MS+6-BA0.01mg/L+NAA0.05mg/L.
Table 2 hormone concentration and the impact of combination on maiden China pink adventitious bud proliferation thereof
Test shows, maiden China pink no matter at Initial culture or in squamous subculture, MS+6-BA0.01mg/L+NAA0.05mg/L cultivate in, have facilitation to the growth of Multiple Buds, compare other combinations even more ideal, Multiple Buds quantity is many, seedling is comparatively large, transplants emergence rate best results.
2.3, the selection of root media
By good for growing way in subculture medium seedling, be divided into individual plant and proceed in root media, every bottle graft enters 4 strains, and on observed and recorded different culture media, the situation of taking root of seedling, added up rooting rate after 10 days.
From the culture of rootage result of the test of 10 days, two formation of medium to root all have inducing action, and wherein No. 2 root media rooting efficiencies are better, and rooting rate reaches 88.89%.Base portion of taking root can increase the Multiple Buds of band root newly, on average can reach 3-4.The growth of visible low salt concn to maiden China pink root is favourable, and suitable medium of taking root is 1/2MS+IBA0.05mg/L(table 3).
The impact that table 3 mineral salt are taken root on seedling
Above embodiment object is for illustrating content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
Claims (5)
1. a method for tissue culture for maiden China pink, is characterized in that it is made up of following steps:
Step 1: the stem apex adopting long 0.5-1.0cm on maiden China pink edible tender branch, be inoculated in the MS medium of improvement after sterilization treatment, choose the Multiple Buds of subculture about 10d, Multiple Buds cuts off base portion; Get the stem apex of Multiple Buds, long 0.5-1.0cm, as the explant of regeneration;
Step 2: explant is inoculated in just on inducing culture MS+6-BA0.01mg/L+NAA0.05mg/L, differentiation-inducing go out Multiple Buds;
Step 3: the Multiple Buds of just generation induction was cultivated for 3 generations on subculture medium MS+6-BA0.01mg/L+NAA0.05mg/L, then its Multiple Buds was cultivated for 2 generations on the MS medium of improvement, after finally above-mentioned Multiple Buds being cultivated a generation on subculture medium, transferred on root media 1/2MS+IBA0.05mg/L and cultivated;
Step 4: seedling of being taken root by the maiden China pink after above-mentioned root induction is transplanted,
Wherein, the MS medium of improvement is the ionic equilibrium solution changing part mineral salt and ion concentration in MS medium, and for the minimal medium that maiden China pink shoot proliferation is cultivated, it specifically consists of:
Macroelement: KNO
31900mg/L; NH
4nO
3413mg/L; CaCl
22H
2o440mg/L; MgSO
47H
2o370mg/L; KH
2pO
4310mg/L
Trace element: KI0.83mg/L; H
3bO
36.2mg/L; MnSO
44H
2o22.3mg/L; ZnSO
47H
2o8.6mg/L; Na
2moO
42H
2o0.25mg/L; CuSO
45H
2o0.25mg/L; CoCl
26H
2o0.025mg/L
Molysite: FeSO
47H
2o27.8mg/L; Na
2-EDTA2H
2o37.3mg/L
Organic substance: inositol 100mg/L; Nicotinic acid 0.5mg/L; Puridoxine hydrochloride (vitamin B6) 0.5mg/L; Thiamine hydrochloride (vitamin B1) 0.1mg/L; Glycine 2.0mg/L6-BA1.0mg/L; NAA0.5mg/L.
2. cultural method according to claim 1, is characterized in that, the condition of culture of each step is: cultivation temperature 25 ± 1 DEG C, intensity of illumination 2500Lx, except natural lighting on daytime, night light filling 4 hours/day.
3. cultural method according to claim 1, it is characterized in that, the sterilisation process of step 1 is: on super-clean bench, put into sterile chamber, drip in the alcohol of 75% of " Tween " at often liter of dropping 2-3 and soak 10 seconds, use the mercuric chloride solution process 5 minutes of 0.15% again, aseptic washing 2 times, suck dry moisture on last aseptic filter paper.
4. cultural method according to claim 1, is characterized in that, in step 1 seeded process, maiden China pink spray stem apex leaf primordium otch inserts medium.
5. cultural method according to claim 1, is characterized in that, the matrix that step 4 transplants rear cultivation is made up of vermiculite, sheep excrement, diammonium phosphate.
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| CN107155604A (en) * | 2017-06-16 | 2017-09-15 | 内蒙古蒙草生态环境（集团）股份有限公司 | The cultivation management method of " girl in red carnation " |
| CN110810245B (en) * | 2019-11-29 | 2022-08-30 | 蒙草生态环境(集团)股份有限公司 | Tissue culture method of floral rod |
| CN112400696B (en) * | 2020-12-28 | 2022-04-08 | 内蒙古蒙草生态环境（集团）股份有限公司 | Tissue culture method of evergreen common selfheal fruit-spike bamboo |
| 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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| CN102257967A (en) * | 2011-07-11 | 2011-11-30 | 大连好地农业有限公司 | A kind of carnation hybrid seed production method |
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| CN102257967A (en) * | 2011-07-11 | 2011-11-30 | 大连好地农业有限公司 | A kind of carnation hybrid seed production method |
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