CN103704134B - Method for inducing day lily red rum callus tissues by taking filaments as explant - Google Patents
Method for inducing day lily red rum callus tissues by taking filaments as explant Download PDFInfo
- Publication number
- CN103704134B CN103704134B CN201310682991.1A CN201310682991A CN103704134B CN 103704134 B CN103704134 B CN 103704134B CN 201310682991 A CN201310682991 A CN 201310682991A CN 103704134 B CN103704134 B CN 103704134B
- Authority
- CN
- China
- Prior art keywords
- culture
- filigree
- callus
- medium
- explant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a method for inducing day lily red rum callus tissues by taking filaments as an explant. The method comprises the steps of collecting and sterilizing the explant, acquiring and inoculating filaments, performing induction culture on the callus tissues, subculturing, and performing rooting culture. According to the method, the defects that the current day lily is low in division propagation and long in period ca be overcome, and the method has the advantages that material availing quantity is higher, the cost is saved, the sterilization effect is good, and callus tissue induction efficiency are higher compared with the conventional method taking rachis as an explant, and the induction rate of the method is higher than the rachis induction rate by about one time. The method can provide related technical support for other research (such as the culture of somatic embryo) on the basis of day lily filaments.
Description
Technical field
The present invention relates to tissue culture technique, specifically, relating to a kind of is the explant induction tawny daylily ' method of Red Rum ' callus with filigree.
Background technology
Tawny daylily is Liliaceae hemerocallis flowering perennial, is traditional famous flower of China.Tawny daylily floral leaf is held concurrently beautiful, and pattern flower shape is abundant, sight is high, viewing period long, strong adaptability, and application mode is various, is especially widely used in the urban afforestation such as flower bed, flower border and landscape architecture as ground cover plant.Along with the continuous progress of breeding technique, the new varieties of tawny daylily also emerge in an endless stream.Tawny daylily ' Red Rum ' is the kind that Holland introduces, robust plant, and have very high sight, mid-June buddings, at the bottom of June to mid-July be full-bloom stage, be Flowers ending to mid-August at the bottom of July.Plant height about 50cm, scape height 50cm, leaf green, the long 60cm of leaf, wide 3cm, blossom average 20, red, gorgeous, Hua Jing about 10cm.Can be widely used in urban landscaping as a kind of excellent ground.But ' Red Rum ' Natural seed setting rate is low, and traditional division propagation method breeding cycle is long, breeding rate is low, is difficult to the needs meeting factorial praluction, limits its quick application in afforestation.Tissue cultures, as a kind of quick propagating technology, is the effective way addressed this problem.
Recent year is also more about the group training research of tawny daylily, but tissue culture method is similar, conventional explant kind also focuses mostly on the materials such as stem section, bud, and (such as, He Qiuyue etc. 2011 have been that explant induction goes out callus to the bud of ' Yu Yihuang '; Li Xiuhua, Song Yang, Lan Liting etc. have induced callus by stem section), minority can pass through young leaves, ovary, petal acquisition callus, and then acquisition group trains seedling, and (Zhiwu Li etc. have induced callus by spire in 2010; An Fengxia etc. have successfully induced the callus of safflower tawny daylily for 2008 by root segment).But the group of the tawny daylily training stage is seldom studied filigree.Focus mostly in body embryo culture field to the research of filigree in tawny daylily, in non-embryonic callus, a situation arises carries out contrast experiment for the callus to the different explants of ' golden doll ' tawny daylily in 2009 such as Gao Shuying, after wherein filigree cultivates a period of time in the medium containing variable concentrations 6-BA, KT, IBA hormone, although filigree has and expands distortion, but all do not have callus to produce, after withered death gradually.
Have not yet to see and obtain non embryogenic callus about by tawny daylily filigree, the research report of further acquisition group training seedling.
Summary of the invention
The object of this invention is to provide a kind of is the explant induction tawny daylily ' method of Red Rum ' callus with filigree.
In order to realize the object of the invention, of the present invention with filigree be explant induction tawny daylily ' method of Red Rum ' callus, comprises the following steps:
1) collection of explant and sterilizing: acquisition length is the normal tawny daylily that the grows ' Red Rum ' bud of 1.0 ~ 2.5cm, the liquid detergent solution dipping low concentration with writing brush carries out preliminary surface cleaning, then rinse with water, be placed in the preferred 15s of 75% ~ 85% alcohol (preferably 75% alcohol) 10 ~ 20s(successively), the preferred 10min of 8 ~ 15min(in 1%NaClO solution), carry out sterilizing, obtain aseptic bud;
2) acquisition of filigree and inoculation: in an aseptic environment, excise holder part bottom above-mentioned aseptic bud, cut perianth open, take out filigree, remove the part that the part that is connected with holder of filigree bottom and filigree top are connected with flower pesticide simultaneously, get the filigree in stage casing as explant; The morphology lower end of the filigree as explant is inoculated in inducing culture downwards and cultivates, by the partial insertion medium of filigree bottom about 1/3;
3) Fiber differentiation of callus: above-mentioned inducing culture is the agar that with the addition of 6-benzyl purine, the dichlorphenoxyacetic acid of 0.5 ~ 2.0mg/L, the sucrose of 30g/L and 7.0g/L that concentration is 0 ~ 1.0mg/L in MS medium, pH value 6.15, is cultured to callus and occurs;
4) shoot proliferation is cultivated: be transferred to by callus in proliferated culture medium, carry out Multiplying culture, every 28 ~ 30 days (preferably 30 days) subcultures once, and subculture one to three time (preferred subculture three times);
5) culture of rootage: well-grown group of training seedling after shoot proliferation is removed after proceeding to after callus and cultivating two weeks in blank MS medium, then proceed in root media and carry out culture of rootage.
Wherein, step 3)-5) in condition of culture be: 23 ± 1 DEG C, intensity of illumination 2000-3000Lx, periodicity of illumination 16/8h.
In step 4), proliferated culture medium is the agar that with the addition of 6-benzyl purine, the methyl α-naphthyl acetate of 0.01 ~ 0.1mg/L, the sucrose of 30g/L and 7.0g/L that concentration is 0.5 ~ 2.0mg/L in MS medium, pH value 6.15.
In step 5), root media is the agar that with the addition of indolebutyric acid, the methyl α-naphthyl acetate of 0 ~ 0.2mg/L, the sucrose of 30g/L and 7.0g/L that concentration is 0 ~ 0.2mg/L in 1/2MS medium, pH value 6.15.
Step 2) in the culture vessel that uses of the inoculation culture dish that is 12cm for diameter, the medium thickness wherein added is 0.7 ~ 1.0cm; Step 3)-5) in the culture vessel that the uses conical flask that is 100mL, medium thickness is 0.7 ~ 1.0cm.
Preferably, the inducing culture used in step 3) is: MS+1.0mg/L6-benzyl purine+0.5mg/L dichlorphenoxyacetic acid+30g/L sucrose+7.0g/L agar, pH value 6.15.
The proliferated culture medium used in step 4) is: MS+1.0mg/L6-benzyl purine+0.01mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar, pH value 6.15; Or MS+2.0mg/L6-benzyl purine+0.01mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar, pH value 6.15.
The root media used in step 5) is: 1/2MS+0.1mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar.
The medium of MS described in the present invention is:
Macroelement: NH
4nO
31650mg/L, KNO
31900mg/L, CaC1
22H
2o440mg/L, MgSO
47H
2o370mg/L, KH
2pO
4170mg/L;
Trace element: KI0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o22.3mg/L, ZnSO
47H
208.6mg/L, Na
2mnO
42H
2o0.25mg/L, CuSO
45H
2o0.025mg/L, CoC1
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, hydrochloric acid adjoin the alcohol 0.5mg/L that trembles, thiamine hydrochloride 0.1mg/L, glycine 2.0mg/L.
The present invention is also provided for being that ' medium of Red Rum ' callus, comprises above-mentioned three kinds of medium to explant induction tawny daylily, i.e. the inducing culture of callus, subculture multiplication medium and root media with filigree.
The present invention is directed to the blank of domestic tawny daylily in filigree tissue culture technology, thering is provided a kind of is that explant carries out the tawny daylily ' method of Red Rum ' callus induction with filigree, the method has the advantage that the amount of drawing materials is larger, cost-saving, sterilization effect is good, callus induction efficiency is higher, and its inductivity exceeds about one times than the inductivity of scape.
Method of the present invention is that ' Red Rum ' 1.0 ~ 2.5cm filigree is that explant carries out tissue cultures, obtains the method for plantlet in vitro, comprises the steps: collection and the sterilizing of (1) explant with tawny daylily; (2) acquisition of filigree and inoculation; (3) Fiber differentiation of callus; (4) Multiplying culture of callus; (5) culture of rootage of plantlet in vitro.
The collection of step (1) explant and sterilizing: tawny daylily ' in Red Rum ' starts to budding in the last ten-days period in June, select sunny noon, at the normotrophic bud of field acquisition length at about 1.0 ~ 2.5cm, in the liquid detergent solution of low concentration, preliminary surface cleaning is carried out with writing brush, then tap water 2h is used, then transfer on superclean bench, adopt 75% alcohol vibration sterilizing 20s, aseptic water washing 2 times, then the NaClO sterilizing 10min that concentration is 1.0% is put into, adopt combinations thereof sterilizing methods, the aseptic bud of final acquisition.
The acquisition of step (2) filigree and inoculation: in an aseptic environment, with scalpel by holder Partial Resection bottom aseptic bud, cut perianth open, carefully filigree is taken out with tweezers, the part that part that filigree bottom is connected with holder is connected with flower pesticide with filigree top to be removed simultaneously, obtain corresponding filigree as explant.The filigree of acquisition is carefully inoculated on corresponding inducing culture, the morphology lower end of filigree will be noted during inoculation to be downwards inserted in medium cultivate.By in the partial insertion medium of filigree bottom about 1/3, all do not insert.
The Fiber differentiation of step (3) callus: being seeded in by the filigree that previous step obtains and with the addition of concentration is the 6-benzyl purine (6-BA) of 0 ~ 1.0mg/L, the dichlorphenoxyacetic acid (2 of 0.5 ~ 2.0mg/L, 4-D), cultivate in the sucrose of 30g/L and the MS medium of 7.0g/L agar, more callus within 60 days, can be had later to occur.When treating to have grown more callus bottom filigree and callus have more bud point occur, callus is transferred on proliferated culture medium and carry out follow-up Multiplying culture.
The Multiplying culture of step (4) callus: the filigree that previous step obtains is seeded in methyl α-naphthyl acetate (NAA), the sucrose of 30g/L and the MS medium of 7.0g/L agar that with the addition of 6-benzyl purine (6-BA) that concentration is 0.5 ~ 2.0mg/L and 0.01 ~ 0.1mg/L and carries out Multiplying culture, every 30 days subcultures once.
The culture of rootage of step (5) plantlet in vitro: growing state is organized preferably training seedling and remove to proceed in blank MS medium after callus and cultivate two weeks, to take the photograph the impact of hormone before eliminating.Then proceed in root media and carry out culture of rootage.
Experiment proves, when the hormone concentration selected is respectively 6-BA1.0mg/L and 2,4-D0.5mg/L, the inductivity of the callus of acquisition is the highest, therefore in step (3), best inducing culture is MS+1.0mg/L6-BA+0.5mg/L2,4-D+30g/L sucrose+7.0g/L agar.
When the hormone concentration selected is 6-BA1.0mg/L and 0.01mg/L NAA, or during 6-BA2.0mg/L and 0.01mg/L NAA, the highest rate of increase can be obtained.Therefore, in step (4), optimum multiplication medium is MS+1.0mg/L6-BA+0.01mg/L NAA+30g/L sucrose+6.5g/L agar or MS+2.0mg/L6-BA+0.01mg/L NAA+30g/L sucrose+6.5g/L agar.In step (5), best root media is 1/2MS+NAA0.1mg/L+ sucrose 30g/L+ agar 6.5g/L.
In step (3), cultivation through callus induction finds, after callus produces a period of time, can differentiation sprout a little gradually, later stage bud point and callus grow simultaneously, therefore do not need the induction of the differentiation carrying out independent Multiple Buds, and directly can carry out the Multiplying culture of Multiple Buds.
In the above-mentioned Primary culture stage, the medium of employing is solid culture medium, and culture vessel is culture dish, and medium thickness is at about 1.0cm; In the Multiplying culture stage, the medium of employing is solid sterile medium, and culture vessel is the conical flask of 100mL.All the other culture environment are temperature 23 ± 1 DEG C, intensity of illumination 2000-3000Lx, periodicity of illumination 16/8h.
Inventor through a large amount of Experimental comparison, by tawny daylily, ' Red Rum ' carries out the group training research of hormon, different explants, has found out the group training approach being carried out the new tawny daylily of of callus induction by filigree.
Advantage of the present invention is the scape that the amount of drawing materials is relatively traditional, and have the advantage that the amount of drawing materials is larger, sterilization effect is good, callus induction efficiency is higher, its inductivity exceeds about one times than the inductivity of scape.Meanwhile, the cultivation base unit weight needed for the initial stage is less, is about 1/12 of medium needed for scape, decreases the step of configuration medium and rolling bottle, therefore more saves labour.Meanwhile, this technology can provide reference for the otherwise group of training research being explant with tawny daylily filigree from now on.
Method of the present invention, also can carry out further acclimatization and transplants after step (5), due to the existing research of these parts, does not therefore repeat in the present invention.
Accompanying drawing explanation
Fig. 1-2 is that ' upgrowth situation when 60 days cultivated by the filigree of Red Rum ' to tawny daylily of the present invention in Primary culture base, can see that filigree has the generation of callus in various degree in the medium, and have the appearance of young tender shoots.
Fig. 3 is the tawny daylily ' spray of Red Rum ' growing formation after callus induction that field of the present invention gathers.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
Embodiment 1 take filigree as the explant induction tawny daylily ' method of Red Rum ' callus
(1) collection of explant and sterilizing
The last ten-days period in June tawny daylily ' at the beginning of Red Rum ', spend period, select sunny noon, take length at the normotrophic bud of about 1 ~ 2.5cm, soak about 5 minutes in the liquid detergent solution of low concentration, and carry out preliminary surface cleaning with writing brush, then explant is placed in bottle, with tap water 2 ~ 3h.After completing preliminary cleaning, bud is transferred on superclean bench, adopt combination sterilizing methods, by one group, 5 buds, put into the conical flask filling 80mL75% alcohol, shake 20s gently, pour out alcohol, add 60mL aseptic water washing 2 times, after pouring out sterile water, then the NaClO solution adding 80mL1.0% in conical flask is thought, sterilizing 10min, period can shake conical flask gently, to ensure that bud surface fully contacts with solution.
(2) acquisition of filigree and inoculation
In an aseptic environment, the NaClO solution in previous step conical flask is poured out, carefully takes out bud with tweezers, notice that bud does not touch taper bottleneck.Bud is placed on aseptic filter paper, with scalpel by holder Partial Resection bottom bud, then cuts perianth open, carefully take out filigree with tweezers, obtain corresponding filigree as explant.It should be noted that obtain complete filigree as far as possible, the part that part that filigree bottom is connected with holder is connected with flower pesticide with filigree top to be removed simultaneously.The filigree of acquisition is carefully inoculated on corresponding inducing culture, the morphology lower end of filigree during inoculation, will be noted downwards to be inserted in medium.By in the partial insertion medium of filigree bottom about 1/3, all do not insert.Each diameter is inoculate 12 filigrees in the culture dish of 12cm.
(3) Fiber differentiation of callus
The filigree that previous step obtains is seeded in 6-benzyl purine (6-BA) (concentration is respectively 0mg/L, 1.0mg/L), the dichlorphenoxyacetic acid (2 that with the addition of variable concentrations, in the medium of 4-D) (concentration is respectively 0.5mg/L, 1.0mg/L, 2.0mg/L) and methyl α-naphthyl acetate (NAA) (concentration is respectively 0mg/L, 0.2mg/L), cultivate.The hormone added in callus inducing medium is as shown in table 1.
The hormone-content added in table 1 callus inducing medium
Observing after 60 days, with the addition of in 6-benzyl purine (6-BA) that concentration is 1.0mg/L, the dichlorphenoxyacetic acid (2,4-D) of 0.5mg/L, the sucrose of 30g/L and the medium of 7.0g/L agar, having more callus to occur.(table 2, Fig. 1-2)
The situation of callus is formed in the different callus inducing medium of table 2
In this kind of inducing culture, the callus incidence that filigree obtains can reach 82.32%.
When treating to have grown more callus bottom filigree and callus have more bud point occur, callus is transferred on proliferated culture medium and carry out follow-up Multiplying culture.Through observing, the callus obtained by filigree can turn out normal group training seedling.
Culture environment is temperature 23 ± 1 DEG C, intensity of illumination 2000-3000L, periodicity of illumination 16/8h.
(4) Multiplying culture of Multiple Buds
In previous step, the generation of callus and the appearance of Multiple Buds are almost simultaneously.So the Multiple Buds obtained in previous step being tested is transferred in proliferated culture medium and is carried out Multiplying culture.The Multiplying culture of Multiple Buds adopts two factor three horizontal completely random district group experimental designs, take MS as minimal medium, select 6-AB, NAA two kinds of hormones to carry out the cultivation of Multiple Buds shoot proliferation, 6-BA select 0.5,1.0,2.0mg/L, NAA select 0.01,0.05,0.1mg/L tri-level.Often kind of culture medium inoculated 6 bottles, every bottle of callus block with bud including 3 pieces of growing states and be similar to.Every surrounding subculture once, after three months, add up respectively from proliferation times and the indefinite bud growing way of sprouting by continuous culture.(table 3)
From proliferation times and the indefinite bud growing way situation of sprouting in the different proliferated culture medium of table 3
Can be found out by table 3, the propagation of 6-BA and NAA on Multiple Buds has remarkable impact.Along with the increase of NAA concentration, the proliferation times of Multiple Buds declines, and when concentration is 0.01mg/L, the proliferation times of Multiple Buds is the highest.The change of 6-BA concentration has larger impact to adventitious buds proliferation multiple, and with the increase of 6-BA concentration, the proliferation times of Multiple Buds first rises, rear decline, and when 6-BA concentration is 1.0mg/l, the proliferation times of Multiple Buds is the highest.Higher with J7 and J4 of average proliferation multiple, and in these two kinds of medium, Multiple Buds is all comparatively healthy and strong.In addition, except J9, in each proliferated culture medium, Multiple Buds is after three subcultures, and proliferation times has decline to a certain degree, finally all reaches about 2.1.
Comprehensive above situation, MS+1.0mg/L6-BA+0.01mg/L NAA+30g/L sucrose+6.5g/L agar and MS+2.0mg/L6-BA+0.01mg/L NAA+30g/L sucrose+6.5g/L agar are optimum multiplication medium.
(5) culture of rootage
Proceed in blank MS medium after the comparatively healthy and strong plantlet in vitro obtained after propagation is removed callus and cultivate two weeks, to take the photograph the impact of hormone before eliminating.Then to proceed in root media (G1-G6 be all be minimal medium with 1/2MS).All root medias all add sucrose and the 6.5g/Ld agar of 30g/L.Within 20 days, add up situation of taking root afterwards, result is as shown in table 4.
Table 4 culture of rootage situation
Note: rooting rate=plantlet in vitro number of having taken root/plantlet in vitro sum; Average individual plant radical=root sum/plantlet in vitro number of having taken root; Average individual plant root length=root overall length/seedling sum of taking root; Average single length=root overall length/root sum.
As can be seen from Table 4, in plant rooting process, NAA to the facilitation of taking root apparently higher than IBA.The 1/2MS that with the addition of NAA is higher than the rooting rate of plantlet in vitro in blank 1/2MS and MS, illustrates that NAA is formed with significant facilitation to root.When NAA concentration is 0.1mg/L, rooting rate is the highest, and plant takes root, number is all maximum with root length.IBA is when concentration is 0.05mg/L, and the inductivity of root is also higher than blank 1/2MS, and when concentration raises, the inductivity of root starts lower than blank 1/2MS, illustrates that IBA only could promote the formation of root when low concentration, and concentration is high has inhibitory action on the contrary.From taking root the time used, with the addition of in the medium of NAA, plantlet in vitro time used of taking root is obviously shorter.In addition, the root of plantlet in vitro in blank MS than 1/2MS in growing state better, illustrate at the initial stage of taking root, sufficient macroelement also can promote the growth of root to a certain extent.All in all, no matter be the angle from rooting rate, radical or single length, the second medium has comparatively significantly advantage, and therefore the best root media of Hemerocallis'Red Rum' is 1/2MS+NAA0.1mg/L+ sucrose 30g/L+ agar 6.5g/L.
(6) also to tawny daylily, ' scape of Red Rum ' has carried out Study on tissue culture (Gao Feng, 2012) in the present invention.At the stage of development of callus, have employed different medium.Experimental conditions is as follows: adopting Three factors-levels orthogonal, take MS as minimal medium, 6-BA and KT select 0 respectively, 1.0,2.0mg/L tri-levels, NAA selects 0.05,0.10,0.20mg/L tri-levels.The filigree of tawny daylily, scape are cultivated.Observe statistics after 4 weeks, find that filigree all can not produce callus, final withered death in all medium.The callus that scape produces in the medium containing 1.0mg/L6-BA and 0.2mg/L NAA is maximum, and Callus induction rate is 44.4%.
About the research of these two kinds of dissimilar explants, can one group of contrast be passed through, understand filigree carries out callus induction advantage (table 5) as explant further.
The scape (Fig. 3) of four buds is contained for one:
Table 5 callus induction situation compares
| Contrast project | Scape | Filigree |
| Available explant number | 3 | 18～24 |
| Explant survival rate after sterilizing | 62.26% | 100.00% |
| Starting rate | 44.4% | 82.3% |
| Start-up time | 18 days | 62 days |
| Cultivation stage container used | Conical flask | 12cm culture dish |
| The educable explant number of each container | 3 | 12 |
| The explant number that every 50mL medium can be cultivated | 3 | 12 |
By relatively finding out, when using filigree as explant, the explant of greater number can be obtained, obtain higher Callus induction rate and survival rate.Startup stage, the medium needed for filigree is less, therefore can greatly reduce the preparation of medium, reduces rolling bottle number of times, therefore more economizes on resources and manpower.Postmenstruation Germicidal efficacy, the callus that filigree obtains, after Multiplying culture, can grow normal group training seedling.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (3)
1. be the method for explant induction tawny daylily " Red Rum " tissue cultures with filigree, it is characterized in that, comprise the following steps:
1) collection of explant and sterilizing: acquisition length is the normal tawny daylily that the grows ' Red Rum ' bud of 1.0 ~ 2.5cm, the liquid detergent solution dipping low concentration with writing brush carries out preliminary surface cleaning, then rinse with water, be placed in 75% ~ 85% alcohol 10 ~ 20s successively, 8 ~ 15min in 1%NaClO solution, carry out sterilizing, obtain aseptic bud;
2) acquisition of filigree and inoculation: in an aseptic environment, excise holder part bottom above-mentioned aseptic bud, cut perianth open, take out filigree, remove the part that the part that is connected with holder of filigree bottom and filigree top are connected with flower pesticide simultaneously, get the filigree in stage casing as explant; The morphology lower end of the filigree as explant is inoculated in inducing culture downwards and cultivates, by the partial insertion medium of filigree bottom about 1/3;
3) Fiber differentiation of callus: above-mentioned inducing culture is MS+1.0mg/L 6-benzyl purine+0.5mg/L dichlorphenoxyacetic acid+30g/L sucrose+7.0g/L agar, pH value 6.15, is cultured to callus and occurs;
4) shoot proliferation is cultivated: be transferred to by callus in proliferated culture medium, carry out Multiplying culture, every 28 ~ 30 days subcultures once, and subculture one to three time;
5) culture of rootage: well-grown group of training seedling after shoot proliferation is removed after proceeding to after callus and cultivating two weeks in blank MS medium, then proceed in root media and carry out culture of rootage;
Step 3)-5) in condition of culture be: 23 ± 1 DEG C, intensity of illumination 2000 ~ 3000Lx, periodicity of illumination 16/8h;
Step 4) in use proliferated culture medium be: MS+1.0mg/L 6-benzyl purine+0.01mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar, pH value 6.15; Or MS+2.0mg/L6-benzyl purine+0.01mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar, pH value 6.15;
Step 5) in use root media be: 1/2MS+0.1mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar;
Described MS medium is:
Macroelement: NH
4nO
31650mg/L, KNO
31900mg/L, CaC1
22H
2o440mg/L, MgSO
47H
2o 370mg/L, KH
2pO
4170mg/L;
Trace element: KI 0.83mg/L, H
3bO
36.2mg/L, MnSO
44H
2o 22.3mg/L, ZnSO
47H
208.6mg/L, Na
2mnO
42H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoC1
26H
2o 0.025mg/L;
Molysite: FeSO
47H
2o 27.8mg/L, Na
2-EDTA2H
2o 37.3mg/L;
Organic substance: inositol 100mg/L, nicotinic acid 0.5mg/L, hydrochloric acid adjoin the alcohol 0.5mg/L that trembles, thiamine hydrochloride 0.1mg/L, glycine 2.0mg/L.
2. method according to claim 1, is characterized in that, step 2) in the culture vessel that uses of the inoculation culture dish that is 12cm for diameter, the medium thickness wherein added is 0.7 ~ 1.0cm; Step 3)-5) in the culture vessel that the uses conical flask that is 100mL, medium thickness is 0.7 ~ 1.0cm.
3. the medium for taking filigree as explant induction tawny daylily " Red Rum " tissue cultures, is characterized in that, comprise the inducing culture of callus, subculture multiplication medium and root media;
Wherein, the inducing culture of callus is: MS+1.0mg/L 6-benzyl purine+0.5mg/L dichlorphenoxyacetic acid+30g/L sucrose+7.0g/L agar, pH value 6.15;
Subculture multiplication medium is: MS+1.0mg/L 6-benzyl purine+0.01mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar, pH value 6.15; Or MS+2.0mg/L 6-benzyl purine+0.01mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar, pH value 6.15;
Root media is: 1/2MS+0.1mg/L methyl α-naphthyl acetate+30g/L sucrose+6.5g/L agar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310682991.1A CN103704134B (en) | 2013-12-12 | 2013-12-12 | Method for inducing day lily red rum callus tissues by taking filaments as explant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310682991.1A CN103704134B (en) | 2013-12-12 | 2013-12-12 | Method for inducing day lily red rum callus tissues by taking filaments as explant |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103704134A CN103704134A (en) | 2014-04-09 |
| CN103704134B true CN103704134B (en) | 2015-05-06 |
Family
ID=50397812
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310682991.1A Expired - Fee Related CN103704134B (en) | 2013-12-12 | 2013-12-12 | Method for inducing day lily red rum callus tissues by taking filaments as explant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103704134B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113170732A (en) * | 2021-05-31 | 2021-07-27 | 上海应用技术大学 | Culture medium and method for obtaining day lily callus through inducing mature embryo |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH104811A (en) * | 1996-06-19 | 1998-01-13 | Gunze Ltd | Proliferation of large amount of plant of genus hemerocallis |
| CN102265785B (en) * | 2010-06-02 | 2013-03-06 | 上海上房园艺有限公司 | Tissue culturing method of hemerocallis middendorfii poinsettia |
| CN102172224A (en) * | 2011-03-24 | 2011-09-07 | 黑龙江生态工程职业学院 | Red flower orange daylily tissue culture formula and culture method |
-
2013
- 2013-12-12 CN CN201310682991.1A patent/CN103704134B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103704134A (en) | 2014-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101647393B (en) | Fast tissue culture reproducing method of actinidia eriantha | |
| CN103444552B (en) | A kind of method of inducing eggplant flower pesticide regeneration haplobiont | |
| CN103190347B (en) | Teapot dates tissue culturing method | |
| CN102499080B (en) | Plant fast propagating method using fagopyrum tataricum leaf stalks as explants | |
| CN110604058B (en) | Tissue culture seedling raising method for camellia chekiangoleosa immature embryos | |
| CN102422810A (en) | In-vitro regeneration culture method for tea clones | |
| CN103430844B (en) | Method for gardenia tissue culture | |
| CN104012417B (en) | High-efficiency and rapid micropropagation method for toxicodendron vernicifluum | |
| CN103583358A (en) | Method for in vitro culturing of regenerated plant of dendrobium officinale | |
| CN104381131B (en) | A kind of Pinus tabuliformis somatic embryo occurs and plant regeneration method | |
| CN109392712A (en) | A kind of tissue culture and rapid propagation method of tara vine kind | |
| CN101946703A (en) | Method for regenerating plants of Chinese rose by using leaves as explants | |
| JPH08112045A (en) | Method for mass production of seed and seedling of plant of genus fritillaria | |
| CN105265316A (en) | Rapid propagation method for plateaus of alliums | |
| CN113331059B (en) | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants | |
| CN108770690B (en) | Method for establishing efficient and stable regeneration system by using dendrocalamus malabaricus bud tips | |
| CN105028193B (en) | A kind of utilization blueberry Lai Gexi blades induction produces the mating system of micro adventitious bud | |
| CN102283130A (en) | Method for culturing and fast propagating tissue of chimaera hosta plantaginea plant | |
| CN106106178A (en) | A kind of method for tissue culture of confection Rhizoma Iridis Tectori | |
| CN103477988A (en) | Culture in vitro and rapid propagation method for syzygium grijsii | |
| CN105265317A (en) | Rapid propagation method of allium victorialis | |
| CN103155869A (en) | Sweet cherry rootstock Colt tissue culture method | |
| CN106305430A (en) | Tissue culture quick breeding method of Cerasus jamasakura Fencai | |
| CN103704134B (en) | Method for inducing day lily red rum callus tissues by taking filaments as explant | |
| CN111280054A (en) | Hydrangea paniculata tissue culture proliferation and rooting integrated seedling method and used culture medium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150506 Termination date: 20211212 |