CN104542285B - A kind of method of hemerocailis middendorffi leaf tissue culture - Google Patents
A kind of method of hemerocailis middendorffi leaf tissue culture Download PDFInfo
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- CN104542285B CN104542285B CN201410843997.7A CN201410843997A CN104542285B CN 104542285 B CN104542285 B CN 104542285B CN 201410843997 A CN201410843997 A CN 201410843997A CN 104542285 B CN104542285 B CN 104542285B
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Abstract
The present invention relates to a kind of method of hemerocailis middendorffi leaf tissue culture, it is concretely comprised the following steps, using the young tender internal layer blade of hemerocailis middendorffi open country growth as explant, by disinfecting, induced synthesis callus in inducing culture is inoculated in, through callus differentiation and proliferation, bud induction and rooting induction, forms the complete seedling of hemerocailis middendorffi, hemerocailis middendorffi seedling is transplanted in matrix, grows up to normal hemerocailis middendorffi plant.The present invention carries out tissue cultures using tawny daylily blade, overcomes tawny daylily and can only carry out tissue cultures using flower portion organ at the florescence and expands numerous limitation, it can be achieved that the anniversary breeds, and is conducive to carry out that breeding is numerous soon, the species of tawny daylily in abundant landscape application.
Description
Technical field
The invention belongs to field of plant tissue culture, and in particular to the hemerocailis middendorffi young leaflet tablet tissue cultures of open country growth
Expand numerous method.
Background technology
Hemerocailis middendorffi is Hemerocallidaceae hemerocallis perennial herb, is important Perennial Flowers, Chinese tawny daylily germ plasm resource
Very abundant, finds that the category shares 14 kinds, just there are 11 kind hemerocailis middendorffis in China in the world.On the basis of wild tawny daylily germplasm
On, the hemerocailis middendorffi horticultural gardening Breeds by artificial hybridization selection and breeding, the kind logged at present has kind more than 80,000.Great Hua
The emerald green long and narrow base life of tawny daylily leaf, lines up two row in banding, has fusiform fleshy root and fibrous root.Short shape of stem, flower tongue are taken out from middle part
Go out, helical form cyme, each titbit can blossom tens of.Bud is opened such as funnel, sliver turnup, for lily shape flower like wearing in one's hair
Hat, petal apex split.Perianth base portion symphysis, is cylindrical in shape, 6 pieces of stamen, and 3 length 3 are short, and flower pesticide is carried on filigree top in T-shaped
End, Hua Jing change greatly, and flower-shape is different, and the florescence is mainly in summer.
Hemerocailis middendorffi is a kind of excellent Horticultural crops.But at present in landscape application hemerocailis middendorffi it is of less types,
Main cause is influenced by breeding.Hemerocailis middendorffi in landscape application mainly realizes breeding by tissue cultures mode,
The common explant of tissue cultures has scape, a petal, ovary, stem apex etc., thus breed can only at the florescence, limit new varieties and
The cultivation of improved seeds.And blade materials influence from the florescence, it is possible to achieve the annual of tissue cultures explant materials time,
Achieve the purpose that tissue-culturing rapid propagation.So if the tissue that hemerocailis middendorffi can be carried out by the use of the blade of outdoor cropping as explant is trained
Support, realize the annual of tawny daylily tissue cultures explant materials time, then it is numerous soon to be advantageously implemented breeding, greatly enriches in gardens
The species of hemerocailis middendorffi.
The content of the invention
It is an object of the present invention in view of the deficiencies of the prior art, there is provided one kind carries out tissue using hemerocailis middendorffi blade
The method for cultivating fast seedling-breeding, realizes the annual of tawny daylily tissue cultures explant materials time.
To achieve the above object, the present invention adopts the following technical scheme that：
1) selection of explant and disinfect；
2) induction of callus：Explant after disinfection is inoculated into inducing culture, to formation callus, institute
The formula for stating inducing culture is：0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines are added in MS culture mediums；
3) bud induces：The callus is inoculated into young shoot culture medium, culture to the regrowth for obtaining hemerocailis middendorffi,
The formula of the young shoot culture medium is：0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines are added in MS culture mediums；
4) culture of rootage：The regrowth is transferred in root media, short of white and length occurs to regrowth base portion
Go out complete root system, obtain hemerocailis middendorffi seedling, the formula of the root media is：0.1mg/L naphthalenes are added in 1/2MS culture mediums
Acetic acid, 3% sucrose, 0.6% agar, the 1/2MS culture mediums are to halve a great number of elements concentration in MS culture mediums；
5) plantlet of transplant：The hemerocailis middendorffi seedling is planted into cultivation matrix, greenhouse hardening, obtain hemerocailis middendorffi children
Seedling.
In the present invention, the selection of the explant selects the extraction a fairly large number of hemerocailis middendorffi single plant of young leaves specifically, choosing,
The more consistent young leaflet tablet of interception stem apex above leaf age is required explant, and the young leaves is less than the leaf of 12 days for leaf age
Son.
It is described to disinfect specifically, explant flowing water is rinsed 3-5 minutes, then with 70% alcoholometer in the present invention
Face sterilizes 20-30s, aseptic water washing 3-4 time, then with 2% NaClO immersion 10-15min, then with aseptic water washing 5-6
It is secondary.
In the present invention, the induction of the callus the step of be：By the explant after processing in superclean bench
Blockage is torn into tweezers, it is paraxial to be inoculated in up in inducing culture；After inoculation at 23-27 DEG C of temperature, dark condition
Culture.
In the present invention, the condition of culture of the bud induction is：25 DEG C, intensity of illumination 2000-3000lx of temperature, light application time
12h/d。
In the present invention, the condition of the culture of rootage is：25 DEG C, intensity of illumination 2000-3000lx of temperature, light application time
Cultivated under 12h/d.
In the present invention, the hemerocailis middendorffi seedling refers to the differentiation for completing bud and root, can independently carry out autophyting growth
Young plant.
Further, there is provided tawny daylily tissue cultures more specifically step is as follows in the present invention：
1) selection of explant and disinfect：The selection extraction a fairly large number of hemerocailis middendorffi single plant of young leaves, intercepts stem apex
The more consistent young leaflet tablet of above leaf age, ensures the uniformity of explant as far as possible, and explant is rinsed 3-5 minutes with flowing water, then
With 70% alcohol surface sterilizing 20-30s, aseptic water washing three times, then soaks 10-15min with 2% NaClO, then with sterile
Water rinses 5-6 times；
2) induction of callus：In superclean bench by the blade after processing in step 1) with tweezers be torn into 1cm ×
The pane of 1cm sizes, paraxial to be inoculated in up on inducing culture, the formula of inducing culture is to be added in MS culture mediums
0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines are added in MS culture mediums, in 25 DEG C of temperature, dark condition after inoculation
There is callus down toward blade edge；
3) bud induces：The callus obtained in step 2) is inoculated into young shoot culture medium, the formula of young shoot culture medium
To add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines in MS culture mediums, in 25 DEG C of temperature after inoculation, illumination is strong
2000-3000lx is spent, is cultivated under light application time 12h/d, is germinateed to callus, obtains the regrowth of hemerocailis middendorffi；
4) culture of rootage：The tawny daylily seedling obtained in step 3) is transferred in root media, the formula of root media
To add 0.1mg/L methyl α-naphthyl acetates, 3% sucrose, 0.6% agar in 1/2MS culture mediums, in 25 DEG C of temperature, intensity of illumination 2000-
Cultivated under 3000lx, light application time 12h/d, cultivate and short of white occur to seedling base portion and grow complete root system；
5) plantlet of transplant：The complete hemerocailis middendorffi seedling of taking root obtained in step 4) is planted to perlite and turf 1:1
In matrix, greenhouse hardening, obtains normal hemerocailis middendorffi seedling.
The present invention carries out tissue cultures using tawny daylily blade and realizes quick breeding, the meaning with following two aspects：(1)
For day lily mainly based on mitogenetic breeding, breeding coefficient is low, long the time required to breeding, is unfavorable for the numerous and good of hemerocailis middendorffi
The culture of kind.The tissue cultures time is short, and increment frequency is high, can improve the reproductive efficiency of hemerocailis middendorffi.(2) with the big of open country growth
Flower tawny daylily young leaflet tablet is drawn materials as explant, from the limitation of materials time at florescence, extends the explant materials time, favorably
In the application and popularization of hemerocailis middendorffi.
Brief description of the drawings
Fig. 1：Tawny daylily new varieties " Red Triangle region " callus tissue culture picture；
Fig. 2：Tawny daylily new varieties " Red Triangle region " bud induces picture；
Fig. 3：Tawny daylily new varieties " Red Triangle region " are taken root picture；
Fig. 4：Wild tawny daylily (Hemerocallis fulva) callus tissue culture picture；
Fig. 5：Wild tawny daylily (Hemerocallis fulva) bud induces picture；
Fig. 6：Wild tawny daylily (Hemerocallis fulva) picture of taking root；
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.
1 hemerocailis middendorffi leaf tissue culture of embodiment
The present embodiment selection tawny daylily new varieties " Red Triangle region " carry out leaf tissue culture.
1) selection of explant and disinfect：The selection extraction a fairly large number of hemerocailis middendorffi single plant of young leaves, is therefrom selected
The leaf age young leaves of 9-10 days, interception stem apex is with the uniformity of the young leaflet tablet of upper bit, as far as possible guarantee explant.By explant with
Flowing water rinses 3-5 minutes, then three times, is then soaked with 70% alcohol surface sterilizing 30s, aseptic water washing with 2% NaClO
10-15min, then with aseptic water washing 5-6 times.
2) induction of callus：In superclean bench by the blade after processing in step 1) with tweezers be torn into 1cm ×
The pane of 1cm sizes, paraxial to be laid in up on inducing culture, the formula of inducing culture is to be added in MS culture mediums
0.1mg/L NAA, 1.0mg/L6- benayl aminopurines.In 25 DEG C of temperature after inoculation, 18 days rear blade edges are cultivated under dark condition
There is callus (such as Fig. 1).
3) bud induces：The callus obtained in step 2) is inoculated into young shoot culture medium, the formula of young shoot culture medium
To add 0.1mg/L NAA, 1.0mg/L 6-benzyl aminopurines in MS culture mediums, in 25 DEG C of temperature, intensity of illumination 2000-
Cultivate, cultivate 40-50 days under 3000lx, light application time 12h/d, grow budding (such as Fig. 2) to hemerocailis middendorffi, obtain tawny daylily regeneration
Seedling.
4) culture of rootage：The tawny daylily regrowth obtained in step 3) is transferred in root media, root media is matched somebody with somebody
Side is that 0.1mg/L methyl α-naphthyl acetates, 3% sucrose, 0.6% agar are added in 1/2MS culture mediums.In 25 DEG C of temperature, intensity of illumination 2000-
Cultivated under 3000lx, light application time 12h/d.There is short of white to seedling base portion and grows complete root system for 15 days in culture, obtains
Tawny daylily seedling (such as Fig. 3).
5) plantlet of transplant：The hemerocailis middendorffi seedling obtained in step 4) is planted to perlite and turf 1:In 1 matrix, temperature
Room hardening, obtains normal hemerocailis middendorffi seedling.
The survival rate for the hemerocailis middendorffi regrowth that the present invention obtains is 98%.
2 hemerocailis middendorffi leaf tissue culture of embodiment
The present embodiment selects a kind of blade of wild hemerocailis middendorffi Hemerocallis fulva to carry out tissue cultures.
1) selection of explant and disinfect：The selection extraction a fairly large number of hemerocailis middendorffi single plant of young leaves, is therefrom selected
The leaf age young leaves of 9-10 days, interception stem apex is with the uniformity of the young leaflet tablet of upper bit, as far as possible guarantee explant.By explant with
Flowing water rinses 3-5 minutes, then three times, is then soaked with 70% alcohol surface sterilizing 30s, aseptic water washing with 2% NaClO
10-15min, then with aseptic water washing 5-6 times.
2) induction of callus：In superclean bench by the blade after processing in step 1) with tweezers be torn into 1cm ×
The pane of 1cm sizes, paraxial to be laid in up on inducing culture, the formula of inducing culture is to be added in MS culture mediums
0.2mg/L NAA, 1.0mg/L6- benayl aminopurines.In 25 DEG C of temperature after inoculation, cultivated 3 weeks under dark condition, blade edge goes out
Existing callus (such as Fig. 4).
3) bud induces：The callus obtained in step 2) is inoculated into young shoot culture medium, the formula of young shoot culture medium
To add 0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines in MS culture mediums in 25 DEG C of temperature, intensity of illumination 2000-
Cultivate, cultivate 40-60 days under 3000lx, light application time 12h/d, grow budding (such as Fig. 5) to hemerocailis middendorffi, obtain tawny daylily regeneration
Seedling.
4) culture of rootage：The tawny daylily regrowth obtained in step 3) is transferred in root media, root media is matched somebody with somebody
Side is that 0.1mg/L methyl α-naphthyl acetates, 3% sucrose, 0.6% agar are added in 1/2MS culture mediums.In 25 DEG C of temperature, intensity of illumination 2000-
Cultivated under 3000lx, light application time 12h/d.There is short of white to seedling base portion and grows complete root system (such as 15-30 days in culture
Fig. 6).
5) plantlet of transplant：The complete hemerocailis middendorffi seedling of taking root obtained in step 4) is planted to perlite and turf 1:1
In matrix, greenhouse hardening, obtains normal hemerocailis middendorffi seedling.
The survival rate for the hemerocailis middendorffi regrowth that the present invention obtains is 96%.
Comparative example 1：The influence that hormon and concentration induce blade callus
It is different according to type of culture medium growing state using blade as in the callus Induction Process of explant, be probably divided into
Lower four kinds of situations：1. plant tissue moves back green, the nearly dead state of white is presented, and (inoculation blade chooses the tender and lovely blade of plant internal layer, connects
It is yellow during kind)；2. plant tissue wound produces brown material；3. plant tissue wound grows white particles shape callus
Organize and tissue expands；4. plant tissue expands or does not occur any change.Experimental data is as follows
As can be seen from the above table, the hormone concentration of the callus tissue culture base of the offer of the present invention is provided, is more advantageous to big
Flower tawny daylily calli induction.
The influence that the selection of 2 explant of comparative example induces blade callus
Compared with embodiment 1, it is differed only in, and explant selects leaf age to find to adopt in experiment for the blade of 15-16 days
Can not induced synthesis callus with this explant.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (5)
- A kind of 1. method of hemerocailis middendorffi leaf tissue culture, it is characterised in that this method comprises the following steps：1) selection extraction young leaves and leaf age are less than 12 days, the more hemerocailis middendorffi single plant of blade quantity, intercept stem apex above leaf age More consistent young leaflet tablet, as explant, carry out disinfection the explant processing；2) induction of callus：Explant after disinfection is inoculated into inducing culture, it is described to lure to formation callus The formula for leading culture medium is：0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines are added in MS culture mediums；3) bud induces：The callus is inoculated into young shoot culture medium, in 25 DEG C, intensity of illumination 2000-3000lx of temperature, Light application time 12h/d, cultivates to callus and sprouts, and forms regrowth；The formula of the young shoot culture medium is：In MS culture mediums Add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines；4) culture of rootage：The regrowth is transferred in root media, in 25 DEG C, intensity of illumination 2000-3000lx of temperature, light Under conditions of time 12h/d, cultivate and short of white occur to regrowth base portion and grow complete root system, it is small to obtain hemerocailis middendorffi Seedling, the formula of the root media are：0.1mg/L methyl α-naphthyl acetates, 3% sucrose, 0.6% agar, institute are added in 1/2MS culture mediums 1/2MS culture mediums are stated as a great number of elements concentration in MS culture mediums is halved；5) plantlet of transplant：The hemerocailis middendorffi seedling is planted into cultivation matrix, greenhouse hardening, obtain hemerocailis middendorffi seedling.
- 2. according to the method described in claim 1, it is characterized in that, described disinfect specifically, explant is rushed with flowing water Wash 3-5 minutes, then with 70% alcohol surface sterilizing 20-30s, aseptic water washing 3-4 times, then soaks 10- with 2% NaClO 15min, then with aseptic water washing 5-6 times.
- 3. according to the method described in claim 1, it is characterized in that, the induction of the callus the step of be：Ultra-clean The explant after processing is torn into blockage with tweezers in workbench, it is paraxial to be inoculated in up in inducing culture；After inoculation Cultivated at 23-27 DEG C of temperature, dark condition.
- 4. according to the method described in claim 1, it is characterized in that, the hemerocailis middendorffi seedling refers to point for completing bud and root Change, can independently carry out the young plant of autophyting growth.
- 5. according to the method described in claim 1, it is characterized in that, specifically comprise the following steps：1) selection of explant and disinfect：The selection extraction a fairly large number of hemerocailis middendorffi single plant of young leaves, intercepts more than stem apex The more consistent young leaflet tablet of leaf age, ensures the uniformity of explant as far as possible, and explant is rinsed 3-5 minutes with flowing water, then uses 70% alcohol surface sterilizing 20-30s, aseptic water washing three times, then soak 10-15min, then use sterile water with 2% NaClO Rinse 5-6 times；2) induction of callus：It is big that the blade after processing in step 1) with tweezers is torn into 1cm × 1cm in superclean bench Small pane, paraxial to be inoculated in up on inducing culture, the formula of inducing culture is that MS trainings are added in MS culture mediums Support and 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines added in base, in 25 DEG C of temperature after inoculation, dark condition down toward There is callus in blade edge；3) bud induces：The callus obtained in step 2) is inoculated into young shoot culture medium, the formula of young shoot culture medium is MS 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurines are added in culture medium, in 25 DEG C of temperature, intensity of illumination after inoculation Cultivated under 2000-3000lx, light application time 12h/d, obtain the regrowth of hemerocailis middendorffi；4) culture of rootage：The tawny daylily seedling obtained in step 3) is transferred in root media, the formula of root media is 1/ 0.1mg/L methyl α-naphthyl acetates, 3% sucrose, 0.6% agar are added in 2MS culture mediums, in 25 DEG C of temperature, intensity of illumination 2000- Cultivated under 3000lx, light application time 12h/d, cultivate and short of white occur to seedling base portion and grow complete root system；5) plantlet of transplant：The complete hemerocailis middendorffi seedling of taking root obtained in step 4) is planted to perlite and turf 1:1 matrix In, greenhouse hardening, obtains normal hemerocailis middendorffi seedling.
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| CN105706926A (en) * | 2016-02-04 | 2016-06-29 | 河南农业大学 | Day lily rooting culture method |
| CN109717075A (en) * | 2017-10-27 | 2019-05-07 | 上海市农业科学院 | A method of hemerocailis middendorffi regeneration plant is obtained by evoking adventive bud |
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| CN102668986A (en) * | 2012-05-30 | 2012-09-19 | 唐山师范学院 | Direct rooting method for tissue culture cluster seedlings of hemerocallis fulva |
| CN103461135A (en) * | 2013-09-18 | 2013-12-25 | 宁波市农业科学研究院 | Method for propagating hemerocallis hybridus |
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|---|---|---|---|---|
| CN102668986A (en) * | 2012-05-30 | 2012-09-19 | 唐山师范学院 | Direct rooting method for tissue culture cluster seedlings of hemerocallis fulva |
| CN103461135A (en) * | 2013-09-18 | 2013-12-25 | 宁波市农业科学研究院 | Method for propagating hemerocallis hybridus |
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| 大花萱草不同外植体诱导愈伤组织的比较研究;王晓娟等;《生命科学研究》;20051231;第9卷(第3期);摘要，第1.2节 * |
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