CN104542285A - Method for tissue culture by applying leaves of hemerocallis middendorfii - Google Patents

Method for tissue culture by applying leaves of hemerocallis middendorfii Download PDF

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CN104542285A
CN104542285A CN201410843997.7A CN201410843997A CN104542285A CN 104542285 A CN104542285 A CN 104542285A CN 201410843997 A CN201410843997 A CN 201410843997A CN 104542285 A CN104542285 A CN 104542285A
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medium
explant
callus
seedling
hemerocailis middendorffi
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CN104542285B (en
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高亦珂
朱琳
王叶
任毅
张启翔
王佳
程堂仁
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Beijing Forestry University
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Beijing Forestry University
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Abstract

The invention relates to a method for tissue culture by applying leaves of hemerocallis middendorfii. The method comprises the following specific steps: taking the tender inner leaves of the hemerocallis middendorfii growing on the open ground as explants, performing disinfection treatment, inoculating in an inducing medium to induce and form callus tissues, forming complete hemerocallis middendorfii seedlings through differentiation and proliferation, bud induction and root induction of the callus tissues, transplanting the hemerocallis fulva seedlings into a substrate and growing into normal hemerocallis middendorfii plants. According to the method, as the leaves of the hemerocallis middendorfii are applied to perform the tissue culture, the limit that the hemerocallis middendorfii only can adopt floral organs during the flower period to perform tissue culture propagation is overcome, the annual propagation can be realized, the rapid propagation of improved varieties is facilitated, and the varieties of the hemerocallis middendorfii applied to gardens are enriched.

Description

A kind of method that hemerocailis middendorffi leaf tissue is cultivated
Technical field
The invention belongs to field of plant tissue culture, the hemerocailis middendorffi young leaflet tablet tissue cultures being specifically related to open country growth expands numerous method.
Background technology
Hemerocailis middendorffi is Hemerocallidaceae hemerocallis perennial herb, is important Perennial Flowers, and the tawny daylily germ plasm resource of China is very abundant, and find that this genus has 14 kinds in the world, just there are 11 kind hemerocailis middendorffis in China.On the basis of wild tawny daylily kind matter, through the hemerocailis middendorffi horticultural gardening Breeds of artificial hybridization seed selection, the kind logged at present has kind more than 80,000.The emerald green long and narrow base of hemerocailis middendorffi leaf is raw, lines up two row, tool fusiform fleshy root and fibrous root in band shape.Stem short shape, flower is extracted out from middle part by tongue, and helical form cyme, each titbit can be blossomed tens of.Bud is like hairpin, and open as funnel, sliver turnup, be lily shape corolla, petal tip splits.The symphysis of perianth base portion, in tubular, 6 pieces, stamen, 3 length 3 are short, and flower pesticide is that T-shaped is carried on filigree top, and greatly, flower shape is different, and the florescence is mainly in summer in Hua Jing change.
Hemerocailis middendorffi is a kind of excellent Horticultural crops.But at present hemerocailis middendorffi of less types in Landscape Application, main cause is the impact of being bred.Hemerocailis middendorffi in Landscape Application mainly realizes breeding by tissue cultures mode, and the explant that tissue cultures is conventional has scape, petal, ovary, stem apex etc., and therefore breeding at the florescence, can only limit the cultivation of new varieties and improved seeds.And blade is drawn materials and do not affected by the florescence, tissue cultures explant can be realized and to draw materials the annual of time, reach the object of tissue-culturing rapid propagation.If therefore can carry out the tissue cultures of hemerocailis middendorffi as explant with the blade of outdoor cropping, realize tawny daylily tissue cultures explant and to draw materials the annual of time, be then conducive to realizing breeding numerous soon, greatly enrich the kind of hemerocailis middendorffi in gardens.
Summary of the invention
The object of the invention is to, for the deficiencies in the prior art, provide a kind of method utilizing hemerocailis middendorffi blade to carry out the quick numerous seedling of tissue cultures, realize tawny daylily tissue cultures explant and to draw materials the annual of time.
For achieving the above object, the present invention adopts following technical scheme:
1) explant selection and disinfect;
2) induction of callus: be inoculated in inducing culture by the explant after sterilization, to forming callus, the formula of described inducing culture is: add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine in MS medium;
3) bud inducement: described callus is inoculated in young shoot medium, be cultured to the regrowth obtaining hemerocailis middendorffi, the formula of described young shoot medium is: add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine in MS medium;
4) culture of rootage: described regrowth is proceeded in root media, there is short of white to regrowth base portion and grow complete root system, obtain hemerocailis middendorffi seedling, the formula of described root media is: add 0.1mg/L methyl α-naphthyl acetate, 3% sucrose, 0.6% agar in 1/2MS medium, and described 1/2MS medium is the macroelement concentration in MS medium reduced by half;
5) plantlet of transplant: plant in cultivation matrix by described hemerocailis middendorffi seedling, greenhouse hardening, obtains hemerocailis middendorffi seedling.
In the present invention, the selection of described explant is specially, choosing, selects and extracts a fairly large number of hemerocailis middendorffi individual plant of young leaves out, and the young leaflet tablet intercepting the above leaf age of stem apex more consistent is required explant, and described young leaves is the leaf that leaf age is less than 12 days.
In the present invention, described in disinfect and be specially, by explant running water 3-5 minute, then use 70% alcohol surface sterilizing 20-30s, aseptic water washing 3-4 time, then soak 10-15min with the NaClO of 2%, then use aseptic water washing 5-6 time.
In the present invention, the induction of described callus step be: in superclean bench, the explant tweezers after process are torn into blockage, adaxial and its surface is inoculated in inducing culture upward; At temperature 23-27 DEG C after inoculation, cultivate under dark condition.
In the present invention, the condition of culture of described bud inducement is: temperature 25 DEG C, intensity of illumination 2000-3000lx, light application time 12h/d.
In the present invention, the condition of described culture of rootage is: temperature 25 DEG C, cultivates under intensity of illumination 2000-3000lx, light application time 12h/d.
In the present invention, described hemerocailis middendorffi seedling refers to the differentiation completing bud and root, independently can carry out the young plant of autophyting growth.
Further, step is as follows more specifically to provide tawny daylily tissue cultures in the present invention:
1) explant selection and disinfect: select to extract young leaves a fairly large number of hemerocailis middendorffi individual plant out, intercept the young leaflet tablet that the above leaf age of stem apex is more consistent, ensure the uniformity of explant as far as possible, by explant with running water 3-5 minute, use 70% alcohol surface sterilizing 20-30s again, aseptic water washing three times, then soaks 10-15min with the NaClO of 2%, then uses aseptic water washing 5-6 time;
2) induction of callus: by step 1 in superclean bench) in blade tweezers after process be torn into the pane of 1cm × 1cm size, adaxial and its surface is inoculated on inducing culture upward, the formula of inducing culture is add in MS medium in MS medium to add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine, temperature 25 DEG C after inoculation, there is callus down to blade edge in dark condition;
3) bud inducement: by step 2) in the callus that obtains be inoculated in young shoot medium, the formula of young shoot medium is add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine in MS medium, temperature 25 DEG C after inoculation, intensity of illumination 2000-3000lx, cultivate under light application time 12h/d, germinate to callus, obtain the regrowth of hemerocailis middendorffi;
4) culture of rootage: by step 3) in the tawny daylily seedling that obtains proceed in root media, the formula of root media is add 0.1mg/L methyl α-naphthyl acetate, 3% sucrose, 0.6% agar in 1/2MS medium, temperature 25 DEG C, intensity of illumination 2000-3000lx, cultivate under light application time 12h/d, be cultured to seedling base portion and occur short of white and grow complete root system;
5) plantlet of transplant: by step 4) in the complete hemerocailis middendorffi seedling of taking root that obtains plant in the matrix of perlite and peat composed of rotten mosses 1:1, greenhouse hardening, obtains normal hemerocailis middendorffi seedling.
The present invention utilizes tawny daylily blade to carry out tissue cultures and realizes Fast-propagation, there is the meaning of following two aspects: (1) day lily is mainly based on mitogenetic breeding, reproduction coefficient is low, and breeding required time is long, is unfavorable for the cultivation of the numerous of hemerocailis middendorffi and breeding.The tissue cultures time is short, and increment frequency is high, can improve the reproductive efficiency of hemerocailis middendorffi.(2) draw materials as explant using the hemerocailis middendorffi young leaflet tablet of open country growth, not draw materials the restriction of time by the florescence, extend explant and draw materials the time, be conducive to application and the popularization of hemerocailis middendorffi.
Accompanying drawing explanation
Fig. 1: tawny daylily new varieties " Red Triangle region " callus tissue culture picture;
Fig. 2: tawny daylily new varieties " Red Triangle region " bud inducement 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 inducement picture;
Fig. 6: wild tawny daylily (Hemerocallis fulva) picture of taking root;
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 hemerocailis middendorffi leaf tissue is cultivated
The present embodiment selects tawny daylily new varieties " Red Triangle region " to carry out leaf tissue cultivation.
1) explant selection and disinfect: select to extract young leaves a fairly large number of hemerocailis middendorffi individual plant out, therefrom select the young leaves of leaf age 9-10 days, intercept the young leaflet tablet at the above position of stem apex, ensure the uniformity of explant as far as possible.By explant with running water 3-5 minute, then use 70% alcohol surface sterilizing 30s, aseptic water washing three times, then soak 10-15min with the NaClO of 2%, then use aseptic water washing 5-6 time.
2) induction of callus: by step 1 in superclean bench) in blade tweezers after process be torn into the pane of 1cm × 1cm size, adaxial and its surface is laid on inducing culture upward, and the formula of inducing culture is add 0.1mg/L NAA, 1.0mg/L6-benayl aminopurine in MS medium.Temperature 25 DEG C after inoculation, cultivate 18 days rear blade edges under dark condition and occur callus (as Fig. 1).
3) bud inducement: by step 2) in the callus that obtains be inoculated in young shoot medium, the formula of young shoot medium is add 0.1mg/L NAA, 1.0mg/L 6-benzyl aminopurine in MS medium, temperature 25 DEG C, intensity of illumination 2000-3000lx, cultivate under light application time 12h/d, cultivate 40-50 days, grow bud (as Fig. 2) to hemerocailis middendorffi, obtain tawny daylily regrowth.
4) culture of rootage: by step 3) in the tawny daylily regrowth that obtains proceed in root media, the formula of root media is add 0.1mg/L methyl α-naphthyl acetate, 3% sucrose, 0.6% agar in 1/2MS medium.Temperature 25 DEG C, cultivate under intensity of illumination 2000-3000lx, light application time 12h/d.Cultivate and occur white short and grow complete root system to seedling base portion in 15 days, obtain tawny daylily seedling (as Fig. 3).
5) plantlet of transplant: by step 4) in the hemerocailis middendorffi seedling that obtains plant in the matrix of perlite and peat composed of rotten mosses 1:1, greenhouse hardening, obtains normal hemerocailis middendorffi seedling.
The survival rate of the hemerocailis middendorffi regrowth that the present invention obtains is 98%.
Embodiment 2 hemerocailis middendorffi leaf tissue is cultivated
The present embodiment selects the blade of a kind of wild hemerocailis middendorffi Hemerocallis fulva to carry out tissue cultures.
1) explant selection and disinfect: select to extract young leaves a fairly large number of hemerocailis middendorffi individual plant out, therefrom select the young leaves of leaf age 9-10 days, intercept the young leaflet tablet at the above position of stem apex, ensure the uniformity of explant as far as possible.By explant with running water 3-5 minute, then use 70% alcohol surface sterilizing 30s, aseptic water washing three times, then soak 10-15min with the NaClO of 2%, then use aseptic water washing 5-6 time.
2) induction of callus: by step 1 in superclean bench) in blade tweezers after process be torn into the pane of 1cm × 1cm size, adaxial and its surface is laid on inducing culture upward, and the formula of inducing culture is add 0.2mg/L NAA, 1.0mg/L6-benayl aminopurine in MS medium.Temperature 25 DEG C after inoculation, cultivating 3 weeks under dark condition, there is callus (as Fig. 4) in blade edge.
3) bud inducement: by step 2) in the callus that obtains be inoculated in young shoot medium, the formula of young shoot medium is add 0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine in MS medium temperature 25 DEG C, intensity of illumination 2000-3000lx, cultivate under light application time 12h/d, cultivate 40-60 days, grow bud (as Fig. 5) to hemerocailis middendorffi, obtain tawny daylily regrowth.
4) culture of rootage: by step 3) in the tawny daylily regrowth that obtains proceed in root media, the formula of root media is add 0.1mg/L methyl α-naphthyl acetate, 3% sucrose, 0.6% agar in 1/2MS medium.Temperature 25 DEG C, cultivate under intensity of illumination 2000-3000lx, light application time 12h/d.Cultivate and occur white short and grow complete root system (as Fig. 6) to seedling base portion in 15-30 days.
5) plantlet of transplant: by step 4) in the complete hemerocailis middendorffi seedling of taking root that obtains plant in the matrix of perlite and peat composed of rotten mosses 1:1, greenhouse hardening, obtains normal hemerocailis middendorffi seedling.
The survival rate of the hemerocailis middendorffi regrowth that the present invention obtains is 96%.
Comparative example 1: hormon and concentration are on the impact of blade callus of induce
Be in the callus of induce process of explant with blade, different according to type of culture medium growing state, probably be divided into following four kinds of situations: 1. plant tissue moves back green, present the nearly dead state of white (inoculation blade chooses the tender and lovely blade of plant internal layer, is yellow during inoculation); 2. plant tissue wound produces brown material; 3. plant tissue wound grows white particles shape callus and organizes and 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 provided of the present invention is provided, is more conducive to hemerocailis middendorffi calli induction.
The selection of comparative example 2 explant is on the impact of blade callus of induce
Compare with embodiment 1, its difference is only, explant selection leaf age is the blade of 15-16 days, finds that this explant of employing cannot induced synthesis callus in experiment.
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 (8)

1. a method for hemerocailis middendorffi leaf tissue cultivation, it is characterized in that, the method comprises the steps:
1) explant selection and disinfect;
2) induction of callus: be inoculated in inducing culture by the explant after sterilization, to forming callus, the formula of described inducing culture is: add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine in MS medium;
3) bud inducement: be inoculated in young shoot medium by described callus, is cultured to callus and sprouts, and forms regrowth; The formula of described young shoot medium is: add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine in MS medium;
4) culture of rootage: described regrowth is proceeded in root media, there is short of white to regrowth base portion and grow complete root system, obtain hemerocailis middendorffi seedling, the formula of described root media is: add 0.1mg/L methyl α-naphthyl acetate, 3% sucrose, 0.6% agar in 1/2MS medium, and described 1/2MS medium is the macroelement concentration in MS medium reduced by half;
5) plantlet of transplant: plant in cultivation matrix by described hemerocailis middendorffi seedling, greenhouse hardening, obtains hemerocailis middendorffi seedling.
2. method according to claim 1, is characterized in that, the selection of described explant is specially, select to extract a fairly large number of hemerocailis middendorffi individual plant of young leaves out, intercept the tender young leaves of children that the above leaf age of stem apex is more consistent, be required explant, described young leaves is the leaf that leaf age is less than 12 days.
3. method according to claim 1, is characterized in that, described in disinfect and be specially, by explant running water 3-5 minute, then use 70% alcohol surface sterilizing 20-30s, aseptic water washing 3-4 time, then soak 10-15min with the NaClO of 2%, then use aseptic water washing 5-6 time.
4. method according to claim 1, is characterized in that, the induction of described callus step be: in superclean bench, the explant tweezers after process are torn into blockage, adaxial and its surface is inoculated in inducing culture upward; At temperature 23-27 DEG C after inoculation, cultivate under dark condition.
5. method according to claim 1, is characterized in that, the condition of culture of described bud inducement is: temperature 25 DEG C, intensity of illumination 2000-3000lx, light application time 12h/d.
6. method according to claim 1, is characterized in that, the condition of described culture of rootage is: temperature 25 DEG C, intensity of illumination 2000-3000lx, light application time 12h/d.
7. method according to claim 1, is characterized in that, described hemerocailis middendorffi seedling refers to the differentiation completing bud and root, independently can carry out the young plant of autophyting growth.
8. method according to claim 1, is characterized in that, specifically comprises the steps:
1) explant selection and disinfect: select to extract young leaves a fairly large number of hemerocailis middendorffi individual plant out, intercept the young leaflet tablet that the above leaf age of stem apex is more consistent, ensure the uniformity of explant as far as possible, by explant with running water 3-5 minute, use 70% alcohol surface sterilizing 20-30s again, aseptic water washing three times, then soaks 10-15min with the NaClO of 2%, then uses aseptic water washing 5-6 time;
2) induction of callus: by step 1 in superclean bench) in blade tweezers after process be torn into the pane of 1cm × 1cm size, adaxial and its surface is inoculated on inducing culture upward, the formula of inducing culture is add in MS medium in MS medium to add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine, temperature 25 DEG C after inoculation, there is callus down to blade edge in dark condition;
3) bud inducement: by step 2) in the callus that obtains be inoculated in young shoot medium, the formula of young shoot medium is add 0.1-0.2mg/L NAA, 1.0mg/L 6-benzyl aminopurine in MS medium, temperature 25 DEG C after inoculation, intensity of illumination 2000-3000lx, cultivate under light application time 12h/d, obtain the regrowth of hemerocailis middendorffi;
4) culture of rootage: by step 3) in the tawny daylily seedling that obtains proceed in root media, the formula of root media is add 0.1mg/L methyl α-naphthyl acetate, 3% sucrose, 0.6% agar in 1/2MS medium, temperature 25 DEG C, intensity of illumination 2000-3000lx, cultivate under light application time 12h/d, be cultured to seedling base portion and occur short of white and grow complete root system;
5) plantlet of transplant: by step 4) in the complete hemerocailis middendorffi seedling of taking root that obtains plant in the matrix of perlite and peat composed of rotten mosses 1:1, greenhouse hardening, obtains normal hemerocailis middendorffi seedling.
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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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