CN102783418A - Tissue culture method for pyrethrum cinerariifolium - Google Patents
Tissue culture method for pyrethrum cinerariifolium Download PDFInfo
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Abstract
A tissue culture method for pyrethrum cinerariifolium includes using unopened buds of pyrethrum cinerariifolium as explant material, subjecting the buds to sterilization, inoculating receptacles of the buds to callus induction medium, culturing the receptacles to obtain green callus, transferring the obtained callus to callus differentiation medium, culturing to obtain a seedling clump of pyrethrum cinerariifolium, subjecting the obtained seedling clump of pyrethrum cinerariifolium to aseptic seedling multiplication to obtain mass seedling clumps of pyrethrum cinerariifolium, transferring the seedling clumps of pyrethrum cinerariifolium to rooting medium, culturing to obtain rooted seedlings of pyrethrum cinerariifolium, exercising the obtained rooted seedlings of pyrethrum cinerariifolium, transplanting the seedlings to a field, and growing flowers of pyrethrum cinerariifolium. The seedlings obtained by the tissue culture method for pyrethrum cinerariifolium can retain excellent characters of female parents of pyrethrum cinerariifolium and are fast in breeding and high in survival rate. Hydroponic seedling exercising is simple to operate, management is simple and easy, cost is low, and survival rate of seedling exercising and transplanting is high.
Description
Technical field
The invention belongs to the agricultural biotechnologies application, relate to the Dalmatian chrysanthemum method for tissue culture.
Background technology
Dalmatian chrysanthemum is a kind of composite family platymiscium, contains insecticide active substance pyrethrin efficiently in its flower; In recent years along with countries in the world to the attention degree of food security and the raising day by day of renovation dynamics, the plantation secondary industry of Dalmatian chrysanthemum has had and has developed rapidly.Its modes of reproduction of growing and cultivation Dalmatian chrysanthemum has two kinds of seminal propagation and vegetative propagations; Seminal propagation can not keep Dalmatian chrysanthemum original excellent proterties; Therefore, Dalmatian chrysanthemum mainly adopts vegetative mode to carry out growing and cultivation in the industrialization planting process of reality, and method commonly used in the vegetative propagation is an offshoot; Its reproduction speed is slow, survival rate is low, can not satisfy the needs of Dalmatian chrysanthemum industrialization cultivating and growing.
Summary of the invention
In prior art; When adopting the offshoot vegetative propagation to carry out the industrialization cultivating and growing of Dalmatian chrysanthemum, have that reproduction speed is slow, survival rate is low, can not satisfy industrialization plantation needs problem; The present invention provides a kind of Dalmatian chrysanthemum method for tissue culture, and its technical scheme may further comprise the steps:
(a) explant is drawn materials and sterilized: choose the bud do not opened in the Dalmatian chrysanthemum improved seeds as explant, selected explant bud requires healthy and strong, anosis, no insect pest, and the bud of choosing requires petal expansion as yet;
Selected bud is put under the running water flowing water flushing 5-10 minute, and will wash the bud of getting well then, to be placed on mass percent concentration be to soak among 15% the liquor natrii hypochloritis, in immersion process, constantly stirs and vibrate; Soak time is 10-20 minute; Soak after the completion, bud is taken out from the liquor natrii hypochloritis, use the aseptic water washing bud then; Washing time is 3-5 time, and the bud inoculation that flushing is good is subsequent use;
(b) callus induction: will wash good bud and be put in the superclean bench, and, cut the petal on the bud with scissors then with the sepal of tweezers strip off bud; Expose the holder of bud, downcut holder, the holder that scales off is inoculated in the I blake bottle that callus inducing medium is housed with vaccinating lancet; There is the I blake bottle of holder to put inoculation then and cultivates indoor cultivation; The warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lex, light application time 12hr/d; Cultivated with this understanding 30-40 days, and be inoculated into holder in the I blake bottle and expand and become green callus;
Said callus inducing medium prescription is: MS+6-BA (0.5-1.0mg/L)+2,4-D (0.5-1.0mg/L)+caseinhydrolysate (20mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
(c) callus differentiation: the callus differential medium of in the II blake bottle, packing into; The callus of the green that obtains in the I blake bottle is cut the callus lines that becomes diameter 0.5cm; Then callus lines is transferred on the callus differential medium in the II blake bottle, has inoculation the II blake bottle of callus lines to put in the culturing room, the warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature; Intensity of illumination 1000-1200Lex; Light application time 12hr/d cultivated 20-30 days with this understanding, and the callus lines differentiation back that is inoculated in the II blake bottle obtains Dalmatian chrysanthemum from giving birth to seedling;
Said callus differentiation culture based formulas is: MS+6-BA (2.0-3.0mg/L)+NAA (0.1-1.0mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
(d) aseptic seedling propagation: the aseptic seedling of in the III blake bottle, packing into proliferated culture medium; The Dalmatian chrysanthemum that obtains differentiation in the II blake bottle is transferred on the aseptic seedling proliferated culture medium the III blake bottle from giving birth to seedling; Have inoculation Dalmatian chrysanthemum to put the culturing room from the III blake bottle of giving birth to seedling, the warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lex; Light application time 12hr/d; After cultivating 30 days with this understanding, the Dalmatian chrysanthemum that is inoculated in the III blake bottle is expanded and numerously obtains more Dalmatian chrysanthemum from giving birth to seedling from giving birth to the seedling differentiation, in the III blake bottle Dalmatian chrysanthemum from give birth to seedling break up at every turn expand numerous speed be 4-5 doubly;
The prescription of said aseptic seedling proliferated culture medium is: MS+6-BA (1.0-3.0mg/L)+NAA (0.1-1.0mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
Repeat after the above-mentioned aseptic seedling breeding, can expand and numerously obtain large batch of Dalmatian chrysanthemum from giving birth to seedling;
(e) take root: the Dalmatian chrysanthemum root media of in the IV blake bottle, packing into, be cut into individual plant seedling from giving birth to seedling with vaccinating lancet with expanding numerous Dalmatian chrysanthemum that obtains in the III blake bottle, then the individual plant seedling that is cut into is transferred on the Dalmatian chrysanthemum root media in the IV blake bottle; There is inoculation the IV blake bottle of Dalmatian chrysanthemum individual plant seedling to be placed in the culturing room; The warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lex, light application time 12hr/d; After cultivating 15-20 days with this understanding; In the IV blake bottle, obtain the Dalmatian chrysanthemum seedling of taking root, the quantity of taking root of every young plant is the 3-5 bar, and the length of every root is 1.5-2.0cm;
The prescription of said Dalmatian chrysanthemum root media is: 1/2MS+NAA (0.1-0.5mg/L)+PP
333(0.5-2.0 mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
(f) refining seedling: carry out water planting refining seedling earlier; The Dalmatian chrysanthemum seedling of taking root is taken out from the IV blake bottle; The take root medium of shoot root portion of flush away Dalmatian chrysanthemum is placed into the Dalmatian chrysanthemum of washing medium off the shoot root of taking root up in the pallet towards inferior lobe, in pallet, injects the take root rhizome base portion of seedling of 1/8MS solution to the liquid level submergence Dalmatian chrysanthemum of injecting solution then; Be placed into and have the indoor of light scattering being loaded with the take root pallet of seedling of Dalmatian chrysanthemum; After placing 7-10 days under field conditions (factors), its leaf look of seedling of taking root of the Dalmatian chrysanthemum in the pallet is deepened, and water planting refining seedling finishes;
After finishing, water planting refining seedling carries out floating refining seedling; As seedling medium, be loaded into seedling medium in the floating dish after peat and perlite mixed according to volume ratio 2:1, then water planting refined Dalmatian chrysanthemum that the pallet middle period look that obtains after seedling finishes the deepens seedling of taking root and plant in the seedling medium of floating dish; There is plantation the take root floating dish of seedling of Dalmatian chrysanthemum to be placed into and to carry out floating refining seedling in the floating pond that is loaded with floating mill water culture nutrient solution; Said floating mill water culture nutrient solution is a 1/8MS solution, and floating refining seedling is thrown in N, P, K proportioning during to 10-12 days in floating pond be composite fertilizer and the metalaxyl-mn-zn of 15:15:15, and the input concentration of composite fertilizer is 0.8-1.0g/L; The input concentration of metalaxyl-mn-zn is 0.1-0.15g/L; Continue floating refining seedling then after 40-50 days, floating refining seedling finishes, and obtains being fit to transplant the Dalmatian chrysanthemum tissue cultivating seedling in land for growing field crops; The survival rate of water planting refining seedling and the survival rate of field-transplanting all reach 98%; After the refining seedling finishes, the Dalmatian chrysanthemum tissue cultivating seedling that the refining seedling obtains is transplanted to big Tanaka, through the flower of results Dalmatian chrysanthemum after the growing and cultivation management.
MS described in above-mentioned steps (b), (c), (d), (e), (f) is international prescription, and 6-BA is a 6-benzyl aminopurine, 2,4-D is 2, the 4-dichlorphenoxyacetic acid, and NAA is a NAA, PP
333Be paclobutrazol.
The present invention adopts the vegetative mode of tissue culture to carry out the seedling breeding of Dalmatian chrysanthemum; The seedling that obtains has vegetative advantage; Can keep the properties and characteristics consistent with the Dalmatian chrysanthemum maternal plant, and have that tissue culture propagating speed is fast, seedling resistance, high, the neat characteristics of plant growing way of output.
The present invention adopts on the Dalmatian chrysanthemum plant in flowering stage unopened bud as explant; Plant when drawing materials is ripe; Can identify and select have that growing way is healthy and strong, better resistance, content is high, plant type is good, per unit area yield is high, the neat plant that blooms is as maternal material; The seedling that obtains after breeding through tissue culture can have the high-quality proterties identical with maternal plant, is the clone seedling with merit; Unopened bud is as explant on the employing Dalmatian chrysanthemum florescence plant, and it is few to draw materials, and less to the injury of maternal plant, reproduction speed is fast.
The present invention adopts and carries out water planting refining seedling earlier, carries out the method for floating refining seedling then, and moisture is supplied with abundance in refining seedling process, and dehydration can not appear in refining seedling plant; Do not have seedling-slowing stage, the refining seedling time is short, and survival rate is high; Need not spray water in the refining seedling process, manage simplely, cost is low.
Specific embodiment
Embodiment 1
A kind of Dalmatian chrysanthemum method for tissue culture, its technical scheme may further comprise the steps:
(a) explant is drawn materials and sterilized: choose the bud do not opened in the Dalmatian chrysanthemum improved seeds as explant, selected explant bud requires healthy and strong, anosis, no insect pest, and the bud of choosing requires petal expansion as yet;
Selected bud is put under the running water flowing water flushing 5 minutes, and will wash the bud of getting well then, to be placed on mass percent concentration be to soak among 15% the liquor natrii hypochloritis, in immersion process, constantly stirs and vibrate; Soak time is 10 minutes; Soak after the completion, bud is taken out from the liquor natrii hypochloritis, use the aseptic water washing bud then; Washing time is 3 times, and the bud inoculation that flushing is good is subsequent use;
(b) callus induction: will wash good bud and be put in the superclean bench, and, cut the petal on the bud with scissors then with the sepal of tweezers strip off bud; Expose the holder of bud, downcut holder, the holder that scales off is inoculated in the I blake bottle that callus inducing medium is housed with vaccinating lancet; There is the I blake bottle of holder to put inoculation then and cultivates indoor cultivation; The warm optical condition of regulating control culturing room is: 20 ℃ of temperature, intensity of illumination 1000Lex, light application time 12hr/d; Cultivated with this understanding 30 days, and be inoculated into holder in the I blake bottle and expand and become green callus;
Said callus inducing medium prescription is: MS+6-BA (0.5mg/L)+2,4-D (0.5mg/L)+caseinhydrolysate (20mg/L)+sucrose (30g/L)+agar (3g/L), pH value 5.8;
(c) callus differentiation: the callus differential medium of in the II blake bottle, packing into; The callus of the green that obtains in the I blake bottle is cut the callus lines that becomes diameter 0.5cm; Then callus lines is transferred on the callus differential medium in the II blake bottle, has inoculation the II blake bottle of callus lines to put in the culturing room, the warm optical condition of regulating control culturing room is: 20 ℃ of temperature; Intensity of illumination 1000Lex; Light application time 12hr/d cultivated 20 days with this understanding, and the callus lines differentiation back that is inoculated in the II blake bottle obtains Dalmatian chrysanthemum from giving birth to seedling;
Said callus differentiation culture based formulas is: MS+6-BA (2.0mg/L)+NAA (0.1mg/L)+sucrose (30g/L)+agar (3g/L), pH value 5.8;
(d) aseptic seedling propagation: the aseptic seedling of in the III blake bottle, packing into proliferated culture medium; The Dalmatian chrysanthemum that obtains differentiation in the II blake bottle is transferred on the aseptic seedling proliferated culture medium the III blake bottle from giving birth to seedling; Have inoculation Dalmatian chrysanthemum to put the culturing room from the III blake bottle of giving birth to seedling, the warm optical condition of regulating control culturing room is: 20 ℃ of temperature, intensity of illumination 1000Lex; Light application time 12hr/d; After cultivating 30 days with this understanding, the Dalmatian chrysanthemum that is inoculated in the III blake bottle is expanded and numerously obtains more Dalmatian chrysanthemum from giving birth to seedling from giving birth to the seedling differentiation, in the III blake bottle Dalmatian chrysanthemum from give birth to seedling break up at every turn expand numerous speed be 4-5 doubly;
The prescription of said aseptic seedling proliferated culture medium is: MS+6-BA (1.0mg/L)+NAA (0.1mg/L)+sucrose (30g/L)+agar (3g/L), pH value 5.8;
Repeat after the above-mentioned aseptic seedling breeding, can expand and numerously obtain large batch of Dalmatian chrysanthemum from giving birth to seedling;
(e) take root: the Dalmatian chrysanthemum root media of in the IV blake bottle, packing into, be cut into individual plant seedling from giving birth to seedling with vaccinating lancet with expanding numerous Dalmatian chrysanthemum that obtains in the III blake bottle, then the individual plant seedling that is cut into is transferred on the Dalmatian chrysanthemum root media in the IV blake bottle; There is inoculation the IV blake bottle of Dalmatian chrysanthemum individual plant seedling to be placed in the culturing room; The warm optical condition of regulating control culturing room is: 20 ℃ of temperature, intensity of illumination 1000Lex, light application time 12hr/d; After cultivating 15 days with this understanding; In the IV blake bottle, obtain the Dalmatian chrysanthemum seedling of taking root, the quantity of taking root of every young plant is the 3-5 bar, and the length of every root is 1.5-2.0cm;
The prescription of said Dalmatian chrysanthemum root media is: 1/2MS+NAA (0.1mg/L)+PP
333(0.5 mg/L)+sucrose (30g/L)+agar (3g/L), pH value 5.8;
(f) water planting refining seedling: the Dalmatian chrysanthemum seedling of taking root is taken out from the IV blake bottle; The take root medium of shoot root portion of flush away Dalmatian chrysanthemum; Up be placed in pallet towards inferior lobe the Dalmatian chrysanthemum of washing medium off the shoot root of taking root; In pallet, inject the take root rhizome base portion of seedling of 1/8MS solution to the liquid level submergence Dalmatian chrysanthemum of injecting solution then; Be placed into and have the indoor of light scattering being loaded with the take root pallet of seedling of Dalmatian chrysanthemum, place 7 days under field conditions (factors) after, its leaf look of seedling of taking root of the Dalmatian chrysanthemum in the pallet is deepened;
After peat and perlite mixed according to volume ratio 2:1 as seedling medium; Be loaded into seedling medium in the floating dish; The Dalmatian chrysanthemum of then pallet middle period look the being deepened seedling of taking root is planted in the seedling medium of floating dish, has plantation the take root floating dish of seedling of Dalmatian chrysanthemum to be placed into and to carry out floating refining seedling in the floating pond that is loaded with floating mill water culture nutrient solution, and said floating mill water culture nutrient solution is a 1/8MS solution; In floating pond, throwing in N, P, K proportioning during floating refining seedling to 10 day is composite fertilizer and the metalaxyl-mn-zn of 15:15:15; The input concentration of composite fertilizer is 0.8g/L, and the input concentration of metalaxyl-mn-zn is 0.1g/L, continue floating refining seedling to 40 day then after; Obtain being fit to transplant the Dalmatian chrysanthemum tissue cultivating seedling in land for growing field crops, the survival rate of water planting refining seedling and the survival rate of field-transplanting all reach 98%.
Embodiment 2
A kind of Dalmatian chrysanthemum method for tissue culture, its technical scheme may further comprise the steps:
(a) explant is drawn materials and sterilized: choose the bud do not opened in the Dalmatian chrysanthemum improved seeds as explant, selected explant bud requires healthy and strong, anosis, no insect pest, and the bud of choosing requires petal expansion as yet;
Selected bud is put under the running water flowing water flushing 10 minutes, and will wash the bud of getting well then, to be placed on mass percent concentration be to soak among 15% the liquor natrii hypochloritis, in immersion process, constantly stirs and vibrate; Soak time is 20 minutes; Soak after the completion, bud is taken out from the liquor natrii hypochloritis, use the aseptic water washing bud then; Washing time is 5 times, and the bud inoculation that flushing is good is subsequent use;
(b) callus induction: will wash good bud and be put in the superclean bench, and, cut the petal on the bud with scissors then with the sepal of tweezers strip off bud; Expose the holder of bud, downcut holder, the holder that scales off is inoculated in the I blake bottle that callus inducing medium is housed with vaccinating lancet; There is the I blake bottle of holder to put inoculation then and cultivates indoor cultivation; The warm optical condition of regulating control culturing room is: 25 ℃ of temperature, intensity of illumination 1200Lex, light application time 12hr/d; Cultivated with this understanding 40 days, and be inoculated into holder in the I blake bottle and expand and become green callus;
Said callus inducing medium prescription is: MS+6-BA (0.5-1.0mg/L)+2,4-D (0.5-1.0mg/L)+caseinhydrolysate (20mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
(c) callus differentiation: the callus differential medium of in the II blake bottle, packing into; The callus of the green that obtains in the I blake bottle is cut the callus lines that becomes diameter 0.5cm; Then callus lines is transferred on the callus differential medium in the II blake bottle, has inoculation the II blake bottle of callus lines to put in the culturing room, the warm optical condition of regulating control culturing room is: 25 ℃ of temperature; Intensity of illumination 1200Lex; Light application time 12hr/d cultivated 30 days with this understanding, and the callus lines differentiation back that is inoculated in the II blake bottle obtains Dalmatian chrysanthemum from giving birth to seedling;
Said callus differentiation culture based formulas is: MS+6-BA (2.0-3.0mg/L)+NAA (0.1-1.0mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
(d) aseptic seedling propagation: the aseptic seedling of in the III blake bottle, packing into proliferated culture medium; The Dalmatian chrysanthemum that obtains differentiation in the II blake bottle is transferred on the aseptic seedling proliferated culture medium the III blake bottle from giving birth to seedling; Have inoculation Dalmatian chrysanthemum to put the culturing room from the III blake bottle of giving birth to seedling, the warm optical condition of regulating control culturing room is: 25 ℃ of temperature, intensity of illumination 1200Lex; Light application time 12hr/d; After cultivating 30 days with this understanding, the Dalmatian chrysanthemum that is inoculated in the III blake bottle is expanded and numerously obtains more Dalmatian chrysanthemum from giving birth to seedling from giving birth to the seedling differentiation, in the III blake bottle Dalmatian chrysanthemum from give birth to seedling break up at every turn expand numerous speed be 4-5 doubly;
The prescription of said aseptic seedling proliferated culture medium is: MS+6-BA (1.0-3.0mg/L)+NAA (0.1-1.0mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
Repeat after the above-mentioned aseptic seedling breeding, can expand and numerously obtain large batch of Dalmatian chrysanthemum from giving birth to seedling;
(e) take root: the Dalmatian chrysanthemum root media of in the IV blake bottle, packing into, be cut into individual plant seedling from giving birth to seedling with vaccinating lancet with expanding numerous Dalmatian chrysanthemum that obtains in the III blake bottle, then the individual plant seedling that is cut into is transferred on the Dalmatian chrysanthemum root media in the IV blake bottle; There is inoculation the IV blake bottle of Dalmatian chrysanthemum individual plant seedling to be placed in the culturing room; The warm optical condition of regulating control culturing room is: 25 ℃ of temperature, intensity of illumination 1200Lex, light application time 12hr/d; After cultivating 20 days with this understanding; In the IV blake bottle, obtain the Dalmatian chrysanthemum seedling of taking root, the quantity of taking root of every young plant is the 3-5 bar, and the length of every root is 1.5-2.0cm;
The prescription of said Dalmatian chrysanthemum root media is: 1/2MS+NAA (0.1-0.5mg/L)+PP
333(0.5-2.0 mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
(f) water planting refining seedling: the Dalmatian chrysanthemum seedling of taking root is taken out from the IV blake bottle; The take root medium of shoot root portion of flush away Dalmatian chrysanthemum; Up be placed in pallet towards inferior lobe the Dalmatian chrysanthemum of washing medium off the shoot root of taking root; In pallet, inject the take root rhizome base portion of seedling of 1/8MS solution to the liquid level submergence Dalmatian chrysanthemum of injecting solution then; Be placed into and have the indoor of light scattering being loaded with the take root pallet of seedling of Dalmatian chrysanthemum, place 10 days under field conditions (factors) after, its leaf look of seedling of taking root of the Dalmatian chrysanthemum in the pallet is deepened;
After peat and perlite mixed according to volume ratio 2:1 as seedling medium; Be loaded into seedling medium in the floating dish, the Dalmatian chrysanthemum of then pallet middle period look the being deepened seedling of taking root is planted in the seedling medium of floating dish, has plantation the take root floating dish of seedling of Dalmatian chrysanthemum to be placed into and to carry out floating refining seedling in the floating pond that is loaded with floating mill water culture nutrient solution; Said floating mill water culture nutrient solution is a 1/8MS solution; In floating pond, throwing in N, P, K proportioning during floating refining seedling to 12 day is composite fertilizer and the metalaxyl-mn-zn of 15:15:15, and the input concentration of composite fertilizer is 1.0g/L, and the input concentration of metalaxyl-mn-zn is 0.15g/L; After continuing floating refining seedling to 50 day then; Obtain being fit to transplant the Dalmatian chrysanthemum tissue cultivating seedling in land for growing field crops, the survival rate of water planting refining seedling and the survival rate of field-transplanting all reach 98%, after the refining seedling finishes; The Dalmatian chrysanthemum tissue cultivating seedling that the refining seedling obtains is transplanted to big Tanaka, through the flower of results Dalmatian chrysanthemum after the growing and cultivation management.
Claims (4)
1. Dalmatian chrysanthemum method for tissue culture is characterized in that may further comprise the steps:
(a) explant is drawn materials and sterilized: choose the explant material of Dalmatian chrysanthemum improved seeds, selected explant material requires healthy and strong, anosis, no insect pest;
Selected explant material is put under the running water flowing water flushing 5-10 minute, and will wash the explant material of getting well then, to be placed on mass percent concentration be to soak among 15% the liquor natrii hypochloritis, in immersion process, constantly stirs and vibrate; Soak time is 10-20 minute; Soak after the completion, explant material is taken out from the liquor natrii hypochloritis, use the aseptic water washing explant material then; Washing time is 3-5 time, and the explant material inoculation that flushing is good is subsequent use;
(b) callus induction: will wash good explant material and be put in the superclean bench; With exposing explant behind the tweezers strip off explant material, downcut explant with vaccinating lancet then, the explant that scales off is inoculated in the I blake bottle that callus inducing medium is housed; There is the I blake bottle of explant to put inoculation then and cultivates indoor cultivation; The warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lex, light application time 12hr/d; Cultivated with this understanding 30-40 days, and be inoculated into explant in the I blake bottle and expand and become green callus;
(c) callus differentiation: the callus differential medium of in the II blake bottle, packing into; The callus of the green that obtains in the I blake bottle is cut the callus lines that becomes diameter 0.5cm; Then callus lines is transferred on the callus differential medium in the II blake bottle, has inoculation the II blake bottle of callus lines to put in the culturing room, the warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature; Intensity of illumination 1000-1200Lex; Light application time 12hr/d cultivated 20-30 days with this understanding, and the callus lines differentiation back that is inoculated in the II blake bottle obtains Dalmatian chrysanthemum from giving birth to seedling;
(d) aseptic seedling propagation: the aseptic seedling of in the III blake bottle, packing into proliferated culture medium; The Dalmatian chrysanthemum that obtains differentiation in the II blake bottle is transferred on the aseptic seedling proliferated culture medium the III blake bottle from giving birth to seedling; Have inoculation Dalmatian chrysanthemum to put the culturing room from the III blake bottle of giving birth to seedling, the warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lex; Light application time 12hr/d; After cultivating 30 days with this understanding, the Dalmatian chrysanthemum that is inoculated in the III blake bottle is expanded and numerously obtains more Dalmatian chrysanthemum from giving birth to seedling from giving birth to the seedling differentiation, in the III blake bottle Dalmatian chrysanthemum from give birth to seedling break up at every turn expand numerous speed be 4-5 doubly;
Repeat after the above-mentioned aseptic seedling breeding, can expand and numerously obtain large batch of Dalmatian chrysanthemum from giving birth to seedling;
(e) take root: the Dalmatian chrysanthemum root media of in the IV blake bottle, packing into, be cut into individual plant seedling from giving birth to seedling with vaccinating lancet with expanding numerous Dalmatian chrysanthemum that obtains in the III blake bottle, then the individual plant seedling that is cut into is transferred on the Dalmatian chrysanthemum root media in the IV blake bottle; There is inoculation the IV blake bottle of Dalmatian chrysanthemum individual plant seedling to be placed in the culturing room; The warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lex, light application time 12hr/d; After cultivating 15-20 days with this understanding; In the IV blake bottle, obtain the Dalmatian chrysanthemum seedling of taking root, the quantity of taking root of every young plant is the 20-25 bar, and the length of every root is 1.5-2.0cm;
(f) refining seedling: the Dalmatian chrysanthemum seedling of taking root is taken out from the IV blake bottle; The take root medium of shoot root portion of flush away Dalmatian chrysanthemum; The Dalmatian chrysanthemum of washing medium off the seedling of taking root is refined seedling; After the refining seedling finishes, the Dalmatian chrysanthemum tissue cultivating seedling that the refining seedling obtains is transplanted to big Tanaka, through the flower of results Dalmatian chrysanthemum after the growing and cultivation management.
2. Dalmatian chrysanthemum method for tissue culture as claimed in claim 1 is characterized in that: the Dalmatian chrysanthemum explant material of choosing in the step (a) is not open bud as yet, and the explant that is inoculated in the step (b) on the callus inducing medium is a holder.
3. Dalmatian chrysanthemum method for tissue culture as claimed in claim 1; It is characterized in that: the culture medium prescription of callus induction is described in the step (b): MS+6-BA (0.5-1.0mg/L)+2,4-D (0.5-1.0mg/L)+caseinhydrolysate (20mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8; The culture medium prescription of the differentiation of callus described in the step (c) is: MS+6-BA (2.0-3.0mg/L)+NAA (0.1-1.0mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8; The culture medium prescription of the propagation of aseptic seedling described in the step (d) is: MS+6-BA (1.0-3.0mg/L)+NAA (0.1-1.0mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8; The prescription of Dalmatian chrysanthemum root media is described in the step (e): 1/2MS+NAA (0.1-0.5mg/L)+PP
333(0.5-2.0 mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8.
4. Dalmatian chrysanthemum method for tissue culture as claimed in claim 1; It is characterized in that: refining Miao Shixian carries out water planting refining seedling in the step (f), the Dalmatian chrysanthemum seedling of taking root is taken out the take root medium of shoot root portion of flush away Dalmatian chrysanthemum from the IV blake bottle; Up be placed in pallet towards inferior lobe the Dalmatian chrysanthemum of washing medium off the shoot root of taking root; In pallet, inject the take root rhizome base portion of seedling of 1/8MS solution to the liquid level submergence Dalmatian chrysanthemum of injecting solution then, be placed into and have the indoor of light scattering being loaded with the take root pallet of seedling of Dalmatian chrysanthemum, place 7-10 days under field conditions (factors) after; Dalmatian chrysanthemum in the pallet its leaf look of seedling of taking root is deepened, and water planting refining seedling finishes;
After finishing, water planting refining seedling carries out floating refining seedling; After peat and perlite mixed according to volume ratio 2:1 as seedling medium; Be loaded into seedling medium in the floating dish; Then water planting is refined Dalmatian chrysanthemum that the pallet middle period look that obtains after seedling finishes the deepens seedling of taking root and plant in the seedling medium of floating dish, have plantation the take root floating dish of seedling of Dalmatian chrysanthemum to be placed into and to carry out floating refining seedling in the floating pond that is loaded with floating mill water culture nutrient solution, said floating mill water culture nutrient solution is a 1/8MS solution; Floating refining seedling is thrown in N, P, K proportioning during to 10-12 days in floating pond be composite fertilizer and the metalaxyl-mn-zn of 15:15:15; The input concentration of composite fertilizer is 0.8-1.0g/L, and the input concentration of metalaxyl-mn-zn is 0.1-0.15g/L, continues floating refining seedling then after 40-50 days; Floating refining seedling finishes, and obtains being fit to transplant the Dalmatian chrysanthemum tissue cultivating seedling in land for growing field crops.
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| CN106171907A (en) * | 2016-08-18 | 2016-12-07 | 张玉玲 | A kind of two sections of floating plate cuttage seedling raising methods of Folium Stevlae Rebaudianae |
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| CN110547200A (en) * | 2019-09-30 | 2019-12-10 | 李传传 | Marigold pollen differentiation medium and differentiation culture method |
| CN114303911A (en) * | 2020-12-19 | 2022-04-12 | 李福元 | Off-bottle soilless transplanting and seedling hardening method for sugarcane detoxification tissue culture rooted seedlings |
| CN114431150A (en) * | 2022-03-07 | 2022-05-06 | 福建农林大学 | Jute tissue culture method taking cotyledonary node as explant |
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| CN103141384A (en) * | 2013-02-27 | 2013-06-12 | 上海交通大学 | Rapid tissue culture propagation method of pot chrysanthemum cultivars |
| CN106171907A (en) * | 2016-08-18 | 2016-12-07 | 张玉玲 | A kind of two sections of floating plate cuttage seedling raising methods of Folium Stevlae Rebaudianae |
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| CN114303911A (en) * | 2020-12-19 | 2022-04-12 | 李福元 | Off-bottle soilless transplanting and seedling hardening method for sugarcane detoxification tissue culture rooted seedlings |
| CN114303911B (en) * | 2020-12-19 | 2023-12-05 | 李福元 | Method for hardening seedlings by out-of-bottle soilless transplanting of sugarcane detoxified tissue culture root seedlings |
| CN114431150A (en) * | 2022-03-07 | 2022-05-06 | 福建农林大学 | Jute tissue culture method taking cotyledonary node as explant |
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Inventor after: Liu Kexin Inventor after: Zhu Zheshe Inventor after: Li Xian Inventor after: Liang Zhonglu Inventor before: Liu Kexin Inventor before: Zhu Zheshe Inventor before: Li Xian |
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