CN102783418B - Tissue culture method for pyrethrum cinerariifolium - Google Patents
Tissue culture method for pyrethrum cinerariifolium Download PDFInfo
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- CN102783418B CN102783418B CN 201210298213 CN201210298213A CN102783418B CN 102783418 B CN102783418 B CN 102783418B CN 201210298213 CN201210298213 CN 201210298213 CN 201210298213 A CN201210298213 A CN 201210298213A CN 102783418 B CN102783418 B CN 102783418B
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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 theseedling 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 the original merit of Dalmatian chrysanthemum, therefore, Dalmatian chrysanthemum mainly adopts vegetative mode to carry out growing and cultivation in the industrialization planting process of reality, method commonly used in the vegetative propagation is 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 the problem of industrialization plantation needs, the invention provides a kind of Dalmatian chrysanthemum method for tissue culture, its technical scheme may further comprise the steps:
(a) select bud and sterilization: choose the material that cultivation is organized in the bud conduct of not opening in the Dalmatian chrysanthemum improved seeds, selected bud requires healthy and strong, anosis, no insect pest, and the bud of choosing requires petal to launch as yet;
Selected bud is put under the running water flowing water flushing 5-10 minute, to wash good bud then, to be placed on mass percent concentration be to soak among 15% the liquor natrii hypochloritis, in immersion process, constantly stir and vibrate, soak time is 10-20 minute, after immersion is finished, 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 standby;
(b) callus induction: will wash good bud and be put in the superclean bench, sepal with tweezers strip off bud, cut petal on the bud with scissors then, the holder that exposes bud, downcut holder with vaccinating lancet, the holder that scales off is inoculated in the I blake bottle that callus inducing medium is housed, then inoculation being had the I blake bottle of holder to put in the culturing room cultivates, the warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lux, light application time 12hr/d cultivated 30-40 days with this understanding, was inoculated into holder in the I blake bottle and expanded and become green callus;
Described 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, there is 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 1000-1200Lux, light application time 12hr/d, cultivated with this understanding 20-30 days, and be inoculated into callus lines differentiation back in the II blake bottle and obtain the Dalmatian chrysanthemum seedling of growing thickly;
Described callus differential medium prescription 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 differentiation in the II blake bottle the is obtained seedling of growing thickly is transferred on the aseptic seedling proliferated culture medium in the III blake bottle, there is inoculation the grow thickly III blake bottle of seedling of Dalmatian chrysanthemum to put in the culturing room, the warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lux, light application time 12hr/d, after cultivating 30 days with this understanding, be inoculated into the seedling differentiation of growing thickly of Dalmatian chrysanthemum in the III blake bottle and expand numerous more Dalmatian chrysanthemum seedling of growing thickly that obtains, in the III blake bottle Dalmatian chrysanthemum grow thickly seedling break up at every turn expand numerous speed be 4-5 doubly;
The prescription of described 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 numerous large batch of Dalmatian chrysanthemum seedling of growing thickly that obtains;
(e) take root: the Dalmatian chrysanthemum root media of in the IV blake bottle, packing into, be cut into the individual plant seedling with expanding numerous Dalmatian chrysanthemum that obtains seedling of growing thickly in the III blake bottle with vaccinating lancet, 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 1000-1200Lux, 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 described Dalmatian chrysanthemum root media is: 1/2MS+NAA(0.1-0.5mg/L)+and PP
333(0.5-2.0 mg/L)+sucrose (30g/L)+agar (3-4g/L), pH value 5.8;
(f) hardening: carry out the water planting hardening 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, up be placed in pallet towards inferior lobe the Dalmatian chrysanthemum of washing medium off the shoot root of taking root, inject the take root rhizome base portion of seedling of 1/8MS solution to the liquid level submergence Dalmatian chrysanthemum of injecting solution then in the pallet, 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), Dalmatian chrysanthemum in the pallet its leaf look of seedling of taking root is deepened, and the water planting hardening finishes;
The water planting hardening is carried out floating hardening after finishing, after peat and perlite mixed according to volume ratio 2:1 as seedling medium, seedling medium is loaded in the floating dish, the Dalmatian chrysanthemum that the pallet middle period look that obtains after then the water planting hardening being finished the is deepened seedling of taking root is planted in the seedling medium of floating dish, there is plantation the take root floating dish of seedling of Dalmatian chrysanthemum to be placed in the floating pond that is loaded with floating mill water culture nutrient solution and to carry out floating hardening, described floating mill water culture nutrient solution is 1/8MS solution, floating hardening is thrown in N during to 10-12 days in floating pond, P, the K proportioning is composite fertilizer and the metalaxyl-mn-zn of 15:15:15, 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 hardening then after 40-50 days, floating hardening finishes, obtain being fit to transplant the Dalmatian chrysanthemum group training seedling in land for growing field crops, the survival rate of water planting hardening and the survival rate of field-transplanting all reach 98%, after hardening finishes, big Tanaka is arrived in the Dalmatian chrysanthemum group training transplantation of seedlings that hardening obtains, 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 6-benzyl aminopurine, 2,4-D is 2, the 4-dichlorphenoxyacetic acid, and NAA is α-Nai Yisuan, PP
333Be paclobutrazol.
The present invention adopts tissue to cultivate the seedling breeding that vegetative mode is carried out 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, output height, the neat characteristics of plant growing way.
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 height, plant type is good, per unit area yield is high, the neat plant that blooms is as maternal material, can have the high-quality proterties identical with maternal plant by the seedling that obtains after organizing cultivation to breed, be 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 the water planting hardening earlier, carries out the method for floating hardening then, and moisture is supplied with abundance in the hardening process, and dehydration can not appear in the hardening plant, do not have seedling-slowing stage, the hardening time is short, the survival rate height, need not spray water in the hardening 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) select bud and sterilization: choose the material that cultivation is organized in the bud conduct of not opening in the Dalmatian chrysanthemum improved seeds, selected bud requires healthy and strong, anosis, no insect pest, and the bud of choosing requires petal to launch as yet;
Selected bud is put under the running water flowing water flushing 5 minutes, to wash good bud then, to be placed on mass percent concentration be to soak among 15% the liquor natrii hypochloritis, in immersion process, constantly stir and vibrate, soak time is 10 minutes, after immersion is finished, 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 standby;
(b) callus induction: will wash good bud and be put in the superclean bench, sepal with tweezers strip off bud, cut petal on the bud with scissors then, the holder that exposes bud, downcut holder with vaccinating lancet, the holder that scales off is inoculated in the I blake bottle that callus inducing medium is housed, then inoculation being had the I blake bottle of holder to put in the culturing room cultivates, the warm optical condition of regulating control culturing room is: 20 ℃ of temperature, intensity of illumination 1000Lux, light application time 12hr/d cultivated 30 days with this understanding, was inoculated into holder in the I blake bottle and expanded and become green callus;
Described 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, there is 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 1000Lux, light application time 12hr/d, cultivated with this understanding 20 days, and be inoculated into callus lines differentiation back in the II blake bottle and obtain the Dalmatian chrysanthemum seedling of growing thickly;
Described callus differential medium prescription 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 differentiation in the II blake bottle the is obtained seedling of growing thickly is transferred on the aseptic seedling proliferated culture medium in the III blake bottle, there is inoculation the grow thickly III blake bottle of seedling of Dalmatian chrysanthemum to put in the culturing room, the warm optical condition of regulating control culturing room is: 20 ℃ of temperature, intensity of illumination 1000Lux, light application time 12hr/d, after cultivating 30 days with this understanding, be inoculated into the seedling differentiation of growing thickly of Dalmatian chrysanthemum in the III blake bottle and expand numerous more Dalmatian chrysanthemum seedling of growing thickly that obtains, in the III blake bottle Dalmatian chrysanthemum grow thickly seedling break up at every turn expand numerous speed be 4-5 doubly;
The prescription of described 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 numerous large batch of Dalmatian chrysanthemum seedling of growing thickly that obtains;
(e) take root: the Dalmatian chrysanthemum root media of in the IV blake bottle, packing into, be cut into the individual plant seedling with expanding numerous Dalmatian chrysanthemum that obtains seedling of growing thickly in the III blake bottle with vaccinating lancet, 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 1000Lux, 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 described Dalmatian chrysanthemum root media is: 1/2MS+NAA(0.1mg/L)+and PP
333(0.5 mg/L)+sucrose (30g/L)+agar (3g/L), pH value 5.8;
(f) hardening: 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, inject the take root rhizome base portion of seedling of 1/8MS solution to the liquid level submergence Dalmatian chrysanthemum of injecting solution then in the pallet, 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 days under field conditions (factors), 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, seedling medium is loaded in the floating dish, the Dalmatian chrysanthemum that pallet middle period look the is deepened seedling of taking root is planted in the seedling medium of floating dish then, there is plantation the take root floating dish of seedling of Dalmatian chrysanthemum to be placed in the floating pond that is loaded with floating mill water culture nutrient solution and to carry out floating hardening, described floating mill water culture nutrient solution is 1/8MS solution, in floating pond, throw in N during floating hardening to 10 day, P, the K proportioning is composite fertilizer and the metalaxyl-mn-zn of 15:15:15, the input concentration of composite fertilizer is 0.8g/L, the input concentration of metalaxyl-mn-zn is 0.1g/L, after continuing floating hardening to 40 day then, obtain being fit to transplant the Dalmatian chrysanthemum group training seedling in land for growing field crops, the survival rate of hardening 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) select bud and sterilization: choose the material that cultivation is organized in the bud conduct of not opening in the Dalmatian chrysanthemum improved seeds, selected bud requires healthy and strong, anosis, no insect pest, and the bud of choosing requires petal to launch as yet;
Selected bud is put under the running water flowing water flushing 10 minutes, to wash good bud then, to be placed on mass percent concentration be to soak among 15% the liquor natrii hypochloritis, in immersion process, constantly stir and vibrate, soak time is 20 minutes, after immersion is finished, 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 standby;
(b) callus induction: will wash good bud and be put in the superclean bench, sepal with tweezers strip off bud, cut petal on the bud with scissors then, the holder that exposes bud, downcut holder with vaccinating lancet, the holder that scales off is inoculated in the I blake bottle that callus inducing medium is housed, then inoculation being had the I blake bottle of holder to put in the culturing room cultivates, the warm optical condition of regulating control culturing room is: 25 ℃ of temperature, intensity of illumination 1200Lux, light application time 12hr/d cultivated 40 days with this understanding, was inoculated into holder in the I blake bottle and expanded and become green callus;
Described callus inducing medium prescription is: MS+6-BA (1.0mg/L)+2,4-D(1.0mg/L)+caseinhydrolysate (20mg/L)+sucrose (30g/L)+agar (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, there is 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 1200Lux, light application time 12hr/d, cultivated with this understanding 30 days, and be inoculated into callus lines differentiation back in the II blake bottle and obtain the Dalmatian chrysanthemum seedling of growing thickly;
Described callus differential medium prescription is: MS+6-BA (3.0mg/L)+NAA(1.0mg/L)+sucrose (30g/L)+agar (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 differentiation in the II blake bottle the is obtained seedling of growing thickly is transferred on the aseptic seedling proliferated culture medium in the III blake bottle, there is inoculation the grow thickly III blake bottle of seedling of Dalmatian chrysanthemum to put in the culturing room, the warm optical condition of regulating control culturing room is: 25 ℃ of temperature, intensity of illumination 1200Lux, light application time 12hr/d, after cultivating 30 days with this understanding, be inoculated into the seedling differentiation of growing thickly of Dalmatian chrysanthemum in the III blake bottle and expand numerous more Dalmatian chrysanthemum seedling of growing thickly that obtains, in the III blake bottle Dalmatian chrysanthemum grow thickly seedling break up at every turn expand numerous speed be 4-5 doubly;
The prescription of described aseptic seedling proliferated culture medium is: MS+6-BA (3.0mg/L)+NAA(1.0mg/L)+sucrose (30g/L)+agar 4g/L), and pH value 5.8;
Repeat after the above-mentioned aseptic seedling breeding, can expand numerous large batch of Dalmatian chrysanthemum seedling of growing thickly that obtains;
(e) take root: the Dalmatian chrysanthemum root media of in the IV blake bottle, packing into, be cut into the individual plant seedling with expanding numerous Dalmatian chrysanthemum that obtains seedling of growing thickly in the III blake bottle with vaccinating lancet, 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 1200Lux, 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 described Dalmatian chrysanthemum root media is: 1/2MS+NAA(0.5mg/L)+and PP
333(2.0 mg/L)+sucrose (30g/L)+agar (4g/L), pH value 5.8;
(f) hardening: 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, inject the take root rhizome base portion of seedling of 1/8MS solution to the liquid level submergence Dalmatian chrysanthemum of injecting solution then in the pallet, be placed into and have the indoor of light scattering being loaded with the take root pallet of seedling of Dalmatian chrysanthemum, after placing 10 days under field conditions (factors), 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, seedling medium is loaded in the floating dish, the Dalmatian chrysanthemum that pallet middle period look the is deepened seedling of taking root is planted in the seedling medium of floating dish then, there is plantation the take root floating dish of seedling of Dalmatian chrysanthemum to be placed in the floating pond that is loaded with floating mill water culture nutrient solution and to carry out floating hardening, described floating mill water culture nutrient solution is 1/8MS solution, in floating pond, throw in N during floating hardening to 12 day, P, the K proportioning is composite fertilizer and the metalaxyl-mn-zn of 15:15:15, the input concentration of composite fertilizer is 1.0g/L, the input concentration of metalaxyl-mn-zn is 0.15g/L, after continuing floating hardening to 50 day then, obtain being fit to transplant the Dalmatian chrysanthemum group training seedling in land for growing field crops, the survival rate of hardening and the survival rate of field-transplanting all reach 98%, after hardening finishes, big Tanaka is arrived in the Dalmatian chrysanthemum group training transplantation of seedlings that hardening obtains, through the flower of results Dalmatian chrysanthemum after the growing and cultivation management.
Claims (2)
1. Dalmatian chrysanthemum method for tissue culture may further comprise the steps:
(a) select bud and sterilization: not open colored thunder is as the material of organizing cultivation as yet to choose the Dalmatian chrysanthemum improved seeds, and selected bud requires healthy and strong, anosis, no insect pest;
Selected bud is put under the running water flowing water flushing 5-10 minute, to wash good bud then, to be placed on mass percent concentration be to soak among 15% the liquor natrii hypochloritis, in immersion process, constantly stir and vibrate, soak time is 10-20 minute, after immersion is finished, 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 standby;
(b) callus induction: will wash good bud and be put in the superclean bench, with exposing the holder explant behind the tweezers strip off bud, downcut the holder explant with vaccinating lancet then, the holder explant that scales off is inoculated in the I blake bottle that callus inducing medium is housed, then inoculation being had the I blake bottle of holder explant to put in the culturing room cultivates, the warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lux, light application time 12hr/d, cultivated with this understanding 30-40 days, and be inoculated into holder 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, there is 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 1000-1200Lux, light application time 12hr/d, cultivated with this understanding 20-30 days, and be inoculated into callus lines differentiation back in the II blake bottle and obtain the Dalmatian chrysanthemum seedling of growing thickly;
(d) aseptic seedling propagation: the aseptic seedling of in the III blake bottle, packing into proliferated culture medium, the Dalmatian chrysanthemum that differentiation in the II blake bottle the is obtained seedling of growing thickly is transferred on the aseptic seedling proliferated culture medium in the III blake bottle, there is inoculation the grow thickly III blake bottle of seedling of Dalmatian chrysanthemum to put in the culturing room, the warm optical condition of regulating control culturing room is: 20 ± 5 ℃ of temperature, intensity of illumination 1000-1200Lux, light application time 12hr/d, after cultivating 30 days with this understanding, be inoculated into the seedling differentiation of growing thickly of Dalmatian chrysanthemum in the III blake bottle and expand numerous more Dalmatian chrysanthemum seedling of growing thickly that obtains, in the III blake bottle Dalmatian chrysanthemum grow thickly seedling break up at every turn expand numerous speed be 4-5 doubly;
Repeat after the above-mentioned aseptic seedling breeding, can expand numerous large batch of Dalmatian chrysanthemum seedling of growing thickly that obtains;
(e) take root: the Dalmatian chrysanthemum root media of in the IV blake bottle, packing into, be cut into the individual plant seedling with expanding numerous Dalmatian chrysanthemum that obtains seedling of growing thickly in the III blake bottle with vaccinating lancet, 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 1000-1200Lux, 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;
(f) hardening: 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 carried out hardening, after hardening finishes, big Tanaka is arrived in the Dalmatian chrysanthemum group training transplantation of seedlings that hardening obtains, through the flower of results Dalmatian chrysanthemum after the growing and cultivation management;
It is characterized in that: the culture medium prescription of callus induction is described in the step (b): MS+6-BA+2,4-D+ caseinhydrolysate+sucrose+agar, pH value 5.8, wherein the concentration of 6-BA is 0.5-1.0mg/L, 2, the concentration of 4-D is 0.5-1.0mg/L, the concentration of caseinhydrolysate is 20mg/L, concentration of sucrose is 30g/L, and the concentration of agar is 3-4g/L; The culture medium prescription of the differentiation of callus described in the step (c) is: MS+6-BA+NAA+ sucrose+agar, pH value 5.8, wherein the concentration of 6-BA is 2.0-3.0mg/L, the concentration of NAA is 0.1-1.0mg/L, concentration of sucrose is 30g/L, and the concentration of agar is 3-4g/L; The culture medium prescription of the propagation of aseptic seedling described in the step (d) is: MS+6-BA+NAA+ sucrose+agar, pH value 5.8, wherein the concentration of 6-BA is 1.0-3.0mg/L, the concentration of NAA is 0.1-1.0mg/L, concentration of sucrose is 30g/L, and the concentration of agar is 3-4g/L; The prescription of Dalmatian chrysanthemum root media is described in the step (e): 1/2MS+NAA+PP
333+ sucrose+agar, pH value 5.8, wherein the concentration of NAA is 0.1-0.5mg/L, PP
333Concentration be 0.5-2.0 mg/L, concentration of sucrose is 30g/L, the concentration of agar is 3-4g/L.
2. Dalmatian chrysanthemum method for tissue culture as claimed in claim 1, it is characterized in that: carry out the water planting hardening in the step (f) during hardening 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, up be placed in pallet towards inferior lobe the Dalmatian chrysanthemum of washing medium off the shoot root of taking root, inject the take root rhizome base portion of seedling of 1/8MS solution to the liquid level submergence Dalmatian chrysanthemum of injecting solution then in the pallet, 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), Dalmatian chrysanthemum in the pallet its leaf look of seedling of taking root is deepened, and the water planting hardening finishes;
The water planting hardening is carried out floating hardening after finishing, after peat and perlite mixed according to volume ratio 2:1 as seedling medium, seedling medium is loaded in the floating dish, the Dalmatian chrysanthemum that the pallet middle period look that obtains after then the water planting hardening being finished the is deepened seedling of taking root is planted in the seedling medium of floating dish, there is plantation the take root floating dish of seedling of Dalmatian chrysanthemum to be placed in the floating pond that is loaded with floating mill water culture nutrient solution and to carry out floating hardening, described floating mill water culture nutrient solution is 1/8MS solution, floating hardening is thrown in N during to 10-12 days in floating pond, P, the K proportioning is composite fertilizer and the metalaxyl-mn-zn of 15:15:15, 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 hardening then after 40-50 days, floating hardening finishes, and obtains being fit to transplant the Dalmatian chrysanthemum group training seedling in land for growing field crops.
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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 |
| CN109287489B (en) * | 2018-11-28 | 2021-08-20 | 云南省农业科学院花卉研究所 | Standardized in-vitro culture method of pyrethrum seedlings |
| CN110547200B (en) * | 2019-09-30 | 2021-03-02 | 济南易通城市建设集团股份有限公司 | Marigold pollen differentiation medium and differentiation culture method |
| 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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