CN103430842A - Quick propagation method of hybrid orchid tissue culture - Google Patents

Quick propagation method of hybrid orchid tissue culture Download PDF

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Publication number
CN103430842A
CN103430842A CN2013103396434A CN201310339643A CN103430842A CN 103430842 A CN103430842 A CN 103430842A CN 2013103396434 A CN2013103396434 A CN 2013103396434A CN 201310339643 A CN201310339643 A CN 201310339643A CN 103430842 A CN103430842 A CN 103430842A
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culture
illumination
days
subculture
tissue culture
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CN103430842B (en
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陈和明
吕复兵
朱根发
操君喜
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ENVIRONMENTAL HORTICULTURE RESEARCH INSTITUTE OF GUANGDONG ACADEMY OF AGRICULTURAL SCIENCES
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ENVIRONMENTAL HORTICULTURE RESEARCH INSTITUTE OF GUANGDONG ACADEMY OF AGRICULTURAL SCIENCES
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Abstract

The invention provides a quick propagation method of hybrid orchid tissue culture, which comprises the steps of conducting induction culture on shoots, conducting propagation culture on multiple shoots, conducting subculture on the multiple shoots, conducting rooting culture, obtaining complete plants, getting the plants out of bottles, exercising seedlings and transplanting. The method is easy to operate and low in production cost, does not pollute an environment and can achieve large-scale production, and cultured dendrobium chrysotoxum seedlings are stable in inheritable property, keep the parental characteristic, and have the advantages of invariance, small investment, high yield, short period and the like.

Description

A kind of hybrid orchid quick breeding method for tissue culture
Technical field
The invention belongs to the tissue culture rapid propagation technique field, be specifically related to a kind of hybrid orchid quick breeding method for tissue culture.
Background technology
Hybrid orchid is the novel orchid of a class selected by the blue artificial hybridization of hybrid cymbidium and state.The proterties such as the flower size of hybrid orchid, plant height, plant type, blade size, between hybrid cymbidium and state orchid, have very high ornamental value, and market prospects are wide.From annual flower market in recent years, supply falls short of demand for hybrid orchid, and some elaboration hybrid orchid prices are high, and what have even sells for several thousand yuan, every basin, but the supply of current hybrid orchid mainly relies on import.Therefore, carry out the research of hybrid orchid tissue-culturing quick-propagation, improve sapling multiplication speed, to applying of hybrid orchid, and the demand of satisfying the market is of great practical significance.
Summary of the invention
In view of this, technical problem solved by the invention is to provide a kind of hybrid orchid quick breeding method for tissue culture, approach by Multiple Buds carries out the test-tube plantlet that the cultivation of Dendrobium Chrysotoxum Lindl tissue can obtain a large amount of stabilization characteristics of genetics, keep good parent's characteristic, and possess and comprise consistency, less investment, the many advantages that output is high, the cycle is short.
The present invention solves above-mentioned technical problem by the following technical programs:
A kind of hybrid orchid quick breeding method for tissue culture comprises the following steps:
1) sterilization of explant: get the sprouting that gives birth to then robust growth, high 5~15cm, sprouting is cut by the place of the base portion of maternal plant, remove the leaf that has launched impurity and upper end, only stay the approximately sprout of 4~5cm of total length, flowing water rinses 20min, uses alcohol and 0.1% HgCl on superclean bench 2solution is processed and is realized once sterilizing, then uses aseptic water washing 5~8 times again, then with tweezers, carefully peels off piecewise the outer quilt encased, until expose lateral bud, will have the stem section of lateral bud to cut down, then use 0.1% HgCl 2solution sterilization 6~8min carries out secondary sterilization, then uses aseptic water washing 5~8 times, and aseptic filter paper blots residual moisture, and is inoculated on inducing culture;
2) inducing of Multiple Buds: adopt inducing culture to cultivate 15~20 days, lateral bud turns green increase; Through 30~40 days, sprouting formed and grows again; In incubation, intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
3) propagation of Multiple Buds: the Multiple Buds that utilizes lateral bud culture to obtain, transfer to the adventitious buds proliferation medium after the clip partial blade, cultivate 40~60 days, obtain the propagation Multiple Buds that the propagation multiple is 4.0~6.0; Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
4) subculture is cultivated: adopt subculture medium to carry out the subculture cultivation, within every 40~60 days, shoot proliferation is 1 time, and subculture number is controlled in 15 times; Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
5) culture of rootage: after the subculture seedling reaches suitable quantity, adopt root media to carry out Rooting and hardening-off culture, cultivate and obtain the seedling of taking root in 40~60 days.Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
6) test-tube seedling transplanting: selecting annual 3~May is the season that bottle outlet is transplanted, or provide similar 3~May natural conditions growing environment carry out the bottle outlet transplanting; Before transplanting, test-tube plantlet is positioned over hardening 10-20 days in the greenhouse of tool natural daylight scattering, then from test tube, takes out seedling, cleans the medium of root, and be put in immersion 3-5 minute in liquor potassic permanganate, after taking out, use the import water moss implantation in the plastic cup basin of bore 4-6cm; Keep greenhouse ventilation, humidity is 70~80%, and temperature remains on more than 15 ℃ to room temperature range, must be with blower fans, cascades cooling higher than 30 ℃.
Preferably, described inducing culture composition is 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+NAA (methyl α-naphthyl acetate) 0.05~0.10 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon; The sugary 30g/L of medium, agar 0.7%, pH value 5.5-5.8.
Preferably, described adventitious buds proliferation medium is 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+AD (adenine) 1.0~3.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon.
Preferably, described subculture medium is 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+AD (adenine) 1.0~3.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon.
Preferably, contain 1/2MS+NAA (methyl α-naphthyl acetate) 0.5~1.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon in described culture of rootage based component.
Preferably, in the sterilisation step of described explant, use alcohol and 0.1% HgCl on superclean bench 2solution is processed and is realized that once sterilizing refers on superclean bench and first use the alcohol clean surface, and soaks 15-45s in 75% alcohol, uses 0.1% HgCl 2solution sterilization 8~10min.
Preferably, in described test-tube seedling transplanting, the liquor potassic permanganate that is 0.1% for the liquor potassic permanganate that soaks the seedling root.
Than prior art, the beneficial effect of hybrid orchid quick breeding method for tissue culture of the present invention is: the method processing ease, production cost is low, free from environmental pollution, can accomplish scale production.The hybrid orchid seedling of cultivating by the present invention, its stabilization characteristics of genetics, kept parent's characteristic, possesses and comprise consistency, less investment, the many advantages that output is high, the cycle is short.
Embodiment
In view of this, a kind of hybrid orchid quick breeding method for tissue culture provided in the specific embodiment of the invention specifically comprises the following steps:
1) sterilization of explant: get the sprouting that gives birth to then robust growth, high 5~15cm, sprouting is cut by the place of the base portion of maternal plant, remove the leaf that has launched impurity and upper end, only stay the approximately sprout of 4~5cm of total length, flowing water rinses 20min, uses alcohol and 0.1% HgCl on superclean bench 2solution is processed and is realized once sterilizing, then uses aseptic water washing 5~8 times again, then with tweezers, carefully peels off piecewise the outer quilt encased, until expose lateral bud, will have the stem section of lateral bud to cut down, then use 0.1% HgCl 2solution sterilization 6~8min carries out secondary sterilization, then uses aseptic water washing 5~8 times, and aseptic filter paper blots residual moisture, and is inoculated on inducing culture;
2) inducing of Multiple Buds: adopt inducing culture to cultivate 15~20 days, lateral bud turns green increase; Through 30~40 days, sprouting formed and grows again; In incubation, intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
3) propagation of Multiple Buds: the Multiple Buds that utilizes lateral bud culture to obtain, transfer to the adventitious buds proliferation medium after the clip partial blade, cultivate 40~60 days, obtain the propagation Multiple Buds that the propagation multiple is 4.0~6.0; Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
4) subculture is cultivated: adopt subculture medium to carry out the subculture cultivation, within every 40~60 days, shoot proliferation is 1 time, and subculture number is controlled in 15 times; Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
5) culture of rootage: after the subculture seedling reaches suitable quantity, adopt root media to carry out Rooting and hardening-off culture, cultivate and obtain the seedling of taking root in 40~60 days.Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
6) test-tube seedling transplanting: selecting annual 3~May is the season that bottle outlet is transplanted, or provide similar 3~May natural conditions growing environment carry out the bottle outlet transplanting; Before transplanting, test-tube plantlet is positioned over hardening 10-20 days in the greenhouse of tool natural daylight scattering, then from test tube, takes out seedling, cleans the medium of root, and be put in immersion 3-5 minute in liquor potassic permanganate, after taking out, use the import water moss implantation in the plastic cup basin of bore 4-6cm; Keep greenhouse ventilation, humidity is 70~80%, and temperature remains on more than 15 ℃ to room temperature range, must be with blower fans, cascades cooling higher than 30 ℃.
The composition of processing mode, condition of culture, incubation time and the medium related in above-mentioned each step all can carry out suitable adjustment according to specific needs.
Wherein, each is cultivated in the situation that composition is definite, and the content of each component of wherein using can be adjusted according to actual cultivation situation, and the medium component of using in said method and the content range of each composition are as follows:
Inducing in step of Multiple Buds, contain 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+NAA (methyl α-naphthyl acetate) 0.05~0.10 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon in the inducing culture composition; The sugary 30g/L of medium, agar 0.7%, pH value 5.5-5.8.
In the propagation step of Multiple Buds, the adventitious buds proliferation medium contains 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+AD (adenine) 1.0~3.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon.
In the subculture incubation step, contain 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+AD (adenine) 1.0~3.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon in the subculture medium composition.
In the culture of rootage step, contain 1/2MS+NAA (methyl α-naphthyl acetate) 0.5~1.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon in the culture of rootage based component.
In addition, because incubation is subject to the impact of many factors such as temperature, illumination, humidity, thereby, in each step of the present invention, processing mode, condition of culture, incubation time all can carry out suitable adjustment according to specific needs.
Wherein, in the sterilisation step of explant, use alcohol and 0.1% HgCl on superclean bench 2solution is processed and is realized that once sterilizing refers on superclean bench and first use the alcohol clean surface, and soaks 15-45s in 75% alcohol, uses 0.1% HgCl 2solution sterilization 8~10min.In addition, in described test-tube seedling transplanting, the liquor potassic permanganate that is 0.1% for the liquor potassic permanganate that soaks the seedling root.
For making the present invention easier to understand, below will further set forth specific embodiments of the invention.
Embodiment 1, hybrid orchid tissue-culturing quick-propagation one:
In the present embodiment, select the hybrid orchid of kind one to carry out tissue-culturing quick-propagation, this kind is for being commonly called as the hybrid orchid of " Korea S Miss ".
Get the sprouting that gives birth to then robust growth, high 5-15cm, sprouting is cut by the place of the base portion of maternal plant, remove the leaf that has launched impurity and upper end, only stay the sprout of the about 4-5cm of total length, flowing water rinses 20min, first use the alcohol clean surface on superclean bench, and soak 30s in 75% alcohol, use 0.1% HgCl 2solution sterilization 8min, aseptic water washing 5 times, then carefully peel off with tweezers the outer quilt encased piecewise, until expose lateral bud, will have the stem section of lateral bud to cut down, then use 0.1% HgCl 2solution sterilization 6min, aseptic water washing 5 times, aseptic filter paper blots residual moisture, and be seeded in to induce and cultivate on 1/2MS+6-BA (6-benzyl aminoadenine) 1.0 ㎎/L+NAA (methyl α-naphthyl acetate) 0.05 ㎎/L+10.0% coconut milk+3.0g/L active carbon, intensity of illumination 2000~2500lx, illumination 12 hours/day, 25~28 ℃ of temperature.Cultivate 45~60 days, sprouting forms and grows.The Multiple Buds that utilizes lateral bud culture to obtain, transfer to adventitious buds proliferation medium 1/2MS+6-BA (6-benzyl aminoadenine) 1.0 ㎎/L+AD (adenine) 1.0 ㎎/L+10.0% coconut milk+3.0g/L active carbon after the clip partial blade, at intensity of illumination 2000~2500lx, illumination 12 hours/day, under 25~28 ℃ of conditions of temperature, cultivate 40~60 days, obtain Multiple Buds, the propagation multiple can reach 4.0.Within every 40~60 days, shoot proliferation is 1 time, general subculture number is no more than 15 times, subculture medium is 1/2MS+6-BA (6-benzyl aminoadenine) 1.0 ㎎/L+AD (adenine) 1.0 ㎎/L+10.0% coconut milk+3.0g/L active carbon, at intensity of illumination 2000~2500lx, illumination 12 hours/day, cultivate under 25~28 ℃ of conditions of temperature.After the subculture seedling reaches some, can carry out Rooting and hardening-off culture, the Rooting and hardening-off culture base is 1/2MS+NAA (methyl α-naphthyl acetate) 0.5 ㎎/L+10.0% coconut milk+3.0g/L active carbon, at intensity of illumination 2000~2500lx, illumination 12 hours/day, cultivate under the condition that temperature is 25~28 ℃, cultivate 40~60 days, obtain the seedling of taking root.Be the season that bottle outlet is transplanted spring, before transplanting, test-tube plantlet is positioned in the greenhouse of tool natural daylight scattering hardening 15 days, then from test tube, takes out seedling, clean the medium of root, and be put in 0.1% liquor potassic permanganate and soak 5 minutes, after taking out with the import water moss implantation in the plastic cup basin of bore 4.8cm, and keep greenhouse ventilation, humidity is 70~80%, temperature remains on more than 15 ℃, higher than 30 ℃ of necessary blower fan, cascades used, lowers the temperature, and transplanting survival rate is up to more than 90%.
Embodiment 2, hybrid orchid tissue-culturing quick-propagation two:
In the present embodiment, select the hybrid orchid of kind two to carry out tissue-culturing quick-propagation, this kind is for being commonly called as the hybrid orchid of " Korea S beauty ".
Get the sprouting that gives birth to then robust growth, high 5-15cm, sprouting is cut by the place of the base portion of maternal plant, remove the leaf that has launched impurity and upper end, only stay the sprout of the about 4-5cm of total length, flowing water rinses 20min, first use the alcohol clean surface on superclean bench, and soak 30s in 75% alcohol, use 0.1% HgCl 2solution sterilization 9min, aseptic water washing 6 times, then carefully peel off with tweezers the outer quilt encased piecewise, until expose lateral bud, will have the stem section of lateral bud to cut down, then use 0.1% HgCl 2solution sterilization 7min, aseptic water washing 6 times, aseptic filter paper blots residual moisture, and be seeded in to induce and cultivate on 1/2MS+6-BA (6-benzyl aminoadenine) 2.0 ㎎/L+NAA (methyl α-naphthyl acetate) 0.075 ㎎/L+15.0% coconut milk+4.0g/L active carbon, intensity of illumination 2000~2500lx, illumination 12 hours/day, 25~28 ℃ of temperature.Cultivate 45~60 days, sprouting forms and grows.The Multiple Buds that utilizes lateral bud culture to obtain, transfer to adventitious buds proliferation medium 1/2MS+6-BA (6-benzyl aminoadenine) 2.0 ㎎/L+AD (adenine) 2.0 ㎎/L+15.0% coconut milk+4.0g/L active carbon after the clip partial blade, at intensity of illumination 2000~2500lx, illumination 12 hours/day, under 25~28 ℃ of conditions of temperature, cultivate 40~60 days, obtain Multiple Buds, the propagation multiple can reach 5.0.Within every 40~60 days, shoot proliferation is 1 time, general subculture number is no more than 15 times, subculture medium is 1/2MS+6-BA (6-benzyl aminoadenine) 2.0 ㎎/L+AD (adenine) 2.0 ㎎/L+15.0% coconut milk+4.0g/L active carbon, at intensity of illumination 2000~2500lx, illumination 12 hours/day, cultivate under 25~28 ℃ of conditions of temperature.After the subculture seedling reaches some, can carry out Rooting and hardening-off culture, the Rooting and hardening-off culture base is 1/2MS+NAA (methyl α-naphthyl acetate) 0.75 ㎎/L+15.0% coconut milk+4.0g/L active carbon, at intensity of illumination 2000~2500lx, illumination 12 hours/day, cultivate under the condition that temperature is 25~28 ℃, cultivate 40~60 days, obtain the seedling of taking root.Be the season that bottle outlet is transplanted spring, before transplanting, test-tube plantlet is positioned in the greenhouse of tool natural daylight scattering hardening 15 days, then from test tube, takes out seedling, clean the medium of root, and be put in 0.1% liquor potassic permanganate and soak 5 minutes, after taking out with the import water moss implantation in the plastic cup basin of bore 4.8cm, and keep greenhouse ventilation, humidity is 70~80%, temperature remains on more than 15 ℃, higher than 30 ℃ of necessary blower fan, cascades used, lowers the temperature, and transplanting survival rate is up to more than 90%.
Embodiment 2, hybrid orchid tissue-culturing quick-propagation three:
In the present embodiment, select the hybrid orchid of kind three to carry out tissue-culturing quick-propagation, this kind is for being commonly called as the hybrid orchid of " Dongfanghong ".
Get the sprouting that gives birth to then robust growth, high 5-15cm, sprouting is cut by the place of the base portion of maternal plant, remove the leaf that has launched impurity and upper end, only stay the sprout of the about 4-5cm of total length, flowing water rinses 20min, first use the alcohol clean surface on superclean bench, and soak 30s in 75% alcohol, use 0.1% HgCl 2solution sterilization 10min, aseptic water washing 8 times, then carefully peel off with tweezers the outer quilt encased piecewise, until expose lateral bud, will have the stem section of lateral bud to cut down, then use 0.1% HgCl 2solution sterilization 8min, aseptic water washing 8 times, aseptic filter paper blots residual moisture, and be seeded in to induce and cultivate on 1/2MS+6-BA (6-benzyl aminoadenine) 3.0 ㎎/L+NAA (methyl α-naphthyl acetate) 0.1 ㎎/L+20.0% coconut milk+5.0g/L active carbon, intensity of illumination 2000~2500lx, illumination 12 hours/day, 25~28 ℃ of temperature.Cultivate 45~60 days, sprouting forms and grows.The Multiple Buds that utilizes lateral bud culture to obtain, transfer to adventitious buds proliferation medium 1/2MS+6-BA (6-benzyl aminoadenine) 3.0 ㎎/L+AD (adenine) 3.0 ㎎/L+20.0% coconut milk+5.0g/L active carbon after the clip partial blade, at intensity of illumination 2000~2500lx, illumination 12 hours/day, under 25~28 ℃ of conditions of temperature, cultivate 40~60 days, obtain Multiple Buds, the propagation multiple can reach 6.0.Within every 40~60 days, shoot proliferation is 1 time, general subculture number is no more than 15 times, subculture medium is 1/2MS+6-BA (6-benzyl aminoadenine) 3.0 ㎎/L+AD (adenine) 3.0 ㎎/L+20.0% coconut milk+5.0g/L active carbon, at intensity of illumination 2000~2500lx, illumination 12 hours/day, cultivate under 25~28 ℃ of conditions of temperature.After the subculture seedling reaches some, can carry out Rooting and hardening-off culture, the Rooting and hardening-off culture base is 1/2MS+NAA (methyl α-naphthyl acetate) 0.1 ㎎/L+20.0% coconut milk+5.0g/L active carbon, at intensity of illumination 2000~2500lx, illumination 12 hours/day, cultivate under the condition that temperature is 25~28 ℃, cultivate 40~60 days, obtain the seedling of taking root.Be the season that bottle outlet is transplanted spring, before transplanting, test-tube plantlet is positioned in the greenhouse of tool natural daylight scattering hardening 15 days, then from test tube, takes out seedling, clean the medium of root, and be put in 0.1% liquor potassic permanganate and soak 5 minutes, after taking out with the import water moss implantation in the plastic cup basin of bore 4.8cm, and keep greenhouse ventilation, humidity is 70~80%, temperature remains on more than 15 ℃, higher than 30 ℃ of necessary blower fan, cascades used, lowers the temperature, and transplanting survival rate is up to more than 90%.
Than prior art, the hybrid orchid quick breeding method for tissue culture disclosed in aforesaid way, processing ease, production cost is low, free from environmental pollution, can accomplish scale production.The Dendrobium Chrysotoxum Lindl seedling of cultivating by the present invention, its stabilization characteristics of genetics, kept parent's characteristic, possesses and comprise consistency, less investment, the many advantages that output is high, the cycle is short.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment, the present invention is explained in detail; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.

Claims (7)

1. a hybrid orchid quick breeding method for tissue culture is characterized in that the method comprises the following steps:
1) sterilization of explant: get the sprouting that gives birth to then robust growth, high 5~15cm, sprouting is cut by the place of the base portion of maternal plant, remove the leaf that has launched impurity and upper end, only stay the approximately sprout of 4~5cm of total length, flowing water rinses 20min, uses alcohol and 0.1% HgCl on superclean bench 2solution is processed and is realized once sterilizing, then uses aseptic water washing 5~8 times again, then with tweezers, carefully peels off piecewise the outer quilt encased, until expose lateral bud, will have the stem section of lateral bud to cut down, then use 0.1% HgCl 2solution sterilization 6~8min carries out secondary sterilization, then uses aseptic water washing 5~8 times, and aseptic filter paper blots residual moisture, and is inoculated on inducing culture;
2) inducing of Multiple Buds: adopt inducing culture to cultivate 15~20 days, lateral bud turns green increase; Through 30~40 days, sprouting formed and grows again; In incubation, intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
3) propagation of Multiple Buds: the Multiple Buds that utilizes lateral bud culture to obtain, transfer to the adventitious buds proliferation medium after the clip partial blade, cultivate 40~60 days, obtain the propagation Multiple Buds that the propagation multiple is 4.0~6.0; Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
4) subculture is cultivated: adopt subculture medium to carry out the subculture cultivation, within every 40~60 days, shoot proliferation is 1 time, and subculture number is controlled in 15 times; Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
5) culture of rootage: after the subculture seedling reaches suitable quantity, adopt root media to carry out Rooting and hardening-off culture, cultivate and obtain the seedling of taking root in 40~60 days; Condition of culture is: intensity of illumination 2000~2500lx, illumination 10~12 hours/day, 25~28 ℃ of temperature;
6) test-tube seedling transplanting: selecting annual 3~May is the season that bottle outlet is transplanted, or provide similar 3~May natural conditions growing environment carry out the bottle outlet transplanting; Before transplanting, test-tube plantlet is positioned over hardening 10-20 days in the greenhouse of tool natural daylight scattering, then from test tube, takes out seedling, cleans the medium of root, and be put in immersion 3-5 minute in liquor potassic permanganate, after taking out, use the import water moss implantation in the plastic cup basin of bore 4-6cm; Keep greenhouse ventilation, humidity is 70~80%, and temperature remains on more than 15 ℃ to room temperature range, must be with blower fans, cascades cooling higher than 30 ℃.
2. hybrid orchid quick breeding method for tissue culture as claimed in claim 1, is characterized in that: contain 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+NAA (methyl α-naphthyl acetate) 0.05~0.10 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon in described inducing culture composition; The sugary 30g/L of medium, agar 0.7%, pH value 5.5-5.8.
3. hybrid orchid quick breeding method for tissue culture as claimed in claim 1, it is characterized in that: described adventitious buds proliferation medium contains 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+AD (adenine) 1.0~3.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon.
4. hybrid orchid quick breeding method for tissue culture as claimed in claim 1, is characterized in that: contain 1/2MS+6-BA (6-benzyl aminoadenine) 1.0~3.0 ㎎/L+AD (adenine) 1.0~3.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon in described subculture medium composition.
5. hybrid orchid quick breeding method for tissue culture as claimed in claim 1, is characterized in that: contain 1/2MS+NAA (methyl α-naphthyl acetate) 0.5~1.0 ㎎/L+10.0~20.0% coconut milk+3.0~5.0g/L active carbon in described culture of rootage based component.
6. hybrid orchid quick breeding method for tissue culture as claimed in claim 1, is characterized in that: in the sterilisation step of described explant, use alcohol and 0.1% HgCl on superclean bench 2solution is processed and is realized that once sterilizing refers on superclean bench and first use the alcohol clean surface, and soaks 15-45s in 75% alcohol, uses 0.1% HgCl 2solution sterilization 8~10min.
7. hybrid orchid quick breeding method for tissue culture as claimed in claim 1 is characterized in that: in described test-tube seedling transplanting, and the liquor potassic permanganate that is 0.1% for the liquor potassic permanganate that soaks the seedling root.
CN201310339643.4A 2013-08-07 2013-08-07 A kind of Quick propagation method of hybrid orchid tissue culture Expired - Fee Related CN103430842B (en)

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CN108990799A (en) * 2018-07-25 2018-12-14 杭州市农业科学研究院 A method of hybrid orchid sterilizable material is efficiently obtained using newborn lateral bud
CN109105263A (en) * 2018-11-01 2019-01-01 翁源县天下泽雨农业科技有限公司 A kind of state orchid rhizomes quick breeding method for tissue culture

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