CN105494103A - Method for tissue culture and rapid propagation of high-quality paphiopedilum maudiae seedlings - Google Patents
Method for tissue culture and rapid propagation of high-quality paphiopedilum maudiae seedlings Download PDFInfo
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- CN105494103A CN105494103A CN201610024474.9A CN201610024474A CN105494103A CN 105494103 A CN105494103 A CN 105494103A CN 201610024474 A CN201610024474 A CN 201610024474A CN 105494103 A CN105494103 A CN 105494103A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention discloses a method for tissue culture and rapid propagation of high-quality paphiopedilum maudiae seedlings. According to the method, after a series of pharmaceutical treatment is conducted on paphiopedilum maudiae stock plants, adventitious bud induction, clump shoot regeneration and rooting culture are conducted with newly grown lateral buds as the explants by means of a unique culture medium, and then rapid propagation of high-quality seedlings is achieved. An effective means is provided to meet the market requirement for high-quality paphiopedilum maudiae seedlings. The technique is practicable, and application value is high.
Description
Technical field:
The invention belongs to plant biotechnology field, be specifically related to one and rub the blue high quality seedling quick breeding method for tissue culture of Supreme Being's class pocket.
Background technology:
The Supreme Being's pocket orchid (PaphiopedilumMaudiae) that rubs is by Lloyd's's pocket orchid (P.lawrenceanum) and callosity pocket orchid (P.callosum) cross breeding.Many filial generations have been bred as again for parent with it, blue having with Supreme Being's pocket orchid plesiomorphic class pocket that rubs on market, be referred to as " Supreme Being's class of rubbing pocket orchid (P.Maudiaetype) ", their pattern is very abundant, all have from atropurpureus, bronzing to green white, spot and lines form are also very abundant, are the principal item of commodity pocket orchid on the world, domestic market.The Sterile culture of Supreme Being's class of rubbing pocket orchid can adopt plant division, but reproduction speed is slow, and reproduction rate is low, far can not meet the needs of commercial market.At present, mostly the Supreme Being's class pocket orchid that rubs world market sold is to adopt aseptic seeding.But owing to rubbing, Supreme Being's class pocket orchid is crossbreed, and aseptic seeding offspring is separated greatly, can not keep the seedling that the merit acquired character of parent is unified, seriously constrain its large-scale production.Meanwhile, because pocket is blue when tissue cultures, the reasons such as the difficult and growth rate of explant decontamination is slow, its tissue cultures difficulty is very big, from greenhouse, does not also choose elite plant strain in the world to carry out rubbing as explant the report of the blue tissue-culturing quick-propagation of Supreme Being's class pocket.
Summary of the invention:
The object of this invention is to provide one to rub the blue high quality seedling quick breeding method for tissue culture of Supreme Being's class pocket, thus the seedling that the asexual clonal acquired character of the blue elite plant strain of Supreme Being's class pocket that carries out rubbing is unified.
The blue high quality seedling quick breeding method for tissue culture of Supreme Being's class pocket that rubs of the present invention, is characterized in that, comprise the following steps:
A, the induction of indefinite bud and the propagation of Multiple Buds: cut the lateral bud of Supreme Being's class pocket orchid that rubs as explant, after sterilization, be inoculated in inducing culture, induce indefinite bud, then the indefinite bud induced is transferred on proliferated culture medium and carry out Multiplying culture, obtain Multiple Buds, described inducing culture and proliferated culture medium are all often liter and contain 6-benzyl purine 3.0 ~ 10.0 milligrams, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, every 60 days subcultures once, during to the 6th generation, growth coefficient can reach 2.5 ~ 3.0 times.
B, Rooting and hardening-off culture: the Multiple Buds that propagation obtains is inoculated in strong seedling culture base and carries out strong sprout, obtain sturdy Multiple Buds, then be transferred to after being cut into simple bud in root media and carry out culture of rootage, obtain whole plant, described strong seedling culture base often rises containing 6-benzyl purine 1.0 ~ 2.0 milligrams, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0; Described root media often rises containing 6-benzyl purine 0.3 ~ 0.5 milligram, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, banana homogenate 50 ~ 150 grams, active carbon 0.1 ~ 1.0 gram, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0;
C, whole plant is transferred to natural daylight lower refining seedling, after hardening, clean its root medium and move into and plants metal and stone: cultivate acquisition in the mixed-matrix of bark 1-3:1 in mass ratio and to rub the blue seedling of Supreme Being's class pocket.General maintenance is suitably ventilated and enough humidity, and the survival rate of transplanting all can reach more than 90%.
Described step a is inoculated in inducing culture, and transfer on proliferated culture medium carry out Multiplying culture, being inoculated into by Multiple Buds in strong seedling culture base of step b carries out strong sprout and is transferred in root media after Multiple Buds is cut into simple bud carrying out culture of rootage, its cultivation temperature 24 ~ 28 DEG C, illuminance 1500 ~ 2000lx, illumination 12 ~ 16 hours/day.
The described lateral bud cutting Supreme Being's class pocket orchid that rubs is as explant, and after sterilization, its explant obtains by the following method:
Select plant strain growth gesture strong, Supreme Being's class pocket orchid that rubs of flower pattern rounding is maternal plant, before 35 days that cut explant sterilization, first water once with 500 ~ 800 times of dilute aqueous solutions of carbendazim and spray blade, within 5 days, water once with 500 ~ 800 times of dilute aqueous solutions of 35% G soil bacterium wetting powder and spray blade afterwards, after 5 days, water once with the dilute aqueous solution of 3000 ~ 4000 times of 72% agricultural streptomycin soluble powder and spray blade, then often 5 days are spent, repeat above-mentioned steps once, last 1 time pouring agricultural streptomycin after 5 days, the lateral bud cutting length 1.5 ~ 2.5 centimetres with the sterile scalpel of volume fraction 75% alcohol water blend is explant.
Its sterilization is specially: first used by the explant cut after soaking 10 ~ 30 seconds in volume fraction 75% alcohol water blend on superclean bench, 5 ~ 10 minutes are soaked with the hypochlorite solution of 1.0% available chlorine, aseptic water washing 4 ~ 5 times, again with mass fraction 0.1% mercuric chloride solution sterilization 5 ~ 10 minutes, aseptic water washing 4 ~ 5 times, then cut away blade, be positioned in 1500 ~ 2000 times of dilute aqueous solutions of medical streptomycin and vibrate 24 hours, the explant after being sterilized.Process like this can make sterilization success rate be 50 ~ 60%, thus effectively solves a difficult problem for explant decontamination difficulty.
MS medium is international medium, its composition and collocation method are shown in MurashigeT, SkoogF (1962) (Arevisedmediumforrapidgrowthandbioassaywithtobaccotissue cultures.PhysiolPlant15:473 – 497).1/2MS medium refers to that by macroelement consumption in MS medium be original 1/2, and all the other compositions are constant.
The present invention is carrying out on the basis of a series of chemicals treatment to the blue maternal plant of Supreme Being's class pocket that rubs, with newborn lateral bud for explant, adopt unique medium to carry out method that the induction of indefinite bud, adventitious buds proliferation and culture of rootage obtain high quality seedling Fast-propagation, for the needs meeting the blue high quality seedling market of Supreme Being's class pocket that rubs provide an effective approach.Technology is practical, and using value is high.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the tissue cultures of red Supreme Being's miracle (PaphiopedilumSCBGMiracle) that rub
(1) Material selec-tion and process
Selection plant strain growth gesture Supreme Being's class pocket orchid that rubs [red Supreme Being's miracle (PaphiopedilumSCBGMiracle) of rubbing] that is strong, flower pattern rounding is maternal plant.Before cutting 35 days that explant (lateral bud) sterilizes, first water once with 500 times of dilute aqueous solutions of carbendazim and spray blade, within 5 days, water once with 500 times of dilute aqueous solutions of 35% G soil bacterium wetting powder and spray blade afterwards, after 5 days, water once with 3000 times of dilute aqueous solutions of 72% agricultural streptomycin soluble powder and spray blade.Then often cross 5 days, repeat above-mentioned steps once, last 1 time pouring agricultural streptomycin after 5 days, the lateral bud cutting length 2.5 centimetres with the sterile scalpel of volume fraction 75% alcohol water blend is explant.
(2) explant sterilization
The explant cut first is used by superclean bench after soaking 30 seconds in volume fraction 75% alcohol water blend, 10 minutes are soaked with the hypochlorite solution of 1.0% available chlorine, aseptic water washing 5 times, then sterilize 10 minutes with the mass fraction 0.1% mercuric chloride aqueous solution, aseptic water washing 5 times.Explant after being sterilized, then blade is cut away, after remaining lateral bud is positioned over and vibrates 24 hours in shaking table in 2000 times of dilute aqueous solutions of medical streptomycin, inoculate in inducing culture, cultivation temperature 28 DEG C, illuminance 2000lx, illumination 16 hours/day, described inducing culture is often liter and contains 6-benzyl purine (6-BA) 10.0 milligrams, methyl α-naphthyl acetate (NAA) 1.0 milligrams, coconut milk 200 milliliters, sodium dihydrogen phosphate 150 milligrams, sucrose 20g and agar 6g, surplus is 1/2MS medium, pH5.8 ~ 6.0, its compound method is after being mixed by the composition in above-mentioned medium, sterilization obtains inducing culture (lower same).Sterilization success rate 60%.
(3) adventitious bud inducing and adventitious buds proliferation
At about 10 days, visible significantly brown secretion was formed postvaccinal explant, will proceed to new inducing culture 30 days time, then cultivated and can form indefinite bud on explant in about 25 days.Then indefinite bud is inoculated in proliferated culture medium and carries out shoot proliferation cultivation, described proliferated culture medium is often liter and contains 6-benzyl purine (6-BA) 4.0 milligrams, methyl α-naphthyl acetate (NAA) 1.0 milligrams, coconut milk 200 milliliters, sodium dihydrogen phosphate 150 milligrams, sucrose 20 grams and 6 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, cultivation temperature 28 DEG C, illuminance 2000lx, illumination 16 hours/day, every 60 days subcultures once, during to the 6th generation, growth coefficient can reach 3.0 times, obtains Multiple Buds.
(4) Rooting and hardening-off culture
Multiple Buds is inoculated into strong seedling culture in strong seedling culture base, cultivation temperature 28 DEG C, illuminance 2000lx, illumination 16 hours/day, culture of rootage in root media is forwarded to after the Multiple Buds of height 3.0 centimetres being cut into simple bud after 60 days, cultivation temperature 28 DEG C, illuminance 2000lx, illumination 16 hours/day, with the whole plant forming 6 centimetres high after 60 days, described strong seedling culture base often rises containing 6-benzyl purine (6-BA) 1.5 milligrams, methyl α-naphthyl acetate (NAA) 0.75 milligram, coconut milk 150 milliliters, sodium dihydrogen phosphate 175 milligrams, sucrose 20g and agar 6g, surplus is 1/2MS medium, pH5.8 ~ 6.0, its compound method is after being mixed by the composition in above-mentioned medium, sterilization obtains strong seedling culture base (lower same), described root media often rises containing 6-benzyl purine (6-BA) 0.5 milligram, methyl α-naphthyl acetate (NAA) 1.0 milligrams, coconut milk 200 milliliters, sodium dihydrogen phosphate 200 milligrams, banana homogenate 150 grams, 1.0 grams of active carbons, sucrose 20 grams and 6 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, its compound method is after being mixed by the composition in above-mentioned medium, and sterilization obtains root media (lower same).
(6) test-tube seedling transplanting
Blake bottle to be transferred to by tool 6 cm height whole plant in the greenhouse of tool natural daylight hardening 20 days, then it is taken out from vial, clean the medium of root, metal and stone (Japanese import) is planted in immigration: bark is in mass ratio in the mixed-matrix of 3:1, keep suitably ventilating and enough humidity, the survival rate of transplanting all can reach 95%.
Embodiment 2: the tissue cultures of the monarch (PaphiopedilumSCBGYunzhijun) of the green Supreme Being's cloud that rubs
(1) Material selec-tion and process
Selection plant strain growth gesture Supreme Being's class pocket orchid that rubs [monarch (PaphiopedilumSCBGYunzhijun) of the green Supreme Being's cloud that rubs] that is strong, flower pattern rounding is maternal plant.Before cutting 35 days that explant (lateral bud) sterilizes, first water once with 600 times of dilute aqueous solutions of carbendazim and spray blade, within 5 days, water once with 600 times of dilute aqueous solutions of 35% G soil bacterium wetting powder and spray blade afterwards, after 5 days, water once with 3500 times of dilute aqueous solutions of 72% agricultural streptomycin soluble powder and spray blade.Then often cross 5 days, repeat above-mentioned steps, last 1 time pouring agricultural streptomycin after 5 days, the lateral bud cutting length 2.5 centimetres with the sterile scalpel of volume fraction 75% alcohol water blend is explant.
(2) explant sterilization
The explant cut first is used by superclean bench after soaking 20 seconds in volume fraction 75% alcohol water blend, 7.5 minutes are soaked with the hypochlorite solution of 1.0% available chlorine, aseptic water washing 5 times, then sterilize 7.5 minutes with the mass fraction 0.1% mercuric chloride aqueous solution, aseptic water washing 5 times.Explant after being sterilized, then blade is cut away, after remaining lateral bud is positioned over and vibrates 24 hours in shaking table in 1750 times of dilute aqueous solutions of medical streptomycin, inoculate in inducing culture, cultivation temperature 24 DEG C, illuminance 1500lx, illumination 12 hours/day, described inducing culture is often liter and contains 6-benzyl purine (6-BA) 7.5 milligrams, methyl α-naphthyl acetate (NAA) 0.75 milligram, coconut milk 150 milliliters, sodium dihydrogen phosphate 175 milligrams, sucrose 30 grams and 7 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, its compound method is after being mixed by the composition in above-mentioned medium, sterilization obtains inducing culture (lower same).Sterilization success rate 55%.
(3) adventitious bud inducing and adventitious buds proliferation
At about 10 days, visible significantly brown secretion was formed postvaccinal explant, will proceed to new inducing culture 30 days time, then cultivated and can form indefinite bud on explant in about 28 days.Then indefinite bud is inoculated in proliferated culture medium and carries out shoot proliferation cultivation, described proliferated culture medium is often liter and contains 6-benzyl purine (6-BA) 10 milligrams, methyl α-naphthyl acetate (NAA) 0.5 milligram, coconut milk 100 milliliters, sodium dihydrogen phosphate 200 milligrams, sucrose 30 grams and 7 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, cultivation temperature 24 DEG C, illuminance 1500lx, illumination 12 hours/day, every 60 days subcultures once, during to the 6th generation, growth coefficient can reach 2.8 times, obtains Multiple Buds.
(4) Rooting and hardening-off culture
Multiple Buds is inoculated into strong seedling culture in strong seedling culture base, cultivation temperature 24 DEG C, illuminance 1500lx, illumination 12 hours/day, culture of rootage in root media is forwarded to after the Multiple Buds of height 2.5 centimetres being cut into simple bud after 60 days, cultivation temperature 24 DEG C, illuminance 1500lx, illumination 12 hours/day, with the whole plant forming 5.5 centimetres high after 60 days, described strong seedling culture base often rises containing 6-benzyl purine (6-BA) 2 milligrams, methyl α-naphthyl acetate (NAA) 1 milligram, coconut milk 200 milliliters, sodium dihydrogen phosphate 200 milligrams, sucrose 30 grams and 7 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, described root media often rises containing 6-benzyl purine (6-BA) 0.4 milligram, methyl α-naphthyl acetate (NAA) 0.75 milligram, coconut milk 150 milliliters, sodium dihydrogen phosphate 175 milligrams, banana homogenate 100 grams, 0.5 gram of active carbon, sucrose 30 grams and 7 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, .
(6) test-tube seedling transplanting
Tool 5.5 cm height whole plant to be transferred to the greenhouse of tool natural daylight hardening 15 days from blake bottle, then it is taken out from vial, clean the medium of root, metal and stone (Japanese import) is planted in immigration: bark is in mass ratio in the mixed-matrix of 2:1, keep suitably ventilating and enough humidity, the survival rate of transplanting all can reach 93%.
Embodiment 3: the tissue cultures of Supreme Being's ruby (PaphiopedilumSCBGRedJewel) that rub
(1) Material selec-tion and process
Selection plant strain growth gesture Supreme Being's class pocket orchid that rubs [rub Supreme Being's ruby (PaphiopedilumSCBGRedJewel)] that is strong, flower pattern rounding is maternal plant.Before cutting 35 days that explant (lateral bud) sterilizes, first water once with 800 times of dilute aqueous solutions of carbendazim and spray blade, within 5 days, water once with 800 times of dilute aqueous solutions of 35% G soil bacterium wetting powder and spray blade afterwards, after 5 days, water once with 4000 times of dilute aqueous solutions of 72% agricultural streptomycin soluble powder and spray blade.Then often cross 5 days, repeat above-mentioned steps, last 1 time pouring agricultural streptomycin after 5 days, the lateral bud cutting length 1.5 centimetres with the sterile scalpel of volume fraction 75% alcohol water blend is explant.
(2) explant sterilization
The explant cut first is used by superclean bench after soaking 10 seconds in volume fraction 75% alcohol water blend, 5 minutes are soaked with the hypochlorite solution of 1.0% available chlorine, aseptic water washing 4 times, then sterilize 5 minutes with the mass fraction 0.1% mercuric chloride aqueous solution, aseptic water washing 4 times.Explant after being sterilized, then blade is cut away, after remaining lateral bud is positioned over and vibrates 24 hours in shaking table in 2000 times of dilute aqueous solutions of medical streptomycin, be inoculated in inducing culture, cultivation temperature 26 DEG C, illuminance 1800lx, illumination 14 hours/day, described inducing culture is often liter and contains 6-benzyl purine (6-BA) 3 milligrams, methyl α-naphthyl acetate (NAA) 0.5 milligram, coconut milk 100 milliliters, sodium dihydrogen phosphate 200 milligrams, sucrose 30 grams and 6.5 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, its compound method is after being mixed by the composition in above-mentioned medium, sterilization obtains inducing culture (lower same).Sterilization success rate 50%.
(3) adventitious bud inducing and adventitious buds proliferation
At about 10 days, visible significantly brown secretion was formed postvaccinal explant, will proceed to new inducing culture 30 days time, then cultivated and can form indefinite bud on explant in about 30 days.Then indefinite bud is inoculated in proliferated culture medium and carries out shoot proliferation cultivation, described proliferated culture medium is often liter and contains 6-benzyl purine (6-BA) 3.0 milligrams, methyl α-naphthyl acetate (NAA) 1.0 milligrams, coconut milk 200 milliliters, sodium dihydrogen phosphate 150 milligrams, sucrose 20 grams and 6.5 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, cultivation temperature 26 DEG C, illuminance 1800lx, illumination 14 hours/day, every 60 days subcultures once, during to the 6th generation, growth coefficient can reach 2.5 times, obtains Multiple Buds.
(4) Rooting and hardening-off culture
Multiple Buds is inoculated into strong seedling culture in strong seedling culture base, cultivation temperature 26 DEG C, illuminance 1800lx, illumination 14 hours/day, culture of rootage in root media is forwarded to after the Multiple Buds of height 2 centimetres being cut into simple bud after 60 days, cultivation temperature 26 DEG C, illuminance 1800lx, illumination 14 hours/day, with the whole plant forming 5 centimetres high after 60 days, described strong seedling culture base often rises containing 6-benzyl purine (6-BA) 1 milligram, methyl α-naphthyl acetate (NAA) 0.5 milligram, coconut milk 100 milliliters, sodium dihydrogen phosphate 150 milligrams, sucrose 30 grams and 6 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, described root media often rises containing 6-benzyl purine (6-BA) 0.3 milligram, methyl α-naphthyl acetate (NAA) 0.5 milligram, coconut milk 100 milliliters, sodium dihydrogen phosphate 150 milligrams, banana homogenate 50 grams, 0.1 gram of active carbon, sucrose 30 grams and 6.5 grams, agar, surplus is 1/2MS medium, pH5.8 ~ 6.0, .
(6) test-tube seedling transplanting
Tool 5 cm height whole plant to be transferred to the greenhouse of tool natural daylight hardening 10 days from blake bottle, then it is taken out from vial, clean the medium of root, metal and stone (Japanese import) is planted in immigration: bark is in mass ratio in the mixed-matrix of 1:1, keep suitably ventilating and enough humidity, the survival rate of transplanting all can reach 90%.
Claims (3)
1. the blue high quality seedling quick breeding method for tissue culture of Supreme Being's class of rubbing pocket, is characterized in that, comprise the following steps:
A, the induction of indefinite bud and the propagation of Multiple Buds: cut the lateral bud of Supreme Being's class pocket orchid that rubs as explant, after sterilization, be inoculated in inducing culture, induce indefinite bud, then the indefinite bud induced is transferred on proliferated culture medium and carry out Multiplying culture, obtain Multiple Buds, described inducing culture and proliferated culture medium are all often liter and contain 6-benzyl purine 3.0 ~ 10.0 milligrams, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0,
B, Rooting and hardening-off culture: the Multiple Buds that propagation obtains is inoculated in strong seedling culture base and carries out strong sprout, obtain sturdy Multiple Buds, then be transferred to after being cut into simple bud in root media and carry out culture of rootage, obtain whole plant, described strong seedling culture base often rises containing 6-benzyl purine 1.0 ~ 2.0 milligrams, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0; Described root media often rises containing 6-benzyl purine 0.3 ~ 0.5 milligram, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, banana homogenate 50 ~ 150 grams, active carbon 0.1 ~ 1.0 gram, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0;
C, whole plant is transferred to natural daylight lower refining seedling, after hardening, clean its root medium and move into and plants metal and stone: cultivate acquisition in the mixed-matrix of bark 1-3:1 in mass ratio and to rub the blue seedling of Supreme Being's class pocket.
2. the blue high quality seedling quick breeding method for tissue culture of Supreme Being's class pocket that rubs according to claim 1, it is characterized in that, described step a is inoculated in inducing culture, and transfer on proliferated culture medium carry out Multiplying culture, being inoculated into by Multiple Buds in strong seedling culture base of step b carries out strong sprout and is transferred in root media after Multiple Buds is cut into simple bud carrying out culture of rootage, its cultivation temperature 24 ~ 28 DEG C, illuminance 1500 ~ 2000lx, illumination 12 ~ 16 hours/day.
3. the blue high quality seedling quick breeding method for tissue culture of Supreme Being's class pocket that rubs according to claim 1, is characterized in that, described cutting rubs the lateral bud of Supreme Being's class pocket orchid as explant, and after sterilization, its explant obtains by the following method:
Select plant strain growth gesture strong, Supreme Being's class pocket orchid that rubs of flower pattern rounding is maternal plant, before 35 days that cut explant sterilization, first water once with 500 ~ 800 times of dilute aqueous solutions of carbendazim and spray blade, within 5 days, water once with 500 ~ 800 times of dilute aqueous solutions of 35% G soil bacterium wetting powder and spray blade afterwards, after 5 days, water once with the dilute aqueous solution of 3000 ~ 4000 times of 72% agricultural streptomycin soluble powder and spray blade, then often 5 days are spent, repeat above-mentioned steps once, last 1 time pouring agricultural streptomycin after 5 days, the lateral bud cutting length 1.5 ~ 2.5 centimetres with the sterile scalpel of volume fraction 75% alcohol water blend is explant.
Its sterilization is specially: first used by the explant cut after soaking 10 ~ 30 seconds in volume fraction 75% alcohol water blend on superclean bench, 5 ~ 10 minutes are soaked with the hypochlorite solution of 1.0% available chlorine, aseptic water washing 4 ~ 5 times, again with mass fraction 0.1% mercuric chloride solution sterilization 5 ~ 10 minutes, aseptic water washing 4 ~ 5 times, then cut away blade, be positioned in 1500 ~ 2000 times of dilute aqueous solutions of medical streptomycin and vibrate 24 hours, the explant after being sterilized.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610024474.9A CN105494103B (en) | 2016-01-13 | 2016-01-13 | One kind is rubbed Supreme Being's class pocket orchid high quality seedling quick breeding method for tissue culture |
| JP2016574460A JP6483163B2 (en) | 2016-01-13 | 2016-01-26 | Tissue culture and rapid propagation of seedlings of paphiopedilum / moody type |
| PCT/CN2016/072150 WO2017120986A1 (en) | 2016-01-13 | 2016-01-26 | Rapid high-quality plantlet tissue culture and propagation method for paphiopedilum maudiae orchid |
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| CN201610024474.9A CN105494103B (en) | 2016-01-13 | 2016-01-13 | One kind is rubbed Supreme Being's class pocket orchid high quality seedling quick breeding method for tissue culture |
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| CN105494103A true CN105494103A (en) | 2016-04-20 |
| CN105494103B CN105494103B (en) | 2017-12-29 |
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| CN106688891A (en) * | 2017-01-03 | 2017-05-24 | 黑龙江省林业科学研究所 | Cypripedium macranthum Sw.f. leaf callus regeneration plant induction medium |
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| CN109258463A (en) * | 2018-09-18 | 2019-01-25 | 广西壮族自治区林业科学研究院 | A kind of asexual reproduction method of paphiopedilum armeniacum |
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| CN109937875B (en) * | 2019-03-12 | 2020-12-01 | 中国科学院华南植物园 | Rapid propagation method for paphiopedilum high-quality seedling tissue culture through leaf clumpy buds |
| CN111802201A (en) * | 2020-06-08 | 2020-10-23 | 贵州省植物园 | Pleione bud cutting propagation method |
| WO2021258738A1 (en) * | 2021-01-14 | 2021-12-30 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality hippeastrum vittatum seedlings |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6483163B2 (en) | 2019-03-13 |
| WO2017120986A1 (en) | 2017-07-20 |
| CN105494103B (en) | 2017-12-29 |
| JP2018505648A (en) | 2018-03-01 |
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