CN107581066A - A kind of little Hua all ages orchid species seedling tissue culture propagations - Google Patents
A kind of little Hua all ages orchid species seedling tissue culture propagations Download PDFInfo
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Abstract
A kind of little Hua all ages orchid species seedling tissue culture propagations, including the first step：Explant sterilizes；Second step：Protocorms Fiber differentiation；3rd step：Protocorms Multiplying culture；4th step：Protocorms differentiation culture；5th step：Rooting and hardening-off culture；6th step：Bottle outlet is transplanted.The present invention carries out asexual clonal tissue-culturing quick-propagation seedling using individual plant terminal bud, has the characteristics that reproduction speed is fast, seedling is best in quality, growth cost is low, it is only necessary to which simple tissue culturing equipment can is completed.
Description
Technical field
The invention belongs to plant protection biology and biological technical field, more particularly to a kind of little Hua all ages orchid species seedling tissues
Cultivate propagation method.
Background technology
Little Hua is all ages blue（Vanda coerulescens Griff.）It is orchid family all ages epidendrum, is distributed in India east
The western Zhenkang of the north, Burma, Thailand, and Chinese yunnan, billows deep blue, Mojiang, Simao, Jinghong, Menhai, Menla, Yuanjiang River, Yuanyang etc.
County；Height above sea level 700-1600 rice is born in, is grown nonparasitically upon another plant in sparse woods on trunk.For epiphytic orchid, stem is sturdy, long 8-30 centimetres, Ye Duomei, and two
Row, tip, which has, irregularly to be incised；Raceme 1-2, up to 36 centimetres, tool is spent more, and pattern white is with light blue, florescence 3-5
Month, Chinese Second Class Key Protected Plant, have important breeding, view and admire, scientific research, protection etc. are worth.But due to the felling that people are excessive
And its growing environment changes, all ages orchid species source is rare by the little Hua in field, thus needs to carry out it artificial propagation, a side
On the other hand the seedling of artificial propagation can be returned original producton location and put in a suitable place to breed by face in addition to the needs of meeting market and breeding, scientific research,
Its energy natural propagation is made, expands its population quantity, admirably protects the diversity of the species.
There is no little Hua all ages sapling multiplication patent and document report also in the world at present.It is therefore desirable to design a kind of little Hua
All ages orchid species seedling tissue culture propagation.
The content of the invention
It is an object of the invention to provide a kind of little Hua all ages orchid species seedling tissue culture propagations.
In order to achieve the above objects and other related objects, technical scheme provided by the invention is：A kind of little Hua all ages orchid speciess
Seedling tissue culture propagation, comprises the following steps：
The first step：Explant sterilizes；
Second step：Protocorms Fiber differentiation；
3rd step：Protocorms Multiplying culture；
4th step：Protocorms differentiation culture；
5th step：Rooting and hardening-off culture；
6th step：Bottle outlet is transplanted.
Preferably technical scheme is：Before collection explant 30 days, first little Hua is poured all ages with 500-800 times of carbendazim
Blue plant, and the blade of little Hua all ages blue plant is sprayed, it is soluble with the agricultural streptomycin that mass fraction is 72% again after one week
The aqueous solution of 3000-4000 times of volume of pulvis pours little Hua all ages blue plant, and sprays the blade of little Hua all ages blue plant, so
The terminal bud of the little Hua all ages blue plant is cut with scalpel afterwards, it is explant to obtain the terminal bud that length is 2 ~ 4 centimetres.
Preferably technical scheme is：The explant first is wiped with dry cotton, then the cotton soaked with ethanol wipes,
Then explant is cut into segment, every section includes at least one bud point, then soaked with ethanol, then with sterile water wash, connects down
To use HgCl2Immersion, then with sterile water wash, bract is peelled off after drying up moisture, then use HgCl2Immersion, obtained after drying up moisture
The tender shoots of drying.
Preferably technical scheme is：Under aseptic condition, the tender shoots after the drying is cut, obtain height for 1 ~
The stem section and terminal bud of 2 centimetres of belt segment, are then seeded on protocorms inducing culture, the stem section section position and terminal bud base portion
Section position have axillary bud generation, explant base portion otch expands to form particle projection after, be transferred to new protocorms inducing culture
Cultivated, obtain protocorms propagating materials；Condition of culture is：Temperature is 23 ~ 27 DEG C, and illuminance is 1500~2000lx,
Light irradiation time is 11 ~ 13h/ days；The pH values of the protocorms inducing culture are 5.4-5.6；Every liter of protocorms lure
Lead culture medium contain 1~2g spend precious No. 1,1~2g spend precious No. 2,20~40mg ferrous sulfate, 20~40mg ethylenediamine
Tetraacethyl disodium, 0.5~2g peptone, 50~100mL coconut milk, 80 ~ 120mg inositol, 1.5 ~ 2.5mg sweet ammonia
Acid, 0.05 ~ 0.2mg thiamine hydrochloride, 0.4 ~ 0.8mg puridoxine hydrochloride, 0.4 ~ 0.8mg nicotinic acid, 5.0 ~ 8.0mg
6- benzyl purines, 0.2 ~ 2mg methyl α-naphthyl acetate, 15~30g sucrose, 6~7g agar.
Preferably technical scheme is：The protocorms propagating materials is forwarded into proliferated culture medium to be cultivated, obtained
The protocorms propagating materials of propagation；Condition of culture is：Temperature is 23 ~ 27 DEG C, and illuminance is 1500~2000lx, light irradiation time
For 11 ~ 13h/ days；The pH values of the proliferated culture medium are 5.4-5.6；Every liter of proliferated culture medium contain 1~2g spend treasured 1
Number, 1~2g spend precious No. 2,20~40mg ferrous sulfate, 20~40mg disodium ethylene diamine tetraacetate, 0.5~2g albumen
Peptone, 40~80g mashed potatoes, 80 ~ 120mg inositol, 1.5 ~ 2.5mg glycine, 0.05 ~ 0.2mg thiamine hydrochloride,
0.4 ~ 0.8mg puridoxine hydrochloride, 0.4 ~ 0.8mg nicotinic acid, 2.0 ~ 5.0mg 6- benzyl purines, 0.2 ~ 0.5mg naphthalene second
Acid, 15~30g sucrose, 6~7g agar.
Preferably technical scheme is：The protocorms propagating materials of the propagation is forwarded into differential medium to be trained
Support, be differentiated to form the adventitious bud that height is 3 ~ 5 centimetres；Condition of culture is：25 ~ 29 DEG C, 2000~3000lx of illuminance of temperature, light
According to when a length of 11 ~ 13 hour/day；Every liter of differential medium contain 1~2g spend precious No. 1,1~2g spend it is precious No. 2,20~
40mg ferrous sulfate, 20~40mg disodium ethylene diamine tetraacetate, 0.5~2g peptone, 40~80g mashed potatoes, 50
~100mg activated carbon, 80 ~ 120mg inositol, 1.5 ~ 2.5mg glycine, 0.05 ~ 0.2mg thiamine hydrochloride, 0.4 ~
0.8mg puridoxine hydrochloride, 0.4 ~ 0.8mg nicotinic acid, 3.0 ~ 6.0mg 6- benzyl purines, 0.2 ~ 0.5mg methyl α-naphthyl acetate, 15
~30g sucrose, 6~7g agar；The pH value of the differential medium is 5.4-5.6.
Preferably technical scheme is：The adventitious bud is transferred into Rooting and hardening-off culture base to be cultivated, it is 5 to obtain height
~ 7 centimetres, the test tube seedling that radical is 3 ~ 5；Condition of culture is：25 ~ 29 DEG C, 2000~3000lx of illuminance of temperature, light irradiation time
For 11 ~ 13 hours/day；Every liter of Rooting and hardening-off culture base contain 1~2g spend precious No. 1,1~2g spend it is precious No. 2,20~
40mg ferrous sulfate, 20~40mg disodium ethylene diamine tetraacetate, 0.5~2g peptone, 40~80g mashed potatoes, 500
~1000mg activated carbon, 80 ~ 120mg inositol, 1.5 ~ 2.5mg glycine, 5 ~ 10mg thiamine hydrochloride, 0.5 ~
1.0mg puridoxine hydrochloride, 5 ~ 10mg nicotinic acid, 0.2 ~ 0.5mg methyl α-naphthyl acetate, 15~30g sucrose, 6~7g agar；Institute
The pH value for stating Rooting and hardening-off culture base is 5.4-5.6.
Preferably technical scheme is：Hardening first is carried out to the test tube seedling, then cleans the culture medium of test tube seedling attachment, then
Soaked with potassium permanganate solution, then cultivate test tube seedling in planting matrix, the planting matrix is by the broad-leaved that ferments
Formed after woods bark and wood chip mixing.
Preferably technical scheme is：Volume fraction is used to be carried out disinfection for 75% ethanol before the scalpel use.
Preferably technical scheme is：First the explant is wiped with dry cotton, then with the ethanol that volume fraction is 75%
The cotton of immersion is wiped, and explant then is cut into segment, and every section include at least one bud point, then with volume fraction is 75%
Ethanol soaks 20 ~ 40 seconds, then with sterile water wash at least three times, next with the HgCl that mass fraction is 0.1%2Immersion 4 ~ 6
Minute, then with sterile water wash at least three times, bract is peelled off after drying up moisture, then with the HgCl that mass fraction is 0.1%2Leaching
Bubble 1 ~ 2 minute, dry up the tender shoots dried up after moisture.
Because above-mentioned technical proposal is used, the present invention this have the advantage that compared with prior art：
The little Hua all ages blue high quality seedling tissue culture propagations of the present invention, using the terminal bud of fine individual plant as explant, utilize
Numerous propagation method can greatly improve its breeding rate to excised cotyledon asexual clonal soon, breed that specification is standardized, shape
The high quality seedling of stable and consistent, meet new excellent little Hua all ages blue growth requirements.
Embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this implementation
Content disclosed by example understands other advantages and effect of the present invention easily.
Embodiment 1：A kind of little Hua all ages orchid species seedling tissue culture propagations
（1）Material selects and pretreatment
Spring and summer season selects the little Hua that plant strain growth is healthy and strong, growth potential is strong all ages blue healthy in little Hua all ages blue Germplasm Resources
Strain, it is explant to choose its terminal bud.Before collection terminal bud explant 30 days, first with the carbendazim of 500 times of volumes（It will buy
Powder of carbendazim be diluted with water 500 times）Pour and once and spray blade, with mass fraction be again 72% agricultural chain after a week
The aqueous solution of 3000 times of volumes of mycin soluble powder pours once and sprays blade.Then repeated the above steps in two weeks.
The preceding terminal bud that 2-4 centimetres of length is cut with the sterilized scalpel of 75% alcohol of inoculation is explant.
（2）Explant sterilizes
Explant sterilization is carried out on superclean bench, sterilization method is as follows：Tender shoots, dried cotton wipe 1 time, volume fraction be
The cotton of 75% ethanol immersion wipes 1 time, removes unnecessary blade and be cut into segment, and every section includes bud point, a volume integral
Number fully soaks 0.1% HgCl that 30s, sterile water wash 3 times, mass fraction be for 75% ethanol2Fully immersion 6min, nothing
Bacterium water rinse 3 times, drying excessive moisture after peel off bract, 0.1% HgCl that mass fraction is2Fully immersion 2min, drying are more
It is inoculated with after remaining moisture.Sterilize success rate 70%.
（3）Protocorms Fiber differentiation
Tender shoots after sterilization and drying is cut to the stem section and terminal bud of high 1 ~ 2 centimetre of belt segment under aseptic condition, is inoculated into class original
Bulb inducing culture SB1：Every liter, containing No. 1 1.5g of treasured is spent, is spent No. 2 1.5g of treasured, ferrous sulfate（FeSO4.7H2O）27.85mg,
Disodium ethylene diamine tetraacetate（EDTA-2Na）37.25mg, peptone 2.0g, coconut milk 100mL, inositol 100mg, glycine
2.0mg, thiamine hydrochloride（VB1）0.2mg, puridoxine hydrochloride（VB6）0.5mg, nicotinic acid 0.5mg, 6- benzyl purine（6-BA）5.0
Milligram, methyl α-naphthyl acetate（NAA）0.2mg, sucrose 20g, agar 6g, pH 5.4；After 45 days, stem section section position and terminal bud base portion section position exist
There is axillary bud generation on culture medium, explant base portion otch expands to form particle projection；Material is forwarded into new protocorms to lure
Lead on culture medium SB1, then a large amount of protocorms can be formed by culture in 45 days.Induce the cultivation temperature in protocorms stage
For (25 ± 2) DEG C, illuminance 1500~2000 lx, illumination 12h/d.
（4）Protocorms Multiplying culture
It is VB2 that the protocorms propagating materials of acquisition is forwarded into proliferated culture medium：Every liter, containing No. 1 2.0g of treasured is spent, is spent precious No. 2
2.0g, ferrous sulfate（FeSO4.7H2O）27.85mg disodium ethylene diamine tetraacetate（EDTA-2Na）37.25mg peptone
1.0g, mashed potatoes 60 g, inositol 100mg, glycine 2.0mg, thiamine hydrochloride（VB1）0.2mg, puridoxine hydrochloride（VB6）
3.0 milligrams of 0.5mg, nicotinic acid 0.5 mg, 6- benzyl purine, methyl α-naphthyl acetate（NAA）0.5mg, sucrose 20g, agar 6.0g, pH 5.6,
After 45 d are cultivated on the culture medium, protocorms proliferation times can reach more than 8 times；By 3 generation protocorms Multiplying cultures
Enough propagating materials can be obtained and carry out protocorms differentiation culture.The cultivation temperature of protocorms multiplicative stage is (25 ± 2)
DEG C, illuminance 1500~2000 lx, illumination 12h/d.
（5）Protocorms differentiation culture
It is VB3 that the protocorms propagating materials of acquisition is forwarded into differential medium:Every liter, containing No. 1 1.5g of treasured is spent, is spent precious No. 2
1.5g, ferrous sulfate（FeSO4.7H2O）27.85mg disodium ethylene diamine tetraacetate（EDTA-2Na）37.25mg peptone
2.0g, the g of mashed potatoes 40, activated carbon 100 mg, inositol 100mg, glycine 2.0mg, thiamine hydrochloride（VB1）0.2mg, salt
Sour pyridoxol（VB6）3.5 milligrams of 0.5mg, nicotinic acid 0.5mg, 6- benzyl purine, methyl α-naphthyl acetate（NAA）0.5mg, sucrose 20g, agar
6.0g, pH 5.6.After 45 days, protocorms are differentiated to form high 3 ~ 5 centimetres of adventitious buds on culture medium.Protocorms differential period
Cultivation temperature be (27 ± 2) DEG C, illuminance 2000~3000 lx, illumination 12h/d.
（6）Rooting and hardening-off culture
By high 3 ~ 5 centimetres of adventitious bud, Rooting and hardening-off culture base VR4 is gone to:Every liter, containing No. 1 1.5g of treasured is spent, is spent No. 2 1.5g of treasured,
Ferrous sulfate（FeSO4.7H2O）27.85mg disodium ethylene diamine tetraacetate（EDTA-2Na）37.25mg, peptone 2.0g, potato
Mud 80 g, activated carbon 500mg, inositol 100mg, glycine 2.0mg, thiamine hydrochloride（VB1）10mg, puridoxine hydrochloride
（VB6）1.0mg, nicotinic acid 10mg, methyl α-naphthyl acetate（NAA）0.5mg, sucrose 20g, agar 6g, pH5.6, when cultivating 60 days, height of seedling is reachable
To 5 ~ 7 centimetres, radical 3 ~ 5, rooting rate is more than 100%.The cultivation temperature in strong plantlets and rootage stage is (27 ± 2) DEG C, illuminance
2000~3000 lx, illumination 12h/d.
（7）Bottle outlet is transplanted
By the culture of rootage test tube seedling of 60 days or so after intense light irradiation lower refining seedling 7 days bottle outlet.During transplanting, life is taken out from blake bottle
Offspring, after cleaning the culture medium adhered to, 5 min are soaked with millesimal liquor potassic permanganate, planting matrix is used and sent out
The mixed-matrix of the good broad-leaf forest bark of ferment and wood chip, pay attention to keeping suitable humidity and temperature, be placed at shady and cool ventilation and cultivate, into
Motility rate is up to more than 95%.Plant after 30 d or so are survived can produce new root system, now may move into greenhouse carry out normal water,
Fertilizer, pencil reason.
Embodiment 2:A kind of little Hua all ages orchid species seedling tissue culture propagations
（1）Material selection and processing
Autumn and winter selects the little Hua that plant strain growth is healthy and strong, growth potential is strong all ages blue healthy in little Hua all ages blue Germplasm Resources
Strain, it is explant to choose its terminal bud.Before collection terminal bud explant 30 days, first poured once and sprayed with 800 times of carbendazim
Blade is applied, is poured once with 4000 times of 72% agricultural streptomycin soluble powder again after a week and sprays blade.Then in two weeks
Repeat the above steps.The preceding terminal bud that 2-4 centimetres of length is cut with the sterilized scalpel of 75% alcohol of inoculation is explant.
（2）Explant sterilizes
Explant sterilization is carried out on superclean bench, sterilization method is as follows：Tender shoots, dried cotton wipe 1 time, the leaching of 75% ethanol
The cotton of bubble wipes 1 time, removes unnecessary blade and be cut into segment, and every section is fully soaked comprising a bud point, 75% ethanol
30s, sterile water wash 3 times, 0.1% HgCl2 peel off bud after fully soaking 4min, aseptic water washing 3 times, drying excessive moisture
Leaf, 0.1% HgCl2It is inoculated with after abundant 1min, drying excessive moisture.Sterilize success rate 80%.
（3）Protocorms Fiber differentiation
Tender shoots after sterilization and drying is cut to the stem section and terminal bud of high 1 ~ 2 centimetre of belt segment under aseptic condition, is inoculated into class original
Bulb inducing culture SB1：Every liter, containing No. 1 1.5g of treasured is spent, is spent No. 2 1.5g of treasured, ferrous sulfate（FeSO4.7H2O）27.85mg,
Disodium ethylene diamine tetraacetate（EDTA-2Na）37.25mg, peptone 2.0g, coconut milk 100mL, inositol 100mg, glycine
2.0mg, thiamine hydrochloride（VB1）0.2mg, puridoxine hydrochloride（VB6）5.0 milligrams of 0.5mg, nicotinic acid 0.5mg, 6- benzyl purine,
Methyl α-naphthyl acetate（NAA）0.2mg, sucrose 20g, agar 6g, pH 5.4；After 45 days, stem section section position and terminal bud base portion section position are in culture medium
On have axillary bud generation, explant base portion otch expands to form particle projection；Material is forwarded on same culture medium, then by 45
Its culture can form a large amount of protocorms.The cultivation temperature in induction protocorms stage is (25 ± 2) DEG C, illuminance 1500~
2000 lx, illumination 12h/d.
（4）Protocorms Multiplying culture
It is VB2 that the protocorms propagating materials of acquisition is forwarded into proliferated culture medium：Every liter, containing No. 1 2.0g of treasured is spent, is spent precious No. 2
2.0g, ferrous sulfate（FeSO4.7H2O）27.85mg disodium ethylene diamine tetraacetate（EDTA-2Na）37.25mg peptone
1.0g, mashed potatoes 60 g, inositol 100mg, glycine 2.0mg, thiamine hydrochloride（VB1）0.2mg, puridoxine hydrochloride（VB6）
3.0 milligrams of 0.5mg, nicotinic acid 0.5 mg, 6- benzyl purine, methyl α-naphthyl acetate（NAA）0.5mg, sucrose 20g, agar 6.0g, pH 5.6,
After 45 d are cultivated on the culture medium, protocorms proliferation times can reach more than 8 times；By 3 generation protocorms Multiplying cultures
Enough propagating materials can be obtained and carry out protocorms differentiation culture.The cultivation temperature of protocorms multiplicative stage is (25 ± 2)
DEG C, illuminance 1500~2000 lx, illumination 12h/d.
（5）Protocorms differentiation culture
It is VB3 that the protocorms propagating materials of acquisition is forwarded into differential medium:Every liter, containing No. 1 1.5g of treasured is spent, is spent precious No. 2
1.5g, ferrous sulfate（FeSO4.7H2O）27.85mg disodium ethylene diamine tetraacetate（EDTA-2Na）37.25mg peptone
2.0g, the g of mashed potatoes 40, activated carbon 100 mg, inositol 100mg, glycine 2.0mg, thiamine hydrochloride（VB1）0.2mg, salt
Sour pyridoxol（VB6）3.5 milligrams of 0.5mg, nicotinic acid 0.5mg, 6- benzyl purine, methyl α-naphthyl acetate（NAA）0.5mg, sucrose 20g, agar
6.0g, pH 5.6.After 45 days, protocorms are differentiated to form high 3 ~ 5 centimetres of adventitious buds on culture medium.Protocorms differential period
Cultivation temperature be (27 ± 2) DEG C, illuminance 2000~3000 lx, illumination 12h/d.
（6）Rooting and hardening-off culture
By high 3 ~ 5 centimetres of adventitious bud, Rooting and hardening-off culture base VR4 is gone to:Every liter, containing No. 1 1.5g of treasured is spent, is spent No. 2 1.5g of treasured,
Ferrous sulfate（FeSO4.7H2O）27.85mg disodium ethylene diamine tetraacetate（EDTA-2Na）37.25mg, peptone 2.0g, potato
Mud 80 g, activated carbon 500mg, inositol 100mg, glycine 2.0mg, thiamine hydrochloride（VB1）10mg, puridoxine hydrochloride
（VB6）1.0mg, nicotinic acid 10mg, methyl α-naphthyl acetate（NAA）0.5mg, sucrose 20g, agar 6g, pH5.6, when cultivating 60 days, height of seedling is reachable
To 5 ~ 7 centimetres, radical 3 ~ 5, rooting rate is more than 100%.The cultivation temperature in strong plantlets and rootage stage is (27 ± 2) DEG C, illuminance
2000~3000lx, illumination 12h/d.
（7）Bottle outlet is transplanted
By the culture of rootage test tube seedling of 60 days or so after intense light irradiation lower refining seedling 7 days bottle outlet.During transplanting, life is taken out from blake bottle
Offspring, after cleaning the culture medium adhered to, 5 min are soaked with millesimal liquor potassic permanganate, planting matrix is used and sent out
The mixed-matrix of the good broad-leaf forest bark of ferment and wood chip, pay attention to keeping suitable humidity and temperature, be placed at shady and cool ventilation and cultivate, into
Motility rate is up to more than 98%.Plant after 30 d or so are survived can produce new root system, now may move into greenhouse carry out normal water,
Fertilizer, pencil reason.
Embodiment 3:A kind of little Hua all ages orchid species seedling tissue culture propagations
A kind of little Hua all ages orchid species seedling tissue culture propagations, comprise the following steps：
The first step：Explant sterilizes；
Second step：Protocorms Fiber differentiation；
3rd step：Protocorms Multiplying culture；
4th step：Protocorms differentiation culture；
5th step：Rooting and hardening-off culture；
6th step：Bottle outlet is transplanted.
Preferred embodiment is：Before collection explant 30 days, first little Hua is poured all ages with the carbendazim of 600 times of volumes
Blue plant, and the blade of little Hua all ages blue plant is sprayed, it is soluble with the agricultural streptomycin that mass fraction is 72% again after one week
The aqueous solution of 3800 times of volumes of pulvis pours little Hua all ages blue plant, and sprays the blade of little Hua all ages blue plant, Ran Houyong
Scalpel is cut to the terminal bud of the little Hua all ages blue plant, and it is explant to obtain the terminal bud that length is 2 ~ 4 centimetres.
Preferred embodiment is：The explant first is wiped with dry cotton, then the cotton soaked with ethanol wipes,
Then explant is cut into segment, every section includes at least one bud point, then soaked with ethanol, then with sterile water wash, connects down
To use HgCl2Immersion, then with sterile water wash, bract is peelled off after drying up moisture, then use HgCl2Immersion, obtained after drying up moisture
The tender shoots of drying.
Preferred embodiment is：Under aseptic condition, the tender shoots after the drying is cut, obtain height for 1 ~
The stem section and terminal bud of 2 centimetres of belt segment, are then seeded on protocorms inducing culture, the stem section section position and terminal bud base portion
Section position have axillary bud generation, explant base portion otch expands to form particle projection after, be transferred to new protocorms inducing culture
Cultivated, obtain protocorms propagating materials；Condition of culture is：Temperature is 23 DEG C, illuminance 1500lx, and light irradiation time is
13h/ days；The pH values of the protocorms inducing culture are 5.4；Every liter of protocorms inducing culture contains 1g's
Spend precious No. 1,2g spend precious No. 2,40mg ferrous sulfate, 20mg disodium ethylene diamine tetraacetate, 2g peptone, 50mL coconut palm
Sub- juice, 100mg inositol, 2.5mg glycine, 0.2mg thiamine hydrochloride, 0.6mg puridoxine hydrochloride, 0.6mg cigarette
Acid, 5.7mg 6- benzyl purines, 0.8mg methyl α-naphthyl acetate, 23g sucrose, 7g agar.
Preferred embodiment is：The protocorms propagating materials is forwarded into proliferated culture medium to be cultivated, obtained
The protocorms propagating materials of propagation；Condition of culture is：Temperature is 27 DEG C, illuminance 2000lx, and light irradiation time is 11h/ days；
The pH values of the proliferated culture medium are 5.6；Every liter of proliferated culture medium contain 2g spend precious No. 1,1g spend treasured No. 2,35mg
Ferrous sulfate, 25mg disodium ethylene diamine tetraacetate, 1.5g peptone, 40g mashed potatoes, 100mg inositol, 2mg
Glycine, 0.05mg thiamine hydrochloride, 0.8mg puridoxine hydrochloride, 0.4mg nicotinic acid, 3mg 6- benzyl purines,
0.25mg methyl α-naphthyl acetate, 15g sucrose, 6g agar.
Preferred embodiment is：The protocorms propagating materials of the propagation is forwarded into differential medium to be trained
Support, be differentiated to form the adventitious bud that height is 4 centimetres；Condition of culture is：26 DEG C, illuminance 2500lx of temperature, light irradiation time 12
Hour/day；Every liter of differential medium contain 1.2g spend precious No. 1,1.3g spend precious No. 2,40mg ferrous sulfate, 30mg
Disodium ethylene diamine tetraacetate, 2g peptone, 55g mashed potatoes, 100mg activated carbon, 120mg inositol, 1.5mg it is sweet
Propylhomoserin, 0.15mg thiamine hydrochloride, 0.8mg puridoxine hydrochloride, 0.48mg nicotinic acid, 4mg 6- benzyl purines, 0.25mg
Methyl α-naphthyl acetate, 28g sucrose, 6g agar；The pH value of the differential medium is 5.6.
Preferred embodiment is：The adventitious bud is transferred into Rooting and hardening-off culture base to be cultivated, it is 7 to obtain height
Centimetre, radical be the test tube seedling of 5；Condition of culture is：27 DEG C, illuminance 3000lx of temperature, light irradiation time are 12 hours/day；
Every liter of Rooting and hardening-off culture base contain 2g spend precious No. 1,2g spend precious No. 2,40mg ferrous sulfate, 40mg ethylenediamine
Tetraacethyl disodium, 0.5g peptone, 40g mashed potatoes, 1000mg activated carbon, 120mg inositol, 1.5mg glycine,
10mg thiamine hydrochloride, 1.0mg puridoxine hydrochloride, 7mg nicotinic acid, 0.3mg methyl α-naphthyl acetate, 15g sucrose, 6g fine jade
Fat；The pH value of the Rooting and hardening-off culture base is 5.4.
Preferred embodiment is：Hardening first is carried out to the test tube seedling, then cleans the culture medium of test tube seedling attachment, then
Soaked with potassium permanganate solution, then cultivate test tube seedling in planting matrix, the planting matrix is by the broad-leaved that ferments
Formed after woods bark and wood chip mixing.
Embodiment 4:A kind of little Hua all ages orchid species seedling tissue culture propagations
A kind of little Hua all ages orchid species seedling tissue culture propagations, comprise the following steps：
The first step：Explant sterilizes；
Second step：Protocorms Fiber differentiation；
3rd step：Protocorms Multiplying culture；
4th step：Protocorms differentiation culture；
5th step：Rooting and hardening-off culture；
6th step：Bottle outlet is transplanted.
Preferred embodiment is：Before collection explant 30 days, first little Hua is poured all ages with the carbendazim of 650 times of volumes
Blue plant, and the blade of little Hua all ages blue plant is sprayed, it is soluble with the agricultural streptomycin that mass fraction is 72% again after one week
The aqueous solution of 3500 times of volumes of pulvis pours little Hua all ages blue plant, and sprays the blade of little Hua all ages blue plant, Ran Houyong
Scalpel is cut to the terminal bud of the little Hua all ages blue plant, and it is explant to obtain the terminal bud that length is 3 centimetres；Scalpel
Volume fraction is used to be carried out disinfection for 75% ethanol before use.
Preferred embodiment is：First the explant is wiped with dry cotton, then with the ethanol that volume fraction is 75%
The cotton of immersion is wiped, and explant then is cut into segment, and every section include at least one bud point, then with volume fraction is 75%
Ethanol soaks 25 seconds, then with sterile water wash three times, next with the HgCl that mass fraction is 0.1%2Immersion 5 minutes, then
With sterile water wash three times, bract is peelled off after drying up moisture, then with the HgCl that mass fraction is 0.1%2Immersion 1.5 minutes, drying
The tender shoots dried up after moisture.
Preferred embodiment is：Under aseptic condition, the tender shoots after the drying is cut, obtain height for 1 ~
The stem section and terminal bud of 2 centimetres of belt segment, are then seeded on protocorms inducing culture, the stem section section position and terminal bud base portion
Section position have axillary bud generation, explant base portion otch expands to form particle projection after, be transferred to new protocorms inducing culture
Cultivated, obtain protocorms propagating materials；Condition of culture is：Temperature is 24 DEG C, illuminance 1800lx, and light irradiation time is
11.5h/ days；The pH values of the protocorms inducing culture are 5.4；Every liter of protocorms inducing culture contains 1g
Spend precious No. 1,1g spend precious No. 2,20mg ferrous sulfate, 20mg disodium ethylene diamine tetraacetate, 0.9g peptone, 50mL
Coconut milk, 80mg inositol, 2.5mg glycine, 0.12mg thiamine hydrochloride, 0.45mg puridoxine hydrochloride,
0.78mg nicotinic acid, 7.5mg 6- benzyl purines, 2mg methyl α-naphthyl acetate, 30g sucrose, 6.6g agar.
Preferred embodiment is：The protocorms propagating materials is forwarded into proliferated culture medium to be cultivated, obtained
The protocorms propagating materials of propagation；Condition of culture is：Temperature is 26 DEG C, illuminance 1600lx, and light irradiation time is 13h/ days；
The pH values of the proliferated culture medium are 5.4；Every liter of proliferated culture medium contain 2g spend precious No. 1,2g spend treasured No. 2,40mg
Ferrous sulfate, 40mg disodium ethylene diamine tetraacetate, 2g peptone, 60g mashed potatoes, 100mg inositol, 2mg it is sweet
Propylhomoserin, 0.15 ~ mg thiamine hydrochloride, 0.8mg puridoxine hydrochloride, 0.8mg nicotinic acid, 5.0mg 6- benzyl purines,
0.2mg methyl α-naphthyl acetate, 15g sucrose, 7g agar.
Preferred embodiment is：The protocorms propagating materials of the propagation is forwarded into differential medium to be trained
Support, be differentiated to form the adventitious bud that height is 3 ~ 5 centimetres；Condition of culture is：29 DEG C, illuminance 3000lx of temperature, light irradiation time are
11 hours/day；Every liter of differential medium contain 1g spend precious No. 1,2g spend precious No. 2,40mg ferrous sulfate, 30mg
Disodium ethylene diamine tetraacetate, 0.9g peptone, 60g mashed potatoes, 75mg activated carbon, 100mg inositol, 2.5mg it is sweet
Propylhomoserin, 0.2mg thiamine hydrochloride, 0.8mg puridoxine hydrochloride, 0.8mg nicotinic acid, 6.0mg 6- benzyl purines, 0.5mg
Methyl α-naphthyl acetate, 30g sucrose, 7g agar；The pH value of the differential medium is 5.6.
Preferred embodiment is：The adventitious bud is transferred into Rooting and hardening-off culture base to be cultivated, it is 6 to obtain height
Centimetre, radical be the test tube seedling of 4；Condition of culture is：25 DEG C, illuminance 2000lx of temperature, light irradiation time are 13 hours/day；
Every liter of Rooting and hardening-off culture base contain 2g spend precious No. 1,1g spend precious No. 2,20mg ferrous sulfate, 20mg ethylenediamine
Tetraacethyl disodium, 0.5g peptone, 78g mashed potatoes, 890mg activated carbon, 110mg inositol, 2.5mg glycine,
10mg thiamine hydrochloride, 1.0mg puridoxine hydrochloride, 5mg nicotinic acid, 0.2mg methyl α-naphthyl acetate, 15g sucrose, 7g fine jade
Fat；The pH value of the Rooting and hardening-off culture base is 5.6.
Preferred embodiment is：Hardening first is carried out to the test tube seedling, then cleans the culture medium of test tube seedling attachment, then
Soaked with potassium permanganate solution, then cultivate test tube seedling in planting matrix, the planting matrix is by the broad-leaved that ferments
Formed after woods bark and wood chip mixing.
As described above is only to explain the preferred embodiments of the invention, and it is any formal to be done to the present invention to be not intended to tool
Limitation, therefore all have any modification or change for making the relevant present invention under identical spirit, should all be included in
The invention is intended to the category protected.
Claims (10)
- A kind of 1. little Hua all ages orchid species seedling tissue culture propagations, it is characterised in that：Comprise the following steps：The first step：Explant sterilizes；Second step：Protocorms Fiber differentiation；3rd step：Protocorms Multiplying culture；4th step：Protocorms differentiation culture；5th step：Rooting and hardening-off culture；6th step：Bottle outlet is transplanted.
- 2. little Hua according to claim 1 all ages orchid species seedling tissue culture propagations, it is characterised in that：In collection explant Before body 30 days, little Hua all ages blue plant first are poured with the carbendazim of 500-800 times of volume, and spray the leaf of little Hua all ages blue plant Piece, poured again with the aqueous solution of 3000-4000 times of volume of the agricultural streptomycin soluble powder that mass fraction is 72% after one week Little Hua all ages blue plant are filled, and spray the blade of little Hua all ages blue plant, then with scalpel to the little Hua all ages blue plant Terminal bud cut, obtain length be 2 ~ 4 centimetres terminal bud be explant.
- 3. little Hua according to claim 2 all ages orchid species seedling tissue culture propagations, it is characterised in that：First with drying Cotton wipes the explant, then the cotton soaked with ethanol is wiped, and explant then is cut into segment, and every section includes at least one Individual bud point, then soaked with ethanol, then with sterile water wash, next use HgCl2Immersion, then with sterile water wash, drying Bract is peelled off after moisture, then uses HgCl2Immersion, dry up the tender shoots dried up after moisture.
- 4. little Hua according to claim 3 all ages orchid species seedling tissue culture propagations, it is characterised in that：In aseptic condition Under, the tender shoots after the drying is cut, stem section and terminal bud of the height for 1 ~ 2 centimetre of belt segment is obtained, is then seeded into On protocorms inducing culture, there is axillary bud generation the stem section section position and terminal bud base portion section position, and explant base portion otch expands After forming particle projection, it is transferred to new protocorms inducing culture and is cultivated, obtains protocorms propagating materials；Culture Condition is：Temperature is 23 ~ 27 DEG C, and illuminance is 1500~2000lx, and light irradiation time is 11 ~ 13h/ days；The protocorms lure The pH values for leading culture medium are 5.4-5.6；It is precious No. 1,1~2g that every liter of protocorms inducing culture contains spending for 1~2g Spend precious No. 2,20~40mg ferrous sulfate, 20~40mg disodium ethylene diamine tetraacetate, 0.5~2g peptone, 50~ 100mL coconut milk, 80 ~ 120mg inositol, 1.5 ~ 2.5mg glycine, 0.05 ~ 0.2mg thiamine hydrochloride, 0.4 ~ 0.8mg puridoxine hydrochloride, 0.4 ~ 0.8mg nicotinic acid, 5.0 ~ 8.0mg 6- benzyl purines, 0.2 ~ 2mg methyl α-naphthyl acetate, 15~ 30g sucrose, 6~7g agar.
- 5. little Hua according to claim 4 all ages orchid species seedling tissue culture propagations, it is characterised in that：The class is former Propagation by corm material is forwarded to proliferated culture medium and cultivated, and obtains the protocorms propagating materials of propagation；Condition of culture is：Temperature Spend for 23 ~ 27 DEG C, illuminance is 1500~2000lx, and light irradiation time is 11 ~ 13h/ days；The pH values of the proliferated culture medium are 5.4-5.6；The sulfuric acid for spending treasured 2,20~40mg for spending treasured 1,1~2g that every liter of proliferated culture medium contains 1~2g is sub- Iron, 20~40mg disodium ethylene diamine tetraacetate, 0.5~2g peptone, 40~80g mashed potatoes, 80 ~ 120mg inositol, 1.5 ~ 2.5mg glycine, 0.05 ~ 0.2mg thiamine hydrochloride, 0.4 ~ 0.8mg puridoxine hydrochloride, 0.4 ~ 0.8mg cigarette Acid, 2.0 ~ 5.0mg 6- benzyl purines, 0.2 ~ 0.5mg methyl α-naphthyl acetate, 15~30g sucrose, 6~7g agar.
- 6. little Hua according to claim 5 all ages orchid species seedling tissue culture propagations, it is characterised in that：By the propagation Protocorms propagating materials be forwarded to differential medium and cultivated, be differentiated to form the adventitious bud that height is 3 ~ 5 centimetres；Culture Condition is：25 ~ 29 DEG C, 2000~3000lx of illuminance of temperature, light irradiation time are 11 ~ 13 hours/day；Every liter of differentiation culture Base contain 1~2g spend precious No. 1,1~2g spend precious No. 2,20~40mg ferrous sulfate, 20~40mg ethylenediamine tetra-acetic acid Disodium, 0.5~2g peptone, 40~80g mashed potatoes, 50~100mg activated carbon, 80 ~ 120mg inositol, 1.5 ~ 2.5mg glycine, 0.05 ~ 0.2mg thiamine hydrochloride, 0.4 ~ 0.8mg puridoxine hydrochloride, 0.4 ~ 0.8mg nicotinic acid, 3.0 ~ 6.0mg 6- benzyl purines, 0.2 ~ 0.5mg methyl α-naphthyl acetate, 15~30g sucrose, 6~7g agar；The differentiation training The pH value for supporting base is 5.4-5.6.
- 7. little Hua according to claim 6 all ages orchid species seedling tissue culture propagations, it is characterised in that：Will be described indefinite Bud is transferred to Rooting and hardening-off culture base and cultivated, and acquisition height is 5 ~ 7 centimetres, the test tube seedling that radical is 3 ~ 5；Condition of culture For：25 ~ 29 DEG C, 2000~3000lx of illuminance of temperature, light irradiation time are 11 ~ 13 hours/day；Every liter of Rooting and hardening-off culture Base contain 1~2g spend precious No. 1,1~2g spend precious No. 2,20~40mg ferrous sulfate, 20~40mg ethylenediamine tetra-acetic acid Disodium, 0.5~2g peptone, 40~80g mashed potatoes, 500~1000mg activated carbon, 80 ~ 120mg inositol, 1.5 ~ 2.5mg glycine, 5 ~ 10mg thiamine hydrochloride, 0.5 ~ 1.0mg puridoxine hydrochloride, 5 ~ 10mg nicotinic acid, 0.2 ~ 0.5mg Methyl α-naphthyl acetate, 15~30g sucrose, 6~7g agar；The pH value of the Rooting and hardening-off culture base is 5.4-5.6.
- 8. little Hua according to claim 7 all ages orchid species seedling tissue culture propagations, it is characterised in that：First to the examination Pipe seedling carries out hardening, then cleans the culture medium of test tube seedling attachment, then is soaked with potassium permanganate solution, then plants test tube seedling Train in planting matrix, the planting matrix is formed after being mixed by the broad-leaf forest bark and wood chip fermented.
- 9. little Hua according to claim 2 all ages orchid species seedling tissue culture propagations, it is characterised in that：The scalpel Volume fraction is used to be carried out disinfection for 75% ethanol before use.
- 10. little Hua according to claim 2 all ages orchid species seedling tissue culture propagations, it is characterised in that：First use drying Cotton wipe the explant, then the cotton soaked with the ethanol that volume fraction is 75% wipes, and is then cut into explant small Section, every section include at least one bud point, then with the ethanol immersion that volume fraction is 75% 20 ~ 40 seconds, then with sterile water wash extremely Less three times, next with the HgCl that mass fraction is 0.1%2Immersion 4 ~ 6 minutes, then with sterile water wash at least three times, drying Peel off bract after moisture, then with the HgCl that mass fraction is 0.1%2Immersion 1 ~ 2 minute, dry up the tender shoots dried up after moisture.
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| CN109220811A (en) * | 2018-11-22 | 2019-01-18 | 福建省农业科学院农业生物资源研究所 | A method of the Protocorm based on Chinese pholidota pseudobulb or herb seed |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106472306A (en) * | 2016-09-30 | 2017-03-08 | 南京仙草堂生物科技有限公司 | One kind begins to flourish Herba Dendrobii high quality seedling asexual clonal method for quickly breeding |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106472306A (en) * | 2016-09-30 | 2017-03-08 | 南京仙草堂生物科技有限公司 | One kind begins to flourish Herba Dendrobii high quality seedling asexual clonal method for quickly breeding |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109220811A (en) * | 2018-11-22 | 2019-01-18 | 福建省农业科学院农业生物资源研究所 | A method of the Protocorm based on Chinese pholidota pseudobulb or herb seed |
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