CN106888970B - A kind of tissue culture and rapid propagation method of the point leaf basin away from orchid - Google Patents
A kind of tissue culture and rapid propagation method of the point leaf basin away from orchid Download PDFInfo
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- CN106888970B CN106888970B CN201710120431.5A CN201710120431A CN106888970B CN 106888970 B CN106888970 B CN 106888970B CN 201710120431 A CN201710120431 A CN 201710120431A CN 106888970 B CN106888970 B CN 106888970B
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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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Abstract
The invention discloses a kind of sharp leaf basins away from blue tissue culture and rapid propagation method；Belong to field of plant tissue culture；Be intended to by induction, proliferation, differentiation, acclimatization and transplants and etc. healthy and strong sharp leaf basin has successfully been obtained away from blue test tube seedling, establish sharp leaf basin away from blue quick breeding by group culture technical system；Induction step, amplification step and the differentiation incubation step for the protocorms that the tissue culture and rapid propagation method carries out successively；The induction step of the protocorms is that the bud point of explant is inoculated into inducing culture to cultivate, induced synthesis protocorms；Wherein, the inducing culture includes：The pH of 1/2MS, 0.1~1.0mg/L zeatin, 0.1~0.5mg/L indolebutyric acids, 15~30g/L sucrose, 3.5~6.0g/L agar, 50~100ml/L coconut milk, the inducing culture is 5.4~5.8；The present invention is used for sharp leaf basin away from blue tissue cultures.
Description
Technical field
The present invention relates to field of plant tissue culture more particularly to a kind of sharp leaf basin away from blue tissue culture and rapid propagation method.
Background technology
Sharp leaf basin is orchid family Gastrochilus away from blue (Gastrochilus acutifolius (Lindley) Kuntze.)
A kind of aerial orchid is distributed mainly on the states such as Nepal, Sillim, Bhutan, Northeastern India, Burma, Laos, Vietnam, Thailand, China
Only south of Yunnan is distributed, and is mainly grown on the mountainous region border trunk of 800~1400 meters of height above sea level.
Sharp leaf basin has irregular tasselled, carries away from blue umbel, sepal and petal yellow band puce spot, edge
Fragrance has higher horticultural importance.In addition, in Simao, Yunnan regional, sharp leaf basin is used for scrofula, menstruation not away from orchid
The treatment of the diseases such as tune, traumatic injury, nephritic dropsy, hiccup, stomachache.Sharp leaf basin, to belong to rare kind away from potted orchid, is distributed away from orchid
It is narrow, due to excessive picking and the destruction on habitat ground, it is at Critical Condition, therefore to its artificial propagation and carry out open country
Outer recurrence is very urgent！
Currently, at home and abroad there is no the reports that sharp leaf basin is studied away from blue tissue culture technique, there is not sharp leaf basin away from blue group yet
Knit the application of culture technique patent.
Invention content
Deficiency in view of the above technology, the present invention provides a kind of sharp leaf basins away from blue tissue culture and rapid propagation method, it is therefore intended that
By induction, proliferation, differentiation, acclimatization and transplants and etc. healthy and strong sharp leaf basin has successfully been obtained away from blue test tube seedling, establish sharp leaf basin
Away from blue quick breeding by group culture technical system.
In order to realize above-mentioned technique effect, the technical scheme is that such：A kind of point leaf basin is fast away from blue tissue culture
Breeding method, the method include the induction step, amplification step and differentiation incubation step of the protocorms carried out successively；It is described
The induction steps of protocorms be the bud point of explant is inoculated into inducing culture to cultivate, induced synthesis class protocorm
Stem；Wherein, the inducing culture includes：1/2MS, 0.1~1.0mg/L zeatin, 0.1~0.5mg/L indolebutyric acids, 15
The pH of~30g/L sucrose, 3.5~6.0g/L agar, 50~100ml/L coconut milk, the inducing culture is 5.4~5.8.
It should be noted that the amplification step be by the protocorms of induced synthesis be inoculated into proliferated culture medium into
Row culture；
Wherein, the proliferated culture medium includes：MS, 0.1~1.0mg/L 6- benzyls aminoadenine, 0.1~0.5mg/L
Methyl α-naphthyl acetate, 15~30g/L sucrose, 3.5~6.0g/L agar, 0.1~0.3g/L activated carbons, the pH of the proliferated culture medium are
5.4~5.8.
It should be noted that the differentiation incubation step is that the protocorms of proliferation gained are inoculated into differential medium
In cultivated, differentiate test tube seedling；
Wherein, the differential medium includes：1/2MS, 1.0~1.5mg/L methyl α-naphthyl acetates, 15~30g/L sucrose, 3.5
The pH of~6.0g/L agar, 0.05~0.1g/L activated carbons, the differential medium is 5.4~5.8.
It should be noted that further including hardening and transplant step after the differentiation step, i.e., by the examination of differentiation gained
Pipe seedling carries out hardening and transplants into mixed-matrix, you can obtains sharp leaf basin away from orchid species seedling；
Wherein, the mixed-matrix is：Volume ratio is 1:1 bark and Lan Shi.
It should be noted that the Fiber differentiation temperature is 25~28 DEG C, the Fiber differentiation time is 90~120 days.
It should be noted that the condition of culture of the amplification step is：Incubation time is 20~30 days, and cultivation temperature is
25~28 DEG C, intensity of illumination is 1500~2000lx, and light application time is 12~14 hours/day.
It should be noted that the condition of culture of the differentiation step is：Cultivation temperature is 25~28 DEG C, and intensity of illumination is
1500~2000lx, light application time are 12~14 hours/day, and incubation time is 40~60 days.
It should be noted that the hardening time of the hardening and transplant step is 1~3 day.
It should be noted that further including explant acquisition step before the induction step of the protocorms.
It should be noted that the induction step of the protocorms is：Explant is sterilized through clear water flushing, mercuric chloride,
It is inoculated into that cultivated in inducing culture 90~120 days can induced synthesis protocorms after rinsed with sterile water.
Beneficial effects of the present invention are：
1, the present invention by induction, proliferation, differentiation, acclimatization and transplants and etc. healthy and strong sharp leaf basin have successfully been obtained tried away from orchid
Guan Miao establishes sharp leaf basin away from blue quick breeding by group culture technical system；
2, sharp survival rate of the leaf basin away from orchid species seedling that method through the invention obtains is up to 90% or more.
Specific implementation mode
With reference to embodiment, claims of the present invention is described in further detail, but do not constituted
Any limitation of the invention, it is any to still fall within institute of the present invention making the obtainable technical solution of limited number of time modification to the present invention
Scope of protection.
Embodiment 1
(1) explant acquires：Autumn in 2013 is big to greenhouse away from blue culture transferring by the wild sharp leaf basin for being collected in Simao, Yunnan
In canopy, 2014 spring carefully cut the newborn vegetative bud that leaf sheath is not switched on when sprouting sprouts, with scalpel, with the white yarn of moistening
Laboratory is taken back rapidly after cloth package water conservation processing and is tested in time；
(2) induction of protocorms：The leaf of outside is peelled off after the explant of step (1) is rinsed 30 minutes under tap water
Sheath to only be left last two layers of leaf sheath；It is placed in superclean bench and is sterilized 20 minutes in 0.1% mercuric chloride solution, sterile water
Peel a layer from leaf sheath after rinsing 2 times；It is placed in 0.1% mercuric chloride solution and sterilizes 5 minutes again, with being peelled off after rinsed with sterile water 3 times
Last layer of leaf sheath；And it is immediately placed in 0.1% mercuric chloride solution and sterilizes 1 minute, through using aseptic filter paper after rinsed with sterile water 5 times
Explant is slit longitudinally into the bud point of 0.3~0.5cm or so with sterile scalpel and is inoculated by the moisture for blotting explant surface
Light culture 90 days in inducing culture, cultivation temperature are 25 DEG C, you can induced synthesis protocorms, inductivity are dirty up to 75.3%
Dye rate is down to 10% or less；
The inducing culture is：1/2MS+1.0mg/L zeatin+0.5mg/L indolebutyric acid+15g/L sucrose+
3.5g/L agar+50ml/L coconut milk, pH 5.4；
(3) proliferation of protocorms：Protocorms obtained by step (2) are smash with aseptic nipper and dissipate and be inoculated into proliferation training
It supports and is cultivated 20 days in base, cultivation temperature is 25 DEG C, intensity of illumination 1500lx, and light application time is 12 hours/day, you can is obtained big
The protocorms of amount, growth coefficient reach 5.1；
The proliferated culture medium is：MS+1.0mg/L 6- benzyl aminoadenine+0.5mg/L methyl α-naphthyl acetate+15g/L sucrose+
3.5g/L agar+0.1g/L activated carbons, pH 5.4；
(4) differentiation culture：Class protocorm obtained by step (3) is inoculated into differential medium and is cultivated 40 days, cultivation temperature
It it is 25 DEG C, intensity of illumination 1500lx, light application time is 12 hours/day, can differentiate test tube seedling, differentiation rate reaches
87.4%；
The differential medium is：1/2MS+1.5mg/L methyl α-naphthyl acetate+15g/L sucrose+3.5g/L agar+0.05g/L lives
Property charcoal, pH 5.4；
(5) hardening and transplanting：Natural light by the test tube bottle seedling of stalwartness obtained by step (4), high about 6~9cm in greenhouse
Lower opening tissue culture bottle lid hardening 1 day is cleaned natural light in greenhouse after the culture medium of root and is placed to root system and whiten, in volume
Than being 1:Seedling is cultivated in 1 bark and Lan Shi mixed-matrixes up to sharp leaf basin away from orchid species seedling, survival rate reaches after transplanting 30 days
90%.
Embodiment 2
(1) explant acquires：2015 spring are big to greenhouse away from blue culture transferring by the wild sharp leaf basin for picking up from In Xishuangbanna of Yunnan
In canopy, when sprouting sprouts, the newborn vegetative bud that leaf sheath is not switched on carefully is cut with scalpel, is protected with the white gauze wrapped of moistening
Laboratory is taken back after water process rapidly and is tested in time；
(2) induction of protocorms：The leaf sheath of outside is peelled off after explant is rinsed 50 minutes under tap water to only surplus
Last two layers of leaf sheath down；It is placed in superclean bench and is sterilized 26 minutes in 0.1% mercuric chloride solution, aseptic water washing 3 times
After peel a layer from leaf sheath；It is placed in 0.1% mercuric chloride solution and sterilizes 8 minutes again, with peelling off last layer after rinsed with sterile water 4 times
Leaf sheath；And it is immediately placed in 0.1% mercuric chloride solution and sterilizes 2 minutes, with aseptic filter paper blot explant after rinsed with sterile water 6 times
Explant is slit longitudinally into the bud point of 0.3~0.5cm or so with sterile scalpel and is inoculated into Fiber differentiation by the moisture in body surface face
Light culture 105 days in base, cultivation temperature are 27 DEG C, you can induced synthesis protocorms, up to 82.9%, pollution rate is less than inductivity
6.5%；
The inducing culture is：1/2MS+0.5mg/L zeatin+0.3mg/L indolebutyric acid+22g/L sucrose+
4.0g/L agar+80ml/L coconut milk, pH 5.6；
(3) proliferation of protocorms：Protocorms obtained by step (2) are smash with aseptic nipper and dissipate and be inoculated into proliferation training
It supports and is cultivated 25 days in base, cultivation temperature is 27 DEG C, intensity of illumination 1700lx, and light application time is 13 hours/day, you can is obtained big
The protocorms of amount, growth coefficient 6.0；
The proliferated culture medium is：MS+0.5mg/L 6- benzyl aminoadenine+0.3mg/L methyl α-naphthyl acetate+24g/L sucrose+
4.5g/L agar+0.2g/L activated carbons, pH 5.6；
(4) differentiation culture：Class protocorm obtained by step (3) is inoculated into differential medium and is cultivated 55 days, cultivation temperature
It it is 27 DEG C, intensity of illumination 1700lx, light application time is 13 hours/day, can differentiate test tube seedling, differentiation rate reaches
75.7%；
The differential medium is：1/2MS+1.3mg/L methyl α-naphthyl acetate+21g/L sucrose+5.0g/L agar+0.07g/L lives
Property charcoal, pH 5.6；
(5) hardening and transplanting：Natural light by the test tube bottle seedling of stalwartness obtained by step (4), high about 6~9cm in greenhouse
Lower opening tissue culture bottle lid hardening 2 days is cleaned natural light in greenhouse after the culture medium of root and is placed to root system and whiten, in volume
Than being 1:Seedling is cultivated in 1 bark and Lan Shi mixed-matrixes up to sharp leaf basin away from orchid species seedling, survival rate reaches after transplanting 30 days
93.8%.
Embodiment 3
(1) explant acquires：2016 spring will pick up from the wild sharp leaf basin in Yunnan Pu'er away from blue culture transferring to greenhouse,
When sprouting sprouts, the newborn vegetative bud that leaf sheath is not switched on carefully is cut with scalpel, at the white gauze wrapped water conservation of moistening
Laboratory is taken back after reason rapidly and is tested in time；
(2) induction of protocorms：Explant is rinsed under tap water and peels off the leaf sheath of outside after sixty minutes to only surplus
Last two layers of leaf sheath down；It is placed in superclean bench and is sterilized 30 minutes in 0.1% mercuric chloride solution, aseptic water washing 3 times
After peel a layer from leaf sheath；It is placed in 0.1% mercuric chloride solution and sterilizes 10 minutes again, with peelling off last after rinsed with sterile water 5 times
Layer leaf sheath；And be immediately placed in 0.1% mercuric chloride solution and sterilize 3 minutes, blotted with aseptic filter paper after rinsed with sterile water 7 times it is outer
Explant is slit longitudinally into the bud point of 0.5cm or so with sterile scalpel and is inoculated into inducing culture by the moisture on implant surface
Middle light culture 120 days, cultivation temperature are 28 DEG C, you can induced synthesis protocorms, inductivity reach 69.5%, and pollution rate is less than
1% or less；
The inducing culture is：1/2MS+0.1mg/L zeatin+0.1mg/L indolebutyric acid+30g/L sucrose+
6.0g/L agar+100ml/L coconut milk, pH 5.8；
(3) proliferation of protocorms：Protocorms obtained by step (2) are smash with aseptic nipper and dissipate and be inoculated into proliferation training
It supports and is cultivated 30 days in base, cultivation temperature is 28 DEG C, intensity of illumination 2000lx, and light application time is 14 hours/day, you can is obtained big
The protocorms of amount, growth coefficient 3.8；
The proliferated culture medium is：MS+0.1mg/L 6- benzyl aminoadenine+0.1mg/L methyl α-naphthyl acetate+30g/L sucrose+
6.0g/L agar+0.3g/L activated carbons, pH 5.8；
(4) differentiation culture：Class protocorm obtained by step (3) is inoculated into differential medium and is cultivated 60 days, cultivation temperature
It it is 28 DEG C, intensity of illumination 2000lx, light application time is 14 hours/day, can differentiate test tube seedling, differentiation rate is
68.2%；
The differential medium is：1/2MS+1.0mg/L methyl α-naphthyl acetate+30g/L sucrose+6.0g/L agar+0.1g/L lives
Property charcoal, pH 5.8；
(5) hardening and transplanting：Natural light by the test tube bottle seedling of stalwartness obtained by step (4), high about 6~9cm in greenhouse
Lower opening tissue culture bottle lid hardening 3 days is cleaned natural light in greenhouse after the culture medium of root and is placed to root system and whiten, in volume
Than being 1:Seedling is cultivated in 1 bark and Lan Shi mixed-matrixes up to sharp leaf basin away from orchid species seedling, survival rate is up to 95% after transplanting 30 days
More than.
It is above-described be only presently preferred embodiments of the present invention, it is all within the scope of the spirit and principles in the present invention made by appoint
What modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of point leaf basin, away from blue tissue culture and rapid propagation method, the method includes the induction step of the protocorms carried out successively
Suddenly, amplification step and differentiation incubation step；It is characterized in that, the induction step of the protocorms is by the bud point of explant
It is inoculated into inducing culture and is cultivated, induced synthesis protocorms；
Wherein, the inducing culture includes：1/2MS, 0.1~1.0mg/L zeatin, 0.1~0.5mg/L indolebutyric acids,
15~30g/L sucrose, 3.5~6.0g/L agar, 50~100ml/L coconut milk, the pH of the inducing culture is 5.4~
5.8；
Wherein, the proliferated culture medium includes：MS, 0.1~1.0mg/L 6- benzyls aminoadenine, 0.1~0.5mg/L naphthalene second
The pH of acid, 15~30g/L sucrose, 3.5~6.0g/L agar, 0.1~0.3g/L activated carbons, the proliferated culture medium is 5.4
~5.8；
Wherein, the differential medium includes：1/2MS, 1.0~1.5mg/L methyl α-naphthyl acetates, 15~30g/L sucrose, 3.5~
The pH of 6.0g/L agar, 0.05~0.1g/L activated carbons, the differential medium is 5.4~5.8.
2. point leaf basin according to claim 1 is away from blue tissue culture and rapid propagation method, which is characterized in that the amplification step is
The protocorms of induced synthesis are inoculated into proliferated culture medium and are cultivated.
3. point leaf basin according to claim 1 is away from blue tissue culture and rapid propagation method, which is characterized in that the differentiation culture step
It is rapid to be cultivated for the protocorms of proliferation gained are inoculated into differential medium, differentiate test tube seedling.
4. point leaf basin according to claim 3 is away from blue tissue culture and rapid propagation method, which is characterized in that the differentiation step it
After further include hardening and transplant step, i.e., the test tube seedling of differentiation gained is carried out hardening and transplanted into mixed-matrix, you can
Sharp leaf basin is obtained away from orchid species seedling；
Wherein, the mixed-matrix is：Volume ratio is 1:1 bark and Lan Shi.
5. point leaf basin according to claim 1 is away from blue tissue culture and rapid propagation method, which is characterized in that the Fiber differentiation temperature
Degree is 25~28 DEG C, and the Fiber differentiation time is 90~120 days.
6. point leaf basin according to claim 2 is away from blue tissue culture and rapid propagation method, which is characterized in that the amplification step
Condition of culture is：Incubation time is 20~30 days, and cultivation temperature is 25~28 DEG C, and intensity of illumination is 1500~2000lx, illumination
Time is 12~14 hours/day.
7. point leaf basin according to claim 3 is away from blue tissue culture and rapid propagation method, which is characterized in that the differentiation step
Condition of culture is：Cultivation temperature is 25~28 DEG C, and intensity of illumination is 1500~2000lx, and light application time is 12~14 hours/day,
Incubation time is 40~60 days.
8. point leaf basin according to claim 4 is away from blue tissue culture and rapid propagation method, which is characterized in that the hardening and transplanting
The hardening time of step is 1~3 day.
9. point leaf basin according to claim 1 is away from blue tissue culture and rapid propagation method, which is characterized in that the protocorms
Further include explant acquisition step before induction step.
10. point leaf basin according to claim 1 is away from blue tissue culture and rapid propagation method, which is characterized in that the protocorms
Induction step be：Explant is rinsed through clear water, mercuric chloride sterilizes, is inoculated into inducing culture after rinsed with sterile water and cultivates 90
It~120 days can induced synthesis protocorms.
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