CN106106163A - A kind of iris cultivates propagation method - Google Patents
A kind of iris cultivates propagation method Download PDFInfo
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- CN106106163A CN106106163A CN201610545717.3A CN201610545717A CN106106163A CN 106106163 A CN106106163 A CN 106106163A CN 201610545717 A CN201610545717 A CN 201610545717A CN 106106163 A CN106106163 A CN 106106163A
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- culture
- iris
- seedling
- outer implant
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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
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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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
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- Cultivation Of Plants (AREA)
Abstract
The present invention relates to plant culturing reproduction technique field, it is specifically related to a kind of iris and cultivates propagation method, comprise the following steps: material selection, materials disinfection, inducing culture, proliferation and subculture is cultivated, strong seedling culture and transplanting, iris can be carried out fast culture breeding by the present invention, and the iris Seedling strain sickness rate after Pei Yanging is low, planting percent is high, Seedling strain quality better, strong adaptability after cultivation, by animal nutrition at short notice, meet the demand commercially produced, for enterprise scale, industrialization is cultivated iris and is provided technical support, meet the needs in iris cut-flower market simultaneously.
Description
Technical field
The present invention relates to plant culturing reproduction technique field, be specifically related to a kind of iris and cultivate propagation method.
Background technology
Iris is the famous flower of current international flower bed, belongs to the orchid family Phalaenopsis plant, and original producton location is mainly subtropical zone, heat
Band, natural distributed in Oceania, Asia.Iris has viewing period length, color and luster is abundant, pattern is beautiful and fine figure is slim and graceful
Etc. feature, also it is known as " cattleya queen ", is that one is the most universal in orchid, most widely cultivates kind, have
The highest commercial value, is international four one viewing and admiring greatly hot bandwidth.Iris is single stem aerial orchid, and collateral development is very
Few, therefore it is difficult to routine and carries out division propagation, and seldom form seed, seed development is the most relatively difficult, substantially at nature
Under the conditions of sprout difficulty bigger, the Main Means that therefore iris is efficiently bred is exactly tissue culture.More external prosperities
Country uses industrial breeding technique and tissue culture rapid propagating technology, is possible not only to create huge economic benefit, the most also
Cultivate a lot of improved seeds, but China studied about the fast breeding technique of iris tissue culture and has been still within the primary stage,
Hinder the large-scale production of iris to a great extent.The most increasing iris is applied among cut-flower, and this is just
The method being badly in need of wanting the breeding of a kind of fast culture, thus promote iris commerciality to develop.
Summary of the invention
It is an object of the invention to provide a kind of cultivation and breed iris cultivation propagation method fast, that planting percent is high.
In order to achieve the above object, the technical solution used in the present invention is: a kind of iris cultivates propagation method, including with
Lower step:
(1) material selection: choose the iris grown fine without pest and disease damage, select profile in plant be Radix Crotalariae szemoensis shape, petal and
Bifurcated begin to expose bennet bract, high 50～70cm bennet, cut and save the bennet bud on position in the middle part of bennet as outer implant；
(2) materials disinfection: outer implant is placed in liquid detergent water immersion 10min and carries out pretreatment, then use tap water
Rinse 30min, blot the globule in outer implant with aseptic filter paper after flushing, then wipe with the cotton ball soaked in alcohol that volume fraction is 75%
Wipe outer planting surface, clean 2～3 times with aquesterilisa, then soak outer implant with the mercuric chloride solution that mass fraction is 0.2%,
Soak time is 10min, with aseptic water washing 3～4 times after immersion, peels off bract, molten with the mercuric chloride that mass fraction is 0.1%
Immersion steeps outer implant, and soak time is 8min, use aseptic water washing 3～4 times after immersion；
(3) inducing culture: the two ends otch of outer implant after excision sterilization, is seeded in outer implant in inducing culture and carries out
Preculture, incubation time is 22～25d, and first 10 days is light culture, and remaining time is that light is cultivated, and cultivation temperature is 23～25
DEG C, when light is cultivated, light application time is 12h/d, and intensity of illumination is 2000～2500lx；Described inducing culture is: 1/2MS+1～
3mg/L 6-BA+0.5～1.5mg/L TDZ+0.1～0.3mg/L NAA+0.2～0.3mg/L VC+0.05～0.15mg/L
GA3+22～27g/L white sugar+5～9g/L carrageenan；After preculture, then culture materials is moved entirely into new inducing culture
Middle continuation is cultivated, and incubation time is 15～20d, cultivation temperature 23～25 DEG C, and light application time is 12h/d, and intensity of illumination is 2000
～2500lx；
(4) proliferation and subculture is cultivated: the outer implant after inducing culture is put into and continues in proliferated culture medium to cultivate, described propagation
Culture medium is: 1/2MS+2.5～3.5mg/L 6-BA+0.2～0.4mg/L NAA+0.1～0.3mg/L TDZ+22～27g/L
White sugar+5～9g/L carrageenan+8～12% coconut juice, condition of culture: cultivation temperature 23～25 DEG C, light application time is 12h/d, light
It is 2000～2500lx according to intensity；
(5) strong seedling culture: after proliferation and subculture is cultivated, select have the seedling of obvious projection to proceed in high more than 2cm, outer implant
Cultivating in strong seedling culture base, described strong seedling culture base is: 1/2MS+2.5～3.5mg/L6-BA+0.4～0.6mg/L NAA+22
～27g/L white sugar+5～9g/L carrageenan+8～12% bananas juice, incubation time is 15～20d, cultivation temperature 23～25 DEG C,
Light application time is 12h/d, and intensity of illumination is 2000～2500lx；After incubation time terminates, then add in strong seedling culture base
0.3% activated carbon, cultivation temperature 27～29 DEG C, light application time is 15h/d, and intensity of illumination is 2500～3000lx；
(6) transplant: when test tube Seedling grows to 4～5cm high, root 2～4, leaf 1～when 2, test tube Seedling is placed on nature light
Lower refining seedling 5～7 days, open bottle cap seedling exercising 1～2 days；Then take out cultivation Seedling, clean the culture medium being attached to root with clear water,
It is careful not to during cleaning damage root, then transplants to sphagna and mixed-matrix that fertile soil mass ratio is 1:2, keep humidity
And ventilation, air humidity is 75～85%, and temperature is 20～25 DEG C.
Iris as above a kind of cultivates propagation method, further illustrate into, described inducing culture is: 1/2MS+
2mg/L 6-BA+1mg/L TDZ+0.2mg/L NAA+0.25mg/L VC+0.1mg/L GA3+25g/L white sugar+7g/L OK a karaoke club
Glue.
Iris as above a kind of cultivates propagation method, further illustrate into, described proliferated culture medium is: 1/2MS+
3mg/L 6-BA+0.3mg/L NAA+0.2mg/L TDZ+25g/L white sugar+7g/L carrageenan+10% coconut juice.
Iris as above a kind of cultivates propagation method, further illustrate into, described strong seedling culture base is: 1/2MS+
3mg/L 6-BA+0.5mg/L NAA+25g/L white sugar+7g/L carrageenan+10% bananas juice.
The invention has the beneficial effects as follows: after iris being carried out fast culture breeding, and cultivation by the present invention
Iris Seedling strain sickness rate is low, and planting percent is high, and Seedling strain quality better, strong adaptability after cultivation, by animal nutrition in short-term
In, meet the demand commercially produced, cultivate iris provide technical support, simultaneously for enterprise scale, industrialization
Meet the needs in iris cut-flower market.
Detailed description of the invention
Below the specific embodiment of the invention is further elaborated.
A kind of iris that the present invention provides cultivates propagation method, comprises the following steps:
(1) material selection: choose the iris grown fine without pest and disease damage, select profile in plant be Radix Crotalariae szemoensis shape, petal and
Bifurcated begin to expose bennet bract, high 50～70cm bennet, cut and save the bennet bud on position in the middle part of bennet as outer implant；
Petal and bifurcated are begun to expose bennet bract by us, the high bennet 50～70cm elects sample 1 as, by petal and point
Fork do not expose completely bennet bract, high 50～70cm bennet as sample 2, petal and bifurcated are completely exposed bennet bud
Sheet, the high bennet 50～70cm do outer implant as sample 3, separately sampled the bennet bud going up different parts, are placed on general
Inducing culture test is carried out on logical inducing culture；
Table 1 is that the induction of bennet bud is affected by different sample
As can be seen from Table 1, there is notable difference in the bennet bud induction rate on different samples, the bennet blastogenesis of sample 1 is produced
Power is strong, and its Senescence rate is the lowest, and survival rate and inductivity the highest, be best suitable for doing outer implant induced material；Sample 2 Induction Transformation
Difficulty, Senescence rate is the highest, and survival rate and inductivity the lowest, be not suitable for doing outer implant induced material；Along with bennet in sample 3
Lateral bud and the growth of flower, the bennet on bifurcated and the flower on bennet top, on bennet is gradually transferred in the nutrition in culture medium
Blastogenesis growing way is weak, and Senescence rate is high, and survival rate and inductivity low, be also not suitable for doing induced material；
From table 1 it can also be seen that in same sample, on bennet, the bennet bud inducement of Different node also differs, and base portion becomes
The bennet bud of ripe joint position is the most thin and weak, and growing power is weak, and Senescence rate is higher, and inductivity is relatively low；The bennet of the tender joint position of children, top
Although the bennet bud survival rate that bud compares base portion is high, but the sprouting of children's tender pedicel pumping is the most elongated, and sprout is thin and weak, and afterbody is often sent out
Raw withered；The bennet bud of joint position, middle part, growing power is strong, and Senescence rate is relatively low, and inductivity is high, is best induced material；Therefore select
In plant profile be Radix Crotalariae szemoensis shape, petal and bifurcated begin to expose bennet bract, high 50～70cm bennet, cut joint position in the middle part of bennet
On bennet bud as outer implant, productivity ratio can be effectively improved.
(2) materials disinfection: outer implant is placed in liquid detergent water immersion 10min and carries out pretreatment, then use tap water
Rinse 30min, blot the globule in outer implant with aseptic filter paper after flushing, then wipe with the cotton ball soaked in alcohol that volume fraction is 75%
Wipe outer planting surface, clean 2～3 times with aquesterilisa, then soak outer implant with the mercuric chloride solution that mass fraction is 0.2%,
Soak time is 10min, with aseptic water washing 3～4 times after immersion, peels off bract, molten with the mercuric chloride that mass fraction is 0.1%
Immersion steeps outer implant, and soak time is 8min, use aseptic water washing 3～4 times after immersion；Mercuric chloride solution is used to disappear at twice
Poison, and the mercuric chloride solution concentration of twice sterilization is different, while so preferably can playing sterilization functions and to it
Activity influence is less；
Table 2 is mercuric chloride solution and the number of operations induction impact on bennet bud of different quality mark
Using mercuric chloride solution to sterilize at twice as can be seen from Table 2, its pollution rate is low, and the surviving of bennet bud after sterilization
Rate is high, and the first mercuric chloride solution using mass fraction to be 0.2% soaks outer implant, can effectively kill major part antibacterial, outside making
Implant is effectively protected, and after aseptic water washing, peels off bract, outside soaking with the mercuric chloride solution that mass fraction is 0.1%
Implant, while protecting outer implant activity, sterilizes further.
(3) inducing culture: the two ends otch of outer implant after excision sterilization, is seeded in outer implant in inducing culture and carries out
Preculture, incubation time is 22～25d, and first 10 days is light culture, and remaining time is that light is cultivated, by the dark training of a period of time
Support, can effective Browning control phenomenon to a certain extent, cultivation temperature is 23～25 DEG C, and when light is cultivated, light application time is 12h/
D, intensity of illumination is 2000～2500lx；Described inducing culture is: 1/2MS+1～3mg/L 6-BA+0.5～1.5mg/L
TDZ+0.1～0.3mg/L NAA+0.2～0.3mg/L VC+0.05～0.15mg/L GA3+22～27g/L white sugar+5～
9g/L carrageenan, by adding VC and GA3, can effectively contain brownization of culture materials；After preculture then culture materials is overall
Moving into and continue in new inducing culture to cultivate, incubation time is 15～20d, cultivation temperature 23～25 DEG C, and light application time is
12h/d, intensity of illumination is 2000～2500lx；
Table 3 is inducing culture based component and consumption
As preferably, described inducing culture is: 1/2MS+2mg/L 6-BA+1mg/L TDZ+0.2mg/L NAA+
0.25mg/L VC+0.1mg/L GA3+25g/L white sugar+7g/L carrageenan, is composition and the use of inducing culture 1 in table 3
Amount；
Table 4 is the test relation between Brown degree and dark treatment, and outer implant is seeded in inducing culture in table 3
Cultivate on base 1
As shown in Table 4, As time goes on, all can there is certain browning through different disposal, but through dark
After cultivation, melting brown rate can reduce, it follows that light culture can the generation of Browning control phenomenon to a certain extent.
(4) proliferation and subculture is cultivated: the outer implant after inducing culture is put into and continues in proliferated culture medium to cultivate, described propagation
Culture medium is: 1/2MS+2.5～3.5mg/L 6-BA+0.2～0.4mg/L NAA+0.1～0.3mg/L TDZ+22～27g/L
White sugar+5～9g/L carrageenan+8～12% coconut juice, condition of culture: cultivation temperature 23～25 DEG C, light application time is 12h/d, light
It is 2000～2500lx according to intensity；
Table 5 is enrichment culture based component and consumption
As preferably, described proliferated culture medium is: 1/2MS+3mg/L 6-BA+0.3mg/L NAA+0.2mg/L TDZ+
25g/L white sugar+7g/L carrageenan+10% coconut juice, is composition and the consumption of proliferated culture medium 1 in table 5, uses 1/2MS training
Support base, suitably reduce a great number of elements concentration, be more beneficial for improving the proliferative induction rate of iris bennet bud.
(5) strong seedling culture: after proliferation and subculture is cultivated, select have the seedling of obvious projection to proceed in high more than 2cm, outer implant
Cultivating in strong seedling culture base, described strong seedling culture base is: 1/2MS+2.5～3.5mg/L6-BA+0.4～0.6mg/L NAA+22
～27g/L white sugar+5～9g/L carrageenan+8～12% bananas juice, incubation time is 15～20d, cultivation temperature 23～25 DEG C,
Light application time is 12h/d, and intensity of illumination is 2000～2500lx；After incubation time terminates, then add in strong seedling culture base
0.3% activated carbon, cultivation temperature 27～29 DEG C, light application time is 15h/d, and intensity of illumination is 2500～3000lx；
Table 6 is strong seedling culture based component and consumption
As preferably, described strong seedling culture base is: 1/2MS+3mg/L 6-BA+0.5mg/L NAA+25g/L white sugar+
7g/L carrageenan+10% bananas juice, is composition and the consumption of strong seedling culture base 1 in table 6.
(6) transplant: when test tube Seedling grows to 4～5cm high, root 2～4, leaf 1～when 2, test tube Seedling is placed on nature light
Lower refining seedling 5～7 days, open bottle cap seedling exercising 1～2 days；Then take out cultivation Seedling, clean the culture medium being attached to root with clear water,
It is careful not to during cleaning damage root, then transplants to sphagna and mixed-matrix that fertile soil mass ratio is 1:2, keep humidity
And ventilation, air humidity is 75～85%, and temperature is 20～25 DEG C.Iris can be carried out fast culture by the present invention numerous
Growing, and the iris Seedling strain sickness rate after cultivation is low, planting percent is high, and Seedling strain quality better, strong adaptability after cultivation, by biology
Technological means at short notice, meets the demand commercially produced, and cultivates iris provide for enterprise scale, industrialization
Technical support, meets the needs in iris cut-flower market simultaneously.
The present invention is not limited to examples detailed above, in claims of the present invention limited range, and art technology
Various deformation or amendment that personnel can make without creative work are all protected by this patent.
Claims (4)
1. an iris cultivates propagation method, it is characterised in that comprise the following steps:
(1) material selection: choose the iris grown fine without pest and disease damage, selecting profile in plant is Radix Crotalariae szemoensis shape, petal and bifurcated
Begin to expose bennet bract, high 50～70cm bennet, cut and save the bennet bud on position in the middle part of bennet as outer implant；
(2) materials disinfection: outer implant is placed in liquid detergent water immersion 10min and carries out pretreatment, then rinse with tap water
30min, blots the globule in outer implant, then with outside the cotton ball soaked in alcohol wiping that volume fraction is 75% with aseptic filter paper after flushing
Implant surface, cleans 2～3 times with aquesterilisa, then soaks outer implant with the mercuric chloride solution that mass fraction is 0.2%, soak
Time is 10min, with aseptic water washing 3～4 times after immersion, peels off bract, with the mercuric chloride solution leaching that mass fraction is 0.1%
Steeping outer implant, soak time is 8min, with aseptic water washing 3～4 times after immersion；
(3) inducing culture: the two ends otch of outer implant after excision sterilization, is seeded in outer implant in inducing culture and carries out pre-training
Supporting, incubation time is 22～25d, and first 10 days is light culture, and remaining time is that light is cultivated, and cultivation temperature is 23～25 DEG C, light
During cultivation, light application time is 12h/d, and intensity of illumination is 2000～2500lx；Described inducing culture is: 1/2MS+1～3mg/L
6-BA+0.5～1.5mg/L TDZ+0.1～0.3mg/L NAA+0.2～0.3mg/L VC+0.05～0.15mg/L GA3+22
～27g/L white sugar+5～9g/L carrageenan；After preculture, then culture materials is moved entirely into continuation in new inducing culture
Cultivate, incubation time is 15～20d, cultivation temperature 23～25 DEG C, and light application time is 12h/d, intensity of illumination be 2000～
2500lx；
(4) proliferation and subculture is cultivated: the outer implant after inducing culture is put into and continues in proliferated culture medium to cultivate, described enrichment culture
Base is: 1/2MS+2.5～3.5mg/L 6-BA+0.2～0.4mg/L NAA+0.1～0.3mg/L TDZ+22～27g/L white sand
Sugar+5～9g/L carrageenan+8～12% coconut juice, condition of culture: cultivation temperature 23～25 DEG C, light application time is 12h/d, and illumination is strong
Degree is 2000～2500lx；
(5) strong seedling culture: after proliferation and subculture is cultivated, select have the seedling of obvious projection to proceed to strong sprout in high more than 2cm, outer implant
In culture medium cultivate, described strong seedling culture base is: 1/2MS+2.5～3.5mg/L6-BA+0.4～0.6mg/L NAA+22～
27g/L white sugar+5～9g/L carrageenan+8～12% bananas juice, incubation time is 15～20d, cultivation temperature 23～25 DEG C, light
Being 12h/d according to the time, intensity of illumination is 2000～2500lx；After incubation time terminates, then in strong seedling culture base, add 0.3%
Activated carbon, cultivation temperature 27～29 DEG C, light application time is 15h/d, and intensity of illumination is 2500～3000lx；
(6) transplant: when test tube Seedling grows to 4～5cm high, root 2～4, leaf 1～when 2, test tube Seedling is placed under nature light refining
Seedling 5～7 days, open bottle cap seedling exercising 1～2 days；Then take out cultivation Seedling, clean the culture medium being attached to root with clear water, clean
Time be careful not to damage root, then transplant to sphagna and mixed-matrix that fertile soil mass ratio is 1:2, keep humidity and logical
Wind, air humidity is 75～85%, and temperature is 20～25 DEG C.
A kind of iris the most according to claim 1 cultivates propagation method, it is characterised in that: described inducing culture is:
1/2MS+2mg/L 6-BA+1mg/L TDZ+0.2mg/L NAA+0.25mg/L VC+0.1mg/L GA3+25g/L white sugar+
7g/L carrageenan.
A kind of iris the most according to claim 1 cultivates propagation method, it is characterised in that: described proliferated culture medium is:
1/2MS+3mg/L 6-BA+0.3mg/L NAA+0.2mg/L TDZ+25g/L white sugar+7g/L carrageenan+10% coconut juice.
A kind of iris the most according to claim 1 cultivates propagation method, it is characterised in that: described strong seedling culture base is:
1/2MS+3mg/L 6-BA+0.5mg/L NAA+25g/L white sugar+7g/L carrageenan+10% bananas juice.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107593457A (en) * | 2017-11-10 | 2018-01-19 | 佛山市恒爱网络科技有限公司 | A kind of iris subculture medium |
| CN107637524A (en) * | 2017-11-10 | 2018-01-30 | 佛山市恒爱网络科技有限公司 | A kind of iris root media |
| CN107691221A (en) * | 2017-11-10 | 2018-02-16 | 佛山市恒爱网络科技有限公司 | A kind of iris tissue culture method |
| CN107810851A (en) * | 2017-11-10 | 2018-03-20 | 佛山市恒爱网络科技有限公司 | A kind of iris Initial culture base |
| CN108243944A (en) * | 2018-01-24 | 2018-07-06 | 青州市亚泰农业科技有限公司 | A kind of iris rapid breeding method and fine quality tissue culture propagation |
| CN108496753A (en) * | 2018-03-07 | 2018-09-07 | 山东省烟台市农业科学研究院 | A method of delaying iris cultivation matrix acidification corruption |
| CN110050700A (en) * | 2019-05-24 | 2019-07-26 | 韶关学院 | A kind of iris detoxic seedling culture propagation method |
| CN110896856A (en) * | 2019-11-07 | 2020-03-24 | 郑州师范学院 | Method for inducing complete inflorescence in vitro by axillary bud of inflorescence shaft of phalaenopsis |
| CN113575421A (en) * | 2021-08-27 | 2021-11-02 | 中德润萌生态科技(青岛)有限公司 | Aseptic processing method for tissue culture material of orchidaceae plant |
| CN113973713A (en) * | 2021-10-28 | 2022-01-28 | 北京市昌平职业学校 | Butterfly orchid tissue culture method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107593457A (en) * | 2017-11-10 | 2018-01-19 | 佛山市恒爱网络科技有限公司 | A kind of iris subculture medium |
| CN107637524A (en) * | 2017-11-10 | 2018-01-30 | 佛山市恒爱网络科技有限公司 | A kind of iris root media |
| CN107691221A (en) * | 2017-11-10 | 2018-02-16 | 佛山市恒爱网络科技有限公司 | A kind of iris tissue culture method |
| CN107810851A (en) * | 2017-11-10 | 2018-03-20 | 佛山市恒爱网络科技有限公司 | A kind of iris Initial culture base |
| CN108243944A (en) * | 2018-01-24 | 2018-07-06 | 青州市亚泰农业科技有限公司 | A kind of iris rapid breeding method and fine quality tissue culture propagation |
| CN108496753A (en) * | 2018-03-07 | 2018-09-07 | 山东省烟台市农业科学研究院 | A method of delaying iris cultivation matrix acidification corruption |
| CN110050700A (en) * | 2019-05-24 | 2019-07-26 | 韶关学院 | A kind of iris detoxic seedling culture propagation method |
| CN110050700B (en) * | 2019-05-24 | 2022-02-22 | 韶关学院 | Culture and propagation method of phalaenopsis virus-free seedlings |
| CN110896856A (en) * | 2019-11-07 | 2020-03-24 | 郑州师范学院 | Method for inducing complete inflorescence in vitro by axillary bud of inflorescence shaft of phalaenopsis |
| CN113575421A (en) * | 2021-08-27 | 2021-11-02 | 中德润萌生态科技(青岛)有限公司 | Aseptic processing method for tissue culture material of orchidaceae plant |
| CN113973713A (en) * | 2021-10-28 | 2022-01-28 | 北京市昌平职业学校 | Butterfly orchid tissue culture method |
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