CN106857258A - A kind of quick breeding method for tissue culture blue with leaf pocket - Google Patents

A kind of quick breeding method for tissue culture blue with leaf pocket Download PDF

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Publication number
CN106857258A
CN106857258A CN201710109534.1A CN201710109534A CN106857258A CN 106857258 A CN106857258 A CN 106857258A CN 201710109534 A CN201710109534 A CN 201710109534A CN 106857258 A CN106857258 A CN 106857258A
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culture
orchid
leaf pocket
sucrose
carragheens
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CN106857258B (en
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贾瑞冬
葛红
李秋香
张晓�
周妍慧
徐玉凤
刘娜
王洪云
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008Methods for regeneration to complete plants
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001Culture apparatus for tissue culture

Abstract

The invention provides a kind of method with leaf pocket orchid tissue-culturing quick-propagation, comprise the following steps:(1) pollination self;(2) Fruit pod is plucked;(3) aseptic seeding;(4) proliferation and subculture culture;(5) Rooting and hardening-off culture;(6) take root transplantation of seedlings.It is suitable to the culture medium system with leaf pocket orchid tissue-culturing quick-propagation, including germination medium, proliferated culture medium and Rooting and hardening-off culture base present invention also offers a kind of, it can be used in above-mentioned band leaf pocket orchid tissue culture and rapid propagation method.The present invention still further provides the optimal collecting period with leaf pocket orchid Fruit pod, i.e., pluck Fruit pod within 120 195 days or so after the orchid selfing of band leaf pocket.By the way that the seedling germination rate of method of the present invention culture is high, healthy and strong, well developed root system, growth cycle it is short, meanwhile, the method for the present invention is simple to operation, low production cost, is that band leaf pocket orchid is commercially produced there is provided excellent basis.

Description

A kind of quick breeding method for tissue culture blue with leaf pocket
Technical field
The present invention relates to field of plant tissue culture technique, specifically, the present invention relates to a kind of blue tissue training of band leaf pocket Support rapid propagation method, a kind of culture medium system and one kind being suitable to leaf pocket orchid tissue-culturing quick-propagation and determine band leaf The method of pocket orchid Fruit pod optimal acquisition phase.
Background technology
Paphiopedilum (Paphiopedilum) is one of category of orchid family most original.Compare with general orchid, cypripedium Colored structure has huge change, and lip is in the pocketed of half elliptic, and shape is very special, and therefore named pocket is blue;Again because its shape exactly likes The slippers of old times Europe maiden, also known as slippers are blue.But due to ecological environment increasingly severe in recent years, itself special biology Learn the arteriopathy excavation of characteristic and people, pocket orchid plant has turned into one of plant species in imminent danger in the world, numerous species Endangered, all pocket orchid wild species are put into《CITS》(CITES) annex I and Be prohibited transaction (Lu Sicong,《China blue and cattleya》, Golden Shield publishing house, 1994;Lang Kaiyong, " pocket of China is blue ",《Science and technology is led Report》,2002,20(9):56+2+65-66;Zeng Songjun etc., " pocket orchid aseptic seeding and Research Advances about Tissue Culture ",《Gardening Report》,2007,34(3):793-796).Therefore the protection work of cypripedium and breed research and be increasingly subject to people Pay attention to.
Band leaf pocket orchid (Paphiopedilum hirsutissimum (Lindl.ex Hook.)) originates in China Guangxi, expensive The ground such as state, Yunnan, Vietnam, Laos and Thailand are also distributed.Blade BG such as band, spends the big and florescence long, in the wild usually Cheng Cong Distribution in flakes.During introducing a fine variety, find compared with other pocket orchids, with leaf pocket orchid strong adaptability, to grow fast, self-reproduction energy Power is strong, ornamental value is high, with application prospect very high.
As other orchids, without endosperm, field could sprout band leaf pocket orchid species with mycosymbiosis.Orchid mainly by Tissue-cultured seedling is bred, the quantity of its tissue-cultured seedling account for ornamental plant tissue-cultured seedling total amount 40% (Xiong Li etc.,《Ornamental flower Tissue cultures and large-scale production》, Chemical Industry Press, 2003).But, the blue tissue culture technique difficulty of pocket greatly (king For paddy etc., " present situation and prospect of Guizhou cypripedium ",《Agriculture of Anhui science》,2009,37(6):2469-2470), it is blue One of category being most difficult in section.With the development of tissue culture technique and going deep into for research, made some progress in recent years, but It is that the technology path that the commercialization for carrying out pocket orchid by tissue culture technique is mass produced is still not up to very ripe.
As how simple and easy to do operating technology carries out the large-scale production of low cost to saving endangered band leaf pocket Blue and its exploitation are significant.Therefore, it is necessary to a kind of rapid propagation system blue with leaf pocket is provided, to shorten it Cultivation cycle, raising kind shoot survival percent and seedling quality, a reduces cost, constantly increase so as to meet market to this ornamental flower Demand.
The content of the invention
In order to solve the above problems and overcoming defect of the prior art, the invention provides a kind of blue tissue training of band leaf pocket Support rapid propagation method, it is a kind of be suitable to leaf pocket orchid tissue-culturing quick-propagation culture medium system and there is provided band leaf pocket The optimal acquisition phase of blue Fruit pod.
The first aspect of the present invention provides a kind of method with leaf pocket orchid tissue-culturing quick-propagation, including following step Suddenly:
(1) pollination self:Selecting the band leaf pocket orchid of large flower and brilliant color carries out cross-pollination;
(2) Fruit pod is plucked:120-195 days or so the collection Fruit pods after the orchid selfing of band leaf pocket;
(3) aseptic seeding:Carried out disinfection by explant of the Fruit pod of collection, cut Fruit pod and seed is seeded in sprouting culture Light culture is carried out in base about 40-50 days, treat that 30% seed grows protocorm, switch to periodicity of illumination for 12h is cultivated;
(4) proliferation and subculture culture:The seedling inoculation that will be produced in step (3) in proliferated culture medium, every 4 weeks subcultures one Secondary, appreciation rate is up to 3 times;
(5) Rooting and hardening-off culture:By the seedling of Multiplying culture in step (4) about 8 weeks, robust growth 2cm or so It is transferred in Rooting and hardening-off culture base and is cultivated;
(6) take root transplantation of seedlings:After seedling in step (5) is cultivated about 4 weeks, by the transfer of root growth good stand To natural light lower refining seedling, then it is transplanted in matrix and is cultivated.
In one preferred embodiment, the germination medium for using in the method for the invention is 1/2MS+ 100ml/L Coconut Juice+20.0g/L sucrose+4.6g/L carragheens or the germination medium be 1/2MS+100ml/L Coconut Juices+ 1.0mg/L NAA+1.0g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
In one embodiment, the proliferated culture medium for using in the method for the invention is 1/2MS+0.3mg/L NAA+100ml/L Coconut Juice+1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
In one embodiment, the composition of the Rooting and hardening-off culture base for using in the method for the invention and its contain Amount as above-mentioned proliferated culture medium, i.e., described Rooting and hardening-off culture base be 1/2MS+0.3mg/L NAA+100ml/L Coconut Juices+ 1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
In one preferred embodiment, band leaf pocket orchid selfing after the 100th day or so, the 120th day or so, the 150th Random time point collection Fruit pod between it or so, the 165th day or so, the 195th day or above-mentioned random time point.For example, can be with Band leaf pocket orchid selfing after the 100th day or so, the 105th day or so, the 110th day or so, the 115th day or so, the 120th day or so, 125th day or so, the 130th day or so, the 135th day or so, the 140th day or so, the 145th day or so, the 150th day or so, the 155th It or so, the 160th day or so, the 165th day or so, the 170th day or so, the 175th day or so, the 180th day or so, a 185th day left side Random time point collection Fruit pod right, between the 190th day or so, the 195th day or so or above-mentioned random time point.Preferably, exist The 100th day or so to the 120th day or so, the 120th day or so to the 135th day or so, the 120th day or so after the pocket orchid selfing of band leaf Fruit pod is plucked to the time period of the 150th day or so or the 120th day or so to the 165th day or so, it is more preferably blue certainly in band leaf pocket Pluck Fruit pod within the 120th day or so to the 135th day or so after friendship.Preferably, 120 days or so, 135 days left sides after the orchid selfing of band leaf pocket Right, 150 days or so or 165 days or so collection Fruit pods, more preferably pluck Fruit pod in the 120th day or so after the orchid selfing of band leaf pocket.
In one embodiment, in the method for the invention, the temperature of culturing room's culture is 24 ± 2 DEG C, intensity of illumination It is 1000-15001x, periodicity of illumination is 12h;Dark culturing temperature is 24 ± 2 DEG C.
The second aspect of the present invention provides a kind of for the germination medium with leaf pocket orchid tissue-culturing quick-propagation, institute Germination medium is stated for 1/2MS+100ml/L Coconut Juice+20.0g/L sucrose+4.6g/L carragheens or the germination medium are 1/ 2MS+100ml/L Coconut Juice+1.0mg/L NAA+1.0g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
The third aspect of the present invention provide it is a kind of for leaf pocket orchid tissue-culturing quick-propagation culture medium system, its Comprising germination medium, proliferated culture medium and Rooting and hardening-off culture base, wherein the germination medium is 1/2MS+100ml/L coconut palms Juice+20.0g/L sucrose+4.6g/L carragheens or the germination medium are 1/2MS+100ml/L Coconut Juice+1.0mg/L NAA+ 1.0g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
In one embodiment, the proliferated culture medium in culture medium system of the invention is 1/2MS+0.3mg/L NAA+100ml/L Coconut Juice+1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
In one preferred embodiment, the Rooting and hardening-off culture base in culture medium system of the invention is 1/ 2MS+0.3mg/L NAA+100ml/L Coconut Juice+1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
In one preferred embodiment, culture medium system of the invention is the culture medium system shown in table 1 below, table 1 composition and its consumption for listing the various culture mediums in culture medium system of the present invention.
Table 1. is with the blue cultured in vitro matrix system of leaf pocket
Note:Rooting and hardening-off culture base in table 1, can not only be used for general Rooting and hardening-off culture base, be also of the invention signified Can be used to do the culture medium of long-term subculture.
Definition
For the ease of the understanding of the present invention, to used in culture medium, culture medium used in above-mentioned various aspects Plant hormone is defined.
MS minimal mediums used in the present invention are according to (the Murashige T.and such as Murashige T. F.Skoog.Physiol.Plant,1962,15:473-497) prepare, its composition and each component content are as shown in table 2 below.1/ 2MS be MS minimal mediums in a great number of elements halve, other elements consumption is constant;1/5MS is a large amount of units in MS minimal mediums Element reduces to 1/5th, and other elements consumption is constant.
Composition " NAA " in culture medium herein refers to methyl α-naphthyl acetate.
Each composition of table 2.MS culture mediums and content
Advantageous Effects of the invention
The present invention produce good effect be:
A () explant of the present invention can largely be concentrated in certain fixed time period and obtain and carry out tissue cultures, beat Explant has been broken to be difficult to obtain and pollution rate limitation high;
B () is present invention determine that the blue optimal Fruit pod collecting time of band leaf pocket, germination percentage highest, and seedling early growth is strong;
(c) present invention also offers be suitable to leaf pocket orchid rapid propagation in vitro culture medium system, including germination medium, propagation Culture medium and Rooting and hardening-off culture base, culture medium prescription are simple, and the operating process of rapid propagation in vitro is succinctly easy to operate, produce it is low into This, can carry out commercialization large-scale production.
Foregoing teachings are only schematical and are never intended to be restricted.Except above-mentioned schematic aspect, implementation Mode and feature, by reference to following detailed description, further aspect, implementation method and feature will be more readily understood.
Brief description
By reference to following accompanying drawings, further aspect of the invention, feature will be more readily understood.People in the art Member is it should be understood that these accompanying drawings are only symbolically elaborated according to certain embodiments of the present invention, and be should not be taken as Limitation of the scope of the invention.
Fig. 1 is shown with leaf pocket orchid field grown, greenhouse domestication, the image bloomed with Fruit pod.
Fig. 2 shows the image with the seed of 120 days and 150 days after the blue pollination of leaf pocket.
Fig. 3 shows that the Fruit pod that different times (from 120 days to 195 days) are plucked is divided in culture medium (1) and culture medium (2) Not Pei Yang after 8 weeks and 16 weeks seed germination rate, wherein culture medium (1) for 1/2MS+100ml/L Coconut Juice+20.0g/L sucrose+ 4.6g/L carragheens, culture medium (2) is 1/2MS+100ml/L Coconut Juice+1.0mg/L NAA+1.0g/L activated carbon+20.0g/L sugarcanes Sugar+4.6g/L carragheens.
Fig. 4 shows the tissue culture procedures figure blue with leaf pocket according to one aspect of the invention.
Fig. 5 shows the flow chart with leaf pocket orchid group culturation rapid propagating technology system according to one aspect of the invention.
Fig. 6 is shown with the blue bottle outlet hardening situation of leaf pocket.
Specific embodiment
Hereinafter, some exemplary embodiments are simply just described.As one skilled in the art will recognize that Like that, without departing from the spirit or scope of the present invention, described embodiment can be changed by various different modes. Therefore, accompanying drawing and description is considered essentially illustrative rather than restrictive.
Embodiment 1. is with leaf pocket orchid pollination self and the determination of Fruit pod optimal collecting period
Experiment material:Band leaf pocket orchid is obtained from Vegetable & Flower Inst., Chinese Academy of Agriculture Science greenhouse;The all realities of the present invention The various reagents or medium component that are used in example are applied through commercially available.
Selecting the band leaf pocket orchid of large flower and brilliant color carries out palmification, and growth obtains Fruit pod after pollination.
After pollination, fruit was collected at the 120th day, the 135th day, the 150th day, the 165th day, the 180th day and the 195th day respectively Pod.
After above-mentioned Fruit pod is cleaned with running water respectively, it is placed in 10% liquor natrii hypochloritis and sterilizes 20 minutes, with 70% Alcohol surface sterilization 30 seconds, then sterilized 10 minutes with 0.1% mercuric chloride, finally with aseptic water washing 5 times.Clean Fruit pod is cut Open, seed is inoculated in two kinds of germination mediums (culture medium (1) and culture medium (2)) are up to carry out light culture 40 days respectively, treat 30% seed grows protocorm, switchs to periodicity of illumination for 12h is cultivated.After culture 8 weeks or 16 weeks, seed germination rate is measured.
Culture medium (1) is 1/2MS+100ml/L Coconut Juice+20.0g/L sucrose+4.6g/L carragheens, and culture medium (2) is 1/ 2MS+100ml/L Coconut Juice+1.0mg/L NAA+1.0g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.Condition of culture is: The temperature of culturing room's culture is 24 ± 2 DEG C, and intensity of illumination is 1000-15001x, and periodicity of illumination is 12h;Dark culturing temperature is 24±2℃。
Result finds that after culture medium (2) is cultivated 16 weeks, germination rate reaches 120 days seeds of collection after pollination 67.56%, less than the germination rate 71.44% of culture medium under the same terms (1).Hereafter, seed germination rate is with kernel maturing Rise and decline, it is negatively correlated.After pollination 135 days collection seed and 150 days collection seed culture after 16 weeks in culture medium (1) germination rate in is less than the germination rate after cultivating 8 weeks on culture medium (2).After pollination 135 days afterwards, cultivate it is identical In the case of time, germination rate of the seed on culture medium (2) is above culture medium (1).It will thus be seen that band leaf pocket is blue most suitable The time preferably sprouted may after the pollination 120 days or before, but the later stage find that the seed growth of 165d is more vigorous, this be probably because For the deposit of seed nutritional material needs the regular hour, the excessive growth of initial stage seed may reduce the growing way in later stage (see Fig. 2- 3)。
Tissue-culturing quick-propagation of the embodiment 2. with leaf pocket orchid species
As shown in figs. 4 through 6, the quick breeding method for tissue culture with leaf pocket orchid species of the invention comprises the following steps:
1st, pollination self:Selecting the band leaf pocket orchid of large flower and brilliant color carries out cross-pollination;
2nd, Fruit pod is plucked:120 days or so the collection Fruit pods after the orchid selfing of band leaf pocket;
3rd, aseptic seeding:According to the method described in embodiment 1 with the Fruit pod that gathers as explant carries out disinfection, fruit is cut Be seeded in seed in germination medium (culture medium (1) in embodiment 1) and carry out light culture 40 days by pod, treats that 30% seed grows Protocorm, switchs to periodicity of illumination for 12h is cultivated, and seed germination rate is up to 71.44%;
4th, proliferation and subculture culture:The seedling inoculation that will be produced in step 3 in proliferated culture medium, every 4 weeks subcultures once, increasing Up to 3 times, wherein proliferated culture medium is 1/2MS+0.3mg/L NAA+100ml/L Coconut Juice+1.5g/L activated carbons+20.0g/L to value rate Sucrose+4.6g/L carragheens;
5th, Rooting and hardening-off culture:The seedling of Multiplying culture in step 48 weeks, robust growth 2cm or so is transferred to life Cultivated in root strong seedling culture base, wherein Rooting and hardening-off culture base be 1/2MS+0.3mg/L NAA+100ml/L Coconut Juices+ 1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens;
6th, take root transplantation of seedlings:After seedling in steps of 5 is cultivated 4 weeks, root growth good stand is transferred to nature Light lower refining seedling, is then transplanted in matrix and is cultivated, and survival rate is up to 95%.
Tissue-culturing quick-propagation of the embodiment 3. with leaf pocket orchid species
As shown in figs. 4 through 6, the quick breeding method for tissue culture with leaf pocket orchid species of the invention comprises the following steps:
1st, pollination self:Selecting the band leaf pocket orchid of large flower and brilliant color carries out cross-pollination;
2nd, Fruit pod is plucked:150 days or so the collection Fruit pods after the orchid selfing of band leaf pocket;
3rd, aseptic seeding:Aseptic seeding is carried out according to the method described in embodiment 2 using above-mentioned culture medium (2), seed is sprouted Hair rate is up to 53.89%;
4th, proliferation and subculture culture:Proliferation and subculture culture is carried out according to the method described in embodiment 2;
5th, Rooting and hardening-off culture:Rooting and hardening-off culture is carried out according to the method described in embodiment 2;
6th, take root transplantation of seedlings:After seedling in steps of 5 is cultivated 4 weeks, root growth good stand is transferred to nature Light lower refining seedling, is then transplanted in matrix and is cultivated, and survival rate is up to 95%.
Tissue-culturing quick-propagation of the embodiment 4. with leaf pocket orchid species
As shown in figs. 4 through 6, the quick breeding method for tissue culture with leaf pocket orchid species of the invention comprises the following steps:
1st, pollination self:Selecting the band leaf pocket orchid of large flower and brilliant color carries out cross-pollination;
2nd, Fruit pod is plucked:165 days or so the collection Fruit pods after the orchid selfing of band leaf pocket;
3rd, aseptic seeding:Aseptic seeding is carried out according to the method described in embodiment 2 using above-mentioned culture medium (2), seed is sprouted Hair rate is close to 51.56%;
4th, proliferation and subculture culture:Proliferation and subculture culture is carried out according to the method described in embodiment 2;
5th, Rooting and hardening-off culture:Rooting and hardening-off culture is carried out according to the method described in embodiment 2;
6th, take root transplantation of seedlings:After seedling in steps of 5 is cultivated 4 weeks, root growth good stand is transferred to nature Light lower refining seedling, is then transplanted in matrix and is cultivated, and survival rate is up to 95%.
The above, specific embodiment only of the invention, but protection scope of the present invention is not limited thereto, and it is any Those familiar with the art the invention discloses technical scope in, its various change or replacement can be readily occurred in, These should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the guarantor of the claim Shield scope is defined.

Claims (10)

1. a kind of for the culture medium system with leaf pocket orchid tissue-culturing quick-propagation, it includes germination medium, Multiplying culture Base and Rooting and hardening-off culture base, wherein the germination medium blocks for 1/2MS+100ml/L Coconut Juice+20.0g/L sucrose+4.6g/L Draw glue or the germination medium be 1/2MS+100ml/L Coconut Juice+1.0mg/L NAA+1.0g/L activated carbon+20.0g/L sucrose+ 4.6g/L carragheens.
2. culture medium system as claimed in claim 1, wherein the proliferated culture medium is 1/2MS+0.3mg/L NAA+ 100ml/L Coconut Juice+1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
3. the culture medium system as described in claim any one of 1-2, wherein the Rooting and hardening-off culture base is 1/2MS+ 0.3mg/L NAA+100ml/L Coconut Juice+1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
4. a kind of method with leaf pocket orchid tissue-culturing quick-propagation, comprises the following steps:
(1) pollination self:Selecting the band leaf pocket orchid of large flower and brilliant color carries out cross-pollination;
(2) Fruit pod is plucked:The 120-195 days collection Fruit pods after the orchid selfing of band leaf pocket;
(3) aseptic seeding:Carried out disinfection by explant of the Fruit pod of collection, cut Fruit pod and seed is seeded in germination medium Carry out light culture about 40-50 days, treat that 30% seed grows protocorm, switch to periodicity of illumination for 12h is cultivated;
(4) proliferation and subculture culture:The seedling inoculation that will be produced in step (3) in proliferated culture medium, every 4 weeks subcultures once, increasing Value rate is up to 3 times;
(5) Rooting and hardening-off culture:The seedling of Multiplying culture in step (4) 8-12 weeks, robust growth 2cm or so is transferred to Cultivated in Rooting and hardening-off culture base;
(6) take root transplantation of seedlings:After seedling in step (5) is cultivated 4 weeks, root growth good stand is transferred to natural light Lower refining seedling, is then transplanted in matrix and is cultivated.
5. method as claimed in claim 4, wherein the germination medium is 1/2MS+100ml/L Coconut Juice+20.0g/L sucrose + 4.6g/L carragheens or the germination medium be 1/2MS+100ml/L Coconut Juice+1.0mg/L NAA+1.0g/L activated carbons+ 20.0g/L sucrose+4.6g/L carragheens.
6. method as claimed in claim 4, wherein the proliferated culture medium is 1/2MS+0.3mg/L NAA+100ml/L Coconut Juices + 1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
7. the method as described in claim any one of 4-6, wherein the Rooting and hardening-off culture base is 1/2MS+0.3mg/L NAA + 100ml/L Coconut Juice+1.5g/L activated carbon+20.0g/L sucrose+4.6g/L carragheens.
8. the method as described in claim any one of 4-6, wherein collection in 120-165 days or so is really after the orchid selfing of band leaf pocket Pod.
9. the method as described in claim any one of 4-6, wherein the 120th day or so to the 135th day after the orchid selfing of band leaf pocket Left and right or the 120th day or so to the 150th day or so collection Fruit pod.
10. the method as described in claim any one of 4-6, wherein 120 days or so the collection Fruit pods after the orchid selfing of band leaf pocket.
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CN108633376A (en) * 2018-04-21 2018-10-12 云南省林业科学院 A kind of cultural method of the sub- non-symbiosis germination of Gree pocket orchid species

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