CN109984030A - A kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method - Google Patents
A kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method Download PDFInfo
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- CN109984030A CN109984030A CN201910310848.7A CN201910310848A CN109984030A CN 109984030 A CN109984030 A CN 109984030A CN 201910310848 A CN201910310848 A CN 201910310848A CN 109984030 A CN109984030 A CN 109984030A
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- 241000732800 Cymbidium Species 0.000 title claims abstract description 115
- 238000000034 method Methods 0.000 title claims abstract description 37
- 230000008929 regeneration Effects 0.000 title claims abstract description 26
- 238000011069 regeneration method Methods 0.000 title claims abstract description 26
- 241000196324 Embryophyta Species 0.000 claims abstract description 98
- 238000009396 hybridization Methods 0.000 claims abstract description 21
- 238000009395 breeding Methods 0.000 claims abstract description 18
- 230000001488 breeding effect Effects 0.000 claims abstract description 18
- 238000012216 screening Methods 0.000 claims abstract description 17
- 241000233855 Orchidaceae Species 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims description 26
- 230000006698 induction Effects 0.000 claims description 25
- 238000012360 testing method Methods 0.000 claims description 25
- 230000004069 differentiation Effects 0.000 claims description 16
- 239000000835 fiber Substances 0.000 claims description 16
- 238000005286 illumination Methods 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 229920001817 Agar Polymers 0.000 claims description 15
- 239000008272 agar Substances 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 15
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 14
- 229930006000 Sucrose Natural products 0.000 claims description 14
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 14
- 239000005720 sucrose Substances 0.000 claims description 14
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 12
- 210000001672 ovary Anatomy 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 239000002699 waste material Substances 0.000 claims description 10
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 239000011159 matrix material Substances 0.000 claims description 8
- 230000035784 germination Effects 0.000 claims description 7
- 238000000338 in vitro Methods 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 230000003020 moisturizing effect Effects 0.000 claims description 7
- 230000010152 pollination Effects 0.000 claims description 7
- 239000002689 soil Substances 0.000 claims description 6
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 5
- 230000032823 cell division Effects 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- 230000000249 desinfective effect Effects 0.000 claims description 5
- 239000003599 detergent Substances 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 239000003337 fertilizer Substances 0.000 claims description 5
- 230000003287 optical effect Effects 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 238000007670 refining Methods 0.000 claims description 5
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 5
- 239000008399 tap water Substances 0.000 claims description 5
- 235000020679 tap water Nutrition 0.000 claims description 5
- 235000013399 edible fruits Nutrition 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 2
- 230000035800 maturation Effects 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 230000035755 proliferation Effects 0.000 claims 1
- 230000002950 deficient Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract 1
- 210000003484 anatomy Anatomy 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 240000002853 Nelumbo nucifera Species 0.000 description 2
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 2
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 241000351644 Cymbidium tortisepalum Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000020509 sex determination Effects 0.000 description 1
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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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
-
- 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 foundation of Cymbidium lianpan unisexuality kind vitro Regeneration System and rapid propagation methods, belong to breeding and the multiplication technique field of the rare kind of orchid.This method obtains male and female unisexuality Cymbidium lianpan strain by the unisexuality Cymbidium lianpan individual of pollen activity and the male and female gender of Pistil recipient phase detection screening field discovery.It is deficient for Cymbidium lianpan unisexuality variety seeds, female plant × staminiferous plant is constructed using artificial hybridization；Female plant × both sexes strain；Both sexes strain × combination of staminiferous plant three F1 generation group obtains candidate seed library and the candidate's pod of Cymbidium lianpan unisexual hybrid kind.Tissue culture expanding propagation is carried out to filial generation in conjunction with orchid breeding technique, establishes the rapid propagation system of unisexuality Cymbidium lianpan kind, and difficult technical bottleneck of sprouting and take root for Cymbidium lianpan unisexuality variety seeds, designs accurate reasonable induced medium.This method substantially reduces the breeding cycle of Cymbidium lianpan unisexuality kind, has important scientific research and market value meaning for the Cymbidium lianpan unisexuality kind for finding and selecting new.
Description
Technical field
The invention belongs to the breeding of the rare kind of orchid and multiplication technique fields, and in particular to a kind of Cymbidium lianpan unisexuality kind
Vitro Regeneration System is established and rapid propagation method.
Background technique
Cymbidium lianpan (Cymbidium tortisepalum) be subordinate to orchid family Cymbidium (Cymbidium), flower pattern is changeable, and pattern is rich
It is rich；It is the important orchid family cultivar of the countries and regions such as China, Japan and Southeast Asia.There are male and female in wild population for Cymbidium lianpan
The strain of unisexuality, flower pattern is peculiar, and ornamental value is high, while being also the excellent material of research orchid Sex Determination mechanism.
Summary of the invention
The purpose of the present invention is to provide a kind of foundation of Cymbidium lianpan unisexuality kind vitro Regeneration System and rapid propagation methods.
The rapid propagation system for the unisexuality Cymbidium lianpan kind that this method is established, it is deficient for unisexuality variety seeds, group is constructed using artificial hybridization
Body obtains the candidate seed library of unisexual hybrid kind, and difficult technology bottle of sprouting and take root for Cymbidium lianpan unisexuality variety seeds
Neck, design proprietary culture medium formula carry out tissue culture expanding propagation.The breeding week of Cymbidium lianpan unisexuality kind is substantially reduced using this method
Cymbidium lianpan unisexuality kind germination rate in special culture media of phase, acquisition are higher, and growth of seedling is in good condition.This method is for hair
Now and selects new Cymbidium lianpan unisexuality kind and have important scientific research and market value meaning.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method, passes through the list of the wild male and female gender of screening
Property Cymbidium lianpan plant, obtain male and female unisexuality Cymbidium lianpan strain, utilize artificial hybridization construct F1 generation group, obtain F1 generation pod, structure
Build the candidate seed library of Cymbidium lianpan unisexual hybrid kind；Tissue culture expanding propagation is carried out to filial generation in conjunction with orchid breeding technique, establishes lotus valve
The rapid propagation system of blue unisexuality kind.
A kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method, comprising the following steps:
(1) the candidate seed library building of Cymbidium lianpan unisexual hybrid kind: the unisexuality Cymbidium lianpan by screening wild male and female gender is planted
Strain obtains male and female unisexuality Cymbidium lianpan strain, constructs F1 generation group using artificial hybridization, obtains F1 generation pod, constructs Cymbidium lianpan list
The candidate seed library of property Hybrid；
(2) orchid breeding technique is utilized, Regeneration in Vitro and rapid propagation system are established.
The candidate seed library of above-mentioned steps (1) Cymbidium lianpan unisexual hybrid kind constructs, comprising the following specific steps
1) male and female unisexuality Cymbidium lianpan strain obtains: screening Cymbidium lianpan unisexuality female plant and unisexuality staminiferous plant candidate individual in field, passes through
It tests its pollen activity and Pistil recipient phase screening obtains Cymbidium lianpan unisexuality strain；It is 0 or without massula, column cap by pollen activity
Enumerable infinite set >=both sexes plant be averaged Enumerable infinite set and ovary it is intact individual as candidate female plant strain；By pollen activity >=
Both sexes plant is averaged vigor, and Pistil recipient phase is 0 or staminiferous plant strain of the individual as candidate without ovary；
2) artificial hybridization is pollinated: to the Cymbidium lianpan unisexuality female plant strain and unisexuality staminiferous plant strain filtered out, and the both sexes of cultivation
The individual of strain carries out artificial hybridization pollination, and pollination cross combination includes following three groups:
Female plant × staminiferous plant；Female plant × both sexes strain；Both sexes strain × staminiferous plant
It is mature when the F1 generation pod 80-90% of three cross combinations and still in it is green when be collected preservation, obtain pod to be sowed
Fruit, in addition, the candidate seed library of building Cymbidium lianpan unisexual hybrid kind, by pod with temperature T=20 DEG C, humidity H in drying box
=50% is saved, in case using.
Above-mentioned steps (2) utilize orchid breeding technique, establish Regeneration in Vitro and rapid propagation system, comprising the following steps:
1) material selection and disinfection: picking F1 generation 80-90% maturation pod detergent rinsed clean, and pod is scrubbed with banister brush
Outside fruit, after cleaning up, 15~20min is rinsed under tap water, distilled water dashes 2~3 times, sets in superclean bench and uses
Alcohol is removed after 75vol% alcohol disinfecting 30s, and 5wt% sodium hypochlorite is added and handles 10~12min, waste liquid is poured into waste liquid bottle, nothing
Bacterium water dashes 3~4 times, is inoculated with after blotting surface moisture with sterilized filter paper spare；
2) protocorm Fiber differentiation: the full pod after disinfection treatment of learning from else's experience clamps pod with gun-shaped forceps, scalpel is along pod
Vertical direction is splitted, and is taken out seed in pod, is inoculated in respectively through training in high temperature, autoclaved protocorm induction medium
It supports；Obtain growth protocorm；
3) Multiplying culture: it will be cut into 2-3cm square through the resulting well-grown protocorm of above-mentioned Fiber differentiation, be inoculated in respectively
In bud proliferated culture medium, Multiplying culture seedling is obtained；Light application time 8h/d, 1000~1500lx of intensity of illumination, when Multiplying culture
Between 20-30d；
4) root induction: 2~3 leaves are had by what Multiplying culture came out, plant height 3~4cm unrooted seedling single plant cuts, is transferred to
In root media, root induction；Light application time 10h/d, 1500~2000lx of intensity of illumination, root induction time 20-25d；
5) test tube seedling culture is completed: when root induction culture test tube seedling grows supreme 5-6cm, root 3~5, and 5, leaf, leaf is long by 2~
The germination of Cymbidium lianpan Seed inducement and quickly breeding culture are completed when 3cm；
6) the test tube bottle seedling that culture is completed is placed on natural light lower refining seedling 5~7 days, bottle cap hardening 1-2d is opened, to enhance test tube
Adaptability of the seedling to outdoor environment；Then it is taken out from culture bottle, cleans the remaining medium for being attached to root system, move into mixing
Moisturizing is shaded in culture substrate, and plant adaptability gradually increases after 2-3 weeks, obtains the complete seedling of nature robust growth.
Above-mentioned steps 2) protocorm Fiber differentiation condition are as follows: culture room temperature is 23 ± 2 DEG C, and no optical culture is after 60 days,
3h/d is arranged in light application time within the 70-80 days, and light application time is adjusted to 5h/d after the 90th day, and intensity of illumination is all 500~1000lx,
Obtain growth protocorm；Protocorm induction medium are as follows: contain the 0.5mg/L basic element of cell division (KT) in MS culture medium, 0.5mg/L
Methyl α-naphthyl acetate (NAA), the peptone of 1g/L, 20g/L sucrose, 5.5g/L agar powder, 1.0 g/L active carbons.
Above-mentioned steps 3) in bud proliferated culture medium are as follows: in MS culture medium contain 2.0mg/L 6- benzyl aminoadenine (6-
BA), 1.5g/L peptone, 0.3mg/L methyl α-naphthyl acetate (NAA), 20g/L sucrose, 5.5g/L agar powder, 1.0 g/L active carbons.
Above-mentioned steps 4) in root media are as follows: 1/2 times of MS culture medium, 1.0g/L spend precious No. 2 fertilizer, 0.1mg/L naphthalene
Acetic acid (NAA), 20g/L sucrose, 5.5g/L agar powder, 1.5g/L active carbon.
Above-mentioned steps 6) in be mixed matrix are as follows: fertile soil: water plant: broken bark: blinding press 1:2:2:2 mass ratio
The matrix mixed.
Above-mentioned steps 6) in moisturizing shading condition are as follows: temperature control at 15~30 DEG C, humidity should be maintained at 75%~
85%, avoid direct sunlight.
The present invention has the advantages that
1, deficient for Cymbidium lianpan unisexuality variety seeds, material is established using artificial hybridization constructive system: picking male and female hybridization
F1 generation pod, constructs candidate seed library, and screening maturity is that 80-90% mature and plump does not crack pod；
2, for the sprouting of Cymbidium lianpan unisexuality variety seeds and difficult technical bottleneck of taking root, special protocorm Fiber differentiation is designed
Base, proliferated culture medium and root media carry out tissue culture expanding propagation.
3, light application time and intensity of illumination are rationally controlled, germination rate is improved；Root induction rate is up to 95% or more.
Detailed description of the invention:
Fig. 1 Cymbidium lianpan female plants and male plants.It is left: Cymbidium lianpan female plants；It is right: Cymbidium lianpan male plants.
Fig. 2 Cymbidium lianpan instaminate flower and male flower internal anatomy.It is left: Cymbidium lianpan instaminate flower internal anatomy；It is right: Cymbidium lianpan male flower solution
Cut open figure.
Fig. 3 Cymbidium lianpan female pistil and stamen.It is left: Cymbidium lianpan female pistil；It is right: Cymbidium lianpan stamen Cymbidium lianpan
Female pistil.
Fig. 4 Cymbidium lianpan tissue-cultured seedling and hardening phase seedling.It is left: the tissue-cultured seedling of Cymbidium lianpan；It is right: the seedling of hardening phase.
Specific embodiment
To make the purpose of the present invention, technical solution and advantage are more clearly understood, and will pass through specific embodiment and phase below
Attached drawing is closed, invention is further described in detail:
Embodiment 1
A kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method, comprising the following steps:
(1) the candidate seed library building of Cymbidium lianpan unisexual hybrid kind: the unisexuality Cymbidium lianpan by screening wild tool male and female gender
Plant obtains male and female unisexuality Cymbidium lianpan strain, constructs F1 generation group using artificial hybridization, obtains F1 generation pod, constructs Cymbidium lianpan
The candidate seed library of unisexual hybrid kind；Comprising the following specific steps
1) male and female unisexuality Cymbidium lianpan strain obtains: screening Cymbidium lianpan unisexuality female plant and unisexuality staminiferous plant candidate individual in field, passes through
It tests its pollen activity and Pistil recipient phase obtains Cymbidium lianpan unisexuality strain.It is 0 or without massula by pollen activity, column cap can be awarded
Property be more than or equal to both sexes plant be averaged Enumerable infinite set and ovary it is intact individual as candidate female plant strain；By pollen activity
It is averaged vigor more than or equal to both sexes plant, Pistil recipient phase is 0 or staminiferous plant strain of the individual as candidate without ovary.
2) artificial hybridization is pollinated: to the Cymbidium lianpan unisexuality female plant strain and unisexuality staminiferous plant strain filtered out, and cultivate
The individual of both sexes strain carries out artificial hybridization pollination.Cross combination of pollinating includes following three groups:
Female plant × staminiferous plant；Female plant × both sexes strain；Both sexes strain × staminiferous plant
It is mature when the F1 generation pod 80% of three cross combination and still in it is green when be collected preservation, obtain pod to be sowed,
In addition, the candidate seed library of building Cymbidium lianpan unisexual hybrid kind, by pod with temperature T=20 DEG C in drying box, humidity H=
50% is saved, in case using.
(2) orchid breeding technique is utilized, Regeneration in Vitro and rapid propagation system are established, comprising the following steps:
1) material selection and disinfection: the mature pod detergent rinsed clean of picking F1 generation 80%, and with outside banister brush scrub pod
15min after cleaning up, rinses in portion under tap water, distilled water dashes 2 times, sets and uses 75vol% alcohol disinfecting in superclean bench
Alcohol is removed after 30s, and 5wt% sodium hypochlorite is added and handles 10min, waste liquid is poured into waste liquid bottle, sterile water dashes 3 times, with disinfection
Filter paper blot be inoculated with after surface moisture it is spare.
2) protocorm Fiber differentiation: take it is full it is sterile-processed after pod, clamp pod with gun-shaped forceps, scalpel along
Pod vertical direction is splitted, and is taken out seed in pod, is inoculated in respectively through high temperature, autoclaved protocorm induction medium
In；Condition of culture: culture room temperature is 23 DEG C, and no optical culture is after 60 days, the 70-80 days light application time setting 3h/d, the 90th day
Light application time is adjusted to 5h/d afterwards, and intensity of illumination is all 500lx, obtains growth protocorm；Protocorm Fiber differentiation are as follows: MS culture
Contain the 0.5mg/L basic element of cell division (KT) in base, the methyl α-naphthyl acetate (NAA) of 0.5mg/L, the peptone of 1g/L, 20g/L sucrose,
5.5g/L agar powder, 1.0 g/L active carbons.
3) Multiplying culture: it will be cut into 2cm square through the resulting well-grown protocorm of above-mentioned Fiber differentiation, be inoculated with respectively
In bud proliferated culture medium, light application time 8h/d, intensity of illumination 1000lx, Multiplying culture time 30d obtain Multiplying culture children
Seedling；The culture medium of bud Multiplying culture are as follows: contain 2.0mg/L 6- benzyl aminoadenine (6-BA), 1.5g/L albumen in MS culture medium
Peptone, 0.3mg/L methyl α-naphthyl acetate (NAA), 20g/L sucrose, 5.5g/L agar powder, 1.0 g/L active carbons.
4) root induction: 2, leaf are had by what Multiplying culture came out, plant height 3cm unrooted seedling single plant is cut, and is transferred to life
In root culture medium；Light application time 10h/d, intensity of illumination 1500lx, root induction time 20d；Root media are as follows: 1/2 times of MS
Culture medium, 1.0g/L spend precious No. 2 fertilizer, 0.1mg/L methyl α-naphthyl acetate (NAA), 20g/L sucrose, 5.5g/L agar powder, and 1.5g/
L active carbon.
5) test tube seedling culture is completed: when root induction culture test tube seedling grows supreme 5cm, root 3, and 5, leaf, the long 2cm of leaf
When complete Cymbidium lianpan Seed inducement germination and quickly breeding culture.
6) hardening: culture of rootage test tube bottle seedling will be completed and be placed on natural light lower refining seedling 5 days, open bottle cap hardening 1d, to increase
Adaptability of the strong test tube seedling to outdoor environment；Then it is taken out from culture bottle, cleans the remaining medium for being attached to root system, moved
Enter fertile soil: water plant: broken bark: blinding (1:2:2:2) is mixed in matrix, and temperature is controlled at 15 DEG C, and humidity should be kept
75%, direct sunlight is avoided, carries out moisturizing shading, survival rate is gradually increased up to plant adaptability after 95% or more, 2 week,
Obtain the complete seedling of nature robust growth.
The Cymbidium lianpan female plants and male plants that the present embodiment screening obtains are shown in Fig. 1.Fig. 1 shows: female and male lotus
Valve orchid plant has larger difference on plant forms, and female plant individual is lower, and staminiferous plant individual is sturdy higher.Cymbidium lianpan instaminate flower
Fig. 2 is seen with male flower internal anatomy.Fig. 2 shows: female and male Cymbidium lianpan have larger difference, female flower lip in flower form
Valve is larger, petal lip, and male flower lip is smaller.Cymbidium lianpan female pistil and stamen see Fig. 3.Fig. 3 shows: female pistil
Without massula, ovary is longer；Stamen has massula, and ovary is degenerated shorter.Cymbidium lianpan tissue-cultured seedling and hardening phase seedling are shown in figure
4.Fig. 4 shows: the seedling adaptability of the tissue-cultured seedling well-grown of Cymbidium lianpan, hardening phase is good.
Embodiment 2
A kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method, comprising the following steps:
(1) the candidate seed library building of Cymbidium lianpan unisexual hybrid kind: the unisexuality Cymbidium lianpan by screening wild tool male and female gender
Plant obtains male and female unisexuality Cymbidium lianpan strain, constructs F1 generation group using artificial hybridization, obtains F1 generation pod, constructs Cymbidium lianpan
The candidate seed library of unisexual hybrid kind.Comprising the following specific steps
1) male and female unisexuality Cymbidium lianpan strain obtains: screening Cymbidium lianpan unisexuality female plant and unisexuality staminiferous plant candidate individual in field, passes through
It tests its pollen activity and Pistil recipient phase obtains Cymbidium lianpan unisexuality strain.It is 0 by pollen activity, Pistil recipient phase > both sexes are planted
The strain averagely intact individual of Enumerable infinite set and ovary is as candidate female plant strain；Pollen activity > both sexes plant is averagely living
Power, the individual that Pistil recipient phase is 0 is as candidate staminiferous plant strain.
2) artificial hybridization is pollinated: to the Cymbidium lianpan unisexuality female plant strain and unisexuality staminiferous plant strain filtered out, and cultivate
The individual of both sexes strain carries out artificial hybridization pollination.Cross combination of pollinating includes following three groups:
Female plant × staminiferous plant；Female plant × both sexes strain；Both sexes strain × staminiferous plant
It is mature when the F1 generation pod 80% of three cross combination and still in it is green when be collected preservation, obtain pod to be sowed,
In addition, the candidate seed library of building Cymbidium lianpan unisexual hybrid kind, by pod with temperature T=20 DEG C in drying box, humidity H=
50% is saved, in case using.
(2) orchid breeding technique is utilized, Regeneration in Vitro and rapid propagation system are established, comprising the following steps:
1) material selection and disinfection: the mature pod detergent rinsed clean of picking F1 generation 90%, and with outside banister brush scrub pod
20min after cleaning up, rinses in portion under tap water, distilled water dashes 3 times, sets and uses 75vol% alcohol disinfecting in superclean bench
Alcohol is removed after 30s, and 5wt% sodium hypochlorite is added and handles 12min, waste liquid is poured into waste liquid bottle, sterile water dashes 4 times, with disinfection
Filter paper blot be inoculated with after surface moisture it is spare.
2) protocorm Fiber differentiation: take it is full it is sterile-processed after pod, clamp pod with gun-shaped forceps, scalpel along
Pod vertical direction is splitted, and is taken out seed in pod, is inoculated in respectively through high temperature, autoclaved protocorm induction medium
In；Condition of culture: culture room temperature is 25 DEG C, and no optical culture is after 60 days, and light application time is arranged 3h/ days, the 90th day within the 70-80 days
Light application time is adjusted to 5h/d afterwards, and intensity of illumination is all 1000lx, obtains growth protocorm；Protocorm Fiber differentiation are as follows: MS training
It supports in base and contains the 0.5mg/L basic element of cell division (KT), the methyl α-naphthyl acetate (NAA) of 0.5mg/L, the peptone of 1g/L, 20g/L sugarcane
Sugar, 5.5g/L agar powder, 1.0 g/L active carbons.
3) Multiplying culture: it will be cut into 3cm square through the resulting well-grown protocorm of above-mentioned Fiber differentiation, be inoculated with respectively
In bud proliferated culture medium, light application time 8h/d, intensity of illumination 1500lx, Multiplying culture time 20d obtain Multiplying culture children
Seedling；The culture medium of bud Multiplying culture are as follows: contain 2.0mg/L 6- benzyl aminoadenine (6-BA), 1.5g/L albumen in MS culture medium
Peptone, 0.3mg/L methyl α-naphthyl acetate (NAA), 20g/L sucrose, 5.5g/L agar powder, 1.0 g/L active carbons.
4) root induction: 3, leaf are had by what Multiplying culture was turned out, plant height 4cm unrooted seedling single plant is cut, transfer
Into root media；Light application time 10h/d, intensity of illumination 2000lx, root induction time 20d；Root media are as follows: 1/2
Times MS culture medium, 1.0g/L spend precious No. 2 fertilizer, 0.1mg/L methyl α-naphthyl acetate (NAA), 20g/L sucrose, 5.5g/L agar powder,
1.5g/L active carbon.
5) test tube seedling culture is completed: when root induction culture test tube seedling grows supreme 6cm, root 5, and 5, leaf, the long 3cm of leaf
When complete Cymbidium lianpan Seed inducement germination and quickly breeding culture.
6) hardening: culture of rootage test tube bottle seedling will be completed and be placed on natural light lower refining seedling 7 days, open bottle cap hardening 2d, to increase
Adaptability of the strong test tube seedling to outdoor environment；Then it is taken out from culture bottle, cleans the remaining medium for being attached to root system, moved
Enter fertile soil: water plant: broken bark: blinding (1:2:2:2) is mixed in matrix, and temperature is controlled at 30 DEG C, and humidity should be kept
85%, direct sunlight is avoided, carries out moisturizing shading, survival rate is up to 97% or more.Plant adaptability gradually increases after 2 weeks,
Obtain the complete seedling of nature robust growth.
Embodiment 3
A kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method, comprising the following steps:
(1) the candidate seed library building of Cymbidium lianpan unisexual hybrid kind: the unisexuality Cymbidium lianpan by screening wild tool male and female gender
Plant obtains male and female unisexuality Cymbidium lianpan strain, constructs F1 generation group using artificial hybridization, obtains F1 generation pod, constructs Cymbidium lianpan
The candidate seed library of unisexual hybrid kind.Comprising the following specific steps
1) male and female unisexuality Cymbidium lianpan strain obtains: screening Cymbidium lianpan unisexuality female plant and unisexuality staminiferous plant candidate individual in field, passes through
It tests its pollen activity and Pistil recipient phase obtains Cymbidium lianpan unisexuality strain.Will be without massula, Pistil recipient phase > both sexes plant is flat
Equal Enumerable infinite set and ovary it is intact individual as candidate female plant strain；Pollen activity > both sexes plant is averaged vigor, nothing
The individual of ovary is as candidate staminiferous plant strain.
2) artificial hybridization is pollinated: to the Cymbidium lianpan unisexuality female plant strain and unisexuality staminiferous plant strain filtered out, and cultivate
The individual of both sexes strain carries out artificial hybridization pollination.Cross combination of pollinating includes following three groups:
Female plant × staminiferous plant；Female plant × both sexes strain；Both sexes strain × staminiferous plant
It is mature when the F1 generation pod 85% of three cross combination and still in it is green when be collected preservation, obtain pod to be sowed,
In addition, the candidate seed library of building Cymbidium lianpan unisexual hybrid kind, by pod with temperature T=20 DEG C in drying box, humidity H=
50% is saved, in case using.
(2) orchid breeding technique is utilized, Regeneration in Vitro and rapid propagation system are established, comprising the following steps:
1) material selection and disinfection: the mature pod detergent rinsed clean of picking F1 generation 85%, and with outside banister brush scrub pod
18min after cleaning up, rinses in portion under tap water, distilled water dashes 2 times, sets and uses 75vol% alcohol disinfecting in superclean bench
Alcohol is removed after 30s, and 5wt% sodium hypochlorite is added and handles 11min, waste liquid is poured into waste liquid bottle, sterile water dashes 3 times, with disinfection
Filter paper blot be inoculated with after surface moisture it is spare.
2) protocorm Fiber differentiation: take it is full it is sterile-processed after pod, clamp pod with gun-shaped forceps, scalpel along
Pod vertical direction is splitted, and is taken out seed in pod, is inoculated in respectively through high temperature, autoclaved protocorm induction medium
In；Condition of culture: culture room temperature is 21 DEG C, and no optical culture is after 60 days, the 70-80 days light application time setting 3h/d, the 90th day
Light application time is adjusted to 5h/d afterwards, and intensity of illumination is all 800lx, obtains growth protocorm；Protocorm Fiber differentiation are as follows: MS culture
Contain the 0.5mg/L basic element of cell division (KT) in base, the methyl α-naphthyl acetate (NAA) of 0.5mg/L, the peptone of 1g/L, 20g/L sucrose,
5.5g/L agar powder, 1.0 g/L active carbons.
3) Multiplying culture: it will be cut into 2cm square through the resulting well-grown protocorm of above-mentioned Fiber differentiation, be inoculated with respectively
In bud proliferated culture medium, light application time 8h/d, intensity of illumination 1200lx, Multiplying culture time 25d obtain Multiplying culture children
Seedling；The culture medium of bud Multiplying culture are as follows: contain 2.0mg/L 6- benzyl aminoadenine (6-BA), 1.5g/L albumen in MS culture medium
Peptone, 0.3mg/L methyl α-naphthyl acetate (NAA), 20g/L sucrose, 5.5g/L agar powder, 1.0 g/L active carbons.
4) root induction: 2, leaf are had by what Multiplying culture was turned out, plant height 3cm unrooted seedling single plant is cut, transfer
Into root media；Light application time 10h/d, intensity of illumination 1800lx, root induction time 23d；Root media are as follows: 1/2
Times MS culture medium, 1.0g/L spend precious No. 2 fertilizer, 0.1mg/L methyl α-naphthyl acetate (NAA), 20g/L sucrose, 5.5g/L agar powder,
1.5g/L active carbon.
5) test tube seedling culture is completed: when root induction culture test tube seedling grows supreme 6cm, root 4, and 5, leaf, the long 2cm of leaf
When complete Cymbidium lianpan Seed inducement germination and quickly breeding culture.
6) hardening: culture of rootage test tube bottle seedling will be completed and be placed on natural light lower refining seedling 6 days, open bottle cap hardening 1d, to increase
Adaptability of the strong test tube seedling to outdoor environment；Then it is taken out from culture bottle, cleans the remaining medium for being attached to root system, moved
Enter fertile soil: water plant: broken bark: blinding (1:2:2:2) is mixed in matrix, and temperature is controlled at 23 DEG C, and humidity should be kept
80%, direct sunlight is avoided, carries out moisturizing shading, survival rate is up to 96% or more.Plant adaptability gradually increases after 2 weeks,
Obtain the complete seedling of nature robust growth.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (9)
1. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method, it is characterised in that: wild by screening
The unisexuality Cymbidium lianpan plant of raw male and female gender, obtains male and female unisexuality Cymbidium lianpan strain, constructs F1 generation group using artificial hybridization, obtains
F1 generation pod is obtained, the candidate seed library of Cymbidium lianpan unisexual hybrid kind is constructed；Tissue culture is carried out to filial generation in conjunction with orchid breeding technique
Expand numerous, establishes the rapid propagation system of Cymbidium lianpan unisexuality kind.
2. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System according to claim 1 is established and rapid propagation method,
It is characterized in that, comprising the following steps:
(1) the candidate seed library building of Cymbidium lianpan unisexual hybrid kind: the unisexuality Cymbidium lianpan by screening wild male and female gender is planted
Strain obtains male and female unisexuality Cymbidium lianpan strain, constructs F1 generation group using artificial hybridization, obtains F1 generation pod, constructs Cymbidium lianpan list
The candidate seed library of property Hybrid；
(2) orchid breeding technique is utilized, Regeneration in Vitro and rapid propagation system are established.
3. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System according to claim 2 is established and rapid propagation method,
It is characterized in that, the candidate seed library building of step (1) the Cymbidium lianpan unisexual hybrid kind, comprising the following specific steps
1) male and female unisexuality Cymbidium lianpan strain obtains: screening Cymbidium lianpan unisexuality female plant and unisexuality staminiferous plant candidate individual in field, passes through
It tests its pollen activity and Pistil recipient phase screening obtains Cymbidium lianpan unisexuality strain；It is 0 or without massula, column cap by pollen activity
Enumerable infinite set >=both sexes plant be averaged Enumerable infinite set and ovary it is intact individual as candidate female plant strain；By pollen activity >=
Both sexes plant is averaged vigor, and Pistil recipient phase is 0 or staminiferous plant strain of the individual as candidate without ovary；
2) artificial hybridization is pollinated: to the Cymbidium lianpan unisexuality female plant strain and unisexuality staminiferous plant strain filtered out, and the both sexes of cultivation
The individual of strain carries out artificial hybridization pollination, and pollination cross combination includes following three groups:
Female plant × staminiferous plant；Female plant × both sexes strain；Both sexes strain × staminiferous plant
It is mature when the F1 generation pod 80-90% of three cross combinations and still in it is green when be collected preservation, obtain pod to be sowed
Fruit, in addition, the candidate seed library of building Cymbidium lianpan unisexual hybrid kind, by pod with temperature T=20 DEG C, humidity H in drying box
=50% is saved, in case using.
4. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System according to claim 2 is established and rapid propagation method,
It is characterized in that, the step (2) utilizes orchid breeding technique, establishes Regeneration in Vitro and rapid propagation system, including in detail below
Step:
1) material selection and disinfection: picking F1 generation 80-90% maturation pod detergent rinsed clean, and pod is scrubbed with banister brush
Outside fruit, after cleaning up, tap water undershoot drips 15~20min, and distilled water dashes 2~3 times, sets in superclean bench and uses
Alcohol is removed after 75vol% alcohol disinfecting 30s, and 5wt% sodium hypochlorite is added and handles 10~12min, waste liquid is poured into waste liquid bottle, nothing
Bacterium water dashes 3~4 times, is inoculated with after blotting surface moisture with sterilized filter paper spare；
2) protocorm Fiber differentiation: the full pod after disinfection treatment of learning from else's experience clamps pod with gun-shaped forceps, scalpel is along pod
Vertical direction is splitted, and seed in pod is taken out, and is inoculated in respectively through passing through in high temperature, autoclaved protocorm induction medium
90 d cultivate to obtain growth protocorm；
3) Multiplying culture: it will be cut into 2-3cm square through the resulting well-grown protocorm of above-mentioned Fiber differentiation, be inoculated in respectively
In bud proliferated culture medium, Multiplying culture seedling is obtained；Light application time 8h/d, 1000~1500lx of intensity of illumination, when Multiplying culture
Between 20-30d；
4) root induction: 2~3 leaves are had by what proliferation-inducing was turned out, plant height 3~4cm unrooted seedling single plant is cut, and is turned
It moves on in root media, root induction；Light application time 10h/d, 1500~2000lx of intensity of illumination, root induction time 20-
25d；
5) test tube seedling culture is completed: when root induction culture test tube seedling grows supreme 5-6cm, root 3~5, and 5, leaf, leaf is long by 2~
The germination of Cymbidium lianpan Seed inducement is completed when 3cm and quickly breeds incubation；
6) the test tube bottle seedling that culture is completed is placed on natural light lower refining seedling 5~7 days, bottle cap hardening 1-2d is opened, to enhance test tube
Adaptability of the seedling to outdoor environment；Then it is taken out from culture bottle, cleans the remaining medium for being attached to root system, move into mixing
Culture substrate fertile soil: water plant: broken bark: moisturizing is shaded in the matrix that blinding is mixed by the mass ratio of 1:2:2:2,2-
Plant adaptability gradually increases after 3 weeks, obtains the complete seedling of nature robust growth.
5. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System according to claim 4 is established and rapid propagation method,
It is characterized in that, the condition cultivated in the step 2 protocorm induction medium are as follows: culture room temperature is 23 ± 2 DEG C without optical culture
After 60 days, 3h/d is arranged in the 70-80 days light application times, and light application time is adjusted to 5h/d after the 90th day, and intensity of illumination 500~
1000lx obtains growth protocorm；Protocorm Fiber differentiation are as follows: contain 0.5mg/L basic element of cell division KT in MS culture medium,
The peptone of the methyl α-naphthyl acetate NAA, 1g/L of 0.5mg/L, 20g/L sucrose, 5.5g/L agar powder, 1.0 g/L active carbons.
6. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System according to claim 4 is established and rapid propagation method,
It is characterized in that, bud proliferated culture medium in the step 3) are as follows: contain 2.0mg/L 6- benzyl aminoadenine 6-BA in MS culture medium,
1.5g/L peptone, 0.3mg/L methyl α-naphthyl acetate NAA, 20g/L sucrose, 5.5g/L agar powder, 1.0 g/L active carbons.
7. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System according to claim 4 is established and rapid propagation method,
It is characterized in that, root media in the step 4) are as follows: 1/2 times of MS culture medium, 1.0g/L spend precious No. 2 fertilizer, 0.1mg/L naphthalene
Acetic acid NAA, 20g/L sucrose, 5.5g/L agar powder, 1.5g/L active carbon.
8. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System according to claim 4 is established and rapid propagation method,
Be characterized in that, be mixed matrix in the step 6) are as follows: fertile soil: water plant: broken bark: blinding presses the quality of 1:2:2:2
Than the matrix mixed.
9. a kind of Cymbidium lianpan unisexuality kind vitro Regeneration System according to claim 4 is established and rapid propagation method,
Be characterized in that, above-mentioned steps 6) in moisturizing shading condition are as follows: temperature control at 15~30 DEG C, humidity should be maintained at 75%~
85%, avoid direct sunlight.
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