CN109964817A - The rapid propagation method of high eyebrow groove and tongue orchid - Google Patents
The rapid propagation method of high eyebrow groove and tongue orchid Download PDFInfo
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- CN109964817A CN109964817A CN201910305770.XA CN201910305770A CN109964817A CN 109964817 A CN109964817 A CN 109964817A CN 201910305770 A CN201910305770 A CN 201910305770A CN 109964817 A CN109964817 A CN 109964817A
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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
Abstract
The invention discloses a kind of acquisition of high eyebrow groove and tongue orchid capsule and the methods for obtaining seedling using its seminal propagation, belong to technical field of plant propagation, obtain capsule, setting percentage 100% by the way of carrying out artificial pollination between the plant with certain genetic distance；The rapid propagation system of high eyebrow groove and tongue orchid is established, breeding coefficient reaches 7 times or more, and planting percent of taking root reaches 90% or more, to protect the wild resource of the species to establish scientific and technical basis；The excellent high eyebrow groove and tongue orchid seedling obtained, provides resource base and technical support for its sustainable use, has actively promoted the industrialized development of the plant resources, has particularly important realistic meaning and economic benefit.
Description
Technical field
The invention belongs to technical field of plant propagation, and in particular to the preparation method and its seed of high eyebrow groove and tongue orchid capsule are fast
Fast propagation method.
Background technique
High eyebrow groove and tongue it is blue (Holcoglossum omeiense) be groove and tongue epidendrum in low mountain vegetation type, for me
The endemic species of state's Emeishan are distributed only over the mountains low-to-middle in height location of this area.High eyebrow groove and tongue orchid is extremely narrow due to distributed areas
Narrow, population and individual amount are few, belong to rare and endangered species, be put into " minimum population (narrow domain distribution) protect species ",
" Chinese biological diversity Red List (higher plant volume) ", " Chinese species Red List (plant part) " and " China it is rare
Endangered plants illustrated handbook " in.Currently, high eyebrow groove and tongue orchid is due to being put into country's rescue and having protected for the minimum population wild plant in the whole nation
" the minimum population wild plant rescue protection project planning in the whole nation " that nurse's journey --- the State Administration of Forestry and National Development and Reform Committee print and distribute
In 120 kinds minimum population conservation species, belong to be included in the rescue planning in 11 kinds be distributed in one of the wild plant in Sichuan Province.
High eyebrow groove and tongue orchid is the herbaceos perennial of orchid family groove and tongue Cymbidium, is grown nonparasitically upon another plant in woods on trunk.Current high eyebrow slot
Tongue orchid Wild plant quantity is very few, is Precious, Rare, Endangered, minimum population wild plant, has high scientific research value；In addition, high eyebrow
Groove and tongue orchid form gracefulness is delicate and pretty, the pure white elegance of pattern, has high gardening ornamental value.Currently, with economy and E Mei trip
Development, scientific research and the acquisition of orchid fan of trip, the plant is while its ecological environment is by destroying, also by mistake
The excavation of degree causes great threat to wild stocks.
China extremely lacks about the research of high eyebrow groove and tongue orchid plant, and it is narrow, big only to understand high eyebrow groove and tongue orchid distributed areas
These coarse informations, the researchs about high eyebrow groove and tongue orchid proliferation such as place, the quantity extremely rareness of distribution is caused still to belong to blank,
China scientific research personnel, which fails, at present breeds high eyebrow groove and tongue orchid plant.
Summary of the invention
It is minimum population plant for the plant, resource quantity is few, field can not collect high eyebrow groove and tongue orchid capsule
Fruit lacks system and the quickly propagation method of high eyebrow groove and tongue orchid in the prior art, and it is an object of that present invention to provide a kind of high eyebrows
The method and rapid propagation method that groove and tongue orchid capsule obtains.The present invention obtains healthy and strong high eyebrow slot by obtaining artificial pollination mode
Tongue orchid capsule, and carry out study on reproduction using its seed, its fast breeding technique system is established, to the open country for protecting the blue preciousness of high eyebrow groove and tongue
Production-goods source has important practical significance.
To achieve the goals above, the present invention adopts the following technical scheme:
The rapid propagation method of high eyebrow groove and tongue orchid, comprising the following steps:
The acquisition of a capsule: selection adaptation is strong, grows plant that is vigorous and can smoothly completing a life cycle, within the florescence,
It is respectively that the Parent pollinated carries out artificial pollination using the plant from different population, obtains capsule, capsule is after pollination
Picking when not cracking after 380-520d；
The processing of b capsule: after the high eyebrow groove and tongue orchid capsule of picking is rinsed 5~10min under tap water, water is blotted with filter paper
Point；75% 30~35s of ethanol postincubation of mass concentration, 0.1% mercuric chloride solution of mass concentration, 15~18min of sterilizing are then used respectively, and
Each personal aseptic water washing 4~6 times, finally blots the moisture on surface with aseptic filter paper；Capsule is aseptically cut off to obtain
Seed；
C Fiber differentiation: on the induction medium by the seed uniform broadcasting obtained in step b, protocorm is induced；
D Proliferation, Differentiation culture: the protocorm of green phyllopodium clearly visible in step c is inoculated on Proliferation, Differentiation culture medium
Carry out Proliferation, Differentiation culture；In the Proliferation, Differentiation culture, the dark culture of 10-15d is carried out first；Then intensity of illumination is
1500-2000Lx, light application time 12h/d, incubation time 90-100d, cultivation temperature are 20 ± 1 DEG C；
E culture of rootage: being the tissue-cultured seedling with the molded blade of 4-6 piece, high 1-2cm by protocorm differential growth in step d,
It is shallow to be inserted into root media after cutting off its root system, depth 1-2mm, after being transferred to root media, progress 10-15d's first
Dark culture, subsequent intensity of illumination are 1500-2000Lx, light application time 12h/d, incubation time 120-130d；Cultivation temperature is 20
±1℃；
The cultivation of f hardening: it is long to when having 6-8 piece blade, high 4-5cm, having 4-8 root root system when seedling in step e, it moves to natural light
After hardening 10-15d, bottle cap is opened, continues hardening 4-6d, later from the culture taken out in tissue culture bottle and on completely clean plant
Base dries rear surface planting moisturizing in decomposed pine bark and cultivates；The same day sprays water in tissue culture miaoye after plant, and 4-5d is not spilt later
Water；Every 1d spills 1-2 water in 15-20d later, and subsequent 1-2d spills a water, and incubation time 80-90d is raw to tissue-cultured seedling new root
After out, it is transferred to greenhouse Routine Management.
Further, seed broadcasting mode described in step c are as follows: after sterile capsule is cut off in sterile petri dish, with nothing
Bacterium suction pipe draws sterile water in culture dish, is evenly mixed in seed in sterile water, and it is seed-bearing sterile then to draw mixing
Water uniform broadcasting is on the induction medium.
Further, the composition of induced medium described in step c are as follows: MS basal medium+sucrose 20g/L+ agar
5.5g/L+ banana puree 60g/L, pH 5.3-5.4.
Further, cultivation temperature described in step c is 20 ± 1 DEG C, intensity of illumination 1000-1200Lx, light application time
For 12h/d, incubation time 60-68d.
Further, Proliferation, Differentiation culture medium described in step d forms are as follows: 1/2MS+ 6-BA 0.1mg/L+IBA 0.2-
0.4 mg/L+PVP 1-4g/L+ sucrose 20g/L+ agar 5.5g/L+ coconut milk 40ml/L, pH 5.3-5.4.
Further, 15-20 protocorm of every bottle of inoculation when being inoculated with described in step d.
Further, root media described in step e forms are as follows: 1/2 MS+6-BA 0.1mg/L+IBA 0.5-
0.7 mg/L+PVP 1-4 g/L+sucrose 20g/L+ agar 5.5g/L+ banana puree 60g/L, pH 5.3-5.4.
Further, in transplanting culture described in step f, culture substrate is the pine bark of 10-15cm thickness, diameter 3-5mm,
Soil humidity is maintained at 85%-90%.
Compared with prior art, the beneficial effects of the present invention are:
The invention discloses a kind of acquisition of high eyebrow groove and tongue orchid capsule and the methods for obtaining seedling using its seminal propagation.It is abundant
High eyebrow groove and tongue orchid plant resources are protected, extremely low, the wild capsule for few, the natural germination rate of current high eyebrow groove and tongue orchid wild plant
It is extremely rare, lack the problems such as mating system, the present invention near-earth protect and the growth adaptation Journal of Sex Research of many years after, selection is different
Point, adaptable, eugonic plant are introduced a fine variety, carries out artificial pollination using the plant from different population, secure good health capsule
Fruit；Carry out breeding research using seed in the capsule maturity period, is planted by aseptic seeding sprouting, Proliferation, Differentiation, strengthening seedling and rooting, hardening
The processes such as training, the problems such as overcoming serious browning, obtain the blue healthy seedling of high-quality high eyebrow groove and tongue.The present invention, which uses, has certain heredity
The mode that artificial pollination is carried out between the plant of distance obtains capsule, setting percentage 100%；Establish the fast traditional font of high eyebrow groove and tongue orchid
System, breeding coefficient reach 7 times or more, and planting percent of taking root reaches 90% or more, to protect the wild resource of the species to establish science
Technical foundation；The excellent high eyebrow groove and tongue orchid seedling obtained, provides resource base and technical support for its sustainable use, actively promotes
The industrialized development of the plant resources has particularly important realistic meaning and economic benefit.The present invention overcomes natural items
High eyebrow groove and tongue orchid capsule is rare under part, can not collect the difficulty of capsule, has also broken high eyebrow groove and tongue orchid species and has sprouted item naturally
Part requires limitation high, that germination rate is extremely low, opens a kind of approach that high eyebrow groove and tongue orchid quickly breeds.The present invention is first using something lost
It passes the plant of distance relatively far away from and carries out artificial pollination acquisition capsule, it is then respective by selection in each stage of seed culture
The conditions such as suitable temperature, illumination are cultivated, and promotion seed germination rate is greatly improved, tissue-cultured seedling growth is the rapidest；
Meanwhile browning is particularly evident during the high fast numerous test of eyebrow groove and tongue orchid species, the present invention is in Proliferation, Differentiation culture and takes root
Using the training method of optical culture after first dark culture in culture, and adsorbent appropriate, appropriate is added, greatly reduces tissue-cultured seedling
Browning.The present invention carries out study on reproduction by obtaining high eyebrow groove and tongue orchid capsule, and using seed, establishes its fast breeding technique
System realizes a large amount of artificial breedings of high eyebrow groove and tongue orchid, and for the minimum population, Mount Emei is peculiar, populations of rare or endangered species
Resource base is established in recovery, is laid the foundation for the factorial production of the plant from now on.
Specific embodiment
The present invention will be further described with reference to the examples below, and described embodiment is only present invention a part
Embodiment is not whole embodiment.Based on the embodiments of the present invention, those skilled in the art are not making
Other all embodiments obtained, belong to protection scope of the present invention under the premise of creative work.
Embodiment 1
The acquisition of a capsule: after near-earth protection and the growth adaptation Journal of Sex Research of many years, selection adaptation is strong, eugonic plant
Strain selects the plant from different population within the florescence, carries out artificial pollination, setting percentage 100%, secure good health capsule, awards
Indicate cross combination and pollination time when powder, capsule picking when 385d does not crack after pollination；
The processing of b capsule: the blue uncracked capsule of high eyebrow groove and tongue is picked, is put on superclean bench after tap water rinses 8min
It is spare, aseptically, successively with the ethanol postincubation 35s of mass concentration 75%, the sterilizing of 0.1% mercuric chloride solution of mass concentration
18min, and each personal aseptic water washing 6 times, then blot with aseptic filter paper the moisture on capsule surface；It is cut off in sterile petri dish
After sterile capsule, sterile water is drawn in culture dish with aseptic straw, is evenly mixed in seed in sterile water；
C Fiber differentiation: drawing and mix seed-bearing sterile water uniform broadcasting on the induction medium, and seed is brown ceramic powder at this time
Shape, it is mature；Culture bottle contamination rate is 0%, no microbiological contamination phenomenon；The induced medium is basic culture medium with MS, is added in every liter
Add 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C, and light application time is
12h/d, intensity of illumination is between 1000-1200Lx；It can induce protocorm after cultivating 27d, 60d is until it can be seen that leaf is former for culture
When base, protocorm is chosen, is inoculated on proliferation and differential medium；Seed inducement rate is 90% or more；
D Proliferation, Differentiation culture: Proliferation, Differentiation culture medium is basic culture medium with 1/2MS, added in every liter 0.1mg 6-BA,
0.2mg IBA, 2gPVP, 20g sucrose, 5.5g agar and 40ml coconut milk, for pH between 5.3-5.4, cultivation temperature is 20 ± 1
℃；The dark culture of 10d is first carried out after switching, subsequent light application time is 12h/d, and intensity of illumination is between 1500-2000Lx, inoculation
When every bottle of inoculation 20 Protocorm 35d after protocorm be largely proliferated, growth coefficient 7；Browning situation obtains obviously
Improve, for melting brown rate less than 10%, the transfer protocorm originally formed can be grown to serve as healthy and strong tissue-cultured seedling at 90d days, take out length
At least tissue-cultured seedling of 4 young leaves, high 1-2cm out is transferred on root media after old of excision；
E culture of rootage: root media is basic culture medium with 1/2MS, added in every liter 0.1mg6-BA, 0.5mg IBA,
2gPVP, 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C；After switching first
The dark culture of 10d is carried out, subsequent light application time is 12h/d, and for intensity of illumination between 1500-2000Lx, the tissue-cultured seedling of transfer is shallow
It is inserted into root media, depth 1-2mm；After cultivating 120d, new root production rate reaches 100%, and new root is strong and healthy and brown
Rate is less than 5%；
The cultivation of f hardening: when tissue-cultured seedling it is long to tool 6 and with blade, high 4-5cm, have 4-8 root root system when, moved to nature
After light lower refining seedling 10d, bottle cap is opened, continues hardening 4d；Without microbiological contamination situation after training tissue culture seedling, tissue-cultured seedling is carefully taken out, with certainly
Water rinses the culture medium of root well, and it is 4d after transplanting in the pine bark of 3-5mm that wet decomposed, diameter is planted after drying
It does not sprinkle water；Every 1d spills 2 water in 20d later, and subsequent 1-2d spills a water, incubation time 90d；Tissue-cultured seedling new root production rate
90% or more, it is subsequently transferred to greenhouse Routine Management.
Embodiment 2
The acquisition of a capsule: after near-earth protection and the growth adaptation Journal of Sex Research of many years, selection adaptation is strong, eugonic plant
Strain selects the plant from different population within the florescence, carries out artificial pollination, setting percentage 100%, secure good health capsule；It awards
Indicate cross combination and pollination time when powder, capsule picking when 520d does not crack after pollination.
The processing of b capsule: the blue uncracked capsule of high eyebrow groove and tongue is picked, is put into ultra-clean work after tap water rinses 8min
It is spare on platform.Aseptically, successively with the ethanol postincubation 35s of mass concentration 75%, the sterilizing of 0.1% mercuric chloride solution of mass concentration
18min, and each personal aseptic water washing 6 times, then the moisture on capsule surface is blotted with aseptic filter paper, sterile capsule is splitted with sterilizing knife
Fruit；
C Fiber differentiation: in seed access induced medium, seed is brown powder, full maturity, culture bottle microbiological contamination at this time
Rate is 0%, no microbiological contamination phenomenon；Induced medium is basic culture medium with MS, adds 20g sucrose, 5.5g agar and 60g in every liter
Banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C, light application time 12h/d, intensity of illumination 1000-
Between 1200Lx；It can induce protocorm after cultivating 31d, culture 68d chooses protocorm group, connect until it can be seen that when phyllopodium
In kind to proliferation and differential medium；Seed inducement rate is 90% or more；
D Proliferation, Differentiation culture: proliferation and differential medium are basic culture medium with 1/2MS, added in every liter 0.1mg 6-BA,
0.4mg IBA, 4g PVP, 20g sucrose, 5.5g agar and 40ml coconut milk, for pH between 5.3-5.4, cultivation temperature is 20 ± 1
℃；The dark culture of 14d time is first carried out after switching, subsequent light application time is 12h/d, and intensity of illumination is between 1500-2000Lx；
Protocorm is proliferated after 15 Protocorm 38d of every bottle of inoculation when inoculation, and growth coefficient 5 has 50% or so tissue-cultured seedling
It will appear browning situation；The transfer protocorm originally formed can be grown to serve as healthy and strong tissue-cultured seedling when at 100d days.Take out length
The tissue-cultured seedling of 4 or more young leaves, high 1-2cm out is transferred on root media after old of excision.
E culture of rootage: root media is basic culture medium with 1/2MS, adds 0.1mg6-BA, 0.7mg in every liter
IBA, 2gPVP, 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C；Switching
The dark culture of 14d is first carried out afterwards, and subsequent light application time is 12h/d, and intensity of illumination is between 1500-2000Lx；The tissue culture of transfer
Seedling is shallowly inserted into root media, depth 1-2mm；After cultivating 120d, new root production rate reaches 100%, but root growth situation one
As；Melting brown rate is fine, less than 10%；
The cultivation of f hardening: when tissue-cultured seedling it is long to tool 6 and with blade, high 4-5cm, have 4-8 root root system when, moving to natural light
After lower refining seedling 14d, bottle cap is opened, continues hardening 6d；Tissue-cultured seedling has 10% or so microbiological contamination；The careful tissue-cultured seedling for taking out non-microbiological contamination is used
Tap water rinses the culture medium of root well, and it is in the pine bark of 3-5mm that wet decomposed, diameter is planted after drying.After transplanting
5d does not sprinkle water；Every 1d spills 1-2 water in 20d later, and subsequent 1-2d spills a water.Incubation time is 90d；Tissue-cultured seedling new root generates
Rate is 90% or more, is transferred to greenhouse Routine Management.
Embodiment 3
The acquisition of a capsule: after near-earth protection and the growth adaptation Journal of Sex Research of many years, selection adaptation is strong, eugonic plant
Strain selects the plant from different population within the florescence, carries out artificial pollination, setting percentage 100%, secure good health capsule, awards
Indicate cross combination and pollination time when powder, capsule picking when 500d does not crack after pollination；
The processing of b capsule: the blue uncracked capsule of high eyebrow groove and tongue is picked, is put on superclean bench after tap water rinses 5min
It is spare, aseptically, successively with the ethanol postincubation 30s of mass concentration 75%, the sterilizing of 0.1% mercuric chloride solution of mass concentration
15min, and each personal aseptic water washing 4 times, then blot with aseptic filter paper the moisture on capsule surface；It is cut off in sterile petri dish
After sterile capsule, sterile water is drawn in culture dish with aseptic straw, is evenly mixed in seed in sterile water；
C Fiber differentiation: drawing and mix seed-bearing sterile water uniform broadcasting on the induction medium, and seed is brown ceramic powder at this time
Shape, it is mature；Culture bottle contamination rate is 0%, no microbiological contamination phenomenon；The induced medium is basic culture medium with MS, is added in every liter
Add 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C, and light application time is
12h/d, intensity of illumination is between 1000-1200Lx；It can induce protocorm after cultivating 28d, 65d is until it can be seen that leaf is former for culture
When base, protocorm is chosen, is inoculated on proliferation and differential medium；Seed inducement rate is 90% or more；
D Proliferation, Differentiation culture: Proliferation, Differentiation culture medium is basic culture medium with 1/2MS, added in every liter 0.1mg 6-BA,
0.4mg IBA, 1gPVP, 20g sucrose, 5.5g agar and 40ml coconut milk, for pH between 5.3-5.4, cultivation temperature is 20 ± 1
℃；The dark culture of 13d is first carried out after switching, subsequent light application time is 12h/d, and intensity of illumination is between 1500-2000Lx, inoculation
When every bottle of inoculation 16 Protocorm 35d after protocorm be largely proliferated, growth coefficient 7；Browning situation obtains obviously
Improve, for melting brown rate less than 30%, the transfer protocorm originally formed can be grown to serve as healthy and strong tissue-cultured seedling at 94d days, take out length
At least tissue-cultured seedling of 4 young leaves, high 1-2cm out is transferred on root media after old of excision；
E culture of rootage: root media is basic culture medium with 1/2MS, added in every liter 0.1mg6-BA, 0.6mg IBA,
4gPVP, 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C；After switching first
The dark culture of 15d is carried out, subsequent light application time is 12h/d, and for intensity of illumination between 1500-2000Lx, the tissue-cultured seedling of transfer is shallow
It is inserted into root media, depth 1-2mm；After cultivating 120d, new root production rate reaches 100%, and new root is strong and healthy and brown
Rate is less than 35%；
The cultivation of f hardening: when tissue-cultured seedling it is long to tool 6 and with blade, high 4-5cm, have 4-8 root root system when, moved to nature
After light lower refining seedling 13d, bottle cap is opened, continues hardening 5d；Without microbiological contamination situation after training tissue culture seedling, tissue-cultured seedling is carefully taken out, with certainly
Water rinses the culture medium of root well, and it is 4d after transplanting in the pine bark of 3-5mm that wet decomposed, diameter is planted after drying
It does not sprinkle water；Every 1d spills 2 water in 20d later, and subsequent 1-2d spills a water, incubation time 90d；Tissue-cultured seedling new root production rate
90% or more, it is subsequently transferred to greenhouse Routine Management.
Embodiment 4
The acquisition of a capsule: after near-earth protection and the growth adaptation Journal of Sex Research of many years, selection adaptation is strong, eugonic plant
Strain selects the plant from different population within the florescence, carries out artificial pollination, setting percentage 100%, secure good health capsule, awards
Indicate cross combination and pollination time when powder, capsule picking when 380d does not crack after pollination；
The processing of b capsule: the blue uncracked capsule of high eyebrow groove and tongue is picked, is put into superclean bench after tap water rinses 10min
It is upper spare, aseptically, successively with the ethanol postincubation 32s of mass concentration 75%, the sterilizing of 0.1% mercuric chloride solution of mass concentration
16min, and each personal aseptic water washing 5 times, then blot with aseptic filter paper the moisture on capsule surface；It is cut off in sterile petri dish
After sterile capsule, sterile water is drawn in culture dish with aseptic straw, is evenly mixed in seed in sterile water；
C Fiber differentiation: drawing and mix seed-bearing sterile water uniform broadcasting on the induction medium, and seed is brown ceramic powder at this time
Shape, it is mature；Culture bottle contamination rate is 0%, no microbiological contamination phenomenon；The induced medium is basic culture medium with MS, is added in every liter
Add 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C, and light application time is
12h/d, intensity of illumination is between 1000-1200Lx；It can induce protocorm after cultivating 29d, 66d is until it can be seen that leaf is former for culture
When base, protocorm is chosen, is inoculated on proliferation and differential medium；Seed inducement rate is 90% or more；
D Proliferation, Differentiation culture: Proliferation, Differentiation culture medium is basic culture medium with 1/2MS, added in every liter 0.1mg 6-BA,
0.2mg IBA, 3gPVP, 20g sucrose, 5.5g agar and 40ml coconut milk, for pH between 5.3-5.4, cultivation temperature is 20 ± 1
℃；The dark culture of 15d is first carried out after switching, subsequent light application time is 12h/d, and intensity of illumination is between 1500-2000Lx, inoculation
When every bottle of inoculation 19 Protocorm 35d after protocorm be largely proliferated, growth coefficient 7；Browning situation obtains obviously
Improve, for melting brown rate less than 35%, the transfer protocorm originally formed can be grown to serve as healthy and strong tissue-cultured seedling at 92d days, take out length
At least tissue-cultured seedling of 4 young leaves, high 1-2cm out is transferred on root media after old of excision；
E culture of rootage: root media is basic culture medium with 1/2MS, added in every liter 0.1mg6-BA, 0.7mg IBA,
1gPVP, 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C；After switching first
The dark culture of 13d is carried out, subsequent light application time is 12h/d, and for intensity of illumination between 1500-2000Lx, the tissue-cultured seedling of transfer is shallow
It is inserted into root media, depth 1-2mm；After cultivating 120d, new root production rate reaches 100%, and new root is strong and healthy and brown
Rate not 25%；
The cultivation of f hardening: when tissue-cultured seedling it is long to tool 6 and with blade, high 4-5cm, have 4-8 root root system when, moved to nature
After light lower refining seedling 12d, bottle cap is opened, continues hardening 4d；Without microbiological contamination situation after training tissue culture seedling, tissue-cultured seedling is carefully taken out, with certainly
Water rinses the culture medium of root well, and it is 4d after transplanting in the pine bark of 3-5mm that wet decomposed, diameter is planted after drying
It does not sprinkle water；Every 1d spills 2 water in 20d later, and subsequent 1-2d spills a water, incubation time 90d；Tissue-cultured seedling new root production rate
90% or more, it is subsequently transferred to greenhouse Routine Management.
Embodiment 5
The acquisition of a capsule: after near-earth protection and the growth adaptation Journal of Sex Research of many years, selection adaptation is strong, eugonic plant
Strain selects the plant from different population within the florescence, carries out artificial pollination, setting percentage 100%, secure good health capsule, awards
Indicate cross combination and pollination time when powder, capsule picking when 450d does not crack after pollination；
The processing of b capsule: the blue uncracked capsule of high eyebrow groove and tongue is picked, is put on superclean bench after tap water rinses 7min
It is spare, aseptically, successively with the ethanol postincubation 33s of mass concentration 75%, the sterilizing of 0.1% mercuric chloride solution of mass concentration
17min, and each personal aseptic water washing 6 times, then blot with aseptic filter paper the moisture on capsule surface；It is cut off in sterile petri dish
After sterile capsule, sterile water is drawn in culture dish with aseptic straw, is evenly mixed in seed in sterile water；
C Fiber differentiation: drawing and mix seed-bearing sterile water uniform broadcasting on the induction medium, and seed is brown ceramic powder at this time
Shape, it is mature；Culture bottle contamination rate is 0%, no microbiological contamination phenomenon；The induced medium is basic culture medium with MS, is added in every liter
Add 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C, and light application time is
12h/d, intensity of illumination is between 1000-1200Lx；It can induce protocorm after cultivating 30d, 67d is until it can be seen that leaf is former for culture
When base, protocorm is chosen, is inoculated on proliferation and differential medium；Seed inducement rate is 90% or more；
D Proliferation, Differentiation culture: Proliferation, Differentiation culture medium is basic culture medium with 1/2MS, added in every liter 0.1mg 6-BA,
0.3mg IBA, 2gPVP, 20g sucrose, 5.5g agar and 40ml coconut milk, for pH between 5.3-5.4, cultivation temperature is 20 ± 1
℃；The dark culture of 12d is first carried out after switching, subsequent light application time is 12h/d, and intensity of illumination is between 1500-2000Lx, inoculation
When every bottle of inoculation 18 Protocorm 35d after protocorm be largely proliferated, growth coefficient 7；Browning situation obtains obviously
Improve, for melting brown rate less than 10%, the transfer protocorm originally formed can be grown to serve as healthy and strong tissue-cultured seedling at 96d days, take out length
At least tissue-cultured seedling of 4 young leaves, high 1-2cm out is transferred on root media after old of excision；
E culture of rootage: root media is basic culture medium with 1/2MS, added in every liter 0.1mg6-BA, 0.5mg IBA,
3gPVP, 20g sucrose, 5.5g agar and 60g banana puree, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C；After switching first
The dark culture of 12d is carried out, subsequent light application time is 12h/d, and for intensity of illumination between 1500-2000Lx, the tissue-cultured seedling of transfer is shallow
It is inserted into root media, depth 1-2mm；After cultivating 120d, new root production rate reaches 100%, and new root is strong and healthy and brown
Rate is less than 30%；
The cultivation of f hardening: when tissue-cultured seedling it is long to tool 6 and with blade, high 4-5cm, have 4-8 root root system when, moved to nature
After light lower refining seedling 15d, bottle cap is opened, continues hardening 4d；Without microbiological contamination situation after training tissue culture seedling, tissue-cultured seedling is carefully taken out, with certainly
Water rinses the culture medium of root well, and it is 4d after transplanting in the pine bark of 3-5mm that wet decomposed, diameter is planted after drying
It does not sprinkle water；Every 1d spills 2 water in 20d later, and subsequent 1-2d spills a water, incubation time 90d；Tissue-cultured seedling new root production rate
90% or more, it is subsequently transferred to greenhouse Routine Management.
Comparative example 1
A, after near-earth protection and the growth adaptation Journal of Sex Research of many years, selection adaptation is strong, eugonic plant, at the florescence
It is interior, the plant from different population is selected, artificial pollination is carried out, setting percentage 100% obtains capsule；Hybridization group is indicated when pollination
It closes and pollination time, capsule 370d and picking when not cracking after pollination.
B, the blue uncracked capsule of high eyebrow groove and tongue is picked, is put into after tap water rinses 5min spare on superclean bench.
Aseptically, ethanol postincubation 30s, 0.1% mercuric chloride solution of the mass concentration sterilizing 15min of mass concentration 75% are successively used, and
Each personal aseptic water washing 4 times, then blot with aseptic filter paper the moisture on capsule surface；Sterile capsule is cut off in sterile petri dish
Afterwards, sterile water is drawn in culture dish with aseptic straw, be evenly mixed in seed in sterile water, then draw and be mixed with seed
Sterile water uniform broadcasting on the induction medium.Seed is partial white part brown, not yet full maturity at this time.Culture bottle
Contamination rate is 0%, no microbiological contamination phenomenon.
C, induced medium is basic culture medium with MS, adds 20g sucrose, 5.5g agar, 60g banana puree, pH in every liter
Between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C, and light application time 12h/d, intensity of illumination is between 1500-2000Lx.Training
It can induce protocorm after supporting 37d.68d is cultivated until proliferation being inoculated into and differentiation being trained it can be seen that choose protocorm when phyllopodium
It supports on base.Seed inducement rate is 30% or more.
D, Proliferation, Differentiation culture medium is basic culture medium with 1/2MS, adds 0.1mg 6-BA, 0.2 mg IBA in every liter,
20g sucrose, 5.5g agar, 60g banana puree, 2g active carbon, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C, when illumination
Between be 12h/d, intensity of illumination is between 1500-2000Lx.15 Protocorms of every bottle of inoculation when inoculation.Browning situation is very
Seriously, originally it is white root gradually melanism, then rots.
Comparative example 2
A, after near-earth protection and the growth adaptation Journal of Sex Research of many years, selection adaptation is strong, eugonic plant, at the florescence
It is interior, the plant from different population is selected, artificial pollination is carried out, setting percentage 100% obtains capsule；Hybridization group is indicated when pollination
It closes and pollination time, capsule 370d and picking when not cracking after pollination.
B, the blue uncracked capsule of high eyebrow groove and tongue is picked, is put into after tap water rinses 5min spare on superclean bench.
Aseptically, ethanol postincubation 30s, 0.1% mercuric chloride solution of the mass concentration sterilizing 15min of mass concentration 75% are successively used, and
Each personal aseptic water washing 4 times, then blot with aseptic filter paper the moisture on capsule surface；Sterile capsule is cut off in sterile petri dish
Afterwards, sterile water is drawn in culture dish with aseptic straw, be evenly mixed in seed in sterile water, then draw and be mixed with seed
Sterile water uniform broadcasting on the induction medium.Seed is partial white part brown, not yet full maturity at this time.Culture bottle
Contamination rate is 0%, no microbiological contamination phenomenon.
C, induced medium is basic culture medium with MS, adds 20g sucrose, 5.5g agar, 60g banana puree, pH in every liter
Between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C, and light application time 12h/d, intensity of illumination is between 1500-2000Lx；Training
It can induce protocorm after supporting 37d；68d is cultivated until proliferation being inoculated into and differentiation being trained it can be seen that choose protocorm when phyllopodium
It supports on base.Seed inducement rate is 30% or more；
D, Proliferation, Differentiation culture medium is basic culture medium with 1/2MS, added in every liter 0.1mg 6-BA, 0.2mg IBA, 2gPVP,
20g sucrose, 5.5g agar and 40ml coconut milk, for pH between 5.3-5.4, cultivation temperature is 20 ± 1 DEG C；Light application time is 12h/
D, intensity of illumination is between 1500-2000Lx；15 Protocorms of every bottle of inoculation when inoculation；There is 80% or so tissue-cultured seedling meeting
There is browning situation.
By comparative example 1 it is found that capsule is picked in non-full maturity, Seed inducement rate is extremely low in later period Fiber differentiation.
In comparative example 1, Proliferation, Differentiation culture is carried out by the way of using active carbon in full exposure and culture medium, from cultivation results
From the point of view of, it is originally white root gradually melanism that browning situation is very serious, and therefore, the PVP of the application is in the effect for inhibiting browning
It is significantly better than active carbon.By embodiment 1 and comparative example 2 it is found that light is trained after the first dark culture in Proliferation, Differentiation incubation step
Supporting can be effectively suppressed browning；By comparative example 2 and embodiment 2 it is found that the application uses first dark culture in Proliferation, Differentiation culture
The training method of optical culture afterwards, and adsorbent appropriate, appropriate is added, greatly reduce the browning of tissue-cultured seedling.By implementing
Example 2 is it is found that the present invention equally uses the training method of optical culture after first dark culture in culture of rootage, and adds in right amount, suitably
Adsorbent, further reduced the browning of tissue-cultured seedling.
Disclosed above is only presently preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, therefore makees ground equivalent variations according to the claims in the present invention, be still within the scope of the present invention.
Claims (8)
1. the rapid propagation method of high eyebrow groove and tongue orchid, it is characterised in that: the following steps are included:
The acquisition of a capsule: selection adaptation is strong, grows plant that is vigorous and can smoothly completing a life cycle, within the florescence,
It is respectively that the Parent pollinated carries out artificial pollination using the plant from different population, obtains capsule, capsule is after pollination
Picking when not cracking after 380-520d；
The processing of b capsule: after the high eyebrow groove and tongue orchid capsule of picking is rinsed 5~10min under tap water, water is blotted with filter paper
Point；75% 30~35s of ethanol postincubation of mass concentration, 0.1% mercuric chloride solution of mass concentration, 15~18min of sterilizing are then used respectively, and
Each personal aseptic water washing 4~6 times, finally blots the moisture on surface with aseptic filter paper；Capsule is aseptically cut off to obtain
Seed；
C Fiber differentiation: on the induction medium by the seed uniform broadcasting obtained in step b, protocorm is induced；
D Proliferation, Differentiation culture: the protocorm of green phyllopodium clearly visible in step c is inoculated on Proliferation, Differentiation culture medium
Carry out Proliferation, Differentiation culture；In the Proliferation, Differentiation culture, the dark culture of 10-15d is carried out first；Then intensity of illumination is
1500-2000Lx, light application time 12h/d, incubation time 90-100d, cultivation temperature are 20 ± 1 DEG C；
E culture of rootage: being the tissue-cultured seedling with the molded blade of 4-6 piece, high 1-2cm by protocorm differential growth in step d,
It is shallow to be inserted into root media after cutting off its root system, depth 1-2mm, after being transferred to root media, progress 10-15d's first
Dark culture, subsequent intensity of illumination are 1500-2000Lx, light application time 12h/d, incubation time 120-130d；Cultivation temperature is 20
±1℃；
The cultivation of f hardening: it is long to when having 6-8 piece blade, high 4-5cm, having 4-8 root root system when seedling in step e, it moves to natural light
After hardening 10-15d, bottle cap is opened, continues hardening 4-6d, later from the culture taken out in tissue culture bottle and on completely clean plant
Base dries rear surface planting moisturizing in decomposed pine bark and cultivates；The same day sprays water in tissue culture miaoye after plant, and 4-5d is not spilt later
Water；Every 1d spills 1-2 water in 15-20d later, and subsequent 1-2d spills a water, and incubation time 80-90d is raw to tissue-cultured seedling new root
After out, it is transferred to greenhouse Routine Management.
2. the rapid propagation method of high eyebrow groove and tongue orchid according to claim 1, it is characterised in that: seed described in step c
Broadcast sowing mode are as follows: after sterile capsule is cut off in sterile petri dish, draw sterile water in culture dish with aseptic straw, make seed
It is evenly mixed in sterile water, then draws and mix seed-bearing sterile water uniform broadcasting on the induction medium.
3. the rapid propagation method of high eyebrow groove and tongue orchid according to claim 1, it is characterised in that: induced described in step c
The composition of culture medium are as follows: MS basal medium+sucrose 20g/L+ agar 5.5g/L+ banana puree 60g/L, pH 5.3-5.4.
4. the rapid propagation method of high eyebrow groove and tongue orchid according to claim 1, it is characterised in that: cultivated described in step c
Temperature is 20 ± 1 DEG C, intensity of illumination 1000-1200Lx, light application time 12h/d, incubation time 60-68d.
5. the rapid propagation method of high eyebrow groove and tongue orchid according to claim 1, it is characterised in that: be proliferated described in step d
Differential medium composition are as follows: 1/2MS+ 6-BA 0.1mg/L+IBA 0.2-0.4 mg/L+PVP 1-4g/L+ sucrose 20g/L+
Agar 5.5g/L+ coconut milk 40ml/L, pH 5.3-5.4.
6. the rapid propagation method of high eyebrow groove and tongue orchid according to claim 1, it is characterised in that: be inoculated with described in step d
When 15-20 protocorm of every bottle of inoculation.
7. the rapid propagation method of high eyebrow groove and tongue orchid according to claim 1, it is characterised in that: take root described in step e
Culture medium composition are as follows: 1/2 MS+6-BA 0.1mg/L+IBA 0.5-0.7 mg/L+PVP 1-4 g/L+sucrose 20g/L+
Agar 5.5g/L+ banana puree 60g/L, pH 5.3-5.4.
8. the rapid propagation method of high eyebrow groove and tongue orchid according to claim 1, it is characterised in that: transplanted described in step f
In culture, culture substrate is the pine bark of 10-15cm thickness, diameter 3-5mm, and soil humidity is maintained at 85%-90%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104429965A (en) * | 2014-12-16 | 2015-03-25 | 四川省自然资源科学研究院 | Hormone-free rapid tissue culture and propagation method of Emei roxburgh anoectochilus terminal bud seeds |
| CN104996301A (en) * | 2015-08-06 | 2015-10-28 | 云南省农业科学院花卉研究所 | Rapid propagation method for holcoglossum flavescens |
| CN107980612A (en) * | 2016-10-26 | 2018-05-04 | 黔西南州绿缘动植物科技开发有限公司 | A kind of blue quick breeding of short distance groove and tongue and bloom controlling method |
-
2019
- 2019-04-16 CN CN201910305770.XA patent/CN109964817B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104429965A (en) * | 2014-12-16 | 2015-03-25 | 四川省自然资源科学研究院 | Hormone-free rapid tissue culture and propagation method of Emei roxburgh anoectochilus terminal bud seeds |
| CN104996301A (en) * | 2015-08-06 | 2015-10-28 | 云南省农业科学院花卉研究所 | Rapid propagation method for holcoglossum flavescens |
| CN107980612A (en) * | 2016-10-26 | 2018-05-04 | 黔西南州绿缘动植物科技开发有限公司 | A kind of blue quick breeding of short distance groove and tongue and bloom controlling method |
Non-Patent Citations (3)
| Title |
|---|
| 周丽等: "短距槽舌兰的驯化栽培与快速繁殖", 《植物生理学报》 * |
| 朱习红等: "峨眉槽舌兰栽培的竹炭基质应用分析", 《科学种养》 * |
| 秦小波等: "槽舌兰资源调查及特有品种试验研究", 《农业与技术》 * |
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