CN105340757B - A kind of method for tissue culture of Cymbidium lianpan and its application - Google Patents
A kind of method for tissue culture of Cymbidium lianpan and its application Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a kind of method for tissue culture of Cymbidium lianpan and its application.Cymbidium lianpan aseptic seedling stem apex is explant by the present invention, using proliferated culture medium induction root-like stock propagation, using LED as tissue culture light source, is cultivated with blue light；Then obtained root-like stock is moved into subculture growth medium, the growth of induction Multiple Buds and sprouting and rooting, using LED as tissue culture light source, cultivated with feux rouges；Newborn seedling is connected to root media and carries out strengthening seedling and rooting culture, using LED as tissue culture light source, with proportion 2~4：6~8 proportioning feux rouges mixing blue lights are cultivated.It is low that the present invention effectively solves bud ratio present in conventional tissue culture technique, slow-growing, the problems such as difficulty of taking root；And LED lamp is used as tissue culture light source, there are saving electric resources 30 50%, reduce cost, and improve the efficiency of factorial praluction.
Description
Technical field
The present invention relates to plant tissue fast breeding technique field, the method for tissue culture of more particularly to a kind of Cymbidium lianpan and its should
With.
Background technology
Cymbidium lianpan is the big monoid in Chinese Terrestrial orchid category, main product Yunnan.Because its plant forms is graceful, flower-shape is changeable,
Pattern is abundant, the market price occupies the reasons such as height, causes Cymbidium lianpan wild resource to go to wreck the excavation of formula, causes resource in imminent danger.
Therefore, the research for Cymbidium lianpan tissue cultures and fast breeding technique is particularly important.However, the growth week of orchid inherently
Phase is long, and differentiation efficiency is low, and the problems such as root system quality is not good hinders industrial seedling rearing and the industry development of Cymbidium lianpan always.
Light quality is to influence an important factor of growth and development of plants.In recent years there are some researches show different light qualities are in plant
The different times of growth can play facilitation.Wherein light emitting diode (light-emitting diodes, LED) is near
The high-quality light source that year grows up, it can send the monochromatic light of accurate wave spectrum, and wave-length coverage is narrower, and amplitude is no more than 20nm, again
Light intensity can be controlled by regulation device.Therefore light quality and the usually used plant tissue of the adjustable LED/light source ratio of intensity of illumination
Culture fluorescent lamp, can more be effectively facilitated the photosynthesis of test tube seedling, have wide application prospect in tissue cultures.
It is more applied to the research of Plant Tissue Breeding on LED at present, but all concentrate on rapid propagation system relative maturity
Species, and the application technology of maturation is yet there are no for the application in Cymbidium lianpan tissue cultures.
The content of the invention
The primary and foremost purpose of the present invention is to overcome the shortcoming and deficiency of prior art, and there is provided a kind of tissue cultures of Cymbidium lianpan
Method.
Another object of the present invention is to the application for the method for tissue culture for providing the Cymbidium lianpan.
The purpose of the present invention is achieved through the following technical solutions：A kind of method for tissue culture of Cymbidium lianpan, including following step
Suddenly：
(1) it is explant by Cymbidium lianpan aseptic seedling stem apex, using proliferated culture medium induction root-like stock propagation and Multiple Buds point
Change, condition of culture is as follows：Using LED as tissue culture light source, 440~480nm of blue light, 20~40 μm of o1m of light intensity-2·s-1,
12~16h/d of photoperiod is cultivated 40~80 days；The composition of proliferated culture medium is：1/2MS culture mediums, 6- benzyl purines (6-BA)
1.5~3.5mg/L, methyl α-naphthyl acetate (NAA) 0.1~0.3mg/L, 10~30g/L of sucrose, 0.5~1g/L of activated carbon, 6~8g/ of agar
L, pH=5.6~5.8；
(2) root-like stock for obtaining step (1) moves to subculture growth medium, the growth of induction Multiple Buds and sprouting and rooting,
Condition of culture is as follows：Using LED as tissue culture light source, 620~660nm of feux rouges, 20~40 μm of o1m of light intensity-2·s-1, light
12~16h/d of cycle is cultivated 30~60 days, improves plant growth rate；The composition of subculture growth medium is：1/2MS is cultivated
Base, 0.1~1.0mg/L of 6-BA, 0.1~0.3mg/L of NAA, 10~30g/L of sucrose, 0.5~1g/L of activated carbon, agar 6~
8g/L, pH=5.6~5.8；
(3) newborn seedling is connected to root media and carries out strengthening seedling and rooting culture, condition of culture is as follows：Group is used as using LED
Knit culture light source, feux rouges (620~660nm) and blue light (440~480nm) in proportion 2~4：6~8 proportionings, the μ of light intensity 30~60
mo1·m-2·s-1, 12~16h/d of photoperiod cultures 30~60 days, the growth elongation of promotion root；The composition of root media is：
1/2MS culture mediums, 0.1~0.3mg/L of 6-BA, 0.2~0.5mg/L of NAA, 10~30g/L of sucrose, 0.5~1g/L of activated carbon,
6~8g/L of agar, pH=5.6~5.8.
Above-mentioned culture can be carried out under the conditions of 20~30 DEG C, relative humidity is 40~60%.
Described blue light be preferably wavelength be 450~480nm blue light.
Described feux rouges be preferably wavelength be 630nm feux rouges.
Proliferated culture medium described in step (1) is preferably 1/2MS culture medium+6-BA 2.5mg/L+NAA 0.3mg/L+
6~8g/L of sucrose 15g/L+ activated carbon 0.5g/L+ agar solid medium (pH=5.6~5.8).
Condition of culture described in step (1) is preferably：Using LED as tissue culture light source, 450~480nm of blue light, light
Strong 20 μm of o1m-2·s-1, photoperiod 16h/d cultures 60 days, environment temperature is 1 DEG C of 24 scholar.
Subculture growth medium described in step (2) is preferably 1/2MS culture medium+6-BA 0.5mg/L+NAA
6~8g/L of 0.2mg/L+ sucrose 20g/L+ activated carbon 0.5g/L+ agar solid medium (pH=5.6~5.8).
Condition of culture described in step (2) is preferably：Using LED as tissue culture light source, feux rouges 630nm, the μ of light intensity 20
mo1·m-2·s-1, photoperiod 16h/d cultures 60 days, environment temperature is 1 DEG C of 24 scholar, relative humidity 50-60%.
Newborn seedling described in step (3) is preferably the new long newborn seedling to 2cm of sprouting.
Root media described in step (3) is preferably 1/2MS culture medium+6-BA 0.2mg/L+NAA 0.5mg/L+
6~8g/L of sucrose 20g/L+ activated carbon 0.5g/L+ agar solid medium (pH=5.6~5.8).
Condition of culture described in step (3) is preferably：Using LED as tissue culture light source, feux rouges and blue light ratio 2：
6,40 μm of o1m of light intensity-2·s-1, photoperiod 16h/d cultures 60 days, environment temperature is 1 DEG C of 24 scholar, relative humidity 50-60%.
Application of the method for tissue culture of the Cymbidium lianpan in large-scale cultivation Cymbidium lianpan.
The present invention has the following advantages and effect relative to prior art：
1. present invention determine that root-like stock proliferative induction is to budding differential period in Cymbidium lianpan tissue culture procedures；Multiple Buds
Rapid growth stage, and strengthening seedling and rooting stage different culture media and condition of culture best configuration, effectively facilitate Cymbidium lianpan high
Imitate quick tissue cultures.
2. present invention determine that root-like stock proliferative induction is to budding differential period in Cymbidium lianpan tissue culture procedures；Multiple Buds
Rapid growth stage, and the strengthening seedling and rooting stage optimal culture condition, including LED light matter and light intensity and processing time, have
It is low that effect solves bud ratio present in conventional tissue culture technique, slow-growing, the problems such as difficulty of taking root.
3. in the present invention, using LED lamp as tissue culture light source, there is saving electric resources 30-50%, reduce into
This, and improve the efficiency of factorial praluction.
Embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
The Cymbidium lianpan tissue culture medium (TCM) optimizing components of embodiment 1 are matched
First, proliferated culture medium
It is (big in MS basal mediums in 1/2MS culture mediums with Cymbidium lianpan Cymbidium Lianpan " heavy snow element " seed
Secondary element and trace element halve) on sprout after growth 14 months aseptic seedling be explant, seedling Multiple Buds are cut into list
Strain, every plant is cut into 2cm or so segments and is seeded to Multiplying culture in proliferated culture medium.Culture medium is with 1/2MS culture mediums+agar 7g/L
For control group, 6- benzyl purines (6-BA) 1.5~3.5mg/L is added, methyl α-naphthyl acetate (NAA) 0.1~0.3mg/L, activated carbon 0.5~
1g/L, sucrose 10~30g/L+ agar 7g/L are optimization group, and specific proportioning is as shown in table 1.PH value=5.6~5.8.
Table 1
| Culture medium prescription | 6-BA(mg/L)1 | NAA(mg/L) | Activated carbon (g/L) | Sucrose (g/L) |
| CK | 0 | 0 | 0 | 15 |
| 1 | 1.5 | 0.1 | 0.5 | 10 |
| 2 | 1.5 | 0.3 | 0.75 | 30 |
| 3 | 2.5 | 0.2 | 1 | 20 |
| 4 | 2.5 | 0.3 | 0.5 | 15 |
| 5 | 3.5 | 0.1 | 0.75 | 30 |
| 6 | 3.5 | 0.2 | 0.5 | 20 |
5 buds of every bottle of inoculation, every kind of culture medium prescription is inoculated with 10 bottles.With the white fluorescent of Philip (PHILIPS) general T 8
Fluorescent tube makees light source.It is 16h/d, 20 μm of o1m of light intensity the photoperiod that condition of culture, which is,-2·s-1, environment temperature is 1 DEG C of 24 scholar, relatively
Humidity 50-60%.After seedling inoculation, root-like stock propagation is can induce, continuous culture is differentiated after 60 days newly sprouts, by 80 days even
The root-like stock number of each inoculation simple bud propagation, root-like stock differentiation budding number, sprouting plant height and growth coefficient are detected after continuous culture.
Simple bud growth coefficient refer to averagely each simple bud through expand it is numerous obtain from sprouting containing workable simple bud number.Plant height vernier
Kind of calliper (is accurate to 0.01mm).
Table 2
Statistical result (table 2) is shown, with formula 4 i.e. 1/2MS culture medium+6-BA 2.5mg/L+NAA in Different Optimization combination
0.3mg/L+ activated carbon 0.5g/L+ sucrose 15g/L (pH=5.6~5.8) cultivation effect is optimal, and growth coefficient is trained up to 1/2MS
Support base control group more than 3 times.
2nd, subculture growth medium
Multiple Buds newborn in step one are divided into individual plant, is directly seeded in subculture growth medium and carries out fast-growth
Culture.5 buds of every bottle of inoculation, each 10 bottles of processing.Culture medium is using 1/2MS culture mediums+agar 7g/L as control group, wherein adding
6- benzyl purines (6-BA) 0.1~1mg/L, methyl α-naphthyl acetate (NAA) 0.1~0.3mg/L, 0.5~1g/L of activated carbon, sucrose 10~
30g/L+ agar 7g/L is optimization group, and specific proportioning is as shown in table 3.PH value=5.6~5.8.
Table 3
| Culture medium prescription | 6-BA(mg/L)1 | NAA(mg/L) | Activated carbon (g/L) | Sucrose (g/L) |
| CK | 0 | 0 | 0 | 15 |
| 1 | 0.1 | 0.1 | 0.5 | 10 |
| 2 | 0.1 | 0.3 | 0.75 | 30 |
| 3 | 0.5 | 0.2 | 1 | 20 |
| 4 | 0.5 | 0.3 | 0.5 | 15 |
| 5 | 1 | 0.1 | 0.75 | 30 |
| 6 | 1 | 0.2 | 0.5 | 20 |
Condition of culture is photoperiod 16h/d, 20 μm of o1m of light intensity-2·s-1, environment temperature is 1 DEG C of 24 scholar, relative humidity
50-60%.Test tube seedling is after 60d is continuously cultivated, the average plant height of detection inoculation bud, individual plant mean number of sheets and fresh weight, Yi Jijie
Seed bud increases bud number newly.Plant height vernier caliper measurement (is accurate to 0.01mm).
Table 4
As a result (table 4) is shown, optimization culture based formulas 3 is 1/2MS culture medium+6-BA 0.5mg/L+NAA 0.2mg/L+
Activated carbon 0.5g/L+ sucrose 20g/L (pH=5.6~5.8) rate of rise is most fast, and newly-increased blade quantity is the two of control group
Times, optimal subculture growth medium is elected in 2.5 times more how close than control group of fresh weight increase as.
3rd, strengthening seedling and rooting culture medium
Newborn seedling in step 2 is seeded to root media and carries out rooting induction and strong seedling culture.Every bottle is inoculated with 5
Bud, each 10 bottles of processing.Culture medium is using 1/2MS minimal mediums+agar 7g/L as control group, using agarose 7g/L.Wherein
Add 6- benzyl purines (6-BA) 0.1~0.3mg/L, methyl α-naphthyl acetate (NAA) 0.2~0.5mg/L, 0.5~1g/L of activated carbon, sucrose
10-30g/L+ agar 7g/L is optimization group, and specific proportioning is as shown in table 5.PH value=5.6~5.8.
Table 5
| Culture medium prescription | 6-BA(mg/L)1 | NAA(mg/L) | Active (g/L) | Sucrose (g/L) |
| CK | 0 | 0 | 0 | 15 |
| 1 | 0.1 | 0.3 | 0.5 | 10 |
| 2 | 0.1 | 0.5 | 0.75 | 30 |
| 3 | 0.2 | 0.2 | 1 | 20 |
| 4 | 0.2 | 0.5 | 0.5 | 20 |
| 5 | 0.3 | 0.2 | 0.75 | 30 |
| 6 | 0.3 | 0.4 | 0.5 | 20 |
Condition of culture is photoperiod 16h/d, 20 μm of o1m of light intensity-2·s-1, environment temperature is 1 DEG C of 24 scholar, relative humidity
50-60%.Test tube seedling is after 60d is continuously cultivated, the average plant height of detection inoculation bud, individual plant mean number of sheets and fresh weight, Yi Jijie
Seed bud is taken root number, and root is long.Plant height and root length vernier caliper measurement (being accurate to 0.01mm).
Table 6
As a result (table 6) is shown, optimization culture based formulas 4 is 1/2MS+6-BA 0.2mg/L+NAA 0.5mg/L+ activated carbons
0.5g/L+ sucrose 20g/L (pH=5.6~5.8) rooting efficiency is optimal, can remarkably promote the elongation of root length, and number of taking root is than control
Group is high about 2.2 times, is used as optimal strong sprout growth medium.
The optimal medium of embodiment 2 collocation LED using effect
First, root-like stock induction and budding differentiation culture
1st, proliferated culture medium
1/2MS+6-BA 2.5mg/L+NAA 0.3mg/L+ activated carbon 0.5g/L+ sucrose 15g/L (pH=5.6~5.8)+
Agar 7g/L solid medium.
2nd, vaccination ways
Cymbidium lianpan seedling (aseptic seedling of growth 14 months after Cymbidium lianpan " heavy snow element " sowing is sprouted) Multiple Buds are cut into
Individual plant, every plant is cut into 2cm or so segments and is seeded to Multiplying culture in proliferated culture medium.5 buds of every bottle of inoculation, each handle 10
Bottle.
3rd, condition of culture
Photoperiod is 16h/d, 20 μm of o1m of light intensity-2·s-1, environment temperature is 1 DEG C of 24 scholar.Experiment sets 5 kinds and not shared the same light altogether
The light source (as shown in table 7) of matter ratio, its ratio is respectively：1. 100%R (feux rouges), 2. 100%B (blue light), 3. 75%R ten
25%B, 4. 75%B+25%R, 5. 50%R+50%B.With conventional fluorescent lamps (PHILIPS, the fluorescent tube of Philip general T 8,
White light) handle as control.LED culturing racks use layer stereo structure, and the high 2.38m of culturing rack, each culture sets up 4 layers, often
Floor height 0.59m.LED lamp tube is arranged on to be posted on the glass plate of lucifuge paper per interlayer, and culturing rack side is blocked using gobo.
Table 7
4th, survey item
After seedling inoculation, root-like stock propagation is can induce, continuous culture is differentiated after 30 days newly sprouted, by continuous training in 60 days
After supporting, survey item includes：The root-like stock number of propagation, sprout number, growth coefficient, plant height.Plant height is (accurate with vernier caliper measurement
To 0.01mm).Table 8 is listed average plant height, average simple bud propagation root-like stock number, budding number and simple bud growth coefficient and (referred to average
Each simple bud through expand it is numerous obtain from sprouting containing workable simple bud number).
Table 8
The result in table 8, after optimum multiplication medium proportioning is selected, culture medium is arranged in pairs or groups different LED light matter
Use, the root-like stock proliferation number highest of T2 treatment groups, show relative to common fluorescent to make using 450-480nm blue lights as light source
Cymbidium lianpan root-like stock propagation efficiency is remarkably improved for light source.
2nd, Multiple Buds fast-growth culture
1st, subculture growth medium
1/2MS+6-BA 0.5mg/L+NAA 0.2mg/L+ activated carbon 0.5g/L+ sucrose 20g/L (pH=5.6~5.8)+
Agar 7g/L+ sucrose 20g/L (pH=5.6~5.8)+solid medium.
2nd, vaccination ways
Multiple Buds newborn in step one are divided into individual plant, progress fast-growth training in subculture growth medium is seeded to
Support.5 buds of every bottle of inoculation, each 10 bottles of processing.
3rd, condition of culture
Photoperiod is 16h/d, 20 μm of o1m of light intensity-2·s-1, environment temperature is 1 DEG C of 24 scholar.Experiment sets 5 kinds and not shared the same light altogether
The light source (as shown in table 9) of matter ratio, its ratio is respectively：1. 100%R, 2. 100%B, 3. 25%B of 75%R ten, 4. 75%B
+ 25%R, 5. 50%R+50%B.Control is used as using conventional fluorescent lamps (PHILIPS) processing.LED culturing racks use layer stereo
Structure, the high 2.38m of culturing rack, each culture sets up 4 layers, per floor height 0.59m.LED lamp tube is arranged on posts lucifuge paper per interlayer
Glass plate on, culturing rack side blocked using gobo.
Table 9
4th, survey item
Test tube seedling is after 60d is continuously cultivated, and survey item has：Average plant height, the individual plant number of sheets increases bud number, fresh weight newly.Strain
Height vernier caliper measurement (is accurate to 0.01mm).As a result it is as shown in table 10
Table 10
It can be seen that, after optimal subculture medium proportioning is selected, culture medium different LED light matter of arranging in pairs or groups are used, T1 processing
The root-like stock proliferation number highest of group, shows relative to common fluorescent as light source can significantly to promote using 630nm feux rouges as light source
Enter the elongation of Cymbidium lianpan root-like stock, improve growth rate.
3rd, strengthening seedling and rooting culture
1st, root media
1/2MS+6-BA0.2mg/L+NAA0.5mg/L+ agar 7g/L+ sucrose 20g/L (pH=5.6~5.8)+activated carbon
0.5g/L solid medium.
2nd, vaccination ways
Newborn seedling in step 2 is seeded to root media and carries out rooting induction and strong seedling culture.Every bottle is inoculated with 5
Bud, each 10 bottles of processing.
3rd, condition of culture
Photoperiod is 16h/d, 40 μm of o1-m of light intensity-2·s-1, environment temperature is 1 DEG C of 24 scholar.Experiment sets 5 kinds and not shared the same light altogether
The light source (as shown in table 11) of matter ratio, its ratio is respectively：1. 100%R, 2. 100%B, 3. 25%B of 75%R ten, 4. 75%
B+25%R, 5. 50%R+50%B.Control is used as using conventional fluorescent lamps (PHILIPS) processing.LED culturing racks use layer stereo
Structure, the high 2.38m of culturing rack, each culture sets up 4 layers, per floor height 0.59m.LED lamp tube is arranged on posts lucifuge paper per interlayer
Glass plate on, culturing rack side blocked using gobo.
Table 11
4th, survey item
Test tube seedling is after 60d is continuously cultivated, and survey item has：Plant height, the number of sheets, number of taking root, root is long.Plant height, root length is used
Vernier caliper measurement (is accurate to 0.01mm).As a result it is as shown in table 12
Table 12
It can be seen that, compared with common fluorescent, feux rouges culture can promote plant height, the elongation growth of blade and root length, but number of taking root
Mesh is without significantly improving, and blue light culture can improve Cymbidium lianpan and newly sprout number of taking root, but to blade and the growth of root length without remarkably promoting
Effect, wherein with T4 processing, i.e., with feux rouges and blue light 1：3 mix and match are optimal as light source effect, can remarkably promote Cymbidium lianpan
Elongation growth and quantity of taking root.
Comparative example
First, root-like stock induction and budding differentiation effect of the different culture media using LED/light source
Optimal medium and contrast culture medium is respectively adopted, is cultivated under LED strip part, compares optimal medium propagation effect
Really.Concrete operation step is as follows：
1st, proliferated culture medium is contrasted
Table 13
| Culture medium prescription | 6-BA(mg/L)1 | NAA(mg/L) | Activated carbon (g/L) | Sucrose (g/L) |
| Contrast groups 1 | 1.5 | 0.1 | 0.5 | 10 |
| Contrast groups 2 | 2.5 | 0.3 | 0.75 | 30 |
| Contrast groups 3 | 3.5 | 0.2 | 1 | 20 |
| Optimization group | 2.5 | 0.3 | 0.5 | 15 |
2nd, vaccination ways
Cymbidium lianpan seedling (aseptic seedling of growth 14 months after Cymbidium lianpan " heavy snow element " sowing is sprouted) Multiple Buds are cut into
Individual plant, every plant is cut into 2cm or so segments and is seeded to Multiplying culture in proliferated culture medium.5 buds of every bottle of inoculation, each handle 10
Bottle.
3rd, condition of culture
It is 16h/d the photoperiods that condition of culture, which is, using 450-480nm blue lights as light source, 20 μm of o1m of light intensity-2·s-1,
Environment temperature is 1 DEG C of 24 scholar, relative humidity 50-60%.
4th, survey item
After seedling inoculation, root-like stock propagation is can induce, continuous culture is differentiated after 30 days newly sprouted, by continuous training in 60 days
Differentiate and newly sprout after supporting, the root-like stock number of each inoculation simple bud propagation, root-like stock differentiation are detected after continuous culture in 80 days
Sprout number, sprouting plant height and growth coefficient, and survey item includes：The root-like stock number of propagation, sprout number, growth coefficient, plant height.
Plant height vernier caliper measurement (is accurate to 0.01mm).Table 14 lists average plant height, average simple bud propagation root-like stock number, budding number
And simple bud growth coefficient (refer to averagely each simple bud through expand it is numerous obtain from sprouting containing workable simple bud number).
Table 14
The result in table 14, the growth coefficient highest of base, table are supported in optimizing tissue culture under the conditions of same LED illumination
It is bright to select optimum multiplication medium (1/2MS+6-BA 2.5mg/L+NAA 0.3mg/L+ activated carbon 0.5g/L+ sucrose 15g/
L (pH=5.6~5.8)) proportioning after, arrange in pairs or groups using 450-480nm blue lights as light source, be remarkably improved Cymbidium lianpan root-like stock propagation
Efficiency.
2nd, different subculture mediums carry out Multiple Buds fast-growth culture using LED/light source
Optimal medium and contrast culture medium is respectively adopted, is cultivated under LED strip part, compares optimal medium growth speed
Rate, concrete operation step is as follows：
1st, subculture growth medium is contrasted
Table 15
| Culture medium prescription | 6-BA(mg/L)1 | NAA(mg/L) | Activated carbon (g/L) | Sucrose (g/L) |
| Contrast groups 1 | 0.1 | 0.3 | 0.75 | 30 |
| Contrast groups 2 | 0.5 | 0.3 | 0.5 | 15 |
| Contrast groups 3 | 1 | 0.2 | 0.5 | 20 |
| Optimization group | 0.5 | 0.2 | 0.5 | 20 |
2nd, vaccination ways
Newborn Multiple Buds are divided into individual plant, progress fast-growth culture in subculture growth medium is seeded to.Every bottle connects
Plant 5 buds, each 10 bottles of processing.
3rd, condition of culture
It is 16h/d the photoperiods that condition of culture, which is, using 630nm feux rouges as light source, 20 μm of o1m of light intensity-2·s-1, environment
Temperature is 1 DEG C of 24 scholar, relative humidity 50-60%.
4th, survey item
Test tube seedling is after 60d is continuously cultivated, and survey item has：Average plant height, the individual plant number of sheets increases bud number, fresh weight newly.Strain
Height vernier caliper measurement (is accurate to 0.01mm).As a result it is as shown in table 16
Table 16
The result in table, the speed of growth of the foster base of optimizing tissue culture is most fast under the conditions of same LED illumination, shows
After optimum multiplication medium proportioning is selected, arrange in pairs or groups using 630nm feux rouges as light source, be remarkably improved the increasing of Cymbidium lianpan root-like stock
Grow efficiency.
3rd, different root medias carry out newborn seedling strengthening seedling and rooting contrast culture using LED/light source
Optimal medium and contrast culture medium is respectively adopted, is cultivated under LED strip part, compares optimal medium and takes root effect
Really, concrete operation step is as follows：
1st, root media is contrasted
Table 17
2nd, vaccination ways
Newborn Multiple Buds are divided into individual plant, progress fast-growth culture in subculture growth medium is seeded to.Every bottle connects
Plant 5 buds, each 10 bottles of processing.
3rd, condition of culture
It is 16h/d the photoperiods that condition of culture, which is, using feux rouges and blue light 2：6 mixing light sources, 20 μm of o1m of light intensity-2·s-1, environment temperature is 1 DEG C of 24 scholar, relative humidity 50-60%.
4th, survey item
Test tube seedling is after 60d is continuously cultivated, and survey item has：Plant height, the number of sheets, number of taking root, root is long.Plant height, root length is used
Vernier caliper measurement (is accurate to 0.01mm).As a result as shown in table 18, it is seen then that compared with common fluorescent, feux rouges culture can promote
Plant height, the elongation growth of blade and root length, but number of taking root are without significantly improving, and blue light culture, which can improve Cymbidium lianpan and newly sprout, takes root
Number, but to blade and the growth of root length without effect is remarkably promoted, with feux rouges and blue light 2：6 mixing light source processing, can be remarkably promoted
Cymbidium lianpan elongation growth and quantity of taking root.
Table 18
The result in table, the number of taking root of the foster base of optimizing tissue culture is most under the conditions of same LED illumination and root is long
It is most long, show after optimum multiplication medium proportioning is selected, collocation is with using feux rouges 630nm and blue light 450-480nm 1：3
Mixing can remarkably promote Cymbidium lianpan and take root and the elongation of root length as light source.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
1. a kind of method for tissue culture of Cymbidium lianpan, it is characterised in that comprise the following steps：
(1) it is explant by Cymbidium lianpan aseptic seedling stem apex, using proliferated culture medium induction root-like stock propagation and differentiation, training
The condition of supporting is as follows：Using LED as tissue culture light source, 450~480nm of blue light, 20~40 μm of o1m of light intensity-2·s-1, light week
12~16h/d of phase is cultivated 40~80 days；The composition of proliferated culture medium is：1/2MS culture mediums, 1.5~3.5mg/L of 6-BA, NAA
0.1~0.3mg/L, 10~30g/L of sucrose, 0.5~1g/L of activated carbon, 6~8g/L of agar, pH=5.6~5.8；
(2) root-like stock for obtaining step (1) moves to subculture growth medium, the growth of induction Multiple Buds and sprouting and rooting, culture
Condition is as follows：Using LED as tissue culture light source, feux rouges 630nm, 20~40 μm of o1m of light intensity-2·s-1, the photoperiod 12~
16h/d is cultivated 30~60 days；The composition of subculture growth medium is：1/2MS culture mediums, 0.1~1.0mg/L of 6-BA, NAA
0.1~0.3mg/L, 10~30g/L of sucrose, 0.5~1g/L of activated carbon, 6~8g/L of agar, pH=5.6~5.8；
(3) newborn seedling is connected to root media and carries out strengthening seedling and rooting culture, condition of culture is as follows：Trained using LED as tissue
Support light source, the blue light that wavelength is 630nm feux rouges and wavelength is 450~480nm in proportion 2~4：6~8 proportionings, the μ of light intensity 30~60
mo1·m-2·s-1, 12~16h/d of photoperiod cultures 30~60 days；The composition of root media is：1/2MS culture mediums, 6-BA
0.1~0.3mg/L, 0.2~0.5mg/L of NAA, 10~30g/L of sucrose, 0.5~1g/L of activated carbon, agar 6~8g/L, pH=
5.6~5.8.
2. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterised in that：The culture of step (1)~(3) be
20~30 DEG C, relative humidity be 40~60% under the conditions of carry out.
3. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterised in that：Propagation training described in step (1)
Support base composition be：1/2MS culture mediums, 6-BA 2.5mg/L, NAA 0.3mg/L, sucrose 15g/L, activated carbon 0.5g/L, agar
6~8g/L, pH=5.6~5.8.
4. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterised in that：Culture bar described in step (1)
Part is：Using LED as tissue culture light source, 450~480nm of blue light, 20 μm of o1m of light intensity-2·s-1, photoperiod 16h/d cultures
60 days, environment temperature was 1 DEG C of 24 scholar.
5. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterised in that：Subculture life described in step (2)
The composition of long culture medium is：1/2MS culture mediums, 6-BA 0.5mg/L, NAA 0.2mg/L, sucrose 20g/L, activated carbon 0.5g/L,
6~8g/L of agar, pH=5.6~5.8.
6. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterised in that：Culture bar described in step (2)
Part is：Using LED as tissue culture light source, feux rouges 630nm, 20 μm of o1m of light intensity-2·s-1, photoperiod 16h/d cultures 60 days,
Environment temperature is 1 DEG C of 24 scholar, relative humidity 50-60%.
7. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterised in that：Training of taking root described in step (3)
Support base composition be：1/2MS culture mediums, 6-BA 0.2mg/L, NAA 0.5mg/L, sucrose 20g/L, activated carbon 0.5g/L, agar
6~8g/L, pH=5.6~5.8.
8. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterised in that：Culture bar described in step (3)
Part is：Using LED as tissue culture light source, the blue light that the feux rouges and wavelength that wavelength is 630nm are 450~480nm in proportion 2：6
Proportioning, 40 μm of o1m of light intensity-2·s-1, photoperiod 16h/d cultures 60 days, environment temperature is 1 DEG C of 24 scholar, relative humidity 50-
60%.
9. application of the method for tissue culture of any one of claim 1~8 Cymbidium lianpan in large-scale cultivation Cymbidium lianpan.
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| CN106900551B (en) * | 2017-03-02 | 2019-03-12 | 玉林师范学院 | A kind of Sha aspidistra in vitro culture quick-breeding method |
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| CN116326483A (en) * | 2023-04-24 | 2023-06-27 | 玉林师范学院 | Application of nerolidol in rooting culture of Mo Langen-shaped stems |
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