CN105340757A - Tissue culture method for cymbidium tortisepalum and application thereof - Google Patents
Tissue culture method for cymbidium tortisepalum and application thereof Download PDFInfo
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Abstract
The invention discloses a tissue culture method for cymbidium tortisepalum and application thereof. The method comprises the steps of taking cymbidium tortisepalum aseptic seedling stem tip as an explant, taking a proliferation medium to induce rhizomes to proliferate, taking an LED (light-emitting diode) as a tissue culture light source, and culturing by using blue light; then transferring the obtained rhizomes to a subculture growth medium, inducing multiple shoots to grow and a seedling to quickly develop, taking the LED as the tissue culture light source, and culturing by using red light; inoculating the newly generated seedling to a rooting medium for strong seedling rooting culture, taking the LED as the tissue culture light source, and culturing by using mixed red light and blue light which are matched according to a ratio of (2 to 4):(6 to 8). By adopting the tissue culture method, the problems of low sprouting ratio, slow growth, difficult rooting and the like existing in the prior tissue culture technology are effectively solved; by using LED lamps as the tissue culture light source, 30 to 50 percent of electricity resource is saved, the cost is saved, and the efficiency of industrialized production is improved.
Description
Technical field
The present invention relates to plant tissue fast breeding technique field, particularly a kind of method for tissue culture of Cymbidium lianpan and application thereof.
Background technology
Cymbidium lianpan is the large monoid during Chinese Terrestrial orchid belongs to, main product Yunnan.Because its plant forms is graceful, flower shape is changeable, pattern abundant, the market price occupies high reason, cause Cymbidium lianpan wild resource to go to wreck excavating of formula, cause resource in imminent danger.Therefore, the research for Cymbidium lianpan tissue cultures and fast breeding technique seems particularly important.But the intrinsic growth cycle of orchid is long, and the problems such as differentiation efficiency is low, and root system quality is not good hinder factorial seedling growth and the industry development of Cymbidium lianpan always.
Light quality is the important factor affecting growth and development of plants.In recent years there are some researches show, different light qualities can play facilitation at the different times of plant growth.Wherein light emitting diode (light-emittingdiodes, LED) is the high-quality light source that developed recently gets up, and it can send the monochromatic light of accurate wave spectrum, and wave-length coverage is narrower, and amplitude is no more than 20nm, controls light intensity again by regulation device.Therefore light quality and the adjustable LED light source of intensity of illumination are than normally used Plant Tissue Breeding fluorescent lamp, more effectively can promote the photosynthesis of test-tube plantlet, in tissue cultures, have wide application prospect.
At present be applied to the research of Plant Tissue Breeding about LED more, but all concentrate on the species of rapid propagation system relative maturity, and ripe application technology be yet there are no for the application in Cymbidium lianpan tissue cultures.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides a kind of method for tissue culture of Cymbidium lianpan.
Another object of the present invention is to the application of the method for tissue culture that described Cymbidium lianpan is provided.
Object of the present invention is achieved through the following technical solutions: a kind of method for tissue culture of Cymbidium lianpan, comprises the steps:
(1) be explant by Cymbidium lianpan aseptic seedling stem apex, adopt proliferated culture medium induction root-like stock propagation and differentiation, condition of culture is as follows: using LED as tissue culture light source, blue light 440 ~ 480nm, light intensity 20 ~ 40 μm of o1m
-2s
-1, photoperiod 12 ~ 16h/d cultivates 40 ~ 80 days; Consisting of of proliferated culture medium: 1/2MS medium, 6-benzyl purine (6-BA) 1.5 ~ 3.5mg/L, methyl α-naphthyl acetate (NAA) 0.1 ~ 0.3mg/L, sucrose 10 ~ 30g/L, active carbon 0.5 ~ 1g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8;
(2) root-like stock that step (1) obtains is moved to subculture growth medium, induced bundle is sprouted and is grown and sprouting and rooting, and condition of culture is as follows: using LED as tissue culture light source, ruddiness 620 ~ 660nm, light intensity 20 ~ 40 μm of o1m
-2s
-1, photoperiod 12 ~ 16h/d cultivates 30 ~ 60 days, improves plant growth rate; Consisting of of subculture growth medium: 1/2MS medium, 6-BA0.1 ~ 1.0mg/L, NAA0.1 ~ 0.3mg/L, sucrose 10 ~ 30g/L, active carbon 0.5 ~ 1g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8;
(3) newborn seedling is connected to root media and carries out strengthening seedling and rooting cultivation, condition of culture is as follows: using LED as tissue culture light source, ruddiness (620 ~ 660nm) and blue light (440 ~ 480nm) 2 ~ 4:6 ~ 8 proportioning in proportion, light intensity 30 ~ 60 μm of o1m
-2s
-1, photoperiod 12 ~ 16h/d cultivates 30 ~ 60 days, promotes that the growth of root is extended; Consisting of of root media: 1/2MS medium, 6-BA0.1 ~ 0.3mg/L, NAA0.2 ~ 0.5mg/L, sucrose 10 ~ 30g/L, active carbon 0.5 ~ 1g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8.
Above-mentioned cultivation can 20 ~ 30 DEG C, relative moisture carries out under being 40 ~ 60% conditions.
Described blue light is preferably the blue light that wavelength is 450 ~ 480nm.
Described ruddiness is preferably the ruddiness that wavelength is 630nm.
Proliferated culture medium described in step (1) is preferably the solid culture medium (pH=5.6 ~ 5.8) of 1/2MS medium+6-BA2.5mg/L+NAA0.3mg/L+ sucrose 15g/L+ active carbon 0.5g/L+ agar 6 ~ 8g/L.
Condition of culture described in step (1) is preferably: using LED as tissue culture light source, blue light 450 ~ 480nm, light intensity 20 μm of o1m
-2s
-1, photoperiod 16h/d cultivates 60 days, and environmental temperature is 24 scholar 1 DEG C.
Subculture growth medium described in step (2) is preferably the solid culture medium (pH=5.6 ~ 5.8) of 1/2MS medium+6-BA0.5mg/L+NAA0.2mg/L+ sucrose 20g/L+ active carbon 0.5g/L+ agar 6 ~ 8g/L.
Condition of culture described in step (2) is preferably: using LED as tissue culture light source, ruddiness 630nm, light intensity 20 μm of o1m
-2s
-1, photoperiod 16h/d cultivates 60 days, and environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.
Newborn seedling described in step (3) is preferably newly sprouted and is grown to the newborn seedling of 2cm.
Root media described in step (3) is preferably the solid culture medium (pH=5.6 ~ 5.8) of 1/2MS medium+6-BA0.2mg/L+NAA0.5mg/L+ sucrose 20g/L+ active carbon 0.5g/L+ agar 6 ~ 8g/L.
Condition of culture described in step (3) is preferably: using LED as tissue culture light source, Red and blue light ratio 2:6, light intensity 40 μm of o1m
-2s
-1, photoperiod 16h/d cultivates 60 days, and environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.
The application of method for tissue culture in large-scale cultivation Cymbidium lianpan of described Cymbidium lianpan.
The present invention has following advantage and effect relative to prior art:
1. the present invention to determine in Cymbidium lianpan tissue culture procedures root-like stock proliferative induction to sprouting differential period; Multiple Buds rapid growth stage, and the best configuration of strengthening seedling and rooting stage different culture media and condition of culture, effectively promote the efficient tissue cultures fast of Cymbidium lianpan.
2. the present invention to determine in Cymbidium lianpan tissue culture procedures root-like stock proliferative induction to sprouting differential period; Multiple Buds rapid growth stage, and the optimal culture condition in strengthening seedling and rooting stage, comprise LED light matter and light intensity and processing time, effectively solves the bud ratio existed in tissue culture technique in the past low, poor growth, the problem such as to take root difficult.
3., in the present invention, use LED lamp as tissue culture light source, have saving electric resources 30-50%, reduce cost, and improve the efficiency of factorial praluction.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1 Cymbidium lianpan tissue culture medium (TCM) optimizing components proportioning
One, proliferated culture medium
After the upper sprouting of 1/2MS medium (macroelement in MS basal medium and trace element reduce by half), the aseptic seedling of 14 months is grown for explant with Cymbidium lianpan CymbidiumLianpan " heavy snow element " seed, seedling Multiple Buds is cut into individual plant, and every strain is cut into about 2cm segment and is seeded to Multiplying culture in proliferated culture medium.Medium with 1/2MS medium+agar 7g/L for control group, add 6-benzyl purine (6-BA) 1.5 ~ 3.5mg/L, methyl α-naphthyl acetate (NAA) 0.1 ~ 0.3mg/L, active carbon 0.5 ~ 1g/L, sucrose 10 ~ 30g/L+ agar 7g/L is optimization group, and concrete 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) | Active 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 |
Every bottle graft kind 5 buds, often kind of culture medium prescription inoculates 10 bottles.Light source is made with Philip (PHILIPS) general T 8 white fluorescent fluorescent tube.Condition of culture is the photoperiod is 16h/d, light intensity 20 μm of o1m
-2s
-1, environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.After seedling inoculation, root-like stock can be induced to breed, and continuous culture differentiated after 60 days newly sprouts, and detected the root-like stock number of each inoculation simple bud propagation after continuous culture through 80 days, root-like stock breaks up the number that sprouts, sprouting plant height and growth coefficient.Simple bud growth coefficient refer to average each simple bud through expand numerous obtain from sprout containing spendable simple bud number.Plant height vernier caliper measurement (being accurate to 0.01mm).
Table 2
Statistics (table 2) shows, best with the cultivation effect of 4 i.e. 1/2MS medium+6-BA2.5mg/L+NAA0.3mg/L+ active carbon 0.5g/L+ sucrose 15g/L (pH=5.6 ~ 5.8) that fill a prescription in Different Optimization combination, growth coefficient reaches more than 3 times of 1/2MS medium controls.
Two, subculture growth medium
Multiple Buds newborn in step one is divided into individual plant, is directly seeded in subculture growth medium and carries out quick grown cultures.Every bottle graft kind 5 buds, each process 10 bottles.Medium with 1/2MS medium+agar 7g/L for control group, wherein add 6-benzyl purine (6-BA) 0.1 ~ 1mg/L, methyl α-naphthyl acetate (NAA) 0.1 ~ 0.3mg/L, active carbon 0.5 ~ 1g/L, sucrose 10 ~ 30g/L+ agar 7g/L is optimization group, and concrete 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) | Active 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, light intensity 20 μm of o1m
-2s
-1, environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.Test-tube plantlet, after 60d continuous culture, detects the average plant height of inoculation bud, individual plant mean number of sheets and fresh weight, and inoculation bud increases bud number newly.Plant height vernier caliper measurement (being accurate to 0.01mm).
Table 4
Result (table 4) shows, the optimization culture based formulas 3 i.e. rate of rise of 1/2MS medium+6-BA0.5mg/L+NAA0.2mg/L+ active carbon 0.5g/L+ sucrose 20g/L (pH=5.6 ~ 5.8) is the fastest, newly-increased blade quantity is the twice of control group, fresh weight increases more how close than control group 2.5 times, elects best subculture growth medium as.
Three, strengthening seedling and rooting medium
Newborn seedling in step 2 is seeded to root media and carries out root induction and strong seedling culture.Every bottle graft kind 5 buds, each process 10 bottles.Medium for control group, adopts agarose 7g/L with 1/2MS minimal medium+agar 7g/L.Wherein add 6-benzyl purine (6-BA) 0.1 ~ 0.3mg/L, methyl α-naphthyl acetate (NAA) 0.2 ~ 0.5mg/L, active carbon 0.5 ~ 1g/L, sucrose 10-30g/L+ agar 7g/L is optimization group, and concrete 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, light intensity 20 μm of o1m
-2s
-1, environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.Test-tube plantlet, after 60d continuous culture, detects the average plant height of inoculation bud, individual plant mean number of sheets and fresh weight, and inoculation blastogenesis radical, and root is long.Plant height and root length vernier caliper measurement (being accurate to 0.01mm).
Table 6
Result (table 6) shows, the optimization culture based formulas 4 i.e. rooting efficiency of 1/2MS+6-BA0.2mg/L+NAA0.5mg/L+ active carbon 0.5g/L+ sucrose 20g/L (pH=5.6 ~ 5.8) is best, significantly can promote that root is long to extend, take root number than control group height about 2.2 times, as the best growth medium in strong sprout.
The result of use of embodiment 2 optimal medium collocation LED
One, root-like stock is induced and is gone out Bud polarization and cultivates
1, proliferated culture medium
The solid culture medium of 1/2MS+6-BA2.5mg/L+NAA0.3mg/L+ active carbon 0.5g/L+ sucrose 15g/L (pH=5.6 ~ 5.8)+agar 7g/L.
2, vaccination ways
Cymbidium lianpan seedling (Cymbidium lianpan " heavy snow element " sowing grows the aseptic seedling of 14 months after sprouting) Multiple Buds is cut into individual plant, and every strain is cut into about 2cm segment and is seeded to Multiplying culture in proliferated culture medium.Every bottle graft kind 5 buds, each process 10 bottles.
3, condition of culture
Photoperiod is 16h/d, light intensity 20 μm of o1m
-2s
-1, environmental temperature is 24 scholar 1 DEG C.The light source of 5 kinds of different light medium ratios (as shown in table 7) is established in experiment altogether, and its ratio is respectively: 1. 100%R (ruddiness), 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, Philip general T 8 fluorescent tube, white light) process in contrast.LED culturing rack adopts layer stereo structure, and culturing rack height 2.38m, each cultivation sets up 4 layers, every floor height 0.59m.LED lamp tube is arranged on every interlayer and posts on the glass plate of lucifuge paper, and culturing rack side uses gobo to block.
Table 7
4, survey item
After seedling inoculation, root-like stock can be induced to breed, and continuous culture differentiated after 30 days newly sprouts, and after 60 days continuous culture, survey item comprises: the root-like stock number of propagation, sprout number, growth coefficient, plant height.Plant height vernier caliper measurement (being accurate to 0.01mm).Table 8 list average plant height, average simple bud propagation root-like stock number, sprout number and simple bud growth coefficient (refer to average each simple bud through expand numerous obtain from sprout containing spendable simple bud number).
Table 8
From result in table 8, after selecting optimum multiplication medium proportioning, different LED of being arranged in pairs or groups by medium light quality uses, the root-like stock proliferation number of T2 processed group is the highest, shows can significantly improve Cymbidium lianpan root-like stock proliferate efficiency relative to common fluorescent as light source using 450-480nm blue light as light source.
Two, the quick grown cultures of Multiple Buds
1, subculture growth medium
1/2MS+6-BA0.5mg/L+NAA0.2mg/L+ active carbon 0.5g/L+ sucrose 20g/L (pH=5.6 ~ 5.8)+agar 7g/L+ sucrose 20g/L (pH=5.6 ~ 5.8)+solid culture medium.
2, vaccination ways
Multiple Buds newborn in step one is divided into individual plant, is seeded in subculture growth medium and carries out quick grown cultures.Every bottle graft kind 5 buds, each process 10 bottles.
3, condition of culture
Photoperiod is 16h/d, light intensity 20 μm of o1m
-2s
-1, environmental temperature is 24 scholar 1 DEG C.The light source of 5 kinds of different light medium ratios (as shown in table 9) is established in experiment altogether, and its ratio is respectively: 1. 100%R, 2. 100%B, 3. 75%R ten 25%B, 4. 75%B+25%R, 5. 50%R+50%B.With conventional fluorescent lamps (PHILIPS) process in contrast.LED culturing rack adopts layer stereo structure, and culturing rack height 2.38m, each cultivation sets up 4 layers, every floor height 0.59m.LED lamp tube is arranged on every interlayer and posts on the glass plate of lucifuge paper, and culturing rack side uses gobo to block.
Table 9
4, survey item
Test-tube plantlet is after 60d continuous culture, and survey item has: average plant height, the individual plant number of sheets, newly-increased bud number, fresh weight.Plant height vernier caliper measurement (being accurate to 0.01mm).Result is as shown in table 10
Table 10
Visible, after selecting best subculture medium proportioning, different LED of being arranged in pairs or groups by medium light quality uses, and the root-like stock proliferation number of T1 processed group is the highest, show can significantly promote that Cymbidium lianpan root-like stock extend relative to common fluorescent as light source using 630nm ruddiness as light source, improve growth rate.
Three, strengthening seedling and rooting is cultivated
1, root media
The solid culture medium of 1/2MS+6-BA0.2mg/L+NAA0.5mg/L+ agar 7g/L+ sucrose 20g/L (pH=5.6 ~ 5.8)+active carbon 0.5g/L.
2, vaccination ways
Newborn seedling in step 2 is seeded to root media and carries out root induction and strong seedling culture.Every bottle graft kind 5 buds, each process 10 bottles.
3, condition of culture
Photoperiod is 16h/d, light intensity 40 μm of o1-m
-2s
-1, environmental temperature is 24 scholar 1 DEG C.The light source of 5 kinds of different light medium ratios (as shown in table 11) is established in experiment altogether, and its ratio is respectively: 1. 100%R, 2. 100%B, 3. 75%R ten 25%B, 4. 75%B+25%R, 5. 50%R+50%B.With conventional fluorescent lamps (PHILIPS) process in contrast.LED culturing rack adopts layer stereo structure, and culturing rack height 2.38m, each cultivation sets up 4 layers, every floor height 0.59m.LED lamp tube is arranged on every interlayer and posts on the glass plate of lucifuge paper, and culturing rack side uses gobo to block.
Table 11
4, survey item
Test-tube plantlet is after 60d continuous culture, and survey item has: plant height, the number of sheets, and number of taking root, root is long.Plant height, root length vernier caliper measurement (being accurate to 0.01mm).Result is as shown in table 12
Table 12
Visible, compared with common fluorescent, ruddiness is cultivated can promote plant height, the elongation growth of blade and root length, but number of taking root is without significantly improving, and blue light is cultivated and can be improved Cymbidium lianpan newborn blastogenesis radical order, but to blade and the long-living length of root without remarkable facilitation, wherein with T4 process, namely best using Red and blue light 1:3 mix and match as light source effect, significantly can promote Cymbidium lianpan elongation growth and quantity of taking root.
Comparative example
One, different culture media uses the root-like stock induction of LED light source and differentiation effect of sprouting
Adopt optimal medium and contrast medium respectively, cultivate under LED strip part, compare optimal medium cultivation effect.Concrete operation step is as follows:
1, proliferated culture medium is contrasted
Table 13
| Culture medium prescription | 6-BA(mg/L)1 | NAA(mg/L) | Active 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 |
2, vaccination ways
Cymbidium lianpan seedling (Cymbidium lianpan " heavy snow element " sowing grows the aseptic seedling of 14 months after sprouting) Multiple Buds is cut into individual plant, and every strain is cut into about 2cm segment and is seeded to Multiplying culture in proliferated culture medium.Every bottle graft kind 5 buds, each process 10 bottles.
3, condition of culture
Condition of culture is the photoperiod is 16h/d, using 450-480nm blue light as light source, and light intensity 20 μm of o1m
-2s
-1, environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.
4, survey item
After seedling inoculation, root-like stock can be induced to breed, continuous culture differentiated after 30 days newly sprouts, and differentiates newly sprout through 60 days after continuous culture, detects the root-like stock number of each inoculation simple bud propagation through 80 days after continuous culture, root-like stock breaks up the number that sprouts, sprouting plant height and growth coefficient, survey item comprises: the root-like stock number of propagation, and sprout number, growth coefficient, plant height.Plant height vernier caliper measurement (being accurate to 0.01mm).Table 14 list average plant height, average simple bud propagation root-like stock number, sprout number and simple bud growth coefficient (refer to average each simple bud through expand numerous obtain from sprout containing spendable simple bud number).
Table 14
From result in table 14, under same LED illumination condition, the growth coefficient of the foster base of optimizing tissue cultivation is the highest, show after selecting optimum multiplication medium (1/2MS+6-BA2.5mg/L+NAA0.3mg/L+ active carbon 0.5g/L+ sucrose 15g/L (pH=5.6 ~ 5.8)) proportioning, collocation, using 450-480nm blue light as light source, can significantly improve Cymbidium lianpan root-like stock proliferate efficiency.
Two, different subculture medium uses LED light source to carry out the quick grown cultures of Multiple Buds
Adopt optimal medium and contrast medium respectively, cultivate under LED strip part, compare optimal medium growth rate, concrete operation step is as follows:
1, subculture growth medium is contrasted
Table 15
| Culture medium prescription | 6-BA(mg/L)1 | NAA(mg/L) | Active 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 |
2, vaccination ways
Multiple Buds newborn in embodiment 4 is divided into individual plant, is seeded in subculture growth medium and carries out quick grown cultures.Every bottle graft kind 5 buds, each process 10 bottles.
3, condition of culture
Condition of culture is the photoperiod is 16h/d, using 630nm ruddiness as light source, and light intensity 20 μm of o1m
-2s
-1, environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.
4, survey item
Test-tube plantlet is after 60d continuous culture, and survey item has: average plant height, the individual plant number of sheets, newly-increased bud number, fresh weight.Plant height vernier caliper measurement (being accurate to 0.01mm).Result is shown in table 16
Table 16
From result in table, under same LED illumination condition, optimizing tissue cultivates the growth rate of foster base the soonest, shows after selecting optimum multiplication medium proportioning, arranges in pairs or groups using 630nm ruddiness as light source, can significantly improve Cymbidium lianpan root-like stock proliferate efficiency.
Three, different root media uses LED light source to carry out newborn seedling strengthening seedling and rooting contrast cultivation
Adopt optimal medium and contrast medium respectively, cultivate under LED strip part, compare optimal medium rooting efficiency, concrete operation step is as follows:
1, root media is contrasted
Table 17
2, vaccination ways
Multiple Buds newborn in embodiment 5 is divided into individual plant, is seeded in subculture growth medium and carries out quick grown cultures.Every bottle graft kind 5 buds, each process 10 bottles.
3, condition of culture
Condition of culture is the photoperiod is 16h/d, adopts Red and blue light 2:6 hybrid light source, light intensity 20 μm of o1m
-2s
-1, environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.
4, survey item
Test-tube plantlet is after 60d continuous culture, and survey item has: plant height, the number of sheets, and number of taking root, root is long.Plant height, root length vernier caliper measurement (being accurate to 0.01mm).Result is shown in table 18, visible, compared with common fluorescent, ruddiness is cultivated can promote plant height, the elongation growth of blade and root length, but number of taking root is without significantly improving, blue light is cultivated and can be improved Cymbidium lianpan newborn blastogenesis radical order, but to blade and the long-living length of root without remarkable facilitation, with the process of Red and blue light 2:6 hybrid light source, Cymbidium lianpan elongation growth and quantity of taking root significantly can be promoted.
Table 18
From result in table, under same LED illumination condition optimizing tissue cultivate support base take root number at most and root length the longest, show after selecting optimum multiplication medium proportioning, arrange in pairs or groups to adopt ruddiness 630nm and blue light 450-480nm1:3 to mix as light source, can significantly promote Cymbidium lianpan to take root and the long elongation of root.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a method for tissue culture for Cymbidium lianpan, is characterized in that comprising the steps:
(1) be explant by Cymbidium lianpan aseptic seedling stem apex, adopt proliferated culture medium induction root-like stock propagation and differentiation, condition of culture is as follows: using LED as tissue culture light source, blue light 440 ~ 480nm, light intensity 20 ~ 40 μm of o1m
-2s
-1, photoperiod 12 ~ 16h/d cultivates 40 ~ 80 days; Consisting of of proliferated culture medium: 1/2MS medium, 6-BA1.5 ~ 3.5mg/L, NAA0.1 ~ 0.3mg/L, sucrose 10 ~ 30g/L, active carbon 0.5 ~ 1g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8;
(2) root-like stock that step (1) obtains is moved to subculture growth medium, induced bundle is sprouted and is grown and sprouting and rooting, and condition of culture is as follows: using LED as tissue culture light source, ruddiness 620 ~ 660nm, light intensity 20 ~ 40 μm of o1m
-2s
-1, photoperiod 12 ~ 16h/d cultivates 30 ~ 60 days; Consisting of of subculture growth medium: 1/2MS medium, 6-BA0.1 ~ 1.0mg/L, NAA0.1 ~ 0.3mg/L, sucrose 10 ~ 30g/L, active carbon 0.5 ~ 1g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8;
(3) newborn seedling is connected to root media and carries out strengthening seedling and rooting cultivation, condition of culture is as follows: using LED as tissue culture light source, blue light 2 ~ 4:6 ~ 8 proportioning in proportion of wavelength to be 620 ~ 660nm ruddiness and wavelength be 440 ~ 480nm, light intensity 30 ~ 60 μm of o1m
-2s
-1, photoperiod 12 ~ 16h/d cultivates 30 ~ 60 days; Consisting of of root media: 1/2MS medium, 6-BA0.1 ~ 0.3mg/L, NAA0.2 ~ 0.5mg/L, sucrose 10 ~ 30g/L, active carbon 0.5 ~ 1g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8.
2. the method for tissue culture of Cymbidium lianpan according to claim 1, is characterized in that: the cultivation described in step (1) ~ (3) be 20 ~ 30 DEG C, relative moisture carries out under being 40 ~ 60% conditions.
3. the method for tissue culture of Cymbidium lianpan according to claim 1, is characterized in that:
The blue light of described blue light to be wavelength be 450 ~ 480nm;
The ruddiness of described ruddiness to be wavelength be 630nm.
4. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterized in that: consisting of of the proliferated culture medium described in step (1): 1/2MS medium, 6-BA2.5mg/L, NAA0.3mg/L, sucrose 15g/L, active carbon 0.5g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8.
5. the method for tissue culture of Cymbidium lianpan according to claim 1, is characterized in that: the condition of culture described in step (1) is: using LED as tissue culture light source, blue light 450 ~ 480nm, light intensity 20 μm of o1m
-2s
-1, photoperiod 16h/d cultivates 60 days, and environmental temperature is 24 scholar 1 DEG C.
6. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterized in that: consisting of of the subculture growth medium described in step (2): 1/2MS medium, 6-BA0.5mg/L, NAA0.2mg/L, sucrose 20g/L, active carbon 0.5g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8.
7. the method for tissue culture of Cymbidium lianpan according to claim 1, is characterized in that: the condition of culture described in step (2) is: using LED as tissue culture light source, ruddiness 630nm, light intensity 20 μm of o1m
-2s
-1, photoperiod 16h/d cultivates 60 days, and environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.
8. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterized in that: consisting of of the root media described in step (3): 1/2MS medium, 6-BA0.2mg/L, NAA0.5mg/L, sucrose 20g/L, active carbon 0.5g/L, agar 6 ~ 8g/L, pH=5.6 ~ 5.8.
9. the method for tissue culture of Cymbidium lianpan according to claim 1, it is characterized in that: the condition of culture described in step (3) is: using LED as tissue culture light source, the blue light 2:6 proportioning in proportion of wavelength to be the ruddiness of 630nm and wavelength be 450 ~ 480nm, light intensity 40 μm of o1m
-2s
-1, photoperiod 16h/d cultivates 60 days, and environmental temperature is 24 scholar 1 DEG C, relative moisture 50-60%.
10. the application of method for tissue culture in large-scale cultivation Cymbidium lianpan of Cymbidium lianpan described in any one of claim 1 ~ 9.
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| CN106900551A (en) * | 2017-03-02 | 2017-06-30 | 玉林师范学院 | A kind of Sha aspidistra cultured in vitro quick-breeding method |
| CN106942065A (en) * | 2017-05-12 | 2017-07-14 | 玉林师范学院 | One kind set aspidistra cultured in vitro quick-breeding method |
| CN107646684A (en) * | 2017-10-23 | 2018-02-02 | 广东省农业科学院环境园艺研究所 | A kind of breeding method of purpleback murdannia herb and its application |
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| CN106942065B (en) * | 2017-05-12 | 2018-11-27 | 玉林师范学院 | A kind of set aspidistra in vitro culture quick-breeding method |
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| CN109105263A (en) * | 2018-11-01 | 2019-01-01 | 翁源县天下泽雨农业科技有限公司 | A kind of state orchid rhizomes quick breeding method for tissue culture |
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