CN105165631B - A kind of blue tissue culture production method of leaf skill - Google Patents
A kind of blue tissue culture production method of leaf skill Download PDFInfo
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- CN105165631B CN105165631B CN201510715326.7A CN201510715326A CN105165631B CN 105165631 B CN105165631 B CN 105165631B CN 201510715326 A CN201510715326 A CN 201510715326A CN 105165631 B CN105165631 B CN 105165631B
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Abstract
The invention discloses a kind of blue tissue culture production method of leaf skill, it comprises the following steps：The PLB of the blue tissue culture propagation of selection leaf skill is used as culture materials；From after base portion cuts complete PLB, entirely it is inoculated on proliferated culture medium, going out new PLB by complete PLB Surface Differentiations is bred；PLB by screening propagation is transferred on liquid differential medium, concussion and cultivate PLB upper ends differentiation and seedling emergence after 14~21 days；The high band skill seedlings of the 1cm formed on differential medium or so are transferred on root media, culture is taken root for 5~10 days to seedling lower end, continue to cultivate 30~50 days to the healthy and strong plant of formation；When 3~5cm of plant is high, blake bottle is transferred to natural light lower refining seedling 7~10 days, then taken out it from culture vessel, clean the culture medium of root, move into fermented pine bark culture matrix, keep appropriate ventilation and enough humidity, the survival rate of transplanting is up to more than 95%.
Description
Technical field
The present invention relates to field of plant tissue culture technique, and in particular to is related to the tissue culture propagating of leaf skill cymbidium variety
Method.
Background technology
Ye Yilan be one of orchid it is important view and admire species, occur in that a class to appreciate in the seed selection of orchid, domestication, cultivation
Cymbidium variety based on leaf, the blade of these kinds occurs in that the leaf skills such as Phnom Penh, Jin Xin, silver-colored side, galactic center, thin white silk used in ancient China skill, spot skill, has
High ornamental value.The variation of these leaf colors is mostly due to the change that part cytometaplasia causes leaf color, forms chimera
Plant, this character can not be kept by seminal propagation, can only be preserved by vegetative propagation, and how unstable its character is, but
It is more stable that part leaf skill character, which is dragged etc. such as silver-colored side, silver in division propagation,；Its essence of Plant Tissue Breeding is also asexual
Breeding, but in the blue tissue cultures of leaf skill, leaf skill character loss frequency is higher, loses the sight decline of skill plant, directly affects
To the price of commodity.If being reduced in the blue tissue-cultured seedling production of leaf skill and losing skill frequency, production efficiency can be greatly improved.Have at present
The research report of a small amount of this respect, but the problem of still suffer from larger in production practices.
The content of the invention
The purpose of the present invention is to be directed to hybrid cymbidium leaf skill kind the problem of occurring losing skill during tissue culture, passes through adjustment
Culture medium prescription, with reference to the selection of culture early stage form there is provided a kind of blue tissue culture production method of leaf skill, to reduce leaf skill kind group
The frequency of skill is lost in training.
The blue tissue culture production method of the leaf skill of the present invention, its feature comprises the following steps：
Step 1, material selection：The PLB of leaf skill orchid tissue culture propagation（Protocorms）；
Step 2, PLB（Protocorms）Propagation：With carrying out horizontal and vertical cutting not to PLB in conventional PLB Reproduction methods
Together, the present invention is entirely inoculated on proliferated culture medium from after base portion cuts complete PLB, passes through complete PLB Surface Differentiations
Go out new PLB to be bred, in subculture operation, only select viridian PLB to be bred, lose color is dark green with albefaction PLB
Abandon.The present invention does not use the conventionally used enrichment procedure for repeatedly cutting PLB progress and forming fritter, to keep its chimeric shape
State, the frequency that reduction leaf skill is lost.On proliferated culture medium of the present invention, the PLB of material is met in chimerism, albefaction
Color distortion substantially, can be screened most of skill material that loses by color, during subculture under state and normal condition
Dark green and albefaction the PLB of color is abandoned, it is ensured that fertile material is in chimerism.
Step 3, PLB differentiation：PLB by screening propagation is transferred on liquid differential medium, concussion and cultivate 14~
PLB upper ends differentiation and seedling emergence after 21 days.The seedling 85% that is differentiated on differential medium of the present invention Ye Yixing maintained above
Shape.
Step 4, Rooting and hardening-off culture：The high band skill seedlings of the 1cm formed on differential medium or so are transferred to and taken root
On culture medium, 7 days or so visible seedling lower ends of culture are taken root, and healthy and strong plant can be formed in 30~50 days by continuing to cultivate.
Step 5, test tube transplantation of seedlings：When about 3~5cm is high for plant, blake bottle is transferred to natural light lower refining seedling 7~10
My god, then it is taken out from culture vessel, the culture medium of root is cleaned, moved into fermented pine bark culture matrix, is protected
Appropriate ventilation and enough humidity are held, the survival rate of transplanting is up to more than 95%.
Wherein, the condition of culture of step 2 is 22~24 DEG C of cultivation temperature, 1500~2000lx of illuminance, illumination 12 hours/
My god.Condition of culture in step 3 is 25~28 DEG C of cultivation temperature, 1500~2000lx of illuminance, hour/day of illumination 12, shaking table
Rotating speed 100rpm.Condition of culture in step 4 is 25~28 DEG C of cultivation temperature, 1500~2000lx of illuminance, illumination 12 hours/
My god.
Further, proliferated culture medium uses the MS culture mediums that macro-and microelements halve in step 2（1/2MS）For base
Basal culture medium, additional peptone（Peptone）0.5~3g.L-1, caseinhydrolysate（CH）0.2~0.5g.L-1, methyl α-naphthyl acetate
（NAA）0.1~1 mg.L-1, paclobutrazol（PP333）0.1~0.5mg.L-1, banana be homogenized 50~200 g.L-1, sucrose 15
~30g.L-1, 6~7g.L of agar-1, activated carbon（AC）0.5~2 g.L-1；PH 5.4~5.6.
Differential medium uses 1/2MS culture mediums for minimal medium in step 3,0.5~3g.L of additional peptone-1, water
Solve 0.2~0.5g.L of casein-1, thiadiazole phenylurea（TDZ）0.1~0.5 mg.L-1, coconut milk（CM）50~200
ml.L-1, 15~30g.L of sucrose-1；PH 5.4~5.6.
Rooting and hardening-off culture base is precious No. 1 to spend in step 4（Hyponex 1）1.5~3g. L-1, 0.5~3g of peptone
g. L-1, 0.2~0.5g.L of caseinhydrolysate-1, the mg. L of methyl α-naphthyl acetate 0.5~2-1, banana be homogenized 50~200 g. L-1, sucrose
15~30 g. L-1, the g. L of agar 6~7-1, the g. L of activated carbon 0.5~2-1, remaining be MS culture mediums in vitamin and flesh
Alcohol composition；PH 5.4~5.6.
In step 2, the PLB of timely subculture（Protocorms）On differential medium to keep emerald green, minority loses skill more
Material shows as light yellow（Alphosis）Or bottle green.During Multiplying culture subculture, with reference to PLB colors, by obvious albefaction
And the excision of bottle-green explant, only retain emerald green PLB, be greatly improved leaf skill seedling frequency.Bottle green PLB can be used for
The cultivation of unlimited plant, it may have commodity.
The present invention is applied to chimera material caused by leaf color related genes variants, golden-rimmed skill, golden pawl skill during such as state is blue,
Silver-colored side skill, heart skill etc..On the one hand the culture medium that the present invention is invented can ensure leaf skill orchid PLB normal proliferative, on the other hand,
Under the illumination that is provided, temperature conditionss, the PLB of experimental cultivar can be made in band skill and without between skill, obvious aberration occur,
Skill explant is lost so that early stage is removed by morphologic observation, so as to improve production efficiency.
Embodiment
With reference to embodiment, the present invention is described in further detail.
Embodiment 1：Tissue culture kind：Pawl skill Phnom Penh prodigy
Method and step：
Step 1：Multiplying culture
1.1st, the obvious golden-rimmed prodigy's aseptic seedling of pawl skill is chosen, blade and root is cut, is inoculated in proliferated culture medium（1/
2MS+Peptone 1 g. L-1+CH 0.5 g. L-1+NAA1 mg. L-1+PP333 0.2 mg. L-1+ banana homogenate 100
g.L-1+ sucrose 30g.L-1+ agar 6g.L-1+ activated carbon 1g.L-1；pH 5.4）On.
1.2nd, there is grass green PLB in edge of materials after 4-6 weeks, and PLB is cut along base portion and is transferred to new proliferated culture medium
On.
1.3rd, PLB propagation is formed with 5-8 PLB bulk after 4-6 weeks, and PLB is cut along base portion, new culture is transferred to
Breed on base.
1.4th, continuous repeat step 1.3, therebetween, if the PLB formed is bottle green or albefaction（Yellow or white）, then
Being abandoned during subculture should not.Subculture number is no more than 15 generations.
Step 2：Differentiation and culture of rootage
2.1st, PLB is inoculated into liquid differential medium（1/2MS+Peptone 0.5g.L-1+CH 0.5g.L-1+TDZ
0.5 mg.L-1+CM 100 ml.L-1+ sucrose 15g.L-1；pH 5.4）On, 25-28 DEG C, 1500~2000lx of illuminance, light
According to 12 hours/day, 100rpm is cultivated 14-21 weeks, it is seen that PLB formation budlets.
2.2nd, budlet is inoculated into strong seedling culture base（Hyponex 1 2g. L-1+Peptone 1 g. L-1+CH 0.5
g. L-1+NAA 1mg. L-1+ banana is homogenized 100 g.L-1+ sucrose 20g.L-1+ agar 6g.L-1+ activated carbon 1g.L-1；pH
5.4）On, visible new root is grown after cultivating 7 days, and bud grows tall.
2.3rd, continue to cultivate 30-50 days, formation can transplant leaf skill finished product seedling.
Using the present invention, seedling more than 90% is produced for the Ye Yimiao with pawl skill, separately there is a small amount of complete green seedling without skill,
Almost without Albino Seedling, because albefaction PLB is screened out during PLB screenings.
Certainly, it is the concrete application example of the present invention above, the present invention also has other embodiments, all using equivalent
Replacement or the technical scheme of equivalent transformation formation, all fall within protection domain of the presently claimed invention.
Claims (2)
1. the blue tissue culture production method of a kind of leaf skill, it is characterised in that comprise the following steps：
Step 1, material selection：The PLB of the blue tissue culture propagation of selection leaf skill is used as culture materials；
Step 2, PLB propagation：From after base portion cuts complete PLB, entirely it is inoculated on proliferated culture medium, by complete
PLB Surface Differentiations go out new PLB and bred；
In the breeding, screened with reference to PLB colors, obvious albefaction and bottle-green explant is removed, only
Retain emerald green PLB；
The proliferated culture medium is solid medium；
The proliferated culture medium uses 1/2MS culture mediums for minimal medium, the gL of additional peptone 0.5~3-1, hydrolysis junket egg
White 0.2~0.5 gL-1, the mgL of methyl α-naphthyl acetate 0.1~1-1, the mgL of paclobutrazol 0.1~0.5-1, banana homogenate 50~200
g·L-1, the gL of sucrose 15~30-1, the gL of agar 6~7-1, the gL of activated carbon 0.5~2-1；PH 5.4~5.6；
Step 3, PLB differentiation：PLB by screening propagation is transferred on liquid differential medium, concussion and cultivate 14~21 days
PLB upper ends differentiation and seedling emergence afterwards；
The differential medium uses 1/2MS culture mediums for minimal medium, the gL of additional peptone 0.5~3-1, hydrolysis junket egg
White 0.2~0.5 gL-1, the mgL of thiadiazole phenylurea 0.1~0.5-1, the mlL of coconut milk 50~200-1, sucrose 15~30
g·L-1；PH 5.4~5.6；
Step 4, Rooting and hardening-off culture：The high band skill seedlings of the 1cm formed on differential medium or so are transferred to culture of rootage
On base, culture is taken root for 5~10 days to seedling lower end, continues to cultivate 30~50 days to the healthy and strong plant of formation；
The composition of the root media includes：Spend No. 1 1.5~3 gL of treasured-1, the gL of peptone 0.5~3-1, hydrolysis junket egg
White 0.2~0.5 gL-1, the mgL of methyl α-naphthyl acetate 0.5~2-1, banana be homogenized 50~200 gL-1, the gL of sucrose 15~30-1、
The gL of agar 6~7-1, the gL of activated carbon 0.5~2-1, remaining be MS culture mediums in vitamin and inositol composition；pH 5.4
~5.6；
Step 5, test tube transplantation of seedlings：When 3~5cm of plant is high, blake bottle is transferred to natural light lower refining seedling 7~10 days, then
It is taken out from culture vessel, the culture medium of root is cleaned, moved into fermented pine bark culture matrix, keeps appropriate logical
Wind and enough humidity, the survival rate of transplanting is up to more than 95%.
2. the blue tissue culture production method of leaf skill according to claim 1, it is characterised in that：The condition of culture of step 2 is culture
22~24 DEG C of temperature, 1500~2000lx of illuminance, hour/day of illumination 12；The condition of culture of step 3 is cultivation temperature 25~28
DEG C, 1500~2000lx of illuminance, hour/day of illumination 12, shaking speed 100rpm；The condition of culture of step 4 is cultivation temperature
25~28 DEG C, 1500~2000lx of illuminance, hour/day of illumination 12.
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