CN104904603B - Spring begonia tissue culture breeding method - Google Patents
Spring begonia tissue culture breeding method Download PDFInfo
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Abstract
The invention discloses a spring begonia tissue culture breeding method. The spring begonia tissue culture breeding method can purposefully induce formation of adventitious buds of spring begonias by regulating constituents of a plant growth regulator in a culture medium. Additionally, the spring begonia tissue culture breeding method can introduce development of roots, and finally enables test tube plantlets to develop into complete plants. The spring begonia tissue culture breeding method enables transplanting survival rate of the spring begonias to reach above 95%, can preserve and develop the spring begonias which are threatened, and establishes a good foundation for further utilization of the spring begonias in future.
Description
Technical field：
The invention belongs to plant tissue breeds field, and in particular to a kind of spring Flos Begoniae Evansianae tissue culture method for breeding.
Background technology：
Spring Flos Begoniae Evansianae (Begonia coptidifolia) is Begoniaceae (Begoniaceae) Begonia plant.
The streams side being only distributed in the Guangdong Province Yangchun City E Huang Zhang Nature Reserve of China.Begonia plant is generally without decomposite leaf
Or shallow decomposite leaf type, and spring Flos Begoniae Evansianae blade is drastic crack blade profile, with higher ornamental value, and is occupied in systematics important
Status.Due to the characteristic distributions that NATURAL DISTRIBUTION is narrow and special, wild spring Flos Begoniae Evansianae population quantity is few.It is badly in need of carrying out and is somebody's turn to do
The protection planted and breeding process.
The content of the invention：
It is an object of the invention to provide a kind of can be quick, spring Flos Begoniae Evansianae is efficiently bred, so that the endangered plants autumn in spring
The method that Caulis et folium euphorbiae milii is preserved and the spring Flos Begoniae Evansianae tissue culture that develops breeds.
The method that the spring Flos Begoniae Evansianae tissue culture of the present invention breeds, it is characterised in that comprise the following steps
A, explant and its process：Blade using spring Flos Begoniae Evansianae (Begonia coptidifolia) as explant,
By explant disinfection；
The induction of b, adventitious bud：Explant after disinfecting is inoculated on inducing culture, condition of culture is：22~
28 DEG C, 20 μm of ol m of dark culturing or intensity of illumination-2s-1Hereinafter, hour/day of light application time 14, cultivates 60 days, then goes to light
According to 40~100 μm of ol m of intensity-2s-1, hour/day of light application time 6~14, cultivate 30 days, obtain adventitious bud；Described induction training
Per liter of foster base containing kinetins (KT) 0.1~2.0mg or 6- benzyladenines (BA) 0.1~2.0mg or N- phenyl-N ' -1,2,3-
Thiadiazoles -5- ureas (TDZ) 0.1~2.0mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；
C, successive propagation：Adventitious bud is proceeded in propagating culture medium and is cultivated, condition of culture is：22~28 DEG C, intensity of illumination
20~100 μm of ol m-2s-1, hour/day of light application time 14, per liter described of propagating culture medium is containing 6- benzyladenines (BA) 0.1
~1.5mg, α-naphthaleneacetic acid (NAA) 0.01~0.1mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；
D, root culture：The adventitious bud that step c successive propagation is obtained is proceeded to into into root media culture, condition of culture
For 22~28 DEG C, light intensity is 50~80 μm of ol m-2s-1, hour/day of light application time 6~14 so as to take root grows up to test tube seedling, institute
Per liter of the root media stated contains indole -3-butyric acid (IBA) 0.05~0.2mg or α-naphthaleneacetic acid (NAA) 0.05~0.2mg,
And also contain sucrose 30g, agar 6g, remaining composition be 1/2MS culture medium, pH6.0；
The transplanting of e, test tube seedling：It is by volume 1 that test tube seedling is transplanted to into Vermiculitum and sandstone：In 1 substrate, it is placed in wet
Cultivate in the environment of profit, shade, water, the moisture and nutritional need applied fertilizer needed for meet test tube seedling growth, Jing cellar cultures
Cultivation Seedling is obtained afterwards.
Can to grow to 4~5cm in substrate high for test tube seedling after one month, and period is per week to apply 1:1000 compound fertilizer (N:P:K
=1:1:1), between 20-30 DEG C, transplanting survival rate reaches more than 95% to outdoor temperature, ensure that very high survival rate, now
Seedling can be transplanted in basin and be cultivated, fiery ash is comprised only in basin.
It is described explant disinfection is preferably cleaned up in spring Flos Begoniae Evansianae vanes tap water after, first
Sterilization 10 seconds, aseptic water elution 2 times, afterwards in mass fraction 0.1% are cleaned with the alcohol water blend surface of volume fraction 75%
Soak 8 minutes in mercuric chloride aqueous solution, and with aseptic water washing 3 times, finally blade is cut into into 0.6cm2Size, is seeded to induction training
On foster base.
Described MS culture medium is international culture medium, its composition and compound method referring to Tan Wencheng, wear plan and just lead
Compile,《Ornamental plant tissue culture technique》, Beijing：China Forestry Publishing House, 1991, described 1/2MS culture medium is by MS
A great number of elements and trace element halve, other components unchangeds and the culture medium that formed.
Under isolated culture condition, plant growth regulator key effect for cell dedifferentiation plays in culture medium.This
Invention can purposefully induce the shape of spring Flos Begoniae Evansianae adventitious bud by adjusting culture medium implants growth regulator component
Into.In addition the development of root is also can induce, finally enables test tube seedling bud into complete plant.Transplanting survival rate reaches 95%
More than, this endangered plants of spring Flos Begoniae Evansianae can be enable to preserve and develop, it is further to be established using the species from now on
Good basis.
Specific embodiment：
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.
Embodiment 1：Experiment place：South China Botanical Garden Chinese Academy of Sciences tissue culture room
The method that the spring Flos Begoniae Evansianae tissue culture of the present embodiment breeds, it is comprised the following steps that：
A, explant and its process：Stream from the E Huang Zhang Nature Reserve of Guangdong Province Yangchun City collects one plant of sun
Spring and autumn Caulis et folium euphorbiae milii plant, takes spring Flos Begoniae Evansianae blade, Jing after tap water is cleaned up, first with the alcohol water blend table of volume fraction 75%
Sterilization 10 seconds is cleaned in face, and aseptic water elution 2 times soaks 8 minutes afterwards in the mercuric chloride aqueous solution of mass fraction 0.1%, and with
Aseptic water washing 3 times, is finally cut into 0.6cm by blade2Size, the explant being sterilized after processing.
The induction of b, adventitious bud：Explant after disinfecting is inoculated on inducing culture, condition of culture is：22
DEG C, dark culturing is cultivated 60 days, then goes to 40 μm of ol m of intensity of illumination-2s-1, hour/day of light application time 14, culture 30 days,
Obtain adventitious bud；Per liter described of inducing culture contains kinetins (KT) 0.1mg, sucrose 30g, agar 6g, and remaining composition is
MS culture medium, (concrete configuration method is, by mentioned component mix homogeneously, to adjust pH value to pH6.0, is then sterilized standby, is below cultivated
The collocation method of base is identical with this)；
C, successive propagation：Adventitious bud is proceeded in propagating culture medium and is cultivated, condition of culture is：22 DEG C, the μ of intensity of illumination 100
mol m-2s-1, hour/day of light application time 14, per liter described of propagating culture medium is containing 6- benzyladenines (BA) 0.1mg, α-naphthalene
Acetic acid (NAA) 0.1mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；Clump bud is cultivated on propagating culture medium
Every month can breed 3.3 times；
D, root culture：The adventitious bud that step c successive propagation is obtained is proceeded to into into root media culture, condition of culture
For 22 DEG C, light intensity is 80 μm of ol m-2s-1, hour/day of light application time 14 so as to take root, grow up to test tube seedling；Described training of taking root
Per liter of foster base contains indole -3-butyric acid (IBA) 0.05mg, sucrose 30g, agar 6g, and remaining composition is 1/2MS culture medium,
pH6.0。
The transplanting of e, test tube seedling：It is by volume 1 that test tube seedling is transplanted to into Vermiculitum and sandstone：In 1 substrate, it is placed in wet
Cultivate in the environment of profit, shade, water, the moisture and nutritional need applied fertilizer needed for meet test tube seedling growth, Jing cellar cultures
Cultivation Seedling is obtained afterwards；
It is high that test tube seedling can grow to 4~5cm after transplanting one month in substrate, and period is per week to apply 1:1000 compound fertilizer
(N:P:K=1:1:1), between 20-30 DEG C, transplanting survival rate reaches more than 95% to outdoor temperature, ensure that very high surviving
Rate, seedling (cultivation Seedling) now can be transplanted in basin cultivate, and fiery ash is comprised only in basin.
Embodiment 2：Experiment place：In the E Huang Zhang Nature Reserve of Yangchun City
The method that the spring Flos Begoniae Evansianae tissue culture of the present embodiment breeds, it is comprised the following steps that：
A, explant and its process：Stream from the E Huang Zhang Nature Reserve of Guangdong Province Yangchun City collects one plant of sun
Spring and autumn Caulis et folium euphorbiae milii plant, takes spring Flos Begoniae Evansianae blade, Jing after tap water is cleaned up, first with the alcohol water blend table of volume fraction 75%
Sterilization 10 seconds is cleaned in face, and aseptic water elution 2 times soaks 8 minutes afterwards in the mercuric chloride aqueous solution of mass fraction 0.1%, and with
Aseptic water washing 3 times, is finally cut into 0.6cm by blade2Size, the explant being sterilized after processing.
The induction of b, adventitious bud：Explant after disinfecting is inoculated on inducing culture, condition of culture is：28
DEG C, 20 μm of ol m of intensity of illumination-2s-1Hereinafter, hour/day of light application time 14, cultivates 60 days, then goes to 80 μm of ol of intensity of illumination
m-2s-1, hour/day of light application time 6, cultivate 30 days, obtain adventitious bud；Per liter described of inducing culture contains kinetins (KT)
2.0mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；
C, successive propagation：Adventitious bud is proceeded in propagating culture medium and is cultivated, condition of culture is：28 DEG C, the μ of intensity of illumination 20
mol m-2s-1, hour/day of light application time 14；Per liter described of propagating culture medium contains 6- benzyladenines (BA) 1.5mg, α-naphthalene
Acetic acid (NAA) 0.01mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；Clump bud is trained on propagating culture medium
Foster every month can breed 5.5 times；
D, root culture：The adventitious bud that step c successive propagation is obtained is proceeded to into into root media culture, condition of culture
For 28 DEG C, light intensity is 80 μm of ol m-2s-1, hour/day of light application time 6 so as to take root, grow up to test tube seedling；Described root culture
Per liter of base contains indole -3-butyric acid (IBA) 0.2mg, sucrose 30g, agar 6g, and remaining composition is 1/2MS culture medium, pH6.0.
The transplanting of e, test tube seedling：It is by volume 1 that test tube seedling is transplanted to into Vermiculitum and sandstone：In 1 substrate, it is placed in wet
Cultivate in the environment of profit, shade, water, the moisture and nutritional need applied fertilizer needed for meet test tube seedling growth, Jing cellar cultures
Cultivation Seedling is obtained afterwards；
It is high that test tube seedling can grow to 4~5cm after transplanting one month in substrate, and period is per week to apply 1:1000 compound fertilizer
(N:P:K=1:1:1), between 20-30 DEG C, transplanting survival rate reaches more than 95% to outdoor temperature, ensure that very high surviving
Rate, seedling (cultivation Seedling) now can be transplanted in basin cultivate, and fiery ash is comprised only in basin.
Embodiment 3：Experiment place：In the E Huang Zhang Nature Reserve of Yangchun City
The method that the spring Flos Begoniae Evansianae tissue culture of the present embodiment breeds, it is comprised the following steps that：
A, explant and its process：Stream from the E Huang Zhang Nature Reserve of Guangdong Province Yangchun City collects one plant of sun
Spring and autumn Caulis et folium euphorbiae milii plant, takes spring Flos Begoniae Evansianae blade, Jing after tap water is cleaned up, first with the alcohol water blend table of volume fraction 75%
Sterilization 10 seconds is cleaned in face, and aseptic water elution 2 times soaks 8 minutes afterwards in the mercuric chloride aqueous solution of mass fraction 0.1%, and with
Aseptic water washing 3 times, is finally cut into 0.6cm by blade2Size, the explant being sterilized after processing.
The induction of b, adventitious bud：Explant after disinfecting is inoculated on inducing culture, condition of culture is：26
DEG C, dark culturing is cultivated 60 days, then goes to 100 μm of ol m of intensity of illumination-2s-1, hour/day of light application time 14, culture 30
My god, obtain adventitious bud；Per liter described of inducing culture contains 6- benzyladenines (BA) 0.1mg, sucrose 30g, agar 6g, remaining
Composition be MS culture medium, pH6.0；
C, successive propagation：Adventitious bud is proceeded in propagating culture medium and is cultivated, condition of culture is：26 DEG C, the μ of intensity of illumination 50
mol m-2s-1, hour/day of light application time 14；Per liter described of propagating culture medium contains 6- benzyladenines (BA) 0.5mg, α-naphthalene
Acetic acid (NAA) 0.05mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；Clump bud is trained on propagating culture medium
Foster every month can breed 4.3 times；
D, root culture：The adventitious bud that step c successive propagation is obtained is proceeded to into into root media culture, condition of culture
For 26 DEG C, light intensity is 50 μm of ol m-2s-1, hour/day of light application time 14 so as to take root, grow up to test tube seedling；Described training of taking root
Per liter of foster base contains α-naphthaleneacetic acid (NAA) 0.05mg, sucrose 30g, agar 6g, and remaining composition is 1/2MS culture medium, pH6.0.
The transplanting of e, test tube seedling：It is by volume 1 that test tube seedling is transplanted to into Vermiculitum and sandstone：In 1 substrate, it is placed in wet
Cultivate in the environment of profit, shade, water, the moisture and nutritional need applied fertilizer needed for meet test tube seedling growth, Jing cellar cultures
Cultivation Seedling is obtained afterwards；
It is high that test tube seedling can grow to 4~5cm after transplanting one month in substrate, and period is per week to apply 1:1000 compound fertilizer
(N:P:K=1:1:1), between 20-30 DEG C, transplanting survival rate reaches more than 95% to outdoor temperature, ensure that very high surviving
Rate, seedling (cultivation Seedling) now can be transplanted in basin cultivate, and fiery ash is comprised only in basin.
Embodiment 4：Experiment place：In the E Huang Zhang Nature Reserve of Yangchun City
The method that the spring Flos Begoniae Evansianae tissue culture of the present embodiment breeds, it is comprised the following steps that：
A, explant and its process：Stream from the E Huang Zhang Nature Reserve of Guangdong Province Yangchun City collects one plant of sun
Spring and autumn Caulis et folium euphorbiae milii plant, takes spring Flos Begoniae Evansianae blade, Jing after tap water is cleaned up, first with the alcohol water blend table of volume fraction 75%
Sterilization 10 seconds is cleaned in face, and aseptic water elution 2 times soaks 8 minutes afterwards in the mercuric chloride aqueous solution of mass fraction 0.1%, and with
Aseptic water washing 3 times, is finally cut into 0.6cm by blade2Size, the explant being sterilized after processing.
The induction of b, adventitious bud：Explant after disinfecting is inoculated on inducing culture, condition of culture is：25
DEG C, 10 μm of ol m of intensity of illumination-2s-1Hereinafter, hour/day of light application time 14, cultivates 60 days, then goes to 80 μm of ol of intensity of illumination
m-2s-1, hour/day of light application time 14, cultivate 30 days, obtain adventitious bud；Per liter described of inducing culture is fast containing 6- benzyl glands
Purine (BA) 2.0mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；
C, successive propagation：Adventitious bud is proceeded in propagating culture medium and is cultivated, condition of culture is：25 DEG C, the μ of intensity of illumination 80
mol m-2s-1, hour/day of light application time 14；Per liter described of propagating culture medium contains 6- benzyladenines (BA) 1mg, α-naphthalene second
Acid (NAA) 0.08mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；Clump bud is cultivated on propagating culture medium
Every month can breed 8 times；
D, root culture：The adventitious bud that step c successive propagation is obtained is proceeded to into into root media culture, condition of culture
For 25 DEG C, light intensity is 80 μm of ol m-2s-1, hour/day of light application time 10 so as to take root, grow up to test tube seedling；Described training of taking root
Per liter of foster base contains α-naphthaleneacetic acid (NAA) 0.2mg, sucrose 30g, agar 6g, and remaining composition is 1/2MS culture medium, pH6.0.
The transplanting of e, test tube seedling：It is by volume 1 that test tube seedling is transplanted to into Vermiculitum and sandstone：In 1 substrate, it is placed in wet
Cultivate in the environment of profit, shade, water, the moisture and nutritional need applied fertilizer needed for meet test tube seedling growth, Jing cellar cultures
Cultivation Seedling is obtained afterwards；
It is high that test tube seedling can grow to 4~5cm after transplanting one month in substrate, and period is per week to apply 1:1000 compound fertilizer
(N:P:K=1:1:1), between 20-30 DEG C, transplanting survival rate reaches more than 95% to outdoor temperature, ensure that very high surviving
Rate, seedling (cultivation Seedling) now can be transplanted in basin cultivate, and fiery ash is comprised only in basin.
Embodiment 5：
The present embodiment is substantially the same manner as Example 4, simply its inducing culture be per liter containing N- phenyl-N ' -1,2,3-
Thiadiazoles -5- ureas (TDZ) 0.1mg and sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0.Clump bud is in breeding training
Cultivating on foster base can breed 3.5 times every month；
It is high that test tube seedling can grow to 4~5cm after transplanting one month in substrate, and period is per week to apply 1:1000 compound fertilizer
(N:P:K=1:1:1), between 20-30 DEG C, transplanting survival rate reaches more than 95% to outdoor temperature, ensure that very high surviving
Rate, seedling (cultivation Seedling) now can be transplanted in basin cultivate, and fiery ash is comprised only in basin.
Embodiment 6：
The present embodiment is substantially the same manner as Example 4, simply its inducing culture be per liter containing N- phenyl-N ' -1,2,3-
Thiadiazoles -5- ureas (TDZ) 1mg and sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0.The somatic embryo of induction
Cultivate on the propagating culture medium with adventitious bud and only breed 2.5 times every month；
It is high that test tube seedling can grow to 4~5cm after transplanting one month in substrate, and period is per week to apply 1:1000 compound fertilizer
(N:P:K=1:1:1), between 20-30 DEG C, transplanting survival rate only reaches 45% or so to outdoor temperature, now (can plant seedling
Seedlings cultivating) it is transplanted in basin and cultivates, fiery ash is comprised only in basin.
Claims (3)
1. a kind of method that spring Flos Begoniae Evansianae tissue culture breeds, it is characterised in that comprise the following steps
A, explant and its process：Using the blade of spring Flos Begoniae Evansianae (Begonia coptidifolia) as explant, will be outer
Implant disinfection；
The induction of b, adventitious bud：Explant after disinfecting is inoculated on inducing culture, condition of culture is：22~28
DEG C, 20 μm of ol m of dark culturing or intensity of illumination-2s-1Hereinafter, hour/day of light application time 14, cultivates 60 days, then goes to illumination
40~100 μm of ol m of intensity-2s-1, hour/day of light application time 6~14, cultivate 30 days, obtain adventitious bud；Described inducing culture
Per liter of base contains kinetins 0.1~2.0mg or 6- benzyladenine 0.1~2.0mg or N- phenyl-N ' -1,2,3- thiadiazoles -5- ureas
0.1~2.0mg, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；
C, successive propagation：Adventitious bud is proceeded in propagating culture medium and is cultivated, condition of culture is：22~28 DEG C, intensity of illumination 20~
100μmol m-2s-1, hour/day of light application time 14, per liter of described propagating culture medium containing 6- 0.1~1.5mg of benzyladenine,
0.01~0.1mg of α-naphthaleneacetic acid, sucrose 30g, agar 6g, remaining composition be MS culture medium, pH6.0；
D, root culture：The adventitious bud that step c successive propagation is obtained is proceeded to into into root media culture, condition of culture is 22
~28 DEG C, light intensity is 50~80 μm of ol m-2s-1, hour/day of light application time 6~14 so as to take root grows up to test tube seedling, described
Per liter of root media contains 0.05~0.2mg of 0.05~0.2mg of indole -3-butyric acid or α-naphthaleneacetic acid, and also contains sucrose
30g, agar 6g, remaining composition be 1/2MS culture medium, pH6.0；
The transplanting of e, test tube seedling：Test tube seedling is transplanted in substrate, be placed in moistening, shade in the environment of cultivate, water, apply fertilizer with
Meet the moisture and nutritional need needed for test tube seedling growth, cultivation Seedling is obtained Jing after cellar culture.
2. method according to claim 1, it is characterised in that described is by the autumn in spring by explant disinfection
After Caulis et folium euphorbiae milii vanes tap water is cleaned up, first sterilization 10 seconds is cleaned with the alcohol water blend surface of volume fraction 75%, it is aseptic
Water elution 2 times, soaks 8 minutes afterwards in the mercuric chloride aqueous solution of mass fraction 0.1%, and with aseptic water washing 3 times, finally by leaf
Piece is cut into 0.6cm2Size, is seeded on inducing culture.
3. method according to claim 1, it is characterised in that described substrate is Vermiculitum and sandstone are by volume 1：1
The substrate of mixing.
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| CN108935101B (en) * | 2018-08-13 | 2022-06-07 | 云南省农业科学院花卉研究所 | Method for reducing browning rate of begonia leaf tissue culture |
| CN114176007B (en) * | 2021-12-23 | 2023-01-13 | 中国热带农业科学院热带作物品种资源研究所 | Begonia peltata tissue culture breeding method |
| CN115024223B (en) * | 2022-06-27 | 2023-03-14 | 安徽农业大学 | In-vitro preservation method for malus spectabilis germplasm |
| CN116849126A (en) * | 2023-07-31 | 2023-10-10 | 中国科学院华南植物园 | Method for in-vitro preservation of begonia nigra germplasm resources |
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