CN104904603A - 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 and breed field, be specifically related to one begonia tissue culture method for breeding in spring.
Background technology:
Begonia in spring (Begonia coptidifolia) is Begoniaceae (Begoniaceae) Begonia plant.Only be distributed in the limit, streams in Yangchun City, the Guangdong Province E Huang Zhang Nature Reserve of China.Begonia plant is generally without decomposite leaf or shallow decomposite leaf type, and spring, begonia blade was drastic crack blade profile, had higher ornamental value, and occupies critical role in phylogeny.Due to the characteristic distributions that natural distribution is narrow and special, wild spring, begonia population quantity was few.Be badly in need of protection and the breeding process of carrying out this kind.
Summary of the invention:
The object of this invention is to provide a kind of fast, efficiently can breeding begonia in spring, thus make the method that spring, begonia tissue cultures was bred that endangered plants begonia in spring is preserved and develops.
The method that spring, begonia tissue cultures was bred of the present invention, is characterized in that, comprise the following steps
A, explant and process thereof: using the blade in begonia in spring (Begonia coptidifolia) as explant, by explant disinfection;
The induction of b, indefinite bud: be inoculated on inducing culture by the explant after disinfecting, condition of culture is: 22 ~ 28 DEG C, dark culturing or intensity of illumination 20 μm of ol m
-2s
-1below, light application time 14 hours/day, cultivates 60 days, then forwards intensity of illumination 40 ~ 100 μm of ol m to
-2s
-1, light application time 6 ~ 14 hours/day, cultivates 30 days, obtains indefinite bud; Described inducing culture often rises containing kinetin (KT) 0.1 ~ 2.0mg or 6-benzyladenine (BA) 0.1 ~ 2.0mg or N-phenyl-N '-1,2,3-thiadiazoles-5-urea (TDZ) 0.1 ~ 2.0mg, sucrose 30g, agar 6g, all the other compositions are MS medium, pH6.0;
C, successive propagation: proceeded in propagating culture medium by indefinite bud and cultivate, condition of culture is: 22 ~ 28 DEG C, intensity of illumination 20 ~ 100 μm of ol m
-2s
-1, light application time 14 hours/day, described propagating culture medium often rises containing 6-benzyladenine (BA) 0.1 ~ 1.5mg, α-naphthaleneacetic acid (NAA) 0.01 ~ 0.1mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0;
D, culture of rootage: proceeded to by the indefinite bud that step c successive propagation obtains and cultivate to root media, condition of culture is 22 ~ 28 DEG C, and light intensity is 50 ~ 80 μm of ol m
-2s
-1light application time 6 ~ 14 hours/day, make it take root, grow up to test-tube plantlet, described root media often rises containing indole-3-butyric acid (IBA) 0.05 ~ 0.2mg or α-naphthaleneacetic acid (NAA) 0.05 ~ 0.2mg, and also containing sucrose 30g, agar 6g, all the other compositions are 1/2MS medium, pH6.0;
The transplanting of e, test-tube plantlet: test-tube plantlet is transplanted to vermiculite and sandstone by volume in the matrix of 1:1, cultivate under being placed in environment that is moistening, that shade, water, apply fertilizer to meet the moisture needed for test-tube plantlet growth and nutritional need, after cellar culture, obtain cultivation seedling.
After one month, just can to grow to 4 ~ 5cm in matrix high for test-tube plantlet, period is per week executes 1:1000 composite fertilizer (N:P:K=1:1:1), outdoor temperature is between 20-30 DEG C, transplanting survival rate reaches more than 95%, very high survival rate can be ensured, now seedling can be transplanted in basin and cultivate, only containing fire ash in basin.
Described begonia vanes running water in spring is preferably cleaned up by explant disinfection after, first clean sterilization 10 second with volume fraction 75% alcohol water blend surface, sterile water wash-out 2 times, soak 8 minutes in the mass fraction 0.1% mercuric chloride aqueous solution afterwards, and wash 3 times with sterile water, finally blade is cut into 0.6cm
2size, is seeded on inducing culture.
Described MS medium is international medium, its composition and compound method are edited referring to Tan Wencheng, Dai Cegang, " ornamental plants tissue culture technique ", Beijing: China Forestry Publishing House, 1991, described 1/2MS medium is reduced by half at the macroelement in MS and trace element, other components unchanged and the medium formed.
Under isolated culture condition, in medium, plant growth regulator plays key effect for cell dedifferentiation.The present invention, by adjustment medium implants growth regulator component, on purpose can induce the formation of begonia indefinite bud in spring.In addition also can induce the growth of root, finally enable test-tube plantlet bud into complete plant.Transplanting survival rate reaches more than 95%, this endangered plants of begonia in spring can being made to be preserved and develop, laying a good foundation for further utilizing these species from now on.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: experiment place: South China Botanical Garden Chinese Academy of Sciences tissue culture room
The method that spring, begonia tissue cultures was bred of the present embodiment, its concrete steps are as follows:
A, explant and process thereof: collect strain begonia in a spring plant from the stream in the E Huang Zhang Nature Reserve of Yangchun City, Guangdong Province; get begonia blade in spring; after running water cleans up; first clean sterilization 10 second with volume fraction 75% alcohol water blend surface; sterile water wash-out 2 times; soak 8 minutes in the mass fraction 0.1% mercuric chloride aqueous solution afterwards, and wash 3 times with sterile water, finally blade is cut into 0.6cm
2size, the explant after the process that is sterilized.
The induction of b, indefinite bud: be inoculated on inducing culture by the explant after disinfecting, condition of culture is: 22 DEG C, dark culturing, cultivates 60 days, then forwards intensity of illumination 40 μm of ol m to
-2s
-1, light application time 14 hours/day, cultivates 30 days, obtains indefinite bud; Described inducing culture often rises containing kinetin (KT) 0.1mg, sucrose 30g, agar 6g, all the other compositions are MS medium, (to pH6.0 concrete configuration method mixed by mentioned component, adjust pH, then sterilizing is for subsequent use, and the collocation method of following medium is identical therewith);
C, successive propagation: proceeded in propagating culture medium by indefinite bud and cultivate, condition of culture is: 22 DEG C, intensity of illumination 100 μm of olm
-2s
-1, light application time 14 hours/day, described propagating culture medium often rises containing 6-benzyladenine (BA) 0.1mg, α-naphthaleneacetic acid (NAA) 0.1mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0; Clump bud is cultivated every month and can be bred 3.3 times on propagating culture medium;
D, culture of rootage: proceeded to by the indefinite bud that step c successive propagation obtains and cultivate to root media, condition of culture is 22 DEG C, and light intensity is 80 μm of ol m
-2s
-1, light application time 14 hours/day, makes it take root, grows up to test-tube plantlet; Described root media often rises containing indole-3-butyric acid (IBA) 0.05mg, sucrose 30g, agar 6g, and all the other compositions are 1/2MS medium, pH6.0.
The transplanting of e, test-tube plantlet: test-tube plantlet is transplanted to vermiculite and sandstone by volume in the matrix of 1:1, cultivate under being placed in environment that is moistening, that shade, water, apply fertilizer to meet the moisture needed for test-tube plantlet growth and nutritional need, after cellar culture, obtain cultivation seedling;
Test-tube plantlet transplants that just can to grow to 4 ~ 5cm in matrix after one month high, period is per week executes 1:1000 composite fertilizer (N:P:K=1:1:1), outdoor temperature is between 20-30 DEG C, transplanting survival rate reaches more than 95%, very high survival rate can be ensured, now seedling (cultivation seedling) can be transplanted in basin and cultivate, only containing fire ash in basin.
Embodiment 2: experiment place: in the E Huang Zhang Nature Reserve of Yangchun City
The method that spring, begonia tissue cultures was bred of the present embodiment, its concrete steps are as follows:
A, explant and process thereof: collect strain begonia in a spring plant from the stream in the E Huang Zhang Nature Reserve of Yangchun City, Guangdong Province; get begonia blade in spring; after running water cleans up; first clean sterilization 10 second with volume fraction 75% alcohol water blend surface; sterile water wash-out 2 times; soak 8 minutes in the mass fraction 0.1% mercuric chloride aqueous solution afterwards, and wash 3 times with sterile water, finally blade is cut into 0.6cm
2size, the explant after the process that is sterilized.
The induction of b, indefinite bud: be inoculated on inducing culture by the explant after disinfecting, condition of culture is: 28 DEG C, intensity of illumination 20 μm of ol m
-2s
-1below, light application time 14 hours/day, cultivates 60 days, then forwards intensity of illumination 80 μm of ol m to
-2s
-1, light application time 6 hours/day, cultivates 30 days, obtains indefinite bud; Described inducing culture often rises containing kinetin (KT) 2.0mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0;
C, successive propagation: proceeded in propagating culture medium by indefinite bud and cultivate, condition of culture is: 28 DEG C, intensity of illumination 20 μm of olm
-2s
-1, light application time 14 hours/day; Described propagating culture medium often rises containing 6-benzyladenine (BA) 1.5mg, α-naphthaleneacetic acid (NAA) 0.01mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0; Clump bud is cultivated every month and can be bred 5.5 times on propagating culture medium;
D, culture of rootage: proceeded to by the indefinite bud that step c successive propagation obtains and cultivate to root media, condition of culture is 28 DEG C, and light intensity is 80 μm of ol m
-2s
-1, light application time 6 hours/day, makes it take root, grows up to test-tube plantlet; Described root media often rises containing indole-3-butyric acid (IBA) 0.2mg, sucrose 30g, agar 6g, and all the other compositions are 1/2MS medium, pH6.0.
The transplanting of e, test-tube plantlet: test-tube plantlet is transplanted to vermiculite and sandstone by volume in the matrix of 1:1, cultivate under being placed in environment that is moistening, that shade, water, apply fertilizer to meet the moisture needed for test-tube plantlet growth and nutritional need, after cellar culture, obtain cultivation seedling;
Test-tube plantlet transplants that just can to grow to 4 ~ 5cm in matrix after one month high, period is per week executes 1:1000 composite fertilizer (N:P:K=1:1:1), outdoor temperature is between 20-30 DEG C, transplanting survival rate reaches more than 95%, very high survival rate can be ensured, now seedling (cultivation seedling) can be transplanted in basin and cultivate, only containing fire ash in basin.
Embodiment 3: experiment place: in the E Huang Zhang Nature Reserve of Yangchun City
The method that spring, begonia tissue cultures was bred of the present embodiment, its concrete steps are as follows:
A, explant and process thereof: collect strain begonia in a spring plant from the stream in the E Huang Zhang Nature Reserve of Yangchun City, Guangdong Province; get begonia blade in spring; after running water cleans up; first clean sterilization 10 second with volume fraction 75% alcohol water blend surface; sterile water wash-out 2 times; soak 8 minutes in the mass fraction 0.1% mercuric chloride aqueous solution afterwards, and wash 3 times with sterile water, finally blade is cut into 0.6cm
2size, the explant after the process that is sterilized.
The induction of b, indefinite bud: be inoculated on inducing culture by the explant after disinfecting, condition of culture is: 26 DEG C, dark culturing, cultivates 60 days, then forwards intensity of illumination 100 μm of ol m to
-2s
-1, light application time 14 hours/day, cultivates 30 days, obtains indefinite bud; Described inducing culture often rises containing 6-benzyladenine (BA) 0.1mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0;
C, successive propagation: proceeded in propagating culture medium by indefinite bud and cultivate, condition of culture is: 26 DEG C, intensity of illumination 50 μm of ol m
-2s
-1, light application time 14 hours/day; Described propagating culture medium often rises containing 6-benzyladenine (BA) 0.5mg, α-naphthaleneacetic acid (NAA) 0.05mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0; Clump bud is cultivated every month and can be bred 4.3 times on propagating culture medium;
D, culture of rootage: proceeded to by the indefinite bud that step c successive propagation obtains and cultivate to root media, condition of culture is 26 DEG C, and light intensity is 50 μm of ol m
-2s
-1, light application time 14 hours/day, makes it take root, grows up to test-tube plantlet; Described root media often rises containing α-naphthaleneacetic acid (NAA) 0.05mg, sucrose 30g, agar 6g, and all the other compositions are 1/2MS medium, pH6.0.
The transplanting of e, test-tube plantlet: test-tube plantlet is transplanted to vermiculite and sandstone by volume in the matrix of 1:1, cultivate under being placed in environment that is moistening, that shade, water, apply fertilizer to meet the moisture needed for test-tube plantlet growth and nutritional need, after cellar culture, obtain cultivation seedling;
Test-tube plantlet transplants that just can to grow to 4 ~ 5cm in matrix after one month high, period is per week executes 1:1000 composite fertilizer (N:P:K=1:1:1), outdoor temperature is between 20-30 DEG C, transplanting survival rate reaches more than 95%, very high survival rate can be ensured, now seedling (cultivation seedling) can be transplanted in basin and cultivate, only containing fire ash in basin.
Embodiment 4: experiment place: in the E Huang Zhang Nature Reserve of Yangchun City
The method that spring, begonia tissue cultures was bred of the present embodiment, its concrete steps are as follows:
A, explant and process thereof: collect strain begonia in a spring plant from the stream in the E Huang Zhang Nature Reserve of Yangchun City, Guangdong Province; get begonia blade in spring; after running water cleans up; first clean sterilization 10 second with volume fraction 75% alcohol water blend surface; sterile water wash-out 2 times; soak 8 minutes in the mass fraction 0.1% mercuric chloride aqueous solution afterwards, and wash 3 times with sterile water, finally blade is cut into 0.6cm
2size, the explant after the process that is sterilized.
The induction of b, indefinite bud: be inoculated on inducing culture by the explant after disinfecting, condition of culture is: 25 DEG C, intensity of illumination 10 μm of ol m
-2s
-1below, light application time 14 hours/day, cultivates 60 days, then forwards intensity of illumination 80 μm of ol m to
-2s
-1, light application time 14 hours/day, cultivates 30 days, obtains indefinite bud; Described inducing culture often rises containing 6-benzyladenine (BA) 2.0mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0;
C, successive propagation: proceeded in propagating culture medium by indefinite bud and cultivate, condition of culture is: 25 DEG C, intensity of illumination 80 μm of olm
-2s
-1, light application time 14 hours/day; Described propagating culture medium often rises containing 6-benzyladenine (BA) 1mg, α-naphthaleneacetic acid (NAA) 0.08mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0; Clump bud is cultivated every month and can be bred 8 times on propagating culture medium;
D, culture of rootage: proceeded to by the indefinite bud that step c successive propagation obtains and cultivate to root media, condition of culture is 25 DEG C, and light intensity is 80 μm of ol m
-2s
-1, light application time 10 hours/day, makes it take root, grows up to test-tube plantlet; Described root media often rises containing α-naphthaleneacetic acid (NAA) 0.2mg, sucrose 30g, agar 6g, and all the other compositions are 1/2MS medium, pH6.0.
The transplanting of e, test-tube plantlet: test-tube plantlet is transplanted to vermiculite and sandstone by volume in the matrix of 1:1, cultivate under being placed in environment that is moistening, that shade, water, apply fertilizer to meet the moisture needed for test-tube plantlet growth and nutritional need, after cellar culture, obtain cultivation seedling;
Test-tube plantlet transplants that just can to grow to 4 ~ 5cm in matrix after one month high, period is per week executes 1:1000 composite fertilizer (N:P:K=1:1:1), outdoor temperature is between 20-30 DEG C, transplanting survival rate reaches more than 95%, very high survival rate can be ensured, now seedling (cultivation seedling) can be transplanted in basin and cultivate, only containing fire ash in basin.
Embodiment 5:
The present embodiment is substantially the same manner as Example 4, and just its inducing culture be often liter and contains N-phenyl-N '-1,2,3-thiadiazoles-5-urea (TDZ) 0.1mg and sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0.Clump bud is cultivated every month and can be bred 3.5 times on propagating culture medium;
Test-tube plantlet transplants that just can to grow to 4 ~ 5cm in matrix after one month high, period is per week executes 1:1000 composite fertilizer (N:P:K=1:1:1), outdoor temperature is between 20-30 DEG C, transplanting survival rate reaches more than 95%, very high survival rate can be ensured, now seedling (cultivation seedling) can be transplanted in basin and cultivate, only containing fire ash in basin.
Embodiment 6:
The present embodiment is substantially the same manner as Example 4, and just its inducing culture be often liter and contains N-phenyl-N '-1,2,3-thiadiazoles-5-urea (TDZ) 1mg and sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0.The somatic embryo of induction and indefinite bud are cultivated and are only bred 2.5 times on this propagating culture medium every month;
Test-tube plantlet transplants that just can to grow to 4 ~ 5cm in matrix after one month high, period is per week executes 1:1000 composite fertilizer (N:P:K=1:1:1), outdoor temperature is between 20-30 DEG C, transplanting survival rate only reaches about 45%, now seedling (cultivation seedling) can be transplanted in basin and cultivate, only containing fire ash in basin.
Claims (3)
1. the method that spring, begonia tissue cultures was bred, is characterized in that, comprises the following steps
A, explant and process thereof: using the blade in begonia in spring (Begonia coptidifolia) as explant, by explant disinfection;
The induction of b, indefinite bud: be inoculated on inducing culture by the explant after disinfecting, condition of culture is: 22 ~ 28 DEG C, dark culturing or intensity of illumination 20 μm of ol m
-2s
-1below, light application time 14 hours/day, cultivates 60 days, then forwards intensity of illumination 40 ~ 100 μm of ol m to
-2s
-1, light application time 6 ~ 14 hours/day, cultivates 30 days, obtains indefinite bud; Described inducing culture often rises containing kinetin 0.1 ~ 2.0mg or 6-benzyladenine 0.1 ~ 2.0mg or N-phenyl-N '-1,2,3-thiadiazoles-5-urea 0.1 ~ 2.0mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0;
C, successive propagation: proceeded in propagating culture medium by indefinite bud and cultivate, condition of culture is: 22 ~ 28 DEG C, intensity of illumination 20 ~ 100 μm of ol m
-2s
-1, light application time 14 hours/day, described propagating culture medium often rises containing 6-benzyladenine 0.1 ~ 1.5mg, α-naphthaleneacetic acid 0.01 ~ 0.1mg, sucrose 30g, agar 6g, and all the other compositions are MS medium, pH6.0;
D, culture of rootage: proceeded to by the indefinite bud that step c successive propagation obtains and cultivate to root media, condition of culture is 22 ~ 28 DEG C, and light intensity is 50 ~ 80 μm of ol m
-2s
-1, light application time 6 ~ 14 hours/day, makes it take root, grow up to test-tube plantlet, described root media often rises containing indole-3-butyric acid 0.05 ~ 0.2mg or α-naphthaleneacetic acid 0.05 ~ 0.2mg, and also containing sucrose 30g, agar 6g, all the other compositions are 1/2MS medium, pH6.0;
The transplanting of e, test-tube plantlet: be transplanted to by test-tube plantlet in matrix, cultivates under being placed in environment that is moistening, that shade, waters, apply fertilizer to meet the moisture needed for test-tube plantlet growth and nutritional need, obtains cultivation seedling after cellar culture.
2. method according to claim 1, it is characterized in that, described is after being cleaned up by begonia vanes running water in spring by explant disinfection, first clean sterilization 10 second with volume fraction 75% alcohol water blend surface, sterile water wash-out 2 times, soak 8 minutes in the mass fraction 0.1% mercuric chloride aqueous solution afterwards, and wash 3 times with sterile water, finally blade is cut into 0.6cm
2size, is seeded on inducing culture.
3. method according to claim 1, is characterized in that, described matrix is vermiculite and sandstone is the matrix of 1:1 mixing by volume.
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| CN108935101A (en) * | 2018-08-13 | 2018-12-07 | 云南省农业科学院花卉研究所 | A method of reducing lathe work begonia leaf tissue culture melting brown rate |
| CN114176007A (en) * | 2021-12-23 | 2022-03-15 | 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 |
| CN115024223A (en) * | 2022-06-27 | 2022-09-09 | 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108935101A (en) * | 2018-08-13 | 2018-12-07 | 云南省农业科学院花卉研究所 | A method of reducing lathe work begonia leaf tissue culture melting brown rate |
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| 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 |
| CN115024223A (en) * | 2022-06-27 | 2022-09-09 | 安徽农业大学 | In-vitro preservation method for malus spectabilis germplasm |
| 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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| CN104904603B (en) | 2017-05-17 |
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