CN103276014B - Method for improving agribacterium-mediated transformation survival rate of wheat - Google Patents
Method for improving agribacterium-mediated transformation survival rate of wheat Download PDFInfo
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- CN103276014B CN103276014B CN201310242445.6A CN201310242445A CN103276014B CN 103276014 B CN103276014 B CN 103276014B CN 201310242445 A CN201310242445 A CN 201310242445A CN 103276014 B CN103276014 B CN 103276014B
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Abstract
The invention discloses a method for improving the agribacterium-mediated transformation survival rate of wheat. When length cutting sprout leaf-base cushion is used for agrobacterium-mediated transformation, a preservative agent is added into a co-culture medium, so as to restrain the growth of mucedine and improve the survival rate of transformation plants.
Description
Technical field
The invention belongs to wheat transgenic field, relate to and utilize sanitas to improve agriculture bacillus mediated rip cutting seedling phyllopodium seat transformed wheat survival efficiency, and then improve the method for transformation efficiency.
Background technology
Wheat is that ultimate production occupies deputy important food crop, all over the world extensively plantation.Along with the growth of world population and the raising of people's living standard, increasing to the demand of wheat, also more and more higher to the requirement of wheat quality, conventional breeding can not meet the requirement of people to wheat breed and quality.Race's restriction of conventional breeding has been broken in the development of transgenic technology, and many crops such as corn, rape etc. utilize transgenic technology successfully to cultivate high-quality, efficient transgenosis new variety.Compared with other crops, the transgenic breeding of wheat relatively lags behind, and transforms successful example less, and on the one hand, the regeneration frequency of cultivating plant due to Wheat Tissue is low, and the regeneration system of setting up stability and high efficiency is more difficult; On the other hand, wheat is polyploid crop, and genome is large, genetic background complexity, genetic conversion system imperfection.At present, the method for wheat transgenic mainly contains particle bombardment, agrobacterium-mediated transformation, PEG mediated method and pollen tube passage method.
Particle bombardment is the main method of wheat transgenic breeding, in the report of the wheat transgenic plant obtaining, particle bombardment has accounted for the overwhelming majority, this is not subject to the restriction of crop gene type, method relatively simple mainly due to the method, and the wheat transformation system of particle gun mediation is at present fairly perfect.But the cost of particle bombardment cost is high, the cycle is long, is unfavorable for the industrialization of transgenic wheat.PEG method is mainly the method for transformation taking protoplastis as acceptor, due to the protoplast regeneration difficulty of wheat, has limited the use of the method.Pollen tube passage method is the pollen tube that utilizes the rear pollen germination of plant pollination to form, and foreign DNA is imported and still do not possessed in ovum, zygote or the body early embryo cell of normal cell wall.That the method has is simple to operate, it is cheap to expend, do not need through advantages such as loaded down with trivial details tissue culture procedures, but the method is still immature at present, and the importing time of DNA and position tend to affect the setting percentage of plant, are finally difficult to obtain transgenic progeny.The natural host, the cereal crop that are not Agrobacterium due to wheat do not have the reasons such as sentimental reaction to Agrobacterium, it is a difficult problem of pendulum in face of molecular biologist and breeding man that agriculture bacillus mediated wheat transforms always.Although people had realized agriculture bacillus mediated wheat conversion afterwards, but the restrictions such as the genotype of being subject to, conditions of tissue culture, explant type, agrobacterium strains, at present agriculture bacillus mediated wheat transformation efficiency is low, need to be by tissue culture and plant regeneration stage, cycle is long, is unfavorable for the development of industrialization.Agriculture bacillus mediated live body method for transformation does not need, through complicated tissue culture and plant regeneration stage, not only to have the general advantage of agrobacterium mediation converted, and has very high actual operation.
But in the time using rip cutting wheat seedling phyllopodium seat to carry out Agrobacterium-mediated Transformation, in culturing process, the raised growth of mould causes the wheat transformation seedlings overwhelming majority dead altogether, and this kind of phenomenon is very general, has had a strong impact on like this transformation efficiency.Therefore, the present invention, in the time carrying out agriculture bacillus mediated wheat rip cutting seedling phyllopodium seat conversion, adds the growth that sanitas has been contained mould in common culture medium, improves transformed plant survival rate, be increased to 91%-94% by 41%-47%, there is very high actual operation and market popularization value.
Summary of the invention
The present invention aims to provide a kind of method of new rip cutting seedling phyllopodium seat transformed wheat, comprises the following steps:
(1) select suitable wheat seed, reject flat little, with tap water clean, absorb water saturated, the seedling of germination as transform donor;
(2) by the Agrobacterium YEB plate culture medium line activation that contains goal gene, choose single bacterium colony and shake bacterium, get appropriate bacterium liquid coated plate, glass is coated with rod and scrapes Agrobacterium with having added As(200 μ M); KT(0.2mg/L); 2,4-D(1mg/L); DTT(2mM); SIlwet-L77(200 μ l/L) basic liquid nutrient medium suspend, make the OD of suspension bacteria liquid
600value is about 1, pH to 5.5, and gained liquid is conversion fluid;
(3) conversion fluid infects wheat seedling phyllopodium seat;
(4) preparation of culture medium altogether:
In A, every liter of vermiculite, add in advance calcium propionate 1.5-3g;
B, added As(200 μ M), KT(0.2mg/L), 2,4-D(1mg/L), DTT(2mM), SIlwet-L77(200 μ l/L) basic liquid nutrient medium in add again preservative sodium benzoate 0.1-1g/L, potassium sorbate 1.0-5.0 g/L, nipagin esters 0.01-1.0g/L;
C, by A and B mix to humidity be 70-80%, the matrix after mixing is common culture medium.
(5) conversion is imbedded seedling in common culture medium and is carried out common cultivation after finishing, and then field planting is cultivated in booth.
The acquisition of the Agrobacterium that contains goal gene, is that goal gene is inserted to expression vector, then this carrier is imported in Agrobacterium.Conventional expression vector includes but not limited to: pKT serial carrier, pWM serial carrier, pBn serial carrier; Conventional agrobacterium strains includes but not limited to: AGL0, AGL1, GV3101, EHA105, LBA4404 etc.
Wheat described in the present invention is cultivation or Wild Wheats kind, strain, breeding material or intermediate materials.
The present invention has improved the survival efficiency of transformed wheat in the process of agriculture bacillus mediated rip cutting seedling phyllopodium seat transformed wheat after common cultivation stage adds sanitas, survival efficiency has been brought up to 91%-94% by 41%-47%, simultaneously, described sanitas itself is cheap, the little cost of consumption is low, has very large actual application value and marketing prospect.
For the ease of understanding, further annotate the present invention below in conjunction with drawings and Examples.Specific embodiments and the drawings are only in order better to set forth the present invention, not form limitation of the scope of the invention.Obviously those of ordinary skill in the art can, according to explanation herein, make various corrections and change to the present invention within the scope of the invention, and these corrections and change are also included in scope of the present invention.
Brief description of the drawings
Fig. 1 is that wheat adds sanitas in culture medium altogether, seedling normal growth schematic diagram, and survival rate is high.
Fig. 2 is that wheat does not add sanitas in culture medium altogether, and plant stops growing, and grows mould, and survival rate is extremely low, and most conversion seedling are hardened together.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions, and concrete steps can be referring to: " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3
rdedition, 2001, NY, Cold Spring Harbor).
embodiment mono-
1. experiment material
Plasmid: contain anti-Paraquat gene, with the pV1 carrier of antibiotic-screening marker gene, there is paraquat resistance.This gene of pcr amplification is connected to pcambia1300(purchased from Invitrogen company) obtain pV1 carrier on carrier.
Agrobacterium strains: AGL0
Wheat breed for transforming: capital wheat 9158
2. the cultivation of Agrobacterium
The Agrobacterium that what picking glycerine was frozen contain goal gene, setting-out on YEB plate culture medium, 28 DEG C of dark culturing casees are cultivated 2 days.Picking list colony inoculation is in 40ml resistance YEB liquid nutrient medium, and 28 DEG C of dark shaking table 200rpm shake and spend the night, to OD
600≈ 1.Draw above-mentioned bacterium liquid 400 μ l and coat on the YEB solid medium that plate diameter is 15cm, 28 DEG C of dark culturing case cultivations are for subsequent use after 2 days, do not continue to retain in 28 DEG C of dark culturing casees or unloading in 4 DEG C of refrigerators.
3. the preparation of conversion fluid
First according to the basic liquid nutrient medium of following formulated.
Basic liquid nutrient medium preparation (1L):
MgSO
4 0.12209g
KCl 2.95g
NaH
2PO
4·2H
2O 0.15g
Inositol 1.00g
L-glutamic acid 0.876g
Aspartic acid 0.266g
Arginine 0.174g
Casein hydrolysate 0.3g
Sucrose 20g
Iron salt solutions 5ml 1. EDTA-2Na 7.46g/L adjusts pH=8.0
2. add again 4.5ml/L 5M NaCl
③FeSO
4·7H
2O 5.56g/L
CaCl
2solution
4.5ml 25.06g/L
VB
1solution
10ml 1g/L
VB
6solution
1ml 1g/L
Nicotinic acid solution 1ml 0.1g/L
B
5trace 1ml MnSO
4h
2o 7.58g/L
ZnSO
4·7H
2O 2.0g/L
H
3BO
3 3.0g/L
KI 0.75g/L
Na
2MoO
4·2H
2O 0.25g/L
CuSO
4·5H
2O 0.025g/L
CoCl
2·6H
2O 0.025g/L
In the basic liquid nutrient medium of 1L, add following ingredients: As(200 μ M); KT(0.2mg); 2,4-D(1mg); DTT(2mM); SIlwet-L77(200 μ l), suspends scraping the Agrobacterium basic liquid nutrient medium that has added mentioned component, makes the OD of suspension bacteria liquid
600value is about 1, pH to 5.5, and gained bacterium liquid is conversion fluid.
4. seedling is selected to prepare
After tap water washing wheat seed, soak and make it to absorb saturated seedling of tap water, 25 DEG C, 10 hours dark of illumination in 14 hours, every day is each flushing once sooner or later, and seedling to plumular axis grows to 1cm, can be used for transforming.
5. conversion fluid is contaminated wheat seedling phyllopodium seat
(1) dip with the point of a knife of scalpel the central part that conversion fluid inserts phyllopodium seat and transform, blade to base portion direction gently oblique pull go out, rip cutting wound length is 1mm, does not penetrate phyllopodium seat, it is complete making wheat seedling;
(2) wheat seedling of causing injury is immersed in conversion fluid, 28 DEG C, 24 hours dark conditions, 150rpm cultivates 20min.
6. the preparation of culture medium altogether:
In A, every liter of vermiculite, add in advance calcium propionate 1.5g;
B, added As(200 μ M), KT(0.2mg/L), 2,4-D(1mg/L), DTT(2mM), SIlwet-L77(200 μ l/L) basic liquid nutrient medium in add again preservative sodium benzoate 0.1g/L, potassium sorbate 1.0g/L, nipagin esters 0.01g/L;
C, by A and B mix to humidity be 70-80%, the matrix after mixing is common culture medium.
7. will after naturally shady the wheat seedling after transforming doing, imbed in common culture medium, 25 DEG C, under 24 hours dark conditions, cultivate 3d, then field planting is cultivated in booth, makes survival rate 47% be increased to 91% when not adding sanitas by this method.
embodiment bis-
1. experiment material
Plasmid: contain anti-Paraquat gene, with the pV1 carrier of antibiotic-screening marker gene.This gene of pcr amplification is connected to pcambia1300(purchased from Invitrogen company) obtain pV1 carrier on carrier.
Agrobacterium strains: AGL0
Wheat breed for transforming: capital wheat 9158
2. the cultivation of Agrobacterium
The Agrobacterium that what picking glycerine was frozen contain goal gene, setting-out on YEB plate culture medium, 28 DEG C of dark culturing casees are cultivated 2 days.Picking list colony inoculation is in 40ml resistance YEB liquid nutrient medium, and 28 DEG C of dark shaking table 200rpm shake and spend the night, to OD
600≈ 1.Draw above-mentioned bacterium liquid 400 μ l and coat on the YEB solid medium that plate diameter is 15cm, 28 DEG C of dark culturing case cultivations are for subsequent use after 2 days, do not continue to retain in 28 DEG C of dark culturing casees or unloading in 4 DEG C of refrigerators.
3. the preparation of conversion fluid
First according to the basic liquid nutrient medium of following formulated.
Basic liquid nutrient medium preparation (1L):
MgSO
4 0.12209g
KCl 2.95g
NaH
2PO
4·2H
2O 0.15g
Inositol 1.00g
L-glutamic acid 0.876g
Aspartic acid 0.266g
Arginine 0.174g
Casein hydrolysate 0.3g
Sucrose 20g
Iron salt solutions 5ml 1. EDTA-2Na 7.46g/L adjusts pH=8.0
2. add again 4.5ml/L 5M NaCl
③FeSO
4·7H
2O 5.56g/L
CaCl
2solution
4.5ml 25.06g/L
VB
1solution
10ml 1g/L
VB
6solution
1ml 1g/L
Nicotinic acid solution 1ml 0.1g/L
B
5trace 1ml MnSO
4h
2o 7.58g/L
ZnSO
4·7H
2O 2.0g/L
H
3BO
3 3.0g/L
KI 0.75g/L
Na
2MoO
4·2H
2O 0.25g/L
CuSO
4·5H
2O 0.025g/L
CoCl
2·6H
2O 0.025g/L
In the basic liquid nutrient medium of 1L, add following ingredients:
As(200 μ M); KT(0.2mg); 2,4-D(1mg); DTT(2mM); SIlwet-L77(200 μ l), suspends scraping the Agrobacterium basic liquid nutrient medium that has added mentioned component, makes the OD of suspension bacteria liquid
600value is about 1, pH to 5.5, and gained bacterium liquid is conversion fluid.
4. seedling is selected to prepare
After tap water washing wheat seed, soak and make it to absorb saturated seedling of tap water, 25 DEG C, 10 hours dark of illumination in 14 hours, every day is each flushing once sooner or later, and seedling to plumular axis grows to 1cm, can be used for transforming.
5. conversion fluid is contaminated wheat seedling phyllopodium seat
(1) dip with the point of a knife of scalpel the central part that conversion fluid inserts phyllopodium seat and transform, blade to base portion direction gently oblique pull go out, rip cutting wound length is 1mm, does not penetrate phyllopodium seat, it is complete making wheat seedling;
(2) wheat seedling of causing injury is immersed in conversion fluid, 28 DEG C, 24 hours dark conditions, 150rpm cultivates 20min.
6. the preparation of culture medium altogether:
In A, every liter of vermiculite, add in advance calcium propionate 3g;
B, added As(200 μ M), KT(0.2mg/L), 2,4-D(1mg/L), DTT(2mM), SIlwet-L77(200 μ l/L) basic liquid nutrient medium in add again preservative sodium benzoate 1g/L, potassium sorbate 5.0 g/L, nipagin esters 1.0g/L;
C, by A and B mix to humidity be 70-80%, the matrix after mixing is common culture medium.
7. will after naturally shady the wheat seedling after transforming doing, imbed in common culture medium, 25 DEG C, under 24 hours dark conditions, cultivate 3d, then field planting is cultivated in booth, makes survival rate 46% be increased to 93% when not adding sanitas by this method.
embodiment tri-
1. experiment material
Plasmid: contain anti-Paraquat gene, with the pV1 carrier of antibiotic-screening marker gene.This gene of pcr amplification is connected to pcambia1300(purchased from Invitrogen company) obtain pV1 carrier on carrier.
Agrobacterium strains: AGL0
Wheat breed for transforming: capital wheat 9158
2. the cultivation of Agrobacterium
The Agrobacterium that what picking glycerine was frozen contain goal gene, setting-out on YEB plate culture medium, 28 DEG C of dark culturing casees are cultivated 2 days.Picking list colony inoculation is in 40ml resistance YEB liquid nutrient medium, and 28 DEG C of dark shaking table 200rpm shake and spend the night, to OD
600≈ 1.Draw above-mentioned bacterium liquid 400 μ l and coat on the YEB solid medium that plate diameter is 15cm, 28 DEG C of dark culturing case cultivations are for subsequent use after 2 days, do not continue to retain in 28 DEG C of dark culturing casees or unloading in 4 DEG C of refrigerators.
3. the preparation of conversion fluid
First according to the basic liquid nutrient medium of following formulated.
Basic liquid nutrient medium preparation (1L):
MgSO
4 0.12209g
KCl 2.95g
NaH
2PO
4·2H
2O 0.15g
Inositol 1.00g
L-glutamic acid 0.876g
Aspartic acid 0.266g
Arginine 0.174g
Casein hydrolysate 0.3g
Sucrose 20g
Iron salt solutions 5ml 1. EDTA-2Na 7.46g/L adjusts pH=8.0
2. add again 4.5ml/L 5M NaCl
③FeSO
4·7H
2O 5.56g/L
CaCl
2solution
4.5ml 25.06g/L
VB
1solution
10ml 1g/L
VB
6solution
1ml 1g/L
Nicotinic acid solution 1ml 0.1g/L
B
5trace 1ml MnSO
4h
2o 7.58g/L
ZnSO
4·7H
2O 2.0g/L
H
3BO
3 3.0g/L
KI 0.75g/L
Na
2MoO
4·2H
2O 0.25g/L
CuSO
4·5H
2O 0.025g/L
CoCl
2·6H
2O 0.025g/L
In the basic liquid nutrient medium of 1L, add following ingredients:: As(200 μ M); KT(0.2mg); 2,4-D(1mg); DTT(2mM); SIlwet-L77(200 μ l), suspends scraping the Agrobacterium basic liquid nutrient medium that has added mentioned component, makes the OD of suspension bacteria liquid
600value is about 1, pH to 5.5, and gained bacterium liquid is conversion fluid.
4. seedling is selected to prepare
After tap water washing wheat seed, soak and make it to absorb saturated seedling of tap water, 25 DEG C, 10 hours dark of illumination in 14 hours, every day is each flushing once sooner or later, and seedling to plumular axis grows to 1cm, can be used for transforming.
5. conversion fluid is contaminated wheat seedling phyllopodium seat
(1) dip with the point of a knife of scalpel the central part that conversion fluid inserts phyllopodium seat and transform, blade to base portion direction gently oblique pull go out, rip cutting wound length is 1mm, does not penetrate phyllopodium seat, it is complete making wheat seedling;
(2) wheat seedling of causing injury is immersed in conversion fluid, 28 DEG C, 24 hours dark conditions, 150rpm cultivates 20min.
6. the preparation of culture medium altogether:
In A, every liter of vermiculite, add in advance calcium propionate 2.0g;
B, added As(200 μ M), KT(0.2mg/L), 2,4-D(1mg/L), DTT(2mM), SIlwet-L77(200 μ l/L) basic liquid nutrient medium in add again preservative sodium benzoate 0.7g/L, potassium sorbate 3.0 g/L, nipagin esters 0.6g/L;
C, by A and B mix to humidity be 70-80%, the matrix after mixing is common culture medium.
7. the wheat seedling after transforming is imbedded in common culture medium after shady doing naturally, 25 DEG C, under 24 hours dark conditions, cultivate 3d, then field planting is cultivated in booth.Make survival rate 41% be increased to 94% when not adding sanitas by this method.
Claims (7)
1. a method for rip cutting seedling phyllopodium seat transformed wheat, comprises the following steps:
(a) cleaning and dipping wheat seed saturated the seedling that make it to absorb water, the plumular axis that grows seedlings grows to 1cm;
(b) by the Agrobacterium that contains goal gene with containing AS, KT, 2, the basic liquid nutrient medium of 4-D, DTT, Silwet-L77 suspends;
(c) dip the conversion fluid wheat seedling phyllopodium seat of causing injury with scalpel;
(d) wheat seedling after causing injury is immersed in conversion fluid, and under 28 DEG C, 24 hours dark conditions, 150rpm cultivates 20min;
(e) wheat seedling after transforming is imbedded in the common culture medium that contains sanitas after shady doing naturally, 25 DEG C, under 24 hours dark conditions, cultivate 3d, then field planting is cultivated in booth, and the preparation process of wherein said common culture medium comprises:
In A, every liter of vermiculite, add in advance calcium propionate 1.5-3g;
B, add 200 μ M As, 0.2mg/L KT, 1mg/L 2, in the basic liquid nutrient medium of 4-D, 2mM DTT, 200 μ l/L SIlwet-L77, added again preservative sodium benzoate 0.1-1g/L, potassium sorbate 1.0-5.0g/L, nipagin esters 0.01-1.0g/L;
C, by A and B mix to humidity be 70-80%, the matrix after mixing is common culture medium.
2. method according to claim 1, is characterized in that: the described culture condition growing seedlings of step (a) is 10 hours dark of illumination in 25 DEG C, 14 hours.
3. method according to claim 1, it is characterized in that: what step (b) was described contains AS, KT, 2, in the basic liquid nutrient medium of 4-D, DTT, Silwet-L77, the concentration of AS is 200 μ M, 2, the concentration of 4-D is 1mg/L, the concentration of KT is 0.2mg/L, and the concentration of DTT is 2mM, and the concentration of Silwet-L77 is 200 μ L/L.
4. method according to claim 1, is characterized in that, described in contain goal gene Agrobacterium be that goal gene is inserted to expression vector, then this carrier is imported in Agrobacterium.
5. method according to claim 1, is characterized in that, the bacterial strain of described Agrobacterium is AGL0, AGL1, GV3101, EHA105, LBA4404.
6. method according to claim 1, it is characterized in that step (c) described dip the conversion fluid wheat seedling phyllopodium seat of causing injury with scalpel, concrete operations are: the central part that dips conversion fluid insertion phyllopodium seat with the point of a knife of scalpel, blade to base portion direction gently oblique pull go out, rip cutting wound length is 1mm, do not penetrate phyllopodium seat, it is complete making wheat seedling.
7. according to the method described in the arbitrary claim of claim 1-6, it is characterized in that, described wheat is cultivation or Wild Wheats kind, strain, breeding material or intermediate materials.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101687338A (en) * | 2007-01-17 | 2010-03-31 | 詹尼克斯公司 | Preservative compositions for wood and like materials |
| CN102220373A (en) * | 2010-05-17 | 2011-10-19 | 北京未名凯拓作物设计中心有限公司 | Wheat transgene method by slivering seedling leaf bases |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101687338A (en) * | 2007-01-17 | 2010-03-31 | 詹尼克斯公司 | Preservative compositions for wood and like materials |
| CN102220373A (en) * | 2010-05-17 | 2011-10-19 | 北京未名凯拓作物设计中心有限公司 | Wheat transgene method by slivering seedling leaf bases |
Non-Patent Citations (2)
| Title |
|---|
| 冯志敏等.几种防腐剂对青霉菌抑菌效果的观察.《四川省卫生管理干部学院学报》.1997,第16卷(第03期),结果与讨论. |
| 几种防腐剂对青霉菌抑菌效果的观察;冯志敏等;《四川省卫生管理干部学院学报》;19970928;第16卷(第03期);结果与讨论 * |
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