CN108588119A - A kind of favorable genes type inducing the method for asparagus bean transgenosis Hairy root and use - Google Patents
A kind of favorable genes type inducing the method for asparagus bean transgenosis Hairy root and use Download PDFInfo
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- CN108588119A CN108588119A CN201810457019.7A CN201810457019A CN108588119A CN 108588119 A CN108588119 A CN 108588119A CN 201810457019 A CN201810457019 A CN 201810457019A CN 108588119 A CN108588119 A CN 108588119A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The invention discloses a kind of method of induction asparagus bean transgenosis Hairy root and the favorable genes types used, select a variety of asparagus bean Genotypes to carry out hair-like root induction, include the following steps：1) acquisition of explant；2) it infects；3) it is that growing carrier induces Hairy root to be formed to sprout bag using seed；Number and the conversion ratio of taking root that be averaged of each asparagus bean Genotype are counted, and carries out significance difference analysis, the favorable genes type asparagus bean Genotype formed by filtering out induction Hairy root after Integrated comparative.The present invention is at low cost, saves space, reduces pollution rate, improve the convenience of experimental implementation, can screen the favorable genes type with high conversion efficiency.
Description
Technical field
Field of plant genetic of the present invention, especially belong to it is a kind of induction asparagus bean transgenosis Hairy root method and
The favorable genes type used.
Background technology
Cowpea (Vigna unguiculata L.Walp) is that a kind of important legume crop, cowpea are main in world wide
It is divided into asparagus bean (V.unguiculata ssp.Sesquipedalis) and common cowpea (V.unguiculata
Ssp.Unguiculata) two subspecies, wherein asparagus bean are important one of the summer vegetable crops in China, deep by the various regions people
Like；Common cowpea is used mainly as grain, is planted in African Territories more.Cowpea has very strong salt resistance drought resistance, but controls
The gene for making these characters is unclear.Transgenic technology is to study the important means of plant gene function and its mechanism of action,
It is also one of the approach of character improvement, the transgenic method being most widely used at present is mainly agrobacterium-mediated transformation, wherein wrapping
Include agrobacterium tumefaciens-mediated transformation and agrobacterium rhizogenes mediated method.It has been reported that by Agrobacterium tumefaciens mediated cowpea and other more
The transgenosis conversion ratio of number beans is very low (Ilori et al., 2011), the very difficult (Garcia of gene stable conversion
Et al., 1986), therefore using agrobacterium rhizogenes induced synthesis the hairy root of target gene this strategy is carried in soybean
It is used widely in the legumes such as (Kuma et al., 2015), Kidney bean (Colpaert et al., 2008).The technology
General Principle be using agrobacterium rhizogenes induction aboveground vegetation part formed transgenosis Hairy root, to obtain a kind of combination seedling
(composite plants), under ground portion are transformant, and aerial part is non-transformant.At present in common cowpea subspecies
It is that growing carrier successfully obtains transgenosis Hairy root combination seedling, such as attached drawing to have been reported through mineral wool block (rockwool cubes)
1, (Mellor et al., 2012), but there has been no the relevant reports for inducing its Hairy root to be formed in asparagus bean subspecies.One
Aspect asparagus bean has genetic diversity, same process may be different to the changing effect of different genotype with common cowpea,
On the other hand using mineral wool block as the Hairy root transformation system of growing carrier, there are a series of significant deficiencies：Such as to the matter of mineral wool block
Amount requires (especially cleanliness) relatively high；Different batches experiment is very high to mineral wool block coherence request；Mineral wool block is removed after conversion
During be easy to damage root system etc..Therefore, it is necessary to a kind of novel Hairy root transformation system is established for asparagus bean,
It is required that it is easy to operate, of low cost, transformation efficiency is high, reproducible.Sterile seed germination bag is a kind of dedicated for observing, grind
Study carefully the special bags of root growth state after seed is sprouted, it is moderate, it can sterile working.Seed sprouts the above-mentioned advantage of bag
The defect of mineral wool block can just be made up.Therefore, quasi-step matrix sterile seed germination bag of the present invention use with to asparagus bean difference
The screening of Genotype Hairy root inducibility establishes a kind of novel, convenient, the high efficient induction asparagus bean of repeatability and turns base
The method formed by Hairy root.
Invention content
Shortcomings and existing when the present invention for existing cowpea Hairy root genetic conversion system mineral wool block is carrier
The problems such as system did not carried out screening to the transformation efficiency of asparagus bean subspecies different genotype, passes through the optimization of Fiber differentiation system
And genotype screening, a kind of method that efficient induction asparagus bean transgenosis Hairy root that being sprouted bag based on seed is formed is provided, is
Asparagus bean functional genome research provides technical support.
To achieve the goals above, the present invention uses following technical scheme：The method for inducing asparagus bean transgenosis Hairy root
And the favorable genes type used, it selects a variety of asparagus bean Genotypes to carry out hair-like root induction, includes the following steps：
1) acquisition of explant:The asparagus bean seed for selecting ripe no disease and pests harm carries out 10min disinfections with 70% alcohol,
Twice of aseptic water washing is sowed in matrix and sprinkles profoundly water, illumination box culture, and cultivation temperature controlled at 32 DEG C of daytime, night
28 DEG C, the photoperiod is 16h illumination, and 8h is dark；Before growth starts extraction in one week or so to trifoliolate leaf, in asparagus bean plant cotyledon
Plant is cut off along 45 degree of angles obliquely downward at the 2-3cm of section lower section, obtains aerial part explant to be infected；
2) it infects：The 4000 revs/min of centrifugation 10min of agrobacterium rhizogenes that will have been activated, after outwelling supernatant, use Liquid Culture
Agrobacterium rhizogenes is suspended into OD by base again600=0.8 or so, take 15-20mL bacterium solutions to be added in 50mL beakers, by cowpea explant
The stem section notch of body, which is immersed into bacterium solution, infects 1h, and the formula of fluid nutrient medium is：20%MS+0.4mM acetosyringones；
3) it is that growing carrier induces Hairy root to be formed to sprout bag using seed：It is sprouted to seed and is added 30ml's or so in bag
Fluid nutrient medium, the aperture that the explant after infecting is sprouted to bag along seed are inserted into seed sprouting bag, each two explant
One, interval aperture, seed sprout bag be placed on rack, with foam board clamping, sealing, moisturizing, whole process is in asepsis ring
It is operated under border, is subsequently placed in 21 DEG C of biochemical cultivation cases under dark condition and co-cultures 3 days, be transferred to illumination box later, trained
Temperature control is supported 22 DEG C of daytime, the photoperiod is 16h illumination, and 8h is dark, forms Hairy root within two weeks or so, is during which sprouted to seed
The adding liquid culture medium of timely and appropriate discovery in bag.
Preferably, the preparation method of step 1) mesostroma is to mix vermiculite and turf according to 3 to 1 ratio, 121
DEG C, it is used after 20min high pressure sterilizations.
Preferably, the agrobacterium rhizogenes in step 1) is R1000 glycerol stocks, the R1000 glycerol stocks, which carry, contains GUS
The binary expression vector pBI121 of reporter gene, the R1000 glycerol stocks pass through following activation process, are inoculated into 1mL first
YEB (Kan containing 50mg/L) fluid nutrient medium in, with the rotating speed culture 12h of 180rpm in 28 DEG C of constant incubators, then take
200 μ L agrobacterium rhizogenes bacterium solutions are inoculated into 200mL YEB (Kan containing 50mg/L), are shaken in 28 DEG C of shaker overnights, culture
To OD600=0.8-1.2.
Preferably, the detection of transgenosis Hairy root ratio is carried out to the Hairy root that step 3) obtains, using to plant
Root system carries out GUS dyeing detections, and the root system of each plant is cut in the centrifuge tube for being placed on and filling GUS dyeing liquors, centrifuge tube is set
Under dark condition, dyeing liquor is removed after shaking 6-7h on 37 DEG C of shaking tables, with soaked in absolute ethyl alcohol 2min, is repeated 3 times at decoloration
Reason, it is transgenosis Hairy root that final display, which is dyed to the root system of blue,.
Preferably, choose 14 asparagus bean Genotypes, by the difference asparagus bean Genotypes of step pair 14 into
Row hair-like root induction, counts number and the conversion ratio of taking root that be averaged of each genotype, and carries out significance difference analysis, passes through synthesis
The favorable genes type asparagus bean Genotype that induction Hairy root is formed is filtered out more afterwards.The asparagus bean gene that this programme uses
Proximate matter material is A, H60；B, Jiangxi butterfly No. 2；C,II7E0323；D,II7E0812；E,JX6；F, F dragon；G, special early 30；H,108-
R153；I,II7E0813；J,II7E0826；K,44-68；L,50-27；M,II7E0900；N, cowpea 106；O, it is unconverted " special
Early 30 " kinds (negative control).
Preferably, the number of taking root of being averaged of the excellent asparagus bean Genotype filtered out is 16, conversion ratio is
55.76%.
Compared with prior art, the cost of this programme is low, saves space, reduces pollution rate, improve experimental implementation
Convenience.In the present invention, it sprouts bag induction asparagus bean using seed and forms hairy root, can largely save sky
Between, the cost of hairy root induction is reduced, there is higher stability and repeatability.Seed sprouts bag and passes through sterilization treatment,
It greatly reduces experiment and occurs the risk polluted in the process, and sprouting bag using seed can be during entire hairy root induction clearly
Observe to Chu the formational situation of root system.
This programme can screen the favorable genes type with high conversion efficiency, by sprouting bag as growing carrier using seed
The different asparagus bean genotype of hairy root induction method pair 14 screen, obtained more afterwards with higher conversion and taken root
The favorable genes type of rate.The asparagus bean transgenosis Hairy root height that bag is sprouted as growing carrier using seed that the present invention is established for the first time
Effect induction system is combined with favorable genes type, is laid a good foundation efficiently to carry out asparagus bean transgene gene functional study.
Therefore, the present invention has the advantages that：(1) at low cost, space is saved, pollution rate is reduced, improves reality
Test the convenience of operation；(2) the favorable genes type with high conversion efficiency can be screened.
Description of the drawings
Fig. 1 is to induce common cowpea to form hairy root schematic diagram using mineral wool block.
Fig. 2 is that the seed sprouting bag of the present invention is placed on schematic diagram on rack.
Fig. 3 be the present invention seed sprout bag induction Hairy root formed the back side and front diagram (arrow meaning be formed
Hairy root).
Fig. 4 is that the cowpea Genotype " II7E0826 " of the present invention is formed for two weeks or so after agrobacterium rhizogenes is infected
Hairy root.
Fig. 5 is that the different asparagus bean Genotypes of the present invention (carry by agrobacterium rhizogenes R1000 and report base containing GUS
The binary expression vector pBI121 of cause) infect GUS expression of results schematic diagrames in rear Hairy root.
Fig. 6 is wild type and to turn p35S after Osmotic treatment:The phenotype comparison schematic diagram of UP12_8740 gene cowpeas.
Fig. 7 be the present invention transgenosis Hairy root in target gene expression RT-PCR detects schematic diagrams.
Specific implementation mode
The present invention will be further described below in conjunction with the accompanying drawings, and in Fig. 5, genotype is as follows：A,H60；B, Jiangxi butterfly No. 2；
C,II7E0323；D,II7E0812；E,JX6；F, F dragon；G, special early 30；H,108-R153；I,II7E0813；J,II7E0826；
K,44-68；L,50-27；M,II7E0900；N, cowpea 106；O, unconverted " special early 30 " kind (negative control)；In Fig. 6, A
For negative control (the combination seedling that unloaded pCAMBIA1301 is converted)；B is the cowpea combination seedling for turning UP12_8740 genes；Figure
In 7, A is template cDNA EF- α primer amplification results；B is UP12_8740FR primer amplification results；C is UP12_11094FR
Primer amplification result；PCR amplification template is as follows：1、2：Unloaded cDNA；3、4：R1000cDNA；5、6：8740cDNA；7、8：
11094cDNA；9：Sterile water.
As shown in Fig. 2-Fig. 7,
Embodiment one, it is drought-induced expressing gene of the growing carrier to Unknown Function in asparagus bean to sprout bag using seed
p35S:UP12_8740 carries out successful conversion.After Osmotic treatment, the expression in root system improves UP12_8740 genes
7.4 times (Xu et al., 2015)；
1) asparagus bean seed it is sterilized, culture after obtain aerial part explant；
The asparagus bean seed for selecting ripe no disease and pests harm, with 70% alcohol disinfecting 10min, twice of aseptic water washing is broadcast
(the preparation of matrix in matrix：Vermiculite and turf are mixed, 121 DEG C according to 3 to 1 ratio, used after 20min high pressure sterilizations)
And sprinkle profoundly water, it is placed in illumination box and cultivates, 25 DEG C of nocturnal temperature, 28 DEG C of day temperature, photoperiod 16h/8h (light/
Secretly).Before growth is extracted out for one week or so to trifoliolate leaf, plant is cut off along 45 degree of angles obliquely downward at 2-3cm under plant cotyledonary node,
Obtain asparagus bean aerial part explant.
2) aerial part is infected with R1000 agrobacterium rhizogenes, sprouting bag using seed obtains combination seedling；
10 μ L agrobacterium rhizogene strain R1000 glycerol stocks are taken (to carry the binary expression vector containing gus reporter gene
PBI121 it) is inoculated into YEB (Kan containing 50mg/L) fluid nutrient medium of 1mL, with 180rpm's in 28 DEG C of constant incubators
Rotating speed culture 12h.200 μ L agrobacterium rhizogenes bacterium solutions are taken to be inoculated into 200mL YEB (Kan containing 50mg/L) again, in 28 DEG C of shaking tables
Upper concussion overnight, culture to OD600=0.8-1.2.
The above-mentioned agrobacterium rhizogenes bacterium solution activated is centrifuged into 10min under the conditions of 4000 revs/min, outwells supernatant, is used
Precipitation is suspended into OD by fluid nutrient medium CIM (20%MS+0.4mM acetosyringones) again600=0.8 or so.Take 15-20mL
Bacterium solution is added in 50mL small beakers, then soaks the stem section notch of the asparagus bean above-ground plant parts explant obtained in step 1
It is less than in bacterium solution and infects 1h.
The culture solution that 30ml or so is added in bag is sprouted to seed, and the explant after infecting is sprouted into bag along seed
Aperture is inserted into seed and sprouts in bag, one, the interval aperture of each two explant, and seed is sprouted bag and is placed on rack, and foam is used
Board clamping, sealing, moisturizing, whole process operate in an aseptic environment, are subsequently placed in 21 DEG C of biochemical cultivation cases under dark condition altogether
Culture 3 days, is transferred to illumination box later, and 22 DEG C, 16h/8h (light dark) photoperiods cultivate, and form hairy root within two weeks or so,
Period pays attention to sprouting the adding liquid suspension medium of timely and appropriate discovery in bag to seed.
3) transplanting growth is carried out to the combination seedling of acquisition；
The asparagus bean combination seedling that step 2 obtains is transferred to containing sterilizing vermiculite and turf (3：1) (diameter in small flower
10cm, high 9cm), it per 5 young plant of flowerpot, sprinkles profoundly water, makes growth five days or so, keep 28 DEG C of day temperature, nocturnal temperature 25
DEG C, it 16h/8h (light dark) photoperiods, finally secures good health, neat and consistent and the good combination seedling of root growth.
4) Osmotic treatment and result；
To the combination seedling Osmotic treatment two weeks that same a batch obtains, phenotypic difference is observed, finds the plant for converting target gene
After two weeks Osmotic treatments, wilting degree is significantly lower than negative control group (Fig. 6).
5) the RT-PCR detections of transgenosis；
With every basin for a unit, control group and transformation seedlings are extracted with plant RNA extraction kit (TIANGEN, Beijing)
Root system RNA, then with reverse transcription reagent box (TIANGEN, Beijing) by RNA reverse transcriptions be cDNA.With house-keeping gene (Reference
Gene) after the concentration of EF- α (table 2) testing and debuggings cDNA (Fig. 7 A), using the cDNA of the concentration as template, drawn with UP12_8740RF
Object (table 2) carries out semiquantitive PCR amplification, and pcr amplification reaction carries out in PCR instrument, and PCR reaction systems are 25 μ l, including 10-
20ng template DNAs, 1 × PCR buffer (10mM Tris-HCl (pH 8.3), 50mM KCl), 0.1mM dNTP, 0.75-
1.5mM MgCl2, 0.2 μM of primer, 1U Taq polymerases.PCR amplification program is：(1) 94 DEG C of 3min of pre-degeneration；(2) 94 are denaturalized
℃30s；(3) anneal 56 DEG C of 40s；(4) extend 72 DEG C of 30s；(5) 72 DEG C of extension 5min；(2-4 steps are 35 for (6) 4 DEG C of preservations
Amplification cycles).The expression of target fragment is analyzed by 1% agarose gel electrophoresis, as a result, it has been found that by Osmotic treatment
The expression of target fragment is significantly higher than control group (Fig. 7 B) in transformant Hairy root afterwards.
Hair-like root induction is carried out by 14 different asparagus bean Genotypes of step pair, counts the average life of each genotype
Radical and conversion ratio, and significance difference analysis is carried out, the excellent long cowpea formed by filtering out induction hair-like after Integrated comparative
Beans Genotype obtains result such as table 1：
Number and the conversion ratio of taking root that be averaged of 1 different genotype material of table
Note：With the expression significant difference (P without identical lowercase after column data in table<0.05).
As it can be seen that the excellent asparagus bean Genotype of gained is II7E0826, its number of taking root of being averaged is 16, and conversion ratio is
55.76%.
Embodiment two sprouts bag unknown gene p35S peculiar to asparagus bean using seed:UP12_11094 is successfully turned
Change.The gene is after Osmotic treatment, and expression in root system is in medium level up-regulation (Xu et al., 2015)；
1) asparagus bean seed it is sterilized, culture after obtain aerial part explant；
Aerial part explant is obtained using the method in embodiment one
2) aerial part is infected with R1000 agrobacterium rhizogenes, sprouting bag using seed obtains combination seedling；
10 μ L R1000 agrobacterium rhizogenes glycerol stocks are taken (to carry the binary expression vector of UP12_11094 genes
PCAMBIA1301 it) is added in 1mL YEB (Kan containing 50mg/L) fluid nutrient medium, with 180rpm in 28 DEG C of constant incubators
Rotating speed culture 12h, then 200 μ L Agrobacteriums is taken to be added in 200mL YEB (Kan containing 50mg/L), in 28 DEG C, 180rpm shaking tables
Upper concussion overnight, culture to OD600=0.8-1.2.
3) restoration ecosystem is carried out to the combination seedling of acquisition；
It secures good health by step 3 operation in example 1, neat and consistent and the good combination seedling of root growth.
4) Osmotic treatment and result；
To the combination seedling Osmotic treatment two weeks that same a batch obtains, phenotypic difference is observed, finds the plant for converting target gene
After two weeks Osmotic treatments, wilting degree and negative control no significant difference.
5) semiquantitive PCR verifies transformant；
With the plant of each basin for a unit, the root of transformant and negative control is extracted respectively according to step 5 in example 1
It is RNA, reverse transcription cDNA, is adjusted with house-keeping gene (Reference gene) EF-1 alpha tests after the concentration of cDNA (Fig. 7 A),
Using the cDNA of the concentration as template, semiquantitive PCR amplification is carried out with UP12_11094 primers (table 2), then solidifying with 1% agarose
Gel electrophoresis analyzes the expression of target fragment, as a result, it has been found that the middle target fragment in transformant Hairy root after Osmotic treatment
Expression is significantly higher than control group (Fig. 7 C).
The primer sequence such as table 2 that embodiment uses:
2 primer sequence of table
Claims (6)
1. it is a kind of induction asparagus bean transgenosis Hairy root method, which is characterized in that select a variety of asparagus bean Genotypes into
Row hair-like root induction, includes the following steps：
1) acquisition of explant:The asparagus bean seed for selecting ripe no disease and pests harm carries out 10min disinfections with 70% alcohol, sterile
Water rinses twice, is sowed in matrix and sprinkles profoundly water, illumination box culture, and cultivation temperature controlled on 32 DEG C of daytime, 28 DEG C of night,
Photoperiod is 16h illumination, and 8h is dark；Before growth starts extraction in one week or so to trifoliolate leaf, under asparagus bean plant cotyledonary node
Plant is cut off along 45 degree of angles obliquely downward at square 2-3cm, obtains aerial part explant to be infected；
2) it infects：It will with fluid nutrient medium after outwelling supernatant by the 4000 revs/min of centrifugation 10min of agrobacterium rhizogenes activated
Agrobacterium rhizogenes is suspended into OD again600=0.8 or so, take 15-20mL bacterium solutions to be added in 50mL beakers, by cowpea explant
Stem section notch, which is immersed into bacterium solution, infects 1h, and the formula of fluid nutrient medium is：20%MS+0.4mM acetosyringones；
3) it is that growing carrier induces Hairy root to be formed to sprout bag using seed：The liquid that 30ml or so is added in bag is sprouted to seed
Culture medium, the aperture that the explant after infecting is sprouted to bag along seed is inserted into seed sprouting bag, between each two explant
Every an aperture, seed is sprouted bag and is placed on rack, and with foam board clamping, sealing, moisturizing, whole process is in an aseptic environment
Operation, is subsequently placed in 21 DEG C of biochemical cultivation cases under dark condition and co-cultures 3 days, is transferred to illumination box, culture temperature later
22 DEG C of daytime, the photoperiod is 16h illumination, and 8h is dark, forms Hairy root within two weeks or so, is during which sprouted in bag to seed for degree control
The adding liquid culture medium of timely and appropriate discovery.
2. a kind of method of induction asparagus bean transgenosis Hairy root according to claim 1, characterized in that base in step 1)
The preparation method of matter is to mix vermiculite and turf according to 3 to 1 ratio, is used after 121 DEG C, 20min high pressure sterilizations.
3. a kind of method of induction asparagus bean transgenosis Hairy root according to claim 1, characterized in that in step 1)
Agrobacterium rhizogenes is R1000 glycerol stocks, and the R1000 glycerol stocks carry the binary expression vector containing gus reporter gene
PBI121, the R1000 glycerol stocks pass through following activation process, are inoculated into YEB (Kan containing 50mg/L) liquid of 1mL first
In culture medium, with the rotating speed culture 12h of 180rpm in 28 DEG C of constant incubators, then 200 μ L agrobacterium rhizogenes bacterium solutions is taken to be inoculated with
Into 200mL YEB (Kan containing 50mg/L), shaken in 28 DEG C of shaker overnights, culture to OD600=0.8-1.2.
4. a kind of method of induction asparagus bean transgenosis Hairy root according to claim 1, characterized in that obtained to step 3)
The Hairy root obtained carries out the detection of transgenosis Hairy root ratio, GUS dyeing detections is carried out using the root system to plant, by each plant
Root system cut in the centrifuge tube for being placed on and filling GUS dyeing liquors, centrifuge tube is placed under dark condition, shakes 6- on 37 DEG C of shaking tables
Dyeing liquor is removed after 7h, with soaked in absolute ethyl alcohol 2min, is repeated 3 times decolorization, and final display is dyed to the root system of blue i.e.
For transgenosis Hairy root.
5. a kind of method of induction asparagus bean transgenosis Hairy root according to claim 1 or 4, characterized in that choose 14
A asparagus bean Genotype carries out hair-like root induction by 14 different asparagus bean Genotypes of step pair, counts each base
Because of number and the conversion ratio of taking root that be averaged of type, and significance difference analysis is carried out, by filtering out induction Hairy root after Integrated comparative
The favorable genes type asparagus bean Genotype of formation.
6. a kind of method of induction asparagus bean transgenosis Hairy root according to claim 5, characterized in that is filtered out is excellent
Being averaged of different asparagus bean Genotype number of taking root is 16, conversion ratio 55.76%.
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| CN110885852A (en) * | 2019-12-05 | 2020-03-17 | 浙江省农业科学院 | Method for efficiently inducing formation of transgenic hairy roots of bottle gourds |
| CN113106118A (en) * | 2021-05-14 | 2021-07-13 | 浙江大学 | Method for screening cowpea genetic transformants by using glyphosate |
| CN113832178A (en) * | 2021-09-08 | 2021-12-24 | 浙江省农业科学院 | Method for establishing agrobacterium rhizogenes-mediated vegetable pea genetic transformation system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885852A (en) * | 2019-12-05 | 2020-03-17 | 浙江省农业科学院 | Method for efficiently inducing formation of transgenic hairy roots of bottle gourds |
| CN113106118A (en) * | 2021-05-14 | 2021-07-13 | 浙江大学 | Method for screening cowpea genetic transformants by using glyphosate |
| CN113832178A (en) * | 2021-09-08 | 2021-12-24 | 浙江省农业科学院 | Method for establishing agrobacterium rhizogenes-mediated vegetable pea genetic transformation system |
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Application publication date: 20180928 |