CN110317825A - A kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes - Google Patents
A kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes Download PDFInfo
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- CN110317825A CN110317825A CN201910352893.9A CN201910352893A CN110317825A CN 110317825 A CN110317825 A CN 110317825A CN 201910352893 A CN201910352893 A CN 201910352893A CN 110317825 A CN110317825 A CN 110317825A
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Abstract
The present invention discloses a kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes, include the following steps: to choose seedling when true leaf will be unfolded after soybean planting, it is carried out at hypocotyl under cotyledonary node at 0.2-0.8cm crosscutting, cuttage after agrobacterium rhizogenes bacterium solution is dipped in the hypocotyl incision of the seedling with cotyledon, and is poured the induction of hairy roots nutrient solution containing the agrobacterium rhizogenes bacterium solution and is cultivated.Advantages of the present invention saves soybean planting matrix and space；The time of hairy generation shortens, and saves operating procedure and time, especially when studying interaction between rhizobium and soybean, reduces the pollution of other rhizobium.
Description
Technical field
The invention belongs to gene engineering technology fields.Turn more particularly to a kind of hairy root induction mediated with agrobacterium rhizogenes
Hairy method for transformation of change method more particularly to soybean.
Background technique
Currently, the genetic transformation that the method for Genetic Transformation of Soybean technology mainly has Agrobacterium tumefaciems (Ti) to mediate is stablized
Transgenic plant and agrobacterium rhizogenes (Ri) mediate hairy conversion method.Agrobacterium tumefaciems (Ti) mediate genetic transformation its
Deficiency is that conversion ratio is extremely low, and screening transformant is more difficult.The hairy conversion that agrobacterium rhizogenes (Ri) mediates is that one kind is quickly obtained
The method of transgenosis chimera plant, symbiotic nitrogen fixation, the expression of soybean root gene specific between research soybean and rhizobium etc.
It is widely used in research.
The hairy conversion that agrobacterium rhizogenes (Ri) mediates is a kind of method for being quickly obtained transgenosis chimera plant.Mesh
Preceding common Efficient Conversion bacterial strain is K599 in hairy conversion process of soybean, and method for transformation uses hypocotyl puncture method
(Kereszt A, Li D et al (2007) Agrobacterium rhizogenes- mediated
Transformation of soybean to study root biology. Nat Protoc 2:948-952), wherein public
The method opened is as follows: (1) by soya seeds sowing in vermiculite, illumination cultivation carries out percutaneous puncture-inoculation after 5 days.(2) purpose is carried
The agrobacterium rhizogenes bacterium solution of carrier is containing vector selection antibiotic and streptomysin (concentration 50mg/L, similarly hereinafter) solid LB media
In 28 DEG C cultivate 2 days, picking monoclonal be added the LB liquid medium 500ul containing above-mentioned antibiotic stay overnight, take 200ul bacterium solution,
It is applied on LB solid medium, is incubated overnight, collect thallus for puncturing with spreader.(3) not yet it is unfolded to soybean cotyledon
When, soybean hypocotyl (Fig. 1 .a) is punctured with 1ml syringe, then moisturizing illumination cultivation.(4) it after growing transgenic hairy root, cuts
Fall main root (Fig. 1 .b), transplanted, carries out being inoculated with etc. correlative study (Fig. 1 .c) after slow seedling.But this hypocotyl puncture method exists
Following disadvantage: (1) hairy transformation efficiency is influenced by genotype: the efficiency that some soybean varieties generate hairy only has 50%.
(2) it is longer to form hairy time: starting to see hairy generation after puncturing 14 days under normal circumstances, after 30 days substantially no longer
There is hairy new generation.The time that most of kind generates hairy is about 15-30 days or so.(3) it after forming hairy, needs
Main root is cut, is transplanted again, in this course, soybean needs slow seedling and be easy to cause artificial pollution.Also there is research
Hairy is induced using cotyledon, i.e., is cut the soybean cotyledon cultivated 7-10 days, and profoundly counts knife at cotyledonary node, in work
The mode impregnated in the agrobacterium rhizogenes bacterium solution of change induce hairy (ancestor knows autumn etc., agrobacterium rhizogenes inducing soybean hairy
The suggestion of system, Hua Zhong Agriculture University's journal, the 6th the 699-703 pages of phase of volume 31 in 2012), its shortcoming is that being also required to cut
Hairy of lower induction and transplanting culture, it is more stringent to operate the requirement of relatively complicated and condition of culture.
It is, thus, sought for or soybean hairy conversion mediating of the agrobacterium rhizogenes that is more effectively easier to operate to of research and development
Method.
Summary of the invention
The present invention for hypocotyl puncture method there are the shortcomings that, carry out technological improvement to be solved to overcome its disadvantage
Technical problem are as follows: shorten the period of hairy conversion, improve the efficiency of hairy of generation；Operating procedure is reduced, is reduced artificial dirty
Dye.
The present invention use crosscutting soybean seedling hypocotyl, agrobacterium rhizogenes is applied to cutting section, cuttage into vermiculite,
And it is poured agrobacterium rhizogenes bacterium solution, visible incision expands and grows hairy after culture, and the method is referred to as cuttage one-step method.
In order to ensure this method can succeed, the cuttage after crosscutting of the hypocotyl of solution soybean seedling is not survived first
Problem after needing to take crosscutting cuttage to this, needs to guarantee enough humidity, it is secondary guarantee effectively to induce grow it is hairy
On the one hand root will select the thin seedling of the soybean of specific growth phase, while need to choose crosscutting position.
In this regard, present invention accordingly provides a kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes, including
Following steps: the seedling after selection soybean planting when true leaf will be unfolded, the hypocotyl under cotyledonary node at 0.2-0.8 cm
Place carries out crosscutting, dips in cuttage after agrobacterium rhizogenes thallus in the hypocotyl incision of the seedling with cotyledon, and be poured and contain the hair
The induction of hairy roots nutrient solution of root Agrobacterium bacterium solution is cultivated, and 0.5mM MgSO is contained preferably in nutrient solution4；1mM CaCl2；
0.7mM KH2PO4；0.5mM Na2HPO4；0.05mM Fe-EDTA；0.5mM NH4NO3；1×Hougland Minor Salt.
Preferably, wherein the temperature when culture is 20-24 DEG C, 80% or more relative humidity, excellent under cultivation temperature
85% or more is selected, such as 85-90%.In order to keep humidity, take such as under type: by 80% or more light passing rate, preferably 90% material
As to keep humidity, be put on high light passing freshness protection package cover normal illumination culture on culturing rack (such as 16 h illumination, 24 DEG C；8 h are black
Secretly, 20 DEG C).
Preferably, crosscutting position is the 0.4-0.6cm under cotyledonary node, at most preferably 0.45-0.55cm.
Preferably, it is described it is crosscutting be with hypocotyl tilt 40-50 degree angle, preferably 45 degree of angles are cut.
In an embodiment, the cuttage is that the hypocotyl incision of the seedling with cotyledon is dipped in agrobacterium rhizogenes thallus
Afterwards, it is put into the vermiculite of preparatory jack, and is compacted with the vermiculite of side.
Preferably, the agrobacterium rhizogenes is Agrobacterium rhyzogenesK599.
Preferably, hypocotyl incision dips in agrobacterium rhizogenes, i.e., the agrobacterium rhizogenes bacterium cultivated on solid LB media
Body；Thereafter the bacterium solution containing the agrobacterium rhizogenes, concentration OD are poured600=0.05 or more, preferably 0.1 or more, such as
0.1-0.15 is preferred bacterial concentration.
In an opposite specific embodiment, the method for the present invention operating procedure is as follows:
(1) first day, soybean planting: the seed of surface sterilizing is sowed in the vermiculite of sterilizing, depth of planting 1.5cm is used in combination
Sterile water pours sufficient；
(2) the 4th days, Agrobacterium activation culture: carry purpose carrier Agrobacterium rhyzogenesK599 (itself have streptomycin resistance,
Str+ bacterium solution) is saved in solid LB media (containing having purpose carrier antibiotic and streptomysin, streptomysin concentration is 25mg/L)
28 DEG C are incubated overnight；
(3) the 6th days, Agrobacterium mass propgation: the solid LB media culture of 2-3 monoclonal bacterium of inoculation to antibiotic-free
Night；
(4) the 7th days, cuttage conversion: true leaf will be unfolded after the 7th day, and left hand pinches cotyledon, under soybean cotyledon node at 0.5cm
With sterilizing/sharp scalpel blade, 45 degree of angles are tilted with hypocotyl and are cut, hypocotyl incision dips in the thallus of K599, is put into pre-
In the vermiculite of first jack, and it is compacted with the vermiculite of side;
It (5) is OD with 5ml concentration600=0.1 K599 bacterium solution induction of hairy roots nutrient solution (contains 0.5mM MgSO4；1mM CaCl2；
0.7mM KH2PO4；0.5mM Na2HPO4；0.05mM Fe-EDTA；0.5mM NH4NO3；1 × Hougland Minor Salt),
High light passing freshness protection package on cover, be put into illumination cultivation on culturing rack (16 h illumination, 24 DEG C；8 h are dark, and 20 DEG C)；
(6) culture is to growing new hairy: having new root to grow in cutting section, with Rhizobium Inoculation or can carry out other related real
It tests.
The present invention is taking the aforementioned measure referred to by the way of crosscutting hypocotyl, and agrobacterium rhizogenes body is smeared
In cutting section, it is ensured that cuttage survival is poured agrobacterium rhizogenes bacterium solution, directly generating in incision site makes hairy, realizes hair
Quick, efficient, a stepization for shape root conversion is completed.Thus, the present invention has following apparent the utility model has the advantages that (1) saves soybean
Planting matrix and space: the culture of transformation of soybean seedling, every box (8cm*11cm*9cm) can plant 20 soybean, be suitble to big rule
The culture of mould.(2) time of hairy generation shortens: the time for generating hairy using this crosscutting-cuttage is faster than puncture method
5-7 days.(3) do not influenced by genotype: the ratio that adopting said method soybean generates transgenic hairy root reaches 90-100%, no
It is influenced by genotype.(4) it after cuttage of the present invention, does not need to cut main root as piercing method and be transplanted again, saved
Operating procedure and time reduce the pollution of other rhizobium especially when studying interaction between rhizobium and soybean.
Detailed description of the invention
Hairy conversion hypocotyl puncture method of soybean of Fig. 1 prior art
A. syringe punctures soybean hypocotyl, after b. grows transgenic hairy root, cuts main root, c. transplanting.
One soybean varieties Williams of Fig. 2 embodiment, 82 hairy conversion cuttage one-step method processes
A. seedling when true leaf will be unfolded, b. hypocotyl incision dip in agrobacterium rhizogenes, and c. cuttage is into vermiculite, d.
The case where the case where hairy is grown at 10 days, e. the 17th day when, grow hairy.
The 17th day long situation of yellow No. 13 hairy conversion cuttage one-step method in two soybean of Fig. 3 embodiment.
Specific embodiment
Below by specific embodiment, the present invention is further illustrated, and with the understanding present invention arranged, but it is not
It is construed as limiting the invention.Operation such as culture medium without special instruction etc. is in the usual way.
Embodiment one
(1) first day, open kind was sequenced in soybean varieties Williams 82(soybean genome) sowing: by the kind of surface sterilizing
Son sowing is in the vermiculite of sterilizing, depth of planting 1.5cm, and pours abundance with sterile water.
(2) the 4th days, Agrobacterium activation culture: carry purpose carrier Agrobacterium rhyzogenesK599 (itself have streptomysin
Resistance, Str+) saving bacterium solution, (containing having purpose carrier antibiotic and streptomysin, streptomysin concentration is 25mg/ in solid LB media
L it is incubated overnight for 28 DEG C in).
(3) the 6th days, Agrobacterium rhyzogenesK599 mass propgation: the solid LB of 2-3 monoclonal bacterium of inoculation to antibiotic-free
Culture medium overnight incubation.
(4) the 7th days, cuttage conversion: (Fig. 2 .a) will be unfolded in true leaf after soybean planting 7 days, and left hand pinches cotyledon, soybean
45 degree of angles are tilted with hypocotyl and are cut, hypocotyl incision is dipped in sterilizing and sharp scalpel blade at 0.5cm under cotyledonary node
The Agrobacterium rhyzogenesK599 cultivated on solid LB media, i.e., the thallus (Fig. 2 .b) collected from solid medium with spreader,
It is put into the vermiculite of preparatory jack, and is compacted (Fig. 2 .c) with the vermiculite of side.
(5) there is K599 bacterium solution (OD with 5ml600=0.1) induction of hairy roots nutrient solution, the concrete composition 0.5mM of nutrient solution
MgSO4；1mM CaCl2；0.7mM KH2PO4；0.5mM Na2HPO4；0.05mM Fe-EDTA；0.5mM NH4NO3；1×
Hougland Minor Salt, high pressure PE high light passing freshness protection package (with a thickness of 0.03mm, light passing rate 90%) on cover, is put into culturing rack
Upper illumination cultivation；
(6) start to grow new hairy within the 10th day after cuttage: thering is new root to grow (Fig. 2 .d) in hypocotyl incision.After cuttage
15th day, for Mao Xinsheng shape root average length after 5cm or so, cuttage the 17th day, average every plant of new root was 15 or so, average
Root long is up to 9cm(Fig. 2 .e).With Rhizobium Inoculation or other related experiments can be carried out at this time.
Embodiment two
The soybean varieties that the present embodiment uses are middle yellow No. 13, and concrete operations exist as shown in Figure 3 referring to the mode of embodiment one
The long root situation of cutting section after 17th day.
Claims (9)
1. a kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes includes the following steps: to choose soybean planting
Seedling when true leaf will be unfolded afterwards, carry out at the hypocotyl under cotyledonary node at 0.2-0.8cm it is crosscutting, in the children with cotyledon
The hypocotyl incision of seedling dips in cuttage after agrobacterium rhizogenes body, and is poured the induction of hairy roots nutrition containing the agrobacterium rhizogenes bacterium solution
Liquid is cultivated.
2. the hairy root induction method for transformation of soybean as described in claim 1, it is characterised in that: the culture of induction of hairy roots nutrient solution
Base group becomes 0.5mM MgSO4；1mM CaCl2；0.7mM KH2PO4；0.5mM Na2HPO4；0.05mM Fe-EDTA；0.5mM
NH4NO3；1×Hougland Minor Salt.
3. the hairy root induction method for transformation of soybean as described in claim 1, which is characterized in that crosscutting position is in cotyledonary node
At lower 0.4-0.6cm, most preferably 0.45-0.55cm.
4. the hairy root induction method for transformation of soybean as described in claim 1, which is characterized in that it is described it is crosscutting be to incline with hypocotyl
Oblique 40-50 degree angle, preferably 45 degree of angles are cut.
5. the hairy root induction method for transformation of soybean as described in claim 1, which is characterized in that wherein opposite when the culture
Humidity 80% or more, preferably 85% or more, such as 85-90%.
6. the hairy root induction method for transformation of soybean as claimed in claim 5, which is characterized in that excellent by 80% or more light passing rate
It selects on 90% material such as high light passing freshness protection package cover to keep humidity, carries out illumination cultivation, preferred illumination condition is 16 h illumination,
24 degree；8 h are dark, and 20 degree.
7. the hairy root induction method for transformation of soybean as claimed in claim 5, which is characterized in that the cuttage is will be with cotyledon
It after the hypocotyl incision of seedling dips in agrobacterium rhizogenes thallus, is put into the vermiculite of preparatory jack, and is compacted with the vermiculite of side.
8. the hairy root induction method for transformation of soybean as claimed in claim 5, which is characterized in that casting contains the agrobacterium rhizogenes
Bacterial concentration be OD600=0.05 or more, preferably 0.1 or more, such as 0.1-0.15 be preferred bacterial concentration.
9. the hairy root induction method for transformation of soybean as claimed in any one of claims 1 to 8, which is characterized in that the root of hair agriculture
Bacillus is Agrobacterium rhyzogenesK599.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113652446A (en) * | 2021-09-22 | 2021-11-16 | 内蒙古农业大学 | Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermedia |
| 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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| CN101705243A (en) * | 2009-11-13 | 2010-05-12 | 华南农业大学 | Application of method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation |
| CN102206651A (en) * | 2011-04-27 | 2011-10-05 | 东北农业大学 | Soybean cyst nematode resistance gene and application thereof |
| CN102766650A (en) * | 2012-07-03 | 2012-11-07 | 吉林大学 | Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method |
| CN106497971A (en) * | 2017-01-13 | 2017-03-15 | 扬州大学 | A kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently |
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- 2019-04-29 CN CN201910352893.9A patent/CN110317825B/en active Active
Patent Citations (4)
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| CN101705243A (en) * | 2009-11-13 | 2010-05-12 | 华南农业大学 | Application of method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation |
| CN102206651A (en) * | 2011-04-27 | 2011-10-05 | 东北农业大学 | Soybean cyst nematode resistance gene and application thereof |
| CN102766650A (en) * | 2012-07-03 | 2012-11-07 | 吉林大学 | Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method |
| CN106497971A (en) * | 2017-01-13 | 2017-03-15 | 扬州大学 | A kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN113652446A (en) * | 2021-09-22 | 2021-11-16 | 内蒙古农业大学 | Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermedia |
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