CN104988178A - Genetic transformation method utilizing agrobacterium tumefaciens to infect maize immature embryos - Google Patents
Genetic transformation method utilizing agrobacterium tumefaciens to infect maize immature embryos Download PDFInfo
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Abstract
The invention relates to plant tissue culture and genetic transformation, in particular to a genetic transformation method utilizing agrobacterium tumefaciens to infect maize immature embryos. The genetic transformation method comprises the following steps: (1) preparing an agrobacterium tumefaciens infection solution : taking agrobacterium tumefaciens bacterial plaques, dissolving the bacterial plaques in an infection solution with the pH value of 4.8-5.6, slowly shaking for 3-4 h at the room temperature and regulating the OD550 to be 0.5-0.55 to obtain the agrobacterium tumefaciens infection solution; (2) performing infection and co-culture: infesting maize immature embryos with the infection solution, wherein the ratio of the infection solution to the maize immature embryos is 1ml to 100-150, removing the infection solution after finish of infection, adding the agrobacterium tumefaciens infection solution, infecting for 5-15 min in a manner of keeping out of the sun, after finish of infection, placing the immature embryos in a co-culture medium in the way that the shield faces of the immature embryos are upward, and culturing for 1-4 days at 19-23 DEG C. The genetic transformation method is simple and convenient, mature in system, accurate and stable in transformation result and efficient in transformation.
Description
Technical field
The present invention relates to plant tissue culture and genetic transformation, be specifically related to a kind of genetic transforming method utilizing Agrobacterium to infect maize immature embryos.
Background technology
Corn is one of world's Three major grain crops, is also the important source material of modern food industry, medicine industry and chemical industry, and its Study on Genetic Transformation is subject to the attention of various countries scientist always.The main method adopted take maize immature embryos as the Agrobacterium-mediated Transformation method of acceptor now, but corn is monocotyledons, is not the natural host of Agrobacterium, and therefore agriculture bacillus mediated corn transformation difficulty is larger.
Find through retrieval, publication number is a kind of method that the application for a patent for invention of CN103114106 discloses that Agrobacterium infects maize immature embryos, in infection processs, first carry out Heat thermostability to maize immature embryos, then carry out Agrobacterium and infect and Dual culture, that greatly can improve Agrobacterium like this infects efficiency.
Publication number is the corn genetic transformation method that the application for a patent for invention of CN102051377 discloses a kind of non-tissue culture, comprises (1) and strips maize immature embryos; (2) Agrobacterium bacterium liquid is prepared; (3) to infect and Dual culture; (4) transfer-gen plant growth; (5) detection of transfer-gen plant.
But existing genetic transforming method can not ensure transformation efficiency, and the microbiological contamination problem having Agrobacterium to infect excessively to cause.
Summary of the invention
The object of this invention is to provide that a kind of transformation efficiency is stablized, the simple Agrobacterium that utilizes infects the genetic transforming method of maize immature embryos.
For achieving the above object, the technical scheme that concrete employing is following:
Utilize Agrobacterium to infect a genetic transforming method for maize immature embryos, specifically comprise the steps:
(1) prepare Agrobacterium and infect liquid: get Agrobacterium bacterial plaque be dissolved in pH value be 4.8 ~ 5.6 infect in liquid, room temperature (19 ~ 25 DEG C) shakes 3 ~ 4h slowly, adjust OD
550be 0.5 ~ 0.55, obtain Agrobacterium and infect liquid;
(2) to infect and Dual culture: with described in infect liquid maize immature embryos infected, infect liquid: maize immature embryos=1mL:(100 ~ 150) individual, infect removal after terminating and infect liquid, add described Agrobacterium and infect liquid, lucifuge 5 ~ 15min, after infecting end, faces up rataria shield, put into Dual culture substratum, 19 ~ 23 DEG C of light culture 1 ~ 4 day.
In the inventive solutions, the component infecting liquid is as follows: N6 substratum 1.99 ~ 3.98g/L, 2,4-dichlorphenoxyacetic acid 1.0 ~ 1.5mg/L, proline(Pro) 0.5 ~ 0.7g/L, sucrose 68.4 ~ 75g/L, glucose 36 ~ 40g/L, MES 0.3 ~ 0.5g/L, Syringylethanone 100 ~ 150mM/L.
Preferably, the component infecting liquid is as follows: N6 substratum 1.99g/L, 2,4 dichlorophenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 68.4g/L, glucose 36g/L, MES 0.5g/L, Syringylethanone 100mM/L.
Most preferably, when the pH value infecting liquid is 5.2, effectively can promotes that Agrobacterium is active, improve and infect efficiency.
More concrete, the preparation method infecting liquid is: by N6 substratum, 2,4-dichlorphenoxyacetic acid, proline(Pro), sucrose, glucose and MES add in distilled water and thoroughly dissolve, after adjust ph, be cooled to 50 DEG C, add the Syringylethanone that suction filtration sterilizing is preserved, stir afterwards, pour in sterile petri dish, 40-50 ware/L.The preparation method infecting liquid provided by the invention farthest can keep the activity of each component, is conducive to raising and infects efficiency.
In step of the present invention (2), the following N6 substratum of the component 1.99 ~ 3.98g/L of described Dual culture substratum, 2,4 dichlorophenoxyacetic acid 1.0 ~ 1.5mg/L, proline(Pro) 0.5 ~ 0.7g/L, sucrose 30 ~ 35g/L, MES 0.3 ~ 0.5g/L, plant gel 3 ~ 4g/L, Syringylethanone 100 ~ 150mM/L, Silver Nitrate 5 ~ 7mM/L, cysteine hydrochloride 300 ~ 400mg/L, pH value is 5.8 ~ 6.0.
Preferably, the component of described Dual culture substratum is as follows: N6 substratum 1.99g/L, 2,4 dichlorophenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 30g/L, MES 0.5g/L, plant gel 3g/L, Syringylethanone 100mM/L, Silver Nitrate 5mM/L, cysteine hydrochloride 400mg/L, pH value is 5.8.When in this, as Dual culture substratum, the too fast breeding of Agrobacterium in Dual culture process can be prevented, infect excessively, cause rataria dead.
More concrete, the preparation method of Dual culture substratum is: by N6 substratum, 2,4-dichlorphenoxyacetic acid, proline(Pro), sucrose, MES mix, plant gel is added after being settled to pH5.8 ~ 6.0, then autoclave sterilization (being preferably 121 DEG C of autoclave sterilization 30min), finally be cooled to Syringylethanone, Silver Nitrate, cysteine hydrochloride that 50-45 DEG C adds suction filtration sterilizing, magnetic stirring apparatus stir, is dispensed in sterile petri dish and get final product.The preparation method of Dual culture substratum provided by the invention ensure that the activity of each component of substratum can not be lost, and concentration is accurate, aseptic safe.
In the inventive solutions, after infecting end, preferably rataria shield is faced up, put into Dual culture substratum, 19 ~ 23 DEG C of light culture 1 ~ 3 day, be more preferably at 22 DEG C and cultivate 3 days, now can significantly improve the transformation efficiency of maize immature embryos.
In the inventive solutions, the preparation method of Agrobacterium bacterial plaque can be any means of the prior art, the method that preferred employing is following: with transfering loop picking agrobacterium strains in the setting-out of Agrobacterium solid medium, 25 ~ 30 DEG C of light culture 2 ~ 3 days (being preferably 28 DEG C of light culture 2 days), Agrobacterium solid medium is kept in the environment of-80 DEG C, each picking bacterial plaque is applied in Agrobacterium substratum solid medium, and 20 ~ 25 DEG C of light culture can obtain Agrobacterium bacterial plaque after (being preferably 22 DEG C of light culture 3 days) in 2 ~ 4 days.
Wherein, the agrobacterium strains of employing is that EHA101 contains plasmid PTF102, containing bar gene, β-glucuronidase reporter gene, is obtained from transgenic plant environmental safety study seminar of Jilin Academy of Agricultural Science Agricultural biotechnologies institute.
The component of described Agrobacterium solid medium is as follows: peptone 10g/L, NaCl 15g/L, yeast extract 10g/L, agar 15g/L, Rifampin 50mg/L, spectinomycin 50mg/L, kantlex 50mg/L.Yeast extract is optional commercially available prod, such as the Y1625 of sigma company.
More concrete, the preparation method of Agrobacterium solid medium is: by peptone, NaCl, yeast extract, agar mixing, adjust ph (being preferably pH value is 7.0), is cooled to less than 50 DEG C and adds Rifampin, spectinomycin, kantlex 50mg/L after 121 DEG C of autoclave sterilizations.The preparation method of Agrobacterium solid medium provided by the invention ensures that thalline can normal growth, and antibiotic adding effectively screens thalline, ensure that the efficiency that subsequent transformation is tested
The method that strips of maize immature embryos of the present invention is: choose the self-pollination young fringe of 9 ~ 11 days, sterilizing, at the narrow end of young fringe, forceps tips is inserted in young fringe, knife blade is used to ream young grain 1/3 ~ 2/3 from narrow end to wide end, with the wide end feed of the knife back at young grain, to exert oneself picking rataria to narrow end.
The preferred forms that the present invention utilizes Agrobacterium to infect the genetic transforming method of maize immature embryos can make the transformation efficiency of maize immature embryos reach 48.6%, specifically comprises the steps:
(1) prepare Agrobacterium and infect liquid: get Agrobacterium bacterial plaque and be dissolved in and infect in liquid, room temperature shakes 3 ~ 4h slowly, adjust OD
550=0.5 ~ 0.55, described in infect liquid pH value be 5.2;
(2) to infect and Dual culture: with described in infect liquid maize immature embryos infected, infect liquid: maize immature embryos=1mL:(100 ~ 150) individual, infect removal after terminating and infect liquid, add described Agrobacterium and infect liquid, lucifuge 10min infects, and after infecting end, is faced up by rataria shield, put into Dual culture substratum, 22 DEG C of light culture 3 days;
Wherein, described in infect the component of liquid as follows: N6 substratum 1.99g/L, 2,4 dichlorophenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 68.4g/L, glucose 36g/L, MES 0.5g/L, Syringylethanone 100mM/L, pH value is 5.2;
The component of described Dual culture substratum is as follows: N6 substratum 1.99g/L, 2,4-dichlorphenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 30g/L, MES 0.5g/L, plant gel 3g/L, Syringylethanone 100mM/L, Silver Nitrate 5mM/L, cysteine hydrochloride 400mg/L, pH value is 5.8.
The genetic transforming method utilizing Agrobacterium to infect maize immature embryos provided by the invention, specify that and infect liquid, the formula of Dual culture substratum and pH value, the preparation method of Agrobacterium bacterium liquid and the time of Dual culture, its cultural method is easy, system is ripe, and conversion results accurate stable is efficient.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.The raw material adopted in embodiment all can select commercially available product.
Embodiment 1
Maize immature embryos takes from the young fringe of corn inbred line Hi II in the present embodiment, the young fringe of Hi II is provided by transgenic plant environmental safety study seminar of Jilin Academy of Agricultural Science Agricultural biotechnologies institute, agrobacterium strains EHA101 contains plasmid PTF102, containing bar gene, β-glucuronidase reporter gene.
Wherein, the preparation method of maize immature embryos is: choose the self-pollination young fringe of 9 ~ 11 days, strip the bract of young fringe, 7-8 young fringe is put into the measuring cup of sterilizing, add 75% (volume ratio) alcohol, stir with tweezers, remove the bubble be attached on young fringe, sterilizing 10 minutes, pours out alcohol.Pressing from both sides young fringe with aseptic nipper enters in steel disk, inserts in young fringe at the narrow end of young fringe by forceps tips, uses knife blade to ream young grain 1/3 ~ 2/3 from narrow end to wide end, with the wide end feed of the knife back at young grain, to exert oneself picking rataria to narrow end.
The present embodiment utilizes Agrobacterium to infect the genetic transforming method of maize immature embryos, comprises the following steps:
(1) prepare Agrobacterium and infect liquid: Agrobacterium glycerine pipe is taken out from-80 DEG C of refrigerators, transfering loop adjusts bacterium in the setting-out of Agrobacterium substratum solid medium, 28 DEG C, light culture 2 days, substratum is stored in 4 DEG C of refrigerators, each picking bacterial plaque is applied in Agrobacterium substratum solid medium, 19 DEG C, light culture 3 days.What the bacterial plaque of scraping a diameter 3mm was dissolved in 5mL infects in substratum, and room temperature shakes 3 ~ 4 hours slowly, adjusts OD
550=0.5 ~ 0.55, described in infect liquid pH value be 5.2;
(2) to infect and Dual culture: maize immature embryos is put into and infects in the 1.5mL centrifuge tube of liquid containing 1mL, neat about 100 ratarias of collection, centrifuge tube turns upside down 10 times, puts into the static 10min in lucifuge place, goes to infect liquid, add 1mL Agrobacterium and infect liquid, lucifuge 10min, after infecting end, faces up rataria shield, put into Dual culture substratum, 22 DEG C of light culture 3 days.
The described component infecting liquid is as follows: N6 culture medium powder 1.99g/L, 2,4 dichlorophenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 68.4g/L, glucose 36g/L, MES 0.5g/L, Syringylethanone 100mM/L, pH value is 5.2.
The component of described Agrobacterium solid medium is as follows: peptone 10g/L, NaCl 15g/L, yeast extract 10g/L, agar 15g/L, Rifampin 50mg/L, spectinomycin 50mg/L, kantlex 50mg/L, and pH value is 7.0.
The component of described Dual culture substratum is as follows: N6 culture medium powder 1.99g/L, 2,4-dichlorphenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 30g/L, MES 0.5g/L, plant gel 3g/L, Syringylethanone 100mM/L, Silver Nitrate 5mM/L, cysteine hydrochloride 400mg/L.
Embodiment 2
The difference of the present embodiment and embodiment 1 is only: the pH value infecting liquid is in the present embodiment 4.8.
Embodiment 3
The difference of the present embodiment and embodiment 1 is only: the pH value infecting liquid is in the present embodiment 5.6.
Embodiment 4
The difference of the present embodiment and embodiment 1 is only: the component of its Dual culture substratum is as follows in the present embodiment:
N6 culture medium powder 3.98g/L, 2,4 dichlorophenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 30g/L, MES 0.5g/L, plant gel 3g/L, Syringylethanone 100mM/L, Silver Nitrate 5mM/L, cysteine hydrochloride 400mg/L.
Embodiment 5
The difference of the present embodiment and embodiment 1 is only: in the present embodiment, and the time of step (2) light culture is 1 day.
Embodiment 6
The difference of the present embodiment and embodiment 1 is only: in the present embodiment, and the time of step (2) light culture is 2 days.
Embodiment 7
The difference of the present embodiment and embodiment 1 is only: in the present embodiment, and the time of step (2) light culture is 4 days.
Embodiment 8
The difference of the present embodiment and embodiment 1 is only: its component infecting liquid is as follows in the present embodiment: N6 substratum 3.98g/L, 2,4 dichlorophenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 68.4g/L, glucose 36g/L, MES 0.5g/L, Syringylethanone 100mM/L.
Comparative example 1
This comparative example is only with the difference of embodiment 1, and in comparative example 1, its component infecting liquid is identical with the formula infecting substratum in CN103114106A embodiment 1 table 1.
Experimental example:
Utilize β-glucuronidase staining to survey the transformation efficiency of embodiment 1 ~ embodiment 7, concrete steps are: the rataria tweezers after being terminated by light culture take off, and aseptic double-distilled water washes substratum off, suck dry moisture on filter paper; 2ml centrifuge tube is put into respectively in units of ware, often pipe adds 1.5ml β-glucuronidase staining fluid, 37 DEG C of light culture, 16 hours, time as bad in staining conditions, can 25 DEG C cultivate 1-2 days again, use 75% respectively, after the decolouring of 85%, 95% (v/v) alcohol, staining cell is intuitively visible.
The component of described β-glucuronidase staining fluid is as follows: the bromo-4-of 1mM 5-
chloro-3-
indoles-β-D-Glucose glycosides, 100mM pH value is 7.0 sodium phosphate buffers, 10mM disodium ethylene diamine tetraacetate, the 0.5mM Tripotassium iron hexacyanide, 0.5mM yellow prussiate of potash, 0.1% (volume ratio) Triton X-100.
Rataria transformation efficiency=dyeing rataria number/total rataria number, test result is as follows:
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
| Transformation efficiency | 0.486 | 0.261 | 0.275 | 0.213 | 0.124 |
| Embodiment 6 | Embodiment 7 | Embodiment 8 | Comparative example 1 | ||
| Transformation efficiency | 0.321 | 0.107 | 0.203 | 0.143 |
Illustrate, due to Dual culture overlong time, have an appointment in embodiment 7 40% rataria microbiological contamination, cannot β-glucuronidase dyeing be carried out.
Data as can be seen from upper table, embodiment 1-8 transforms for agriculture bacillus mediated maize genetic all has effect, and wherein the effect of embodiment 1 is best.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. utilize Agrobacterium to infect a genetic transforming method for maize immature embryos, it is characterized in that, comprise the steps:
(1) prepare Agrobacterium and infect liquid: get Agrobacterium bacterial plaque be dissolved in pH value be 4.8 ~ 5.6 infect in liquid, room temperature shakes 3 ~ 4h slowly, adjust OD
550be 0.5 ~ 0.55, obtain Agrobacterium and infect liquid;
(2) to infect and Dual culture: with described in infect liquid maize immature embryos infected, infect liquid: maize immature embryos=1mL:(100 ~ 150) individual, infect removal after terminating and infect liquid, add described Agrobacterium and infect liquid, lucifuge 5 ~ 15min infects, and after infecting end, is faced up by rataria shield, put into Dual culture substratum, 19 ~ 23 DEG C of light culture 1 ~ 4 day.
2. the genetic transforming method utilizing Agrobacterium to infect maize immature embryos according to claim 1, it is characterized in that, the described component infecting liquid is as follows: N6 substratum 1.99 ~ 3.98g/L, 2,4 dichlorophenoxyacetic acid 1.0 ~ 1.5mg/L, proline(Pro) 0.5 ~ 0.7g/L, sucrose 68.4 ~ 75g/L, glucose 36 ~ 40g/L, MES 0.3 ~ 0.5g/L, Syringylethanone 100 ~ 150mM/L.
3. the genetic transforming method utilizing Agrobacterium to infect maize immature embryos according to claim 2, it is characterized in that, the described component infecting liquid is as follows: N6 substratum 1.99g/L, 2,4 dichlorophenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 68.4g/L, glucose 36g/L, MES 0.5g/L, Syringylethanone 100mM/L.
4. the Agrobacterium that utilizes according to Claims 2 or 3 infects the genetic transforming method of maize immature embryos, it is characterized in that, described in infect liquid pH value be 5.2.
5. the Agrobacterium that utilizes according to any one of claim 1-4 infects the genetic transforming method of maize immature embryos, it is characterized in that, the component of described Dual culture substratum is as follows: N6 substratum 1.99 ~ 3.98g/L, 2,4-dichlorphenoxyacetic acid 1.0 ~ 1.5mg/L, proline(Pro) 0.5 ~ 0.7g/L, sucrose 30 ~ 35g/L, MES 0.3 ~ 0.5g/L, plant gel 3 ~ 4g/L, Syringylethanone 100 ~ 150mM/L, Silver Nitrate 5 ~ 7mM/L, cysteine hydrochloride 300 ~ 400mg/L, pH value is 5.8 ~ 6.0.
6. the genetic transforming method utilizing Agrobacterium to infect maize immature embryos according to claim 5, is characterized in that, the component of described Dual culture substratum is as follows: N6 substratum 1.99g/L, 2,4-dichlorphenoxyacetic acid 1.5mg/L, proline(Pro) 0.7g/L, sucrose 30g/L, MES 0.5g/L, plant gel 3g/L, Syringylethanone 100mM/L, Silver Nitrate 5mM/L, cysteine hydrochloride 400mg/L, pH value is 5.8.
7. the Agrobacterium that utilizes according to any one of claim 1-4 infects the genetic transforming method of maize immature embryos, it is characterized in that, after step (2) infects end, is faced up by rataria shield, puts into Dual culture substratum, 22 DEG C of light culture 3 days.
8. the Agrobacterium that utilizes according to any one of claim 1-4 infects the genetic transforming method of maize immature embryos, it is characterized in that, the preparation method of the described Agrobacterium bacterial plaque of step (1) is: with transfering loop picking agrobacterium strains in the setting-out of Agrobacterium solid medium, 25 ~ 30 DEG C of light culture 2 ~ 3 days, Agrobacterium solid medium is kept in the environment of-80 DEG C, each picking bacterial plaque is applied in Agrobacterium substratum solid medium, and 20 ~ 25 DEG C of light culture can obtain Agrobacterium bacterial plaque after 2 ~ 4 days.
9. the genetic transforming method utilizing Agrobacterium to infect maize immature embryos according to claim 8, it is characterized in that, the component of described Agrobacterium solid medium is as follows: peptone 10g/L, NaCl 15g/L, yeast extract 10g/L, agar 15g/L, Rifampin 50mg/L, spectinomycin 50mg/L, kantlex 50mg/L.
10. the genetic transforming method utilizing Agrobacterium to infect maize immature embryos according to claim 1, it is characterized in that, the method that strips of described maize immature embryos is: choose the self-pollination young fringe of 9 ~ 11 days, sterilizing, at the narrow end of young fringe, forceps tips is inserted in young fringe, use knife blade to ream young grain 1/3 ~ 2/3 from narrow end to wide end, with the wide end feed of the knife back at young grain, to exert oneself picking rataria to narrow end.
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Application publication date: 20151021 |