CN113736821B - Genetic transformation method for embryonic callus of Korean pine - Google Patents

Genetic transformation method for embryonic callus of Korean pine Download PDF

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CN113736821B
CN113736821B CN202111038703.XA CN202111038703A CN113736821B CN 113736821 B CN113736821 B CN 113736821B CN 202111038703 A CN202111038703 A CN 202111038703A CN 113736821 B CN113736821 B CN 113736821B
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李淑娟
杨玲
沈海龙
侯慧
杨宇宁
董昊
安岩
赵文娜
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Northeast Forestry University
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Abstract

The invention provides a genetic transformation method of Korean pine embryonic callus, belonging to the technical field of plant genetic engineering and comprising the following steps: (1) Korean pine embryonic callusSubculturing the tissue; (2) preparation of an agrobacterium infection solution: transferring agrobacterium strain containing target gene into acetosyringone solution, and re-suspending the strain to OD 600 = 0.4-0.6, namely the dye-leaching solution is obtained for standby; (3) agrobacterium-mediated genetic transformation and co-culture of Korean pine; (4) screening and culturing the resistant callus; and (5) culturing the resistant somatic embryos. The method has simple operation, short period and high genetic transformation efficiency.

Description

Genetic transformation method for Korean pine embryonic callus
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a genetic transformation method of Korean pine embryonic callus.
Background
Korean pine (Pinus koraiensis) is a special precious timber and economic forest tree species in northeast China as a dominant species of broad-leaved red pine forest, and has extremely high economic and ecological values. However, because of large-scale development and utilization, the natural pinus koraiensis is almost completely lost, and in order to recover and expand pinus koraiensis resources and fully play the ecological functions and economic values of pinus koraiensis, artificial afforestation is extremely important. And because the Korean pine has huge genome, high heterozygosity, long growth period and lack of a genetic transformation system, the molecular basic research of a development regulation mechanism is greatly limited.
At present, the modern biotechnology means based on in vitro regeneration and genetic transformation can introduce exogenous genes into conifer genome and make the conifer genome stably inherited to generate directional variation, thereby realizing large-scale genetic improvement and shortening the breeding period. Combining the somatic embryogenesis way with the genetic transformation technology, the resistant callus screened in the genetic transformation process can be further developed into a somatic embryo through the somatic embryogenesis way, and a complete resistant plant is obtained after the somatic embryo germinates. Therefore, in order to recover Korean pine resources and deeply research the molecular basis of a regulation mechanism of development of the Honglin, the development and application of a genetic transformation technology of Korean pine embryogenic callus are very important, but so far, no relevant report about the genetic transformation method of Korean pine embryogenic callus exists.
Disclosure of Invention
The invention aims to provide a genetic transformation method of Korean pine embryonic callus, which is simple and convenient to operate, short in period and high in transformation efficiency.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a genetic transformation method of Korean pine embryonic callus, which comprises the following steps:
(1) Subculturing the embryonic callus of the Korean pine: inoculating the embryonic callus of the Korean pine into a subculture medium, and carrying out dark culture at 24-28 ℃ for 9-18 days to obtain a genetically transformed receptor;
(2) Preparation of agrobacterium infection liquid: transferring agrobacterium strain containing target gene into acetosyringone solution, and re-suspending the strain to OD 600 = 0.4-0.6, namely the dye-leaching solution is obtained for standby;
(3) Agrobacterium-mediated genetic transformation and co-cultivation of pinus koraiensis: soaking a receptor subjected to genetic transformation in an infection solution for infection, draining the infection solution, then inoculating the receptor into a co-culture medium, and carrying out dark culture at 23-27 ℃ for 1-3 d to obtain a co-cultured embryonic callus;
(4) Screening and culturing the resistant callus: transferring the co-cultured embryonic callus into a screening culture medium for culturing for 10-15 d, and continuously screening for 1-3 times to obtain resistant callus;
(5) And (3) culturing a resistant somatic embryo: transferring the resistant callus into a somatic embryo induction culture medium, and carrying out dark culture at 24-27 ℃ for 40-60 d to obtain a resistant somatic embryo.
Preferably, after step (4), further comprising the identification of resistant callus: and (3) performing GUS staining and/or PCR detection on the resistant callus respectively, determining whether the GUS gene and/or the target gene is expressed in the resistant callus according to whether a blue color reaction and/or a target gene band exists, and if the GUS gene and/or the target gene is expressed, indicating that the genetic transformation of the Korean pine is successful.
Preferably, in the step (2), before transferring to the acetosyringone solution, the method further comprises culturing the agrobacterium strain containing the target gene on a solid culture medium, culturing to a logarithmic phase, and collecting the thallus.
Preferably, the agrobacterium is agrobacterium GV3101, and the concentration of the acetosyringone solution is 20-60 mu mol/L.
Preferably, the subculture medium consists of the following components in concentration: 0.1-0.5 mg/L2, 4-D, 0.05-0.1 mg/L6-BA, 340mg/L KNO 3 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、3.1mg/L H 3 BO 3 、4.3mg/L ZnSO 4 ·7H 2 O、8.45mg/L MnSO 4 ·H 2 O、0.125mg/L Na 2 MoO 4 ·2H 2 O、0.415mg/L KI、0.125mg/L CuSO 4 ·5H 2 O、0.0125mg/L CoCl 2 ·6H 2 O、0.0125mg/L NiCl 2 、13.9mg/L FeSO 4 ·7H 2 O、18.65mg/L Na 2 -EDTA、278mg/L Ca(NO 3 ) 2 ·4H 2 O、42.5mg/L CaCl 2 ·2H 2 O, 1mg/L glycine, 0.25mg/L Vitamin B 5 、0.25mg/L Vitamin B 6 、0.5mg/L Vitamin B 1 30g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein and 4g/L of gellan gum; the pH of the subculture medium was 5.9.
Preferably, the solid culture medium is a YEB solid culture medium added with 50mg/L kanamycin and 50mg/L rifampicin.
Preferably, the YEB solid medium consists of the following components in the following concentrations: 5g/L beef extract, 5g/L tryptone, 1g/L yeast extract and 4g/L MgSO 4 ·7H 2 O, 5g/L sucrose and 15g/L agarA lipid; the YEB solid medium pH was 7.4.
Preferably, the co-culture medium is obtained by adding 20-60 mu mol/L acetosyringone on the basis of the subculture medium.
Preferably, the screening culture medium is obtained by adding 10-50 mg/L hygromycin and 200mg/L cefotaxime sodium on the basis of the subculture medium.
Preferably, the somatic embryo induction medium consists of the following components in concentration: 80 mu mol/LABA, 950mg/L KNO 3 、825mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、925mg/L MgSO 4 ·7H 2 O、31mg/L H 3 BO 3 、43mg/L ZnSO 4 ·7H 2 O、21mg/L MnSO 4 ·2H 2 O、1.25mg/L Na 2 MoO 4 ·2H 2 O、4.15mg/L KI、0.5mg/L CuSO 4 ·5H 2 O、0.13mg/L CoCl 2 ·6H 2 O、28mg/L FeSO 4 ·7H 2 O、37mg/L Na 2 -EDTA、11mg/L CaCl 2 ·2H 2 O、0.5mg/L Vitamin B 5 、0.1mg/L Vitamin B 6 、0.1mg/L Vitamin B 1 20g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein, 12g/L of gellan gum and 2g/L of activated carbon; the pH of the somatic embryo induction medium was 5.8.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the invention provides a genetic transformation method of Korean pine embryonic callus for the first time, which comprises the steps of carrying out enrichment culture on the embryonic callus for 9-18 days to obtain a receptor for genetic transformation, then introducing a target gene into the Korean pine embryonic callus by utilizing agrobacterium mediation, and carrying out screening culture to obtain a resistant callus.
Drawings
FIG. 1 is a GUS histochemical staining diagram of wild type embryonic callus of Korean pine;
FIG. 2 is a GUS histochemical staining pattern of the Korean pine resistant callus in example 1;
FIG. 3 is a schematic representation of example 1 after 10d subculture of embryogenic callus of Pinus koraiensis;
FIG. 4 is a schematic representation of example 2 after 15d subculture of embryonic callus of Pinus koraiensis;
FIG. 5 is a schematic representation of the example 1 after the secondary 20d of Korean pine embryogenic callus;
FIG. 6 is a schematic representation of the somatic embryogenesis resistant to Pinus koraiensis of example 3;
FIG. 7 is the schematic diagram of PCR electrophoresis of the Korean pine resistant callus of example 3, wherein M is 2000DNA marker; h is water (negative control); n is wild type Korean pine callus; p is agrobacterium liquid (positive control); 1-8 are resistant callus.
Detailed Description
The invention provides a genetic transformation method of Korean pine embryonic callus, which comprises the following steps:
(1) Subculturing the embryonic callus of the Korean pine: inoculating the embryonic callus of the Korean pine into a subculture medium, and carrying out dark culture at 24-28 ℃ for 9-18 days to obtain a genetically transformed receptor;
(2) Preparation of agrobacterium infection liquid: transferring agrobacterium strain containing target gene into acetosyringone solution, and re-suspending the strain to OD 600 = 0.4-0.6, namely the dye-leaching solution is obtained for standby;
(3) Agrobacterium-mediated genetic transformation and co-cultivation of pinus koraiensis: soaking a receptor subjected to genetic transformation in an infection solution for infection, draining the infection solution, then inoculating the receptor into a co-culture medium, and carrying out dark culture at 23-27 ℃ for 1-3 d to obtain a co-cultured embryonic callus;
(4) Screening and culturing the resistant callus: transferring the co-cultured embryonic callus into a screening culture medium for culturing for 10-15 d, and continuously screening for 1-3 times to obtain resistant callus;
(5) And (3) culturing a resistant somatic embryo: transferring the resistant callus into a somatic embryo induction culture medium, and carrying out dark culture at 24-27 ℃ for 40-60 d to obtain a resistant somatic embryo.
In the invention, the Korean pine embryonic callus is inoculated into a subculture medium and is cultured in the dark at the temperature of 24-28 ℃ for 9-18 days, and the receptor of genetic transformation is obtained. The invention adopts the embryonic callus of the Korean pineHas the characteristics of short culture period, high somatic embryo incidence rate and quick seedling formation. The subculture medium preferably consists of the following components in concentration: 0.1-0.5 mg/L2, 4-D, 0.05-0.1 mg/L6-BA, 340mg/L KNO 3 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、3.1mg/L H 3 BO 3 、4.3mg/L ZnSO 4 ·7H 2 O、8.45mg/L MnSO 4 ·H 2 O、0.125mg/L Na 2 MoO 4 ·2H 2 O、0.415mg/L KI、0.125mg/L CuSO 4 ·5H 2 O、0.0125mg/L CoCl 2 ·6H 2 O、0.0125mg/L NiCl 2 、13.9mg/L FeSO 4 ·7H 2 O、18.65mg/L Na 2 -EDTA、278mg/L Ca(NO 3 ) 2 ·4H 2 O、42.5mg/L CaCl 2 ·2H 2 O, 1mg/L glycine, 0.25mg/L Vitamin B 5 、0.25mg/L Vitamin B 6 、0.5mg/L Vitamin B 1 30g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein and 4g/L of gellan gum; the pH of the secondary culture medium is preferably 5.9. The method for preparing the subculture medium in the present invention is not particularly limited, and any method known in the art may be used.
In the invention, agrobacterium strain containing target gene is transferred into acetosyringone solution, and the strain is re-suspended to OD 600 And (5) obtaining an impregnation liquid for later use, wherein the impregnation liquid is obtained by 0.4-0.6. Before transferring into acetosyringone solution, it is preferable to culture the agrobacterium strain containing the target gene on a solid culture medium, culturing to logarithmic phase, and collecting the strain. The target gene is preferably a beta-glucuronidase gene, the agrobacterium is preferably agrobacterium GV3101, and the concentration of the acetosyringone solution is preferably 20-60 mu mol/L. In the present invention, the solid medium is preferably supplemented with 50mg/L kanamycin and 50mg/L rifampicin on the basis of YEB solid medium. The method for preparing the solid medium of the present invention is not particularly limited, and any method known in the art may be used. In the present invention, the YEB solid medium is preferably composed of the following components in concentrations: 5g/L beef extract and 5gPer L tryptone, 1g/L yeast extract, 4g/L MgSO 4 ·7H 2 O, 5g/L sucrose and 15g/L agar; the YEB solid medium pH is preferably 7.4. The method for preparing the YEB solid medium is not particularly limited in the present invention, and any method known in the art may be used.
In the invention, a receptor of genetic transformation is soaked in the staining solution for infection, and the staining solution is drained. The genetically transformed recipient may be freshly dispersed Korean pine embryogenic callus. When the receptor of genetic transformation is soaked in the staining solution for infection, the receptor is preferably gently shaken for 15-20 min, and the receptor of genetic transformation is fully contacted with the staining solution by gentle shaking. After draining the staining solution, the present invention preferably further comprises draining the genetic transformation receptor of the staining solution on a sterile filter paper to remove the excess bacteria solution on the surface of the genetic transformed embryogenic callus.
In the invention, the callus is inoculated into a co-culture medium and cultured in dark at 23-27 ℃ for 1-3 d, and then the co-cultured embryonic callus is obtained. In the invention, the co-culture medium is preferably added with 20-60 mu mol/L acetosyringone on the basis of the subculture medium. The method for preparing the co-culture medium of the present invention is not particularly limited, and any method known in the art may be used.
In the invention, the embryogenic callus cultured together is transferred into a screening culture medium to be cultured for 10-15 d, and the embryogenic callus is continuously screened for 1-3 times to obtain the resistant callus. The screening culture medium is preferably added with 10-50 mg/L hygromycin and 200mg/L cefotaxime sodium on the basis of the subculture medium. The method for preparing the screening medium of the present invention is not particularly limited, and any method known in the art may be used.
In the present invention, after step (4), preferably the identification of resistant callus is also included: and (3) performing GUS staining and/or PCR detection on the resistant callus, determining whether the GUS gene and/or the target gene is expressed in the resistant callus according to whether a blue color reaction and/or a target gene band exists, and if the GUS gene and/or the target gene is expressed, indicating that the genetic transformation of the Korean pine is successful. According to the invention, the identification of the resistant callus is carried out through GUS staining and PCR detection, so that false positive callus can be eliminated in advance, the workload at the later stage is reduced, and the working efficiency is improved.
In the invention, the resistant callus is transferred into a somatic embryo induction culture medium and cultured in the dark at the temperature of 24-27 ℃ for 40-60 days to obtain the resistant somatic embryo. In the present invention, the somatic embryo induction medium is preferably composed of the following components in concentrations: 80 mu mol/LABA, 950mg/L KNO 3 、825mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、925mg/L MgSO 4 ·7H 2 O、31mg/L H 3 BO 3 、43mg/L ZnSO 4 ·7H 2 O、21mg/L MnSO 4 ·2H 2 O、1.25mg/L Na 2 MoO 4 ·2H 2 O、4.15mg/L KI、0.5mg/L CuSO 4 ·5H 2 O、0.13mg/L CoCl 2 ·6H 2 O、28mg/L FeSO 4 ·7H 2 O、37mg/L Na 2 -EDTA、11mg/L CaCl 2 ·2H 2 O、0.5mg/L Vitamin B 5 、0.1mg/L Vitamin B 6 、0.1mg/L Vitamin B 1 20g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein, 12g/L of gellan gum and 2g/L of activated carbon; the pH of the somatic embryo induction medium is preferably 5.8.
In the present invention, unless otherwise specified, all the raw material components are commercially available products well known to those skilled in the art.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a genetic transformation method of Korean pine embryonic callus, which comprises the following steps:
(1) Subculturing the embryonic callus of the Korean pine: subjecting embryonic callus of Korean pineInoculating into subculture medium, and dark culturing at 25 deg.C for 10d to obtain genetically transformed receptor; wherein the subculture medium consists of the following components in concentration: 0.3 mg/L2, 4-D, 0.07 mg/L6-BA, 340mg/L KNO 3 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、3.1mg/L H 3 BO 3 、4.3mg/L ZnSO 4 ·7H 2 O、8.45mg/L MnSO 4 ·H 2 O、0.125mg/L Na 2 MoO 4 ·2H 2 O、0.415mg/L KI、0.125mg/L CuSO 4 ·5H 2 O、0.0125mg/L CoCl 2 ·6H 2 O、0.0125mg/L NiCl 2 、13.9mg/L FeSO 4 ·7H 2 O、18.65mg/L Na 2 -EDTA、278mg/L Ca(NO 3 ) 2 ·4H 2 O、42.5mg/L CaCl 2 ·2H 2 O, 1mg/L glycine, 0.25mg/L Vitamin B 5 、0.25mg/L Vitamin B 6 、0.5mg/L Vitamin B 1 30g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein and 4g/L of gellan gum; the pH of the subculture medium is 5.9;
(2) Preparation of agrobacterium infection liquid: culturing Agrobacterium GV3101 strain containing beta-glucuronidase gene on solid culture medium, culturing to logarithmic phase, collecting thallus, transferring into 40 μmol/L acetosyringone solution, and resuspending thallus to OD 600 =0.5, namely the dye-leaching solution is obtained for standby; wherein the solid culture medium is prepared by adding 50mg/L kanamycin and 50mg/L rifampicin on the basis of YEB solid culture medium; the YEB solid culture medium consists of the following components in concentration: 5g/L beef extract, 5g/L tryptone, 1g/L yeast extract and 4g/L MgSO 4 ·7H 2 O, 5g/L sucrose and 15g/L agar; the pH of the YEB solid culture medium is 7.4;
(3) Agrobacterium-mediated genetic transformation and co-cultivation of pinus koraiensis: soaking the genetically transformed receptor in an infection solution for infection, draining the infection solution, placing the infected genetically transformed receptor on sterile filter paper, absorbing redundant bacterial liquid on the surface of the infected genetically transformed receptor, then inoculating the infected genetically transformed receptor into a co-culture medium, and carrying out dark culture at 25 ℃ for 2d to obtain a co-cultured embryonic callus; the co-culture medium is added with 40 mu mol/L acetosyringone on the basis of the subculture medium;
(4) Screening and culturing the resistant callus: transferring the co-cultured embryonic callus into a screening culture medium for culturing for 10d, and continuously screening for 3 times to obtain resistant callus; the screening culture medium is added with 30mg/L hygromycin and 200mg/L cefotaxime sodium on the basis of the subculture medium;
(5) Identification of resistant callus: respectively carrying out GUS staining detection on the resistant callus, determining whether the GUS gene expression exists in the resistant callus according to whether a blue color development reaction exists, and if the GUS gene expression exists, indicating that the genetic transformation of the Korean pine succeeds;
(6) And (3) culturing a resistant somatic embryo: transferring the resistant callus into a somatic embryo induction culture medium, and carrying out dark culture at 26 ℃ for 50d to obtain a resistant somatic embryo.
The GUS staining detection method comprises the following steps: and (3) immersing the resistant callus into a proper amount of GUS dye solution, vacuumizing, remaining the resistant callus and the GUS dye solution in a water bath at 37 ℃ for 7 days, and observing and recording the color development condition of the callus. The wild type embryonic callus of the Korean pine is taken as a negative control, the tissue which shows blue is taken as transgenic resistant callus, the tissue which shows colorless, transparent and slightly yellow is taken as control tissue, the GUS histochemical staining map of the wild type embryonic callus of the Korean pine is shown as figure 1, and the GUS histochemical staining map of the resistant embryonic callus of the Korean pine is shown as figure 2.
The result shows that the embryogenic callus state of the 10d subculture is better (see figure 3), 38 resistant calli are obtained after continuous 3 times of resistance screening, 11 calli are blue after GUS staining, and the GUS staining efficiency is 28.9%.
Example 2
This example is the same as example 1 except for the following steps: in step (1), dark culture is carried out at 25 ℃ for 15d, and the rest of the steps in step (1) are the same as in example 1; and (4) transferring the co-cultured embryogenic callus into a screening culture medium for culturing for 15d.
The method of GUS staining detection described in example 1 was used to observe and record the development of calli using the method described in example 1.
The result showed that the embryogenic callus state of the secondary generation 15d of this example was worse than that of example 1 (see fig. 4), and a total of 22 resistant calli were obtained after 3 consecutive resistance screenings, and 5 calli were blue by GUS staining, and the GUS staining efficiency was 22.7%.
Comparative example 1
This comparative example is the same as example 1 except for the following steps: in step (1), dark culture is carried out at 25 ℃ for 20d, and the rest of the steps in step (1) are the same as in example 1; and (4) transferring the co-cultured embryogenic callus into a screening culture medium for culturing for 20d.
The method of GUS staining detection described in example 1 was used to observe and record the development of calli using the method described in example 1.
The results showed that the embryogenic callus of the 20d subculture of comparative example 1 was in a poor state (see FIG. 5), and after 3 consecutive resistance screenings, no resistant callus was obtained, and the tissues were subjected to GUS staining and did not appear blue.
Example 3
The embodiment provides a genetic transformation method of Korean pine embryonic callus, which comprises the following steps:
(1) Subculturing the embryonic callus of the Korean pine: inoculating the embryonic callus of the Korean pine into a subculture medium, and performing dark culture at 25 ℃ for 10d to obtain a genetically transformed receptor; wherein the subculture medium consists of the following components in concentration: 0.3 mg/L2, 4-D, 0.07 mg/L6-BA, 340mg/L KNO 3 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、3.1mg/L H 3 BO 3 、4.3mg/L ZnSO 4 ·7H 2 O、8.45mg/L MnSO 4 ·H 2 O、0.125mg/L Na 2 MoO 4 ·2H 2 O、0.415mg/L KI、0.125mg/L CuSO 4 ·5H 2 O、0.0125mg/L CoCl 2 ·6H 2 O、0.0125mg/L NiCl 2 、13.9mg/L FeSO 4 ·7H 2 O、18.65mg/L Na 2 -EDTA、278mg/L Ca(NO 3 ) 2 ·4H 2 O、42.5mg/L CaCl 2 ·2H 2 O, 1mg/L glycine, 0.25mg/L Vitamin B 5 、0.25mg/L Vitamin B 6 、0.5mg/L Vitamin B 1 30g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein and 4g/L of gellan gum; the pH of the subculture medium is 5.9;
(2) Preparation of agrobacterium infection liquid: culturing Agrobacterium GV3101 strain containing beta-glucuronidase gene on solid culture medium, culturing to logarithmic phase, collecting thallus, transferring into 40 μmol/L acetosyringone solution, and resuspending thallus to OD 600 =0.5, namely obtaining an dye-invasion solution for later use; wherein the solid culture medium is prepared by adding 50mg/L kanamycin and 50mg/L rifampicin on the basis of YEB solid culture medium; the YEB solid culture medium consists of the following components in concentration: 5g/L beef extract, 5g/L tryptone, 1g/L yeast extract and 4g/L MgSO 4 ·7H 2 O, 5g/L sucrose and 15g/L agar; the pH of the YEB solid culture medium is 7.4;
(3) Agrobacterium-mediated genetic transformation and co-cultivation of pinus koraiensis: soaking the genetic transformation embryonic callus in an infection solution for infection, draining the infection solution, putting the infected genetic transformation embryonic callus on sterile filter paper to absorb redundant bacterial liquid on the surface of the genetic transformation embryonic callus, then inoculating the genetic transformation embryonic callus into a co-culture medium, and carrying out dark culture at 25 ℃ for 2d to obtain the co-cultured embryonic callus; the co-culture medium is added with 40 mu mol/L acetosyringone on the basis of the subculture medium;
(4) Screening and culturing the resistant callus: transferring the co-cultured embryonic callus into a screening culture medium for culturing for 10d, and continuously screening for 3 times to obtain resistant callus; the screening culture medium is added with 30mg/L hygromycin and 200mg/L cefotaxime sodium on the basis of the subculture medium;
(5) Identification of resistant callus: respectively carrying out PCR detection on the resistant callus, determining whether the resistant callus has the expression of the beta-glucuronidase gene or not according to whether the beta-glucuronidase gene is expressed or not, and if the beta-glucuronidase gene is expressed, indicating that the genetic transformation of the Korean pine is successful;
(6) And (3) culturing a resistant somatic embryo: the resistant callus was transferred to somatic embryo induction medium and cultured in the dark at 26 ℃ for 50 days to obtain resistant somatic embryos (see FIG. 6).
PCR molecular detection was performed on 8 resistant cell lines randomly selected in this example using Korean pine resistant callus DNA as a template, with the upstream primer being '-CAAAGCAAGTGGATTGATGTAT-3' (SEQ ID NO: 1); the downstream primer is 5. The reaction system is as follows: MIX 10. Mu.L, DNA template and upstream and downstream primers 1. Mu.L, ddH 2 O7. Mu.L. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 42s, and repeating the above processes for 35 times; finally, extension is carried out for 10min at 72 ℃. Agarose gel electrophoresis experiments were performed.
The results are shown in FIG. 7, where 8 resistant cell lines all showed bands of interest with different intensities, and the control callus did not show bands of interest, indicating that the β -glucuronidase gene had been successfully transferred into the genome of Korean pine embryogenic callus.
Example 4
The embodiment provides a genetic transformation method of Korean pine embryonic callus, which comprises the following steps:
(1) Subculturing the embryonic callus of the Korean pine: inoculating Korean pine embryonic callus into subculture medium, dark culturing at 25 deg.C for 9d, and performing proliferation culture to obtain genetically transformed receptor; wherein the subculture medium consists of the following components in concentration: 0.1 mg/L2, 4-D, 0.1 mg/L6-BA, 340mg/L KNO 3 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、3.1mg/L H 3 BO 3 、4.3mg/L ZnSO 4 ·7H 2 O、8.45mg/L MnSO 4 ·H 2 O、0.125mg/L Na 2 MoO 4 ·2H 2 O、0.415mg/L KI、0.125mg/L CuSO 4 ·5H 2 O、0.0125mg/L CoCl 2 ·6H 2 O、0.0125mg/L NiCl 2 、13.9mg/L FeSO 4 ·7H 2 O、18.65mg/L Na 2 -EDTA、278mg/L Ca(NO 3 ) 2 ·4H 2 O、42.5mg/L CaCl 2 ·2H 2 O, 1mg/L GlycineAmino acid, 0.25mg/L Vitamin B 5 、0.25mg/L Vitamin B 6 、0.5mg/L Vitamin B 1 30g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein and 4g/L of gellan gum; the pH of the subculture medium is 5.9;
(2) Preparation of agrobacterium infection liquid: culturing Agrobacterium GV3101 strain containing beta-glucuronidase gene on solid culture medium, culturing to logarithmic phase, collecting thallus, transferring into 60 μmol/L acetosyringone solution, and resuspending thallus to OD 600 =0.6, namely the dye-leaching solution is obtained for standby; wherein the solid culture medium is prepared by adding 50mg/L kanamycin and 50mg/L rifampicin on the basis of YEB solid culture medium; the YEB solid culture medium consists of the following components in concentration: 5g/L beef extract, 5g/L tryptone, 1g/L yeast extract and 4g/L MgSO 4 ·7H 2 O, 5g/L sucrose and 15g/L agar; the pH of the YEB solid culture medium is 7.4;
(3) Agrobacterium-mediated genetic transformation and co-cultivation of pinus koraiensis: soaking the genetic transformation receptor in infection liquid for infection, draining the infection liquid, placing the infected genetic transformation receptor on sterile filter paper, absorbing redundant bacterial liquid on the surface of the infected genetic transformation receptor, then inoculating the infected genetic transformation receptor into a co-culture medium, and carrying out dark culture at 25 ℃ for 2d to obtain co-cultured embryonic callus; the co-culture medium is added with 60 mu mol/L acetosyringone on the basis of the subculture medium;
(4) Screening and culturing the resistant callus: transferring the co-cultured embryogenic callus into a screening culture medium for culturing for 10d, and continuously screening for 2 times to obtain resistant callus; the screening culture medium is added with 10mg/L hygromycin and 200mg/L cefotaxime sodium on the basis of the subculture medium;
(5) Identification of resistant callus: respectively carrying out GUS (glucuronidase) staining detection and PCR (polymerase chain reaction) detection on the resistant callus, determining whether the expression of a GUS gene and the expression of a beta-glucuronidase gene exist in the resistant callus according to the existence of blue color development reaction and the expression of the beta-glucuronidase gene, and indicating that the genetic transformation of the Korean pine is successful if the expression of the GUS gene and the expression of the beta-glucuronidase gene exist;
(6) And (3) culturing a resistant somatic embryo: transferring the resistant callus into a somatic embryo induction culture medium, and carrying out dark culture at 26 ℃ for 60d to obtain a resistant somatic embryo.
Example 5
The embodiment provides a genetic transformation method of Korean pine embryonic callus, which comprises the following steps:
(1) Subculturing the embryonic callus of the Korean pine: inoculating the embryonic callus of the Korean pine into a subculture medium, performing dark culture at 25 ℃ for 18d, and performing propagation culture to obtain a genetically transformed receptor; wherein the subculture medium consists of the following components in concentration: 0.5 mg/L2, 4-D, 0.05 mg/L6-BA, 340mg/L KNO 3 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、3.1mg/L H 3 BO 3 、4.3mg/L ZnSO 4 ·7H 2 O、8.45mg/L MnSO 4 ·H 2 O、0.125mg/L Na 2 MoO 4 ·2H 2 O、0.415mg/L KI、0.125mg/L CuSO 4 ·5H 2 O、0.0125mg/L CoCl 2 ·6H 2 O、0.0125mg/L NiCl 2 、13.9mg/L FeSO 4 ·7H 2 O、18.65mg/L Na 2 -EDTA、278mg/L Ca(NO 3 ) 2 ·4H 2 O、42.5mg/L CaCl 2 ·2H 2 O, 1mg/L glycine, 0.25mg/L Vitamin B 5 、0.25mg/L Vitamin B 6 、0.5mg/L Vitamin B 1 30g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein and 4g/L of gellan gum; the pH of the subculture medium is 5.9;
(2) Preparation of agrobacterium infection liquid: culturing Agrobacterium GV3101 strain containing beta-glucuronidase gene on solid culture medium, culturing to logarithmic phase, collecting thallus, transferring into 20 μmol/L acetosyringone solution, and resuspending thallus to OD 600 =0.4, namely obtaining an dye-invasion solution for later use; wherein the solid culture medium is prepared by adding 50mg/L kanamycin and 50mg/L rifampicin on the basis of YEB solid culture medium; the YEB solid culture medium consists of the following components in concentration: 5g/L beef extract, 5g/L tryptone, 1g/L yeast extract and 4g/L MgSO 4 ·7H 2 O, 5g/L sucrose and 15g/L agar;the pH of the YEB solid culture medium is 7.4;
(3) Agrobacterium-mediated genetic transformation and co-cultivation of pinus koraiensis: soaking the genetically transformed receptor in an infection solution for infection, draining the infection solution, putting the infected genetically transformed receptor on sterile filter paper, absorbing redundant bacterial liquid on the surface of the infected genetically transformed receptor, inoculating the infected genetically transformed receptor into a co-culture medium, and performing dark culture at 25 ℃ for 2d to obtain a co-cultured embryonic callus; adding 20 mu mol/L acetosyringone to the co-culture medium based on the subculture medium;
(4) Screening and culturing the resistant callus: transferring the co-cultured embryonic callus into a screening culture medium for culture for 13d, and continuously screening for 1 time to obtain resistant callus; the screening culture medium is added with 50mg/L hygromycin and 200mg/L cefotaxime sodium on the basis of the subculture medium;
(5) Identification of resistant callus: respectively carrying out GUS staining detection on the resistant callus, determining whether the GUS gene expression exists in the resistant callus according to whether a blue color development reaction exists, and if the GUS gene expression exists, indicating that the genetic transformation of the Korean pine succeeds;
(6) And (3) culturing a resistant somatic embryo: transferring the resistant callus into a somatic embryo induction culture medium, and carrying out dark culture at 26 ℃ for 40d to obtain a resistant somatic embryo.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> genetic transformation method of Korean pine embryonic callus
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Claims (6)

1. A genetic transformation method of Korean pine embryogenic callus is characterized by comprising the following steps:
(1) Subculturing the embryonic callus of the Korean pine: inoculating the embryonic callus of the Korean pine into a subculture medium, and carrying out dark culture at 24-28 ℃ for 9-18 days to obtain a genetically transformed receptor;
(2) Preparation of agrobacterium infection liquid: transferring agrobacterium strain containing target gene into acetosyringone solution, and re-suspending the strain to OD 600 (iii) keeping for 0.4 to 0.6 to obtain an impregnation solution for later use;
(3) Agrobacterium-mediated genetic transformation and co-cultivation of pinus koraiensis: soaking a receptor subjected to genetic transformation in an infection solution for infection, draining the infection solution, then inoculating the receptor into a co-culture medium, and carrying out dark culture at 23-27 ℃ for 1-3 d to obtain a co-cultured embryonic callus;
(4) Screening and culturing the resistant callus: transferring the co-cultured embryogenic callus into a screening culture medium for culturing for 10 to 15d, and continuously screening for 1 to 3 times to obtain a resistant callus;
(5) And (3) culturing a resistant somatic embryo: transferring the resistant callus into a somatic embryo induction culture medium, and carrying out dark culture at 24-27 ℃ for 40-60d to obtain a resistant somatic embryo;
the subculture medium consists of the following components in concentration: 0.1 to 0.5mg/L2, 4-D, 0.05 to 0.1mg/L6-BA, 340mg/L KNO 3 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、3.1mg/L H 3 BO 3 、4.3mg/L ZnSO 4 ·7H 2 O、8.45mg/L MnSO 4 ·H 2 O、0.125mg/L Na 2 MoO 4 ·2H 2 O、0.415mg/L KI、0.125mg/L CuSO 4 ·5H 2 O、0.0125mg/L CoCl 2 ·6H 2 O、0.0125mg/L NiCl 2 、13.9mg/L FeSO 4 ·7H 2 O、18.65mg/L Na 2 -EDTA、278mg/L Ca(NO 3 ) 2 ·4H 2 O、42.5mg/L CaCl 2 ·2H 2 O, 1mg/L glycine, 0.25mg/L Vitamin B 5 、0.25mg/L Vitamin B 6 、0.5mg/L Vitamin B 1 30g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein and 4g/L of gellan gum; the pH of the subculture medium is 5.9;
the co-culture medium is prepared by adding 20 to 60 mu mol/L acetosyringone on the basis of the subculture medium;
the somatic embryo induction culture medium consists of the following components in concentration: 80 mu mol/L ABA and 950mg/L KNO 3 、825mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、925mg/L MgSO 4 ·7H 2 O、31mg/L H 3 BO 3 、43mg/L ZnSO 4 ·7H 2 O、21mg/L MnSO 4 ·2H 2 O、1.25mg/L Na 2 MoO 4 ·2H 2 O、4.15mg/L KI、0.5mg/L CuSO 4 ·5H 2 O、0.13mg/L CoCl 2 ·6H 2 O、28mg/L FeSO 4 ·7H 2 O、37mg/L Na 2 -EDTA、11mg/L CaCl 2 ·2H 2 O、0.5mg/L Vitamin B 5 、0.1mg/L Vitamin B 6 、0.1mg/L Vitamin B 1 20g/L of sucrose, 0.1g/L of inositol, 0.5g/L of glutamine, 0.5g/L of acid hydrolyzed casein, 12g/L of gellan gum and 2g/L of activated carbon; the pH of the somatic embryo induction culture medium is 5.8;
the screening culture medium is formed by adding 10 to 50mg/L of hygromycin and 200mg/L of cefotaxime sodium on the basis of the subculture medium.
2. The genetic transformation method of claim 1, further comprising, after step (4), the identification of resistant callus tissue: and (3) performing GUS staining and/or PCR detection on the resistant callus, determining whether the GUS gene and/or the target gene is expressed in the resistant callus according to whether a blue color reaction and/or a target gene band exists, and if the GUS gene and/or the target gene is expressed, indicating that the genetic transformation of the Korean pine is successful.
3. The genetic transformation method according to claim 1, wherein the step (2) further comprises culturing the Agrobacterium strain containing the desired gene on a solid medium until logarithmic phase before transferring to the acetosyringone solution, and collecting the strain.
4. The genetic transformation method according to claim 1, wherein in the step (2), the Agrobacterium is Agrobacterium GV3101, and the concentration of the acetosyringone solution is 20 to 60 μmol/L.
5. The genetic transformation method according to claim 3, wherein the solid medium is YEB solid medium supplemented with 50mg/L kanamycin and 50mg/L rifampicin.
6. Genetic transformation process according to claim 5, characterized in that said YEB solid medium consists of the following components in concentrations: 5g/L beef extract, 5g/L tryptone, 1g/L yeast extract and 4g/L MgSO 4 ·7H 2 O, 5g/L sucrose and 15g/L agar; the YEB solid medium pH was 7.4.
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