CN114958902B - Method for genetic transformation of fraxinus mandshurica by utilizing embryogenic callus - Google Patents

Method for genetic transformation of fraxinus mandshurica by utilizing embryogenic callus Download PDF

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CN114958902B
CN114958902B CN202210164313.5A CN202210164313A CN114958902B CN 114958902 B CN114958902 B CN 114958902B CN 202210164313 A CN202210164313 A CN 202210164313A CN 114958902 B CN114958902 B CN 114958902B
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CN114958902A (en
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李淑娟
杨玲
沈海龙
安岩
董昊
侯慧
赵文娜
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Northeast Forestry University
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Abstract

The invention provides a method for genetic transformation of fraxinus mandshurica by utilizing embryogenic callus, and relates to the technical field of genetic transformation. The invention combines the somatic embryogenesis approach with the genetic transformation technology, and can further develop the resistant callus screened in the genetic transformation process into somatic embryos by utilizing the somatic embryogenesis approach, and complete resistant plants are obtained after the somatic embryos germinate, so that higher transformation efficiency and more homozygous transformed plants are obtained. By using the method, the fraxinus mandshurica resistant somatic embryo is obtained through transgene for the first time. In the embodiment of the invention, the genetic transformation is carried out by utilizing embryogenic callus of fraxinus mandshurica, and the transformation rate is more than 80 percent and can reach 91.17 percent at most.

Description

Method for genetic transformation of fraxinus mandshurica by utilizing embryogenic callus
Technical Field
The invention belongs to the technical field of genetic transformation, and particularly relates to a method for carrying out genetic transformation on fraxinus mandshurica by utilizing embryogenic callus.
Background
The fraxinus mandshurica (Fraxinus mandshurica rupr.) is one of the third wiggles called as the three precious broad tree species in northeast China, is an excellent material tree species with wider application range in the international market, and has extremely high economic and ecological values. However, the fraxinus mandshurica has long growth period and lacks genetic transformation system, and the molecular basic research of the development regulation mechanism is greatly limited. Meanwhile, the genetic background is complex, particularly the development of the reproductive organs is obviously changed, and the regulation and control modes are different, so that the research of the water yeast Liu Teyi gene and the specific regulation and control channel cannot be broken through.
At present, the modern biotechnology means based on in-vitro regeneration and genetic transformation can introduce exogenous genes into the genome of the fraxinus mandshurica and enable the exogenous genes to be inherited stably, so that directional variation is generated, great genetic improvement is realized, the breeding period is shortened, but the transformation rate is low, and the problem of high-efficiency genetic transformation of the fraxinus mandshurica cannot be solved.
Disclosure of Invention
Therefore, the invention aims to provide a method for genetic transformation of fraxinus mandshurica by utilizing embryogenic callus, which has the advantages of simple operation, short period and high transformation efficiency.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for genetic transformation of fraxinus mandshurica by utilizing embryogenic callus, which comprises the following steps: genetic transformation is carried out on embryogenic callus of fraxinus mandshurica under the mediation of agrobacterium;
co-culturing the embryogenic callus after the genetic transformation, and screening to obtain a resistant callus;
and (3) performing embryo induction on the resistant callus to obtain a resistant embryo.
Preferably, said embryogenic callus is, prior to said genetic transformation, the method also comprises inoculating embryogenic callus into a secondary culture medium for dark culture; the secondary culture medium comprises the following components in concentration: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、17.05mg/L MnSO 4 ·H 2 O、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、27.8mg/L FeSO 4 ·7H 2 O、37.3mg/L Na 2 -EDTA、96mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20-30g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein and 3.5-5.5g/L gellan gum, pH=5.8-6.0.
Preferably, the temperature of the dark culture is 25℃and the time is 15d.
Preferably, said genetic transformation comprises infecting said embryogenic callus with an agrobacterium infection;
the preparation method of the agrobacterium infection solution comprises the following steps: culturing Agrobacterium strain containing target gene in YEB solid culture medium containing antibiotic, picking single colony, inoculating into YEB liquid culture medium containing same antibiotic, culturing to logarithmic phase, collecting thallus, and re-suspending to OD with acetosyringone 600 And (2) obtaining the agrobacterium infection solution by using the method of (1) and (4) with the ratio of (0.4-0.6).
Preferably, the antibiotics comprise Kan and Rif, wherein the concentration of Kan is 50mg/L, and the concentration of Rif is 50mg/L in the YEB solid culture medium and the YEB liquid culture medium.
Preferably, the co-cultivation comprises the step of inoculating the surface liquid of the callus into a co-cultivation medium after sucking the surface liquid of the callus, wherein the co-cultivation medium comprises the following components with the following concentrations: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、17.05mg/L MnSO 4 ·H 2 O、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、27.8mg/L FeSO 4 ·7H 2 O、37.3mg/L Na 2 -EDTA、96mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20-30g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein, 3.5-5.5g/L gellan gum and 20-60 mu M acetosyringone.
Preferably, the co-cultivation is a dark cultivation, and the time of the co-cultivation is 2d.
Preferably, the screening comprises continuous screening culture for 3 times in a screening culture medium, wherein the time of each screening culture is 15-20 d;
the screening medium comprises the following components in concentration: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、17.05mg/L MnSO 4 ·H 2 O、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、27.8mg/L FeSO 4 ·7H 2 O、37.3mg/L Na 2 -EDTA、96mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20-30g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein, 3.5-5.5g/L gellan gum, 10mg/L hygromycin and 250mg/L cefotaxime sodium.
Preferably, the somatic embryo induction comprises inoculating the resistant callus onto a somatic embryo induction medium for dark culture for 40-60 d;
the embryo induction medium comprises the following components in concentration: 1mg/L ABA, 950mg/L KNO 3 、825mg/L NH 4 NO 3 、85mg/L KH 2 PO 4 、185mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、22.3mg/L MnSO4·4H 2 O、0.83mg/L KI、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、0.025mg/L CoCl 2 ·6H 2 O、27.85mg/L FeSO 4 ·7H 2 O、37.25mg/L Na 2 -EDTA、220mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L nicotinic acid, 0.5mg/L Vitamin B6, 0.1mg/L Vitamin B1, 0-100g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein, 4.5-9.5g/L gellan gum and 1-2g/L activated carbon, and pH=5.8-6.0.
Preferably, the temperature of the dark culture is 25 ℃.
The beneficial effects are that: the invention provides a method for carrying out genetic transformation by using embryogenic callus of fraxinus mandshurica, which combines a somatic embryogenesis way with a genetic transformation technology, and can further develop the resistant callus screened in the genetic transformation process into a somatic embryo by using the somatic embryogenesis way, so that a complete resistant plant is obtained after the somatic embryo germinates, thereby obtaining higher transformation efficiency and a more homozygous transformed plant. By using the method, the fraxinus mandshurica resistant somatic embryo is obtained through transgene for the first time. In the embodiment of the invention, the genetic transformation is carried out by utilizing embryogenic callus of fraxinus mandshurica, and the transformation rate is more than 80 percent and can reach 91.17 percent at most.
Drawings
FIG. 1 is a schematic diagram of the culture of the water yeast Liu Pei callus proliferation of example 1;
FIG. 2 is a schematic diagram of the co-culture of the water yeast Liu Pei callus of example 1;
FIG. 3 is a schematic diagram of a screen for resistance to water yeast Liu Pei calli of example 1;
FIG. 4 is a schematic diagram showing GUS histochemical staining of wild embryogenic calli of fraxinus mandshurica in example 1;
FIG. 5 is a schematic diagram showing GUS histochemical staining of fraxinus mandshurica resistant embryogenic callus of example 1;
FIG. 6 is a schematic diagram of embryogenesis of fraxinus mandshurica resistant embryogenic callus of example 1.
Detailed Description
The invention provides a method for genetic transformation of fraxinus mandshurica by utilizing embryogenic callus, which comprises the following steps: genetic transformation is carried out on embryogenic callus of fraxinus mandshurica under the mediation of agrobacterium;
co-culturing the embryogenic callus after the genetic transformation, and screening to obtain a resistant callus;
and (3) performing embryo induction on the resistant callus to obtain a resistant embryo.
The invention carries out genetic transformation on embryogenic callus of the fraxinus mandshurica under the mediation of agrobacterium, and the embryogenic callus is preferably further subjected to dark culture by inoculating the embryogenic callus into a secondary culture medium before carrying out the genetic transformation. The subculture medium of the present invention preferably comprises the following components in concentration: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、17.05mg/L MnSO 4 ·H 2 O、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、27.8mg/L FeSO 4 ·7H 2 O、37.3mg/L Na 2 -EDTA、96mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20g/L sucrose, 100mg/L inositol, 400mg/L acid hydrolyzed casein and 3.5g/L gellan gum, pH=5.8. The temperature of the dark culture according to the invention is preferably 25℃and the time is preferably 15d. The invention preferably selects faint yellow, loose-structure water yeast Liu Pei callus to transfer into the secondary culture medium for the dark culture, so as to multiply embryogenic callus in a secondary way, and provide raw materials for the subsequent genetic transformation of embryogenic callus in different batches. The hydrographic Liu Pei calli described in the present invention were obtained by the method described in reference Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue.
The invention preferably utilizes the embryogenic callus which is dark cultured by the secondary culture medium to be soaked in an agrobacterium infection liquid for infection, and the preparation method of the agrobacterium infection liquid preferably comprises the following steps: culturing Agrobacterium strain containing target gene in YEB solid culture medium containing antibiotic, picking single colony, inoculating into YEB liquid culture medium containing same antibiotic, culturing to logarithmic phase, collecting thallus, and re-suspending to OD with acetosyringone 600 And (2) obtaining the agrobacterium infection solution by using the method of (1) and (4) with the ratio of (0.4-0.6). The temperature of the inverted dark culture according to the present invention is preferably 28℃and the time is preferably 2d. The antibiotics of the present invention preferably include Kan and Rif, wherein the concentration of Kan is 50mg/L, and the concentration of Rif is 50mg/L in the YEB solid medium and the YEB liquid medium. The infestation according to the invention preferably comprises, after the soaking, a gentle shaking at a speed of 60-80r/min for 20min, which brings it into sufficient contact for the infestation.
After the infection, the invention carries out co-culture on the callus and the agrobacterium. Preferably, after the infection, the infection liquid is poured out and drained, placed on sterile filter paper to suck the excessive bacterial liquid on the surface of the callus, and then transferred into a co-culture medium to be cultured for 2d under the condition of dark culture. The co-culture medium is prepared by adding 20-60 mu M acetosyringone based on the secondary culture medium.
Preferably, the callus subjected to co-culture is transferred into a screening culture medium for screening culture for 15-20 d, and the screened resistant tissue is transferred into a new screening culture medium for continuous screening for 15-20 d, and is continuously screened for 3 times to obtain the resistant embryogenic callus. The basic components, the types of the plant hormone and the concentration of the general selection culture medium are the same as those of the subculture culture medium, and only 10mg/L hygromycin and 250mg/L cefotaxime sodium are needed to be added. During operation, the callus on the co-culture medium is directly transferred to the screening medium by forceps.
After the resistant callus is obtained, GUS chemical tissue detection is preferably further included, and in the detection, GUS chemical tissue detection is preferably performed on the resistant callus, non-transformed tissue is used as a negative control, blue-colored tissue is transgenic resistant callus, light yellow tissue is control tissue, and the detection of GUS gene expression in transgenic water yeast Liu Pei callus is indicated.
The invention preferably transfers the detected resistant callus to a somatic embryo induction culture medium, and carries out dark culture for 40-60 d at 25 ℃ to obtain the resistant somatic embryo. The embryo induction medium of the present invention preferably comprises the following components in concentration: 1mg/L ABA, 950mg/L KNO 3 、825mg/L NH 4 NO 3 、85mg/L KH 2 PO 4 、185mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、22.3mg/ LMnSO4·4H 2 O、0.83mg/L KI、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、0.025mg/L CoCl 2 ·6H 2 O、27.85mg/L FeSO 4 ·7H 2 O、37.25mg/L Na 2 -EDTA、220mg/L CaCl 2 ·2H 2 O, 2mg/L glycine,0.5mg/L nicotinic acid, 0.5mg/L Vitamin B6, 0.1mg/L Vitamin B1, 70-100g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein, 4.5-9.5g/L gellan gum and 1-2g/L activated carbon, pH=5.8-6.0.
The method for genetic transformation of fraxinus mandshurica using embryogenic callus provided by the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the embodiment of the invention, the culture mediums used are respectively:
subculture medium: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/LK 2 SO 4 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、17.05mg/L MnSO 4 ·H 2 O、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、27.8mg/L FeSO 4 ·7H 2 O、37.3mg/L Na 2 -EDTA、96mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20g/L sucrose, 100mg/L inositol, 400mg/L acid hydrolyzed casein and 3.5g/L gellan gum, pH=5.8.
Co-culture medium: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、17.05mg/L MnSO 4 ·H 2 O、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、27.8mg/L FeSO 4 ·7H 2 O、37.3mg/L Na 2 -EDTA、96mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20g/L sucrose, 100mg/L inositol, 400mg/L acid hydrolyzed casein, 3.5g/L gellan gum and 20-60 mu M acetosyringone.
Screening the culture medium: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400mg/L NH 4 NO 3 、170mg/L KH 2 PO 4 、370mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、17.05mg/L MnSO 4 ·H 2 O、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、27.8mg/L FeSO 4 ·7H 2 O、37.3mg/L Na 2 -EDTA、96mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20g/L sucrose, 100mg/L inositol, 400mg/L acid hydrolyzed casein, 3.5g/L gellan gum, 10mg/L hygromycin and 250mg/L cefotaxime sodium.
Embryo induction medium: 1mg/L ABA, 950mg/L KNO 3 、825mg/L NH 4 NO 3 、85mg/L KH 2 PO 4 、185mg/L MgSO 4 ·7H 2 O、6.2mg/L H 3 BO 3 、8.6mg/L ZnSO 4 ·7H 2 O、22.3mg/L MnSO4·4H 2 O、0.83mg/L KI、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、0.025mg/L CoCl 2 ·6H 2 O、27.85mg/L FeSO 4 ·7H 2 O、37.25mg/L Na 2 -EDTA、220mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L niacin, 0.5mg/L Vitamin B6, 0.1mg/L Vitamin B1, 70g/L sucrose, 100mg/L inositol, 400mg/L acid hydrolyzed casein, 4.5g/L gellan gum and 1g/L activated carbon, pH=5.8.
Example 1
A method for genetic transformation using fraxinus mandshurica embryogenic callus, comprising:
1. subculture of the water yeast Liu Pei callus:
the water yeast Liu Pei callus was inoculated into a secondary medium and dark-cultured at 25℃for 15d.
2. Preparation of agrobacterium working bacteria liquid
Will containAgrobacterium strains with no load (vector species VB191103-1905 rcy) were dark-cultured for 2d at 28℃in an inverted position on YEB solid medium containing 50mg/L of Kan, rif each. Picking single colony, inoculating into YEB liquid culture medium containing same kind and concentration of antibiotics, culturing until Agrobacterium grows to logarithmic phase, namely OD 600 ＝0.4。
3. Agrobacterium-mediated genetic transformation of fraxinus mandshurica:
and (3) the agrobacterium liquid is centrifuged at 8000r/min for 5min at 4 ℃ to collect thalli. Re-suspending the cells to OD with suspension containing 40. Mu. MAs 600 =0.4, as an invasive solution, the water-curved Liu Pei calli were infected, gently swirled for 20min.
4. Co-culture of callus with Agrobacterium:
excess bacterial liquid on the surface of embryogenic callus is sucked off by sterile filter paper and transferred into a co-culture medium, and co-cultured for 2d under dark culture conditions.
5. Screening and culturing of resistant callus:
transferring the embryogenic callus into a screening culture medium for screening and culturing for 20d, transferring the screened resistant tissue into a new screening culture medium for continuous screening for 20d, and continuously screening for 3 times to obtain the resistant embryogenic callus;
6. detection of resistant callus
And (3) selecting a proper amount of resistant callus, immersing the selected callus in a proper amount of GUS staining solution, and vacuumizing. The tissue was placed in a 37℃water bath with dye solution for one week.
7. Generation of resistant embryos
The water yeast Liu Kang callus was transferred to embryo induction medium and dark cultured at 25℃for 40d.
In example 1, 34 resistant calli were obtained in total after 3 consecutive resistance selections, and 31 calli were blue after GUS staining, with a staining rate of 91.17%.
Example 2
A method for genetic transformation using fraxinus mandshurica embryogenic callus, comprising:
1. subculture of the water yeast Liu Pei callus:
the water yeast Liu Pei callus is inoculated into a secondary culture medium and is subjected to dark culture at 25 ℃ for 20d.
2. Preparation of agrobacterium working bacteria liquid
Agrobacterium strains containing no load (vector species VB191103-1905 rcy) were cultured upside down at 28℃for 2d on YEB solid medium containing 50mg/L each of Kan, rif. Picking single colony, inoculating into YEB liquid culture medium containing same kind and concentration of antibiotics, culturing until Agrobacterium grows to logarithmic phase, namely OD 600 ＝0.6。
3. Agrobacterium-mediated genetic transformation of fraxinus mandshurica:
and (3) the agrobacterium liquid is centrifuged at 8000r/min for 5min at 4 ℃ to collect thalli. Re-suspending the cells to OD with a suspension containing 20. Mu. MAs 600 =0.6, as an invasive solution, the hydrotropic Liu Pei calli were infected and gently shaken for 15min.
4. Co-culture of callus with Agrobacterium:
excess bacterial liquid on the surface of the callus is sucked by sterile filter paper and transferred into a co-culture medium, and co-culture is carried out for 2d under the condition of dark culture.
5. Screening and culturing of resistant callus:
transferring the callus to a screening culture medium for screening culture for 15d, transferring the screened resistant tissue to a new screening culture medium for continuous screening for 15d, and continuously screening for 3 times to obtain the resistant embryogenic callus;
6. detection of resistant callus
And (3) selecting a proper amount of resistant callus, immersing the selected callus in a proper amount of GUS staining solution, and vacuumizing. The tissue was placed in a 37℃water bath with dye solution for one week.
7. Generation of resistant embryos
The water yeast Liu Kang callus was transferred to embryo induction medium and dark cultured at 25℃for 50d.
In example 2, 28 resistant calli were obtained in total after 3 consecutive resistance selections, 24 calli were blue after GUS staining, and the staining rate was 85.71%.
Example 3
A method for genetic transformation using fraxinus mandshurica embryogenic callus, comprising:
1. subculture of the water yeast Liu Pei callus:
the water yeast Liu Pei callus was inoculated into a secondary medium and dark-cultured at 25℃for 25 days.
2. Preparation of agrobacterium working bacteria liquid
Agrobacterium strains containing no load (vector species VB191103-1905 rcy) were cultured upside down at 28℃for 2d on YEB solid medium containing 50mg/L each of Kan, rif. Picking single colony, inoculating into YEB liquid culture medium containing same kind and concentration of antibiotics, culturing until Agrobacterium grows to logarithmic phase, namely OD 600 ＝0.8。
3. Agrobacterium-mediated genetic transformation of fraxinus mandshurica:
and (3) centrifuging the agrobacterium tumefaciens bacterial liquid at 4 ℃ for 10min at 8000r/min to collect thalli. Re-suspending the cells to OD with a suspension containing 60. Mu. MAs 600 =0.4, as an invasive solution, the water-curved Liu Pei calli were infected, gently swirled for 20min.
4. Co-culture of callus with Agrobacterium:
excess bacterial liquid on the surface of embryogenic callus is sucked off by sterile filter paper and transferred into a co-culture medium, and co-cultured for 2d under dark culture conditions.
5. Screening and culturing of resistant callus:
transferring the embryogenic callus into a screening culture medium for screening and culturing for 18d, transferring the screened resistant tissue into a new screening culture medium for continuous screening for 18d, and continuously screening for 3 times to obtain the resistant embryogenic callus;
6. detection of resistant callus
And (3) selecting a proper amount of resistant callus, immersing the selected callus in a proper amount of GUS staining solution, and vacuumizing. The tissue was placed in a 37℃water bath with dye solution for one week.
7. Generation of resistant embryos
The water yeast Liu Kang callus was transferred to embryo induction medium and dark cultured at 25℃for 60d.
In example 3, after 3 consecutive resistance selections, 38 resistant calli were obtained in total, and 32 calli were blue after GUS staining, with a staining rate of 84.21%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (6)

1. A method for genetic transformation of fraxinus mandshurica by utilizing embryogenic callus, which is characterized by comprising the following steps: genetic transformation is carried out on embryogenic callus of fraxinus mandshurica under the mediation of agrobacterium; the embryogenic callus is inoculated into a secondary culture medium for dark culture before the genetic transformation is carried out; the secondary culture medium consists of the following components in concentration: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400 mg/L NH 4 NO 3 、170 mg/L KH 2 PO 4 、370 mg/L MgSO 4 ·7H 2 O、6.2 mg/L H 3 BO 3 、8.6 mg/L ZnSO 4 ·7H 2 O、17.05 mg/L MnSO 4 ·H 2 O、0.25 mg/L Na 2 MoO 4 ·2H 2 O、0.025 mg/L CuSO 4 ·5H 2 O、27.8 mg/L FeSO 4 ·7H 2 O、37.3 mg/L Na 2 -EDTA、96 mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20-30g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein and 3.5-5.5g/L gellan gum, pH=5.8-6.0;
co-culturing the embryogenic callus after the genetic transformation, and screening to obtain a resistant callus; the co-culture comprises the steps of sucking surface liquid of the callus, and inoculating the surface liquid into a co-culture medium, wherein the co-culture medium consists of the following components in concentration: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400 mg/L NH 4 NO 3 、170 mg/L KH 2 PO 4 、370 mg/L MgSO 4 ·7H 2 O、6.2 mg/L H 3 BO 3 、8.6 mg/L ZnSO 4 ·7H 2 O、17.05 mg/L MnSO 4 ·H 2 O、0.25 mg/L Na 2 MoO 4 ·2H 2 O、0.025 mg/L CuSO 4 ·5H 2 O、27.8 mg/L FeSO 4 ·7H 2 O、37.3 mg/L Na 2 -EDTA、96 mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20-30g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein, 3.5-5.5g/L gellan gum and 20-60 mu M acetosyringone; the screening comprises the steps of continuously screening and culturing for 3 times in a screening culture medium, wherein the time of each screening and culturing is 15-20 d;
the screening medium consisted of the following concentrations of components: 0.15 mg/L2, 4-D, 0.1 mg/L6-BA, 990mg/L K 2 SO 4 、400 mg/L NH 4 NO 3 、170 mg/L KH 2 PO 4 、370 mg/L MgSO 4 ·7H 2 O、6.2 mg/L H 3 BO 3 、8.6 mg/L ZnSO 4 ·7H 2 O、17.05 mg/L MnSO 4 ·H 2 O、0.25 mg/L Na 2 MoO 4 ·2H 2 O、0.025 mg/L CuSO 4 ·5H 2 O、27.8 mg/L FeSO 4 ·7H 2 O、37.3 mg/L Na 2 -EDTA、96 mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L Vitamin B5, 0.5mg/L Vitamin B6, 1mg/L Vitamin B1, 20-30g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein, 3.5-5.5g/L gellan gum, 10mg/L hygromycin and 250mg/L cefotaxime sodium;
performing embryo induction on the resistant callus to obtain a resistant embryo; the somatic embryo induction comprises the steps of inoculating the resistant callus onto a somatic embryo induction culture medium for dark culture for 40-60 d;
the somatic embryo induction culture medium consists of the following components in concentration: 1mg/L ABA, 950mg/L KNO 3 、825 mg/L NH 4 NO 3 、85 mg/L KH 2 PO 4 、185 mg/L MgSO 4 ·7H 2 O、6.2 mg/L H 3 BO 3 、8.6 mg/L ZnSO 4 ·7H 2 O、22.3 mg/L MnSO4·4H 2 O、0.83 mg/L KI 、0.25 mg/L Na 2 MoO 4 ·2H 2 O、0.025 mg/L CuSO 4 ·5H 2 O、0.025 mg/L CoCl 2 ·6H 2 O、27.85 mg/L FeSO 4 ·7H 2 O、37.25 mg/L Na 2 -EDTA、220 mg/L CaCl 2 ·2H 2 O, 2mg/L glycine, 0.5mg/L nicotinic acid, 0.5mg/L Vitamin B6, 0.1mg/L Vitamin B1, 70-100g/L sucrose, 100-200mg/L inositol, 400-500mg/L acid hydrolyzed casein, 4.5-9.5g/L gellan gum and 1-2g/L activated carbon, pH=5.8-6.0.
2. The method according to claim 1, wherein the dark culture is carried out at a temperature of 25℃for a period of 15d.
3. The method of claim 1, wherein said genetic transformation comprises infecting said embryogenic callus with agrobacterium tumefaciens liquor;
the preparation method of the agrobacterium infection solution comprises the following steps: culturing Agrobacterium strain containing target gene in YEB solid culture medium containing antibiotic, picking single colony, inoculating into YEB liquid culture medium containing same antibiotic, culturing to logarithmic phase, collecting thallus, and re-suspending to OD with acetosyringone 600 And (2) obtaining the agrobacterium infection solution by using the method of (1) and (4) with the ratio of (0.4-0.6).
4. A method according to claim 3, wherein the antibiotics comprise Kan and Rif, and wherein the concentration of Kan is 50mg/L and the concentration of Rif is 50mg/L in both the YEB solid medium and the YEB liquid medium.
5. The method of claim 1, wherein the co-cultivation is a dark cultivation for a period of 2d.
6. The method according to claim 1, wherein the temperature of the dark culture is 25 ℃.
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