CN113278650A - Genetic transformation method for agrobacterium-infected populus argentea callus - Google Patents
Genetic transformation method for agrobacterium-infected populus argentea callus Download PDFInfo
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Abstract
The invention discloses a genetic transformation method for agrobacterium-infected populus argentea callus, belonging to the technical field of genetic engineering and comprising the following steps: (1) inducing callus; (2) pre-culturing the callus; (3) activating and culturing strains; (4) infection; (5) co-culturing; (6) inducing adventitious buds; (7) bud elongation growth culture; (8) inducing to root and detecting. The method has the advantages of high transformation efficiency, short period, high flux and the like, provides a feasible technical method for poplar transgene, and is a powerful technical means for genetic improvement of forest trees.
Description
Technical Field
The invention relates to a genetic transformation method of a silver glandular poplar callus, in particular to a genetic transformation method of a silver glandular poplar callus infected by agrobacterium, belonging to the technical field of genetic engineering.
Background
The wood is not only the main body of the global land ecosystem, but also an important industrial raw material in the national economic construction and development. China is a large consumption country of wood and wood products, but because forest resources are deficient, the supply and demand relationship of the forest is increasingly tense. China vigorously develops fast-growing and high-quality artificial forests to meet the requirements of ecology and materials. The method improves the yield and quality of wood by genetically improving main afforestation tree species, and is an important direction for breeding the forest trees in China.
The poplar has strong stress resistance and fast growth and plays an important role in wood production. However, due to the limitations of the forest in growth cycle, high heterozygosity and other biological characteristics, the traditional breeding progress is relatively slow, and particularly, the improvement of the wood quality and the resistance meets a great bottleneck. The cultivation of fast-growing high-quality forest varieties can be accelerated by improving the growth and the timber properties of the forest by using modern biotechnology means such as a genetic engineering method.
The good woody plant genetic transformation system not only can provide research materials for further researching development physiology, morphogenesis and other basic theoretical problems, but also lays a foundation for improving poplar varieties by using modern biotechnology, accelerating breeding process, promoting rapid propagation of excellent seedlings and the like. Agrobacterium is a gram-negative Agrobacterium, which inserts its own T-DNA sequence into the plant genome, so that the insertion of a gene of interest into a T-DNA segment allows the transfer of foreign genes of interest and their expression in plant cells, thereby altering the genetic profile of the plant. The agrobacterium tumefaciens mediated method is the most commonly applied and developed mature transgenic method in the current plant transgenic technology.
Most genetic transformation techniques for plants are based on ex vivo regeneration of transformed cells. Common explants of a poplar genetic transformation system are mostly leaves, stems and the like in vitro, and transformation materials are obtained through regeneration of adventitious buds. However, the method has the defects of long culture period, low regeneration frequency and the like.
At present, the transgenic method of the populus argentea mainly comprises a light induction method and a dark induction method. The light induction method refers to a direct differentiation system for directly differentiating adventitious buds of explants (tissues or organs) without callus induction; the dark induction method is a method for obtaining a regeneration plant by transferring an explant to the light for continuous induction after the explant is subjected to dedifferentiation induction to form a callus under the dark condition. The former takes a short time but the transformation efficiency is relatively low, and the latter takes a long time although the transformation efficiency is improved.
Therefore, the technical problem which needs to be solved urgently in the technical field is to provide the agrobacterium-mediated genetic transformation method of the populus argentifolia which takes the callus as the receptor and has short time consumption, high transformation efficiency and stable inheritance.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides the stable genetic transformation method of the silver adenophora populus of the agrobacterium-mediated callus, which has the advantages of short time consumption, high transformation efficiency and stable inheritance.
The above object of the present invention is achieved by the following technical solutions:
a genetic transformation method for infecting poplar with agrobacterium (Populus alba multiplied by P. glandulosa cv. '84K') callus comprises the following steps:
(1) callus induction
Taking the leaf of the tissue culture seedling of the silver adenophora populus, transversely cutting the leaf for 2-3 times along the vein direction by using an operation blade, paving the leaf on a Callus Induction Medium (CIM), and carrying out Induction culture for 30-45 days at the temperature of 24 +/-1 ℃ under the dark condition;
(2) callus preculture
Cutting the callus obtained in the step (1) into small pieces, placing the small pieces in a Co-culture Medium (CM culture Medium), and pre-culturing for 2-6 days for later use;
(3) activation and culture of bacterial species
Dipping agrobacterium tumefaciens strain preserved at ultralow temperature (-80 ℃) by using a sterile toothpick, streaking the agrobacterium tumefaciens strain on an LB (Luria-Bertani) solid culture medium containing antibiotics, and performing inverted culture for 3 days at 28 ℃; picking a single colony, placing the single colony in a sterile test tube, adding 3mL of LB liquid culture medium containing antibiotics, and carrying out shake culture at 200rpm and 28 ℃ overnight; adding 1.0mL of bacterial liquid into 50mL of LB liquid culture medium containing antibiotics, performing shake culture at 200rpm and 28 ℃ to OD600＝0.4-1.0；
(4) Infection by infection
Placing the bacterial liquid obtained in the step (3) in a centrifugal tube, centrifuging for 15 minutes at 4 ℃ under 3500g, removing supernatant, and resuspending the bacteria by using heavy suspension to obtain the infection liquid; placing the callus obtained in the step (2) into an invasion dye solution, and infecting for 10-25 minutes at room temperature;
(5) co-culturing:
taking out the callus, placing on sterile filter paper, blotting off the residual infection liquid on the surface, placing on Co-culture Medium (CM culture Medium), and dark culturing at 24 + -1 deg.C for 2-5 days;
(6) and (3) inducing adventitious buds:
taking out the co-cultured callus, washing with sterile deionized water for 2-3 times, washing with 200mg/L sterile water solution of timentin-deionized water, soaking for 30-60min, sucking dry surface water with sterile filter paper, transferring into SIM (subscriber identity module) (SIM) differentiation medium, culturing at 24 + -1 deg.C under sunlight for 16h for 21 days, and allowing callus to grow adventitious bud;
(7) bud elongation growth culture:
transferring the callus with adventitious buds to an SEM (shoots Elongation Medium) bud Elongation growth medium, wherein the adventitious buds can grow to 1-2 cm after 2 weeks of culture;
(8) inducing rooting and detecting:
cutting off the bottom expanded part of the adventitious bud, transferring the cut-off part into RM Rooting Medium (Rooting Medium) to induce Rooting, cutting leaves and extracting DNA, and performing PCR detection and GUS staining verification.
Preferably, in the step (1), the leaf blade of the tissue culture seedling of the silver poplar is 3 weeks old.
Preferably, in step (1), the callus induction medium has the following composition: WPM (Lloyd & McCown Woody Plant basic Medium w/Vitamins) salt 2.41g/L, sucrose 20g/L, MES (4-morpholino sulfonic acid)0.5g/L, 2,4-D (2,4-dichlorophenoxy carboxylic acid)1.0mg/L, KT (6-furylaminopurine) 0.1mg/L, vegetable gel 5g/L, pH 5.9.
Preferably, in step (2), the pre-incubation time is 6 days.
Preferably, in step (2), the composition of the co-culture medium is: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, acetosyringone AS100 μ M/L, plant gel 5g/L, pH 5.9.
Preferably, in step (3), the agrobacterium is GV3101, which contains expression vector pCAMBIA1301, and the antibiotics and their working concentrations are: rifampicin (34mg/L), gentamicin (50mg/L) and kanamycin (50 mg/L).
Preferably, in step (3), OD600＝0.6。
Preferably, in step (4), the composition of the resuspension is: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, 2, 4-D1.0 mg/L, KT 0.1mg/L, acetosyringone 100 μ M, pH 5.6.
Preferably, in step (4), the infection is for 15 min.
Preferably, in step (6), the composition of the SIM differentiation medium is: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, NAA (naphthylacetic acid)0.05mg/L, 6-BA (6-benzylaminopurine)0.5mg/L, timentin 200mg/L, hygromycin B1.5mg/L, plant gel 5g/L, pH 5.9.
Preferably, in step (7), the composition of the SEM bud elongation growth medium is: 4.43g/L of MS salt, 30g/L of cane sugar, 0.02mg/L of NAA, 0.05mg/L of 6-BA, 200mg/L of timentin, 1.5mg/L of hygromycin, 5g/L of agar and 5.9 of pH.
Preferably, in step (8), the composition of the RM rooting medium is as follows: 2.22g/L of MS salt, 30g/L of cane sugar, 0.02mg/L of NAA, 0.05mg/L of IBA (indoluteric acid), 200mg/L of timentin, 1.5mg/L of hygromycin, 5g/L of agar and 5.9 of pH value.
Has the advantages that:
the invention provides a method for transforming populus argentifolia by agrobacterium with callus as a receptor, which comprises the steps of inducing leaves to dedifferentiate under the combined action of 2,4-D and KT to generate callus, infecting the callus by agrobacterium, redifferentiating the callus to form adventitious buds under the action of 6-BA and NAA, inducing the adventitious buds to elongate and grow under low-concentration hormone, and transferring the adventitious buds into a rooting culture medium to root after the adventitious buds grow to a proper length.
According to the method, poplar leaves are induced into callus tissues and infected, and each callus tissue can develop a plurality of independent transformants, so that the agrobacterium infection efficiency is improved; after the callus is subcultured once, 5-10 times of callus can be induced, and a foundation is provided for large-scale transgenosis; direct infection with callus saves a lot of time compared with the traditional method of infecting the explant first and then inducing the callus. In conclusion, the method has the advantages of high transformation efficiency, high flux, short period and the like, and provides a feasible technical method for large-batch transgenic poplar.
The genetic transformation method of the agrobacterium-infected populus silvery callus takes the callus as a receptor material for genetic transformation, the dedifferentiation and redifferentiation processes are separated by utilizing the culture conditions, the cells of the explant return to the level of meristematic cells after undergoing complete dedifferentiation, the exogenous genes are more easily accepted, the transformation efficiency is relatively higher, more transformants can be obtained, and the genetic transformation method has the advantages of high in-vitro regeneration frequency, short period for obtaining transformed plants and the like. Most plant tissues and organs can generate callus, and the culture mode is generally applicable to plants which can be regenerated through an in vitro culture way. Meanwhile, the callus can be propagated in a large amount in a short time through subculture, so that the use amount of the leaves is greatly reduced, and a large amount of culture space and manpower and material resources can be saved. Therefore, the invention establishes a genetic transformation system with high efficiency, rapidness and high flux by taking the callus induced by the leaf of the populus argentea as an explant and utilizing an agrobacterium-mediated method.
The agrobacterium adopted by the invention is GV3101, which contains an expression vector pCAMBIA1301, the GV3101 has rifampicin resistance, and a Ti plasmid pMP90 carried by the strain has gentamicin resistance; the expression vector pCAMBIA1301 has resistance to kanamycin, and the encoded beta-Glucuronidase (GUS) gene is a commonly used reporter gene in plant transgenes. The hygromycin phosphotransferase gene on the T-DNA of the gene is used as a selective marker gene of transgenic poplar, and the encoded protein has hygromycin resistance. And (3) carrying out PCR detection on the regenerated seedlings with hygromycin resistance by using a specific primer, wherein the size of a product is 653bp, and meanwhile, carrying out GUS (glucuronidase) staining to verify that a target fragment is transferred into the populus argentifolia.
The present invention is further illustrated by the following detailed description and accompanying drawings, which are not meant to limit the scope of the invention.
Drawings
FIG. 1 shows the result of PCR detection at DNA level in example 1 of the present invention.
FIG. 2 shows the result of GUS staining in example 1 of the present invention.
Detailed Description
Unless otherwise specified, the raw materials used in the following examples are commercially available conventional raw materials, and the methods used are those commonly used in the art.
Example 1
A genetic transformation method for infecting poplar with agrobacterium (Populus alba multiplied by P. glandulosa cv. '84K') callus comprises the following steps:
(1) callus induction
Taking 3-week-old leaf of tissue culture seedling of silver glandular poplar, transversely cutting 2-3 times along vein direction with surgical blade, placing in callus induction culture medium CIM to induce callus, and culturing at 24 + -1 deg.C in dark for 37 days to obtain callus;
(2) callus preculture
Cutting the callus into 1cm3The small blocks are placed in a co-culture medium CM and pre-cultured for 6 days for later use;
(3) activation and culture of bacterial species
Dipping agrobacterium strain preserved at ultralow temperature (-80 ℃) by using a sterile toothpick, streaking the agrobacterium strain on an LB solid culture medium containing antibiotics (kanamycin, rifampicin and gentamicin), and performing inverted culture at 28 ℃ for 3 days; picking a single colony, placing the single colony in a sterile test tube, adding 3mL of LB liquid culture medium containing antibiotics, and carrying out shake culture at 200rpm and 28 ℃ overnight; sucking 1.0mL of bacterial liquid, adding 50mL of LB liquid culture medium containing antibiotics, shaking and culturing at 200rpm and 28 ℃ to OD600＝0.6；
(4) Infection by infection
Placing 50mL of the bacterial liquid in the step (3) in a centrifuge tube, centrifuging for 15 minutes at 4 ℃ under 3500g, removing a supernatant, resuspending the bacteria by using 100mL of a heavy suspension to obtain an invasion dye solution, placing the callus in the step (2) in the invasion dye solution, and infecting for 15 minutes at room temperature;
(5) co-culturing:
taking out the callus from the infection solution, placing on sterile filter paper, sucking off the infection solution remained on the surface, placing on a CM co-culture medium, culturing at 24 + -1 deg.C under dark condition for 3 days;
(6) and (3) inducing adventitious buds:
taking out the co-cultured callus, washing with sterile deionized water for 2-3 times, washing with 200mg/L timentin-deionized sterile water solution, soaking for 30-60min, sucking dry surface water on sterile filter paper, transferring to SIM differentiation medium, culturing at 24 + -1 deg.C under sunlight for 16 hr for 21 days, and allowing callus to grow adventitious bud;
(7) bud elongation growth culture:
transferring the callus with adventitious buds to an SEM bud elongation growth medium, and culturing for two weeks until the adventitious buds can grow to 1-2 cm;
(8) inducing rooting and detecting:
cutting off the bottom expanded part of the adventitious bud, transferring the cut-off part into an RM rooting culture medium to induce rooting, cutting leaves, carrying out DNA horizontal PCR detection and GUS staining observation, and verifying the transgenic plant.
As shown in FIG. 1, the result of the DNA level PCR test in example 1 of the present invention is: DNA level PCR detection is carried out by using GUS gene specific primers, and the size of a product is 653 bp.
As shown in FIG. 2, the GUS staining results in example 1 of the present invention are: the leaves become blue after staining, which indicates that the GUS gene is recombined in the plant genome.
Wherein in the step (1), the callus induction culture medium comprises the following components: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, 2, 4-D1.0 mg/L, KT 0.1mg/L, plant gel 5g/L, pH 5.9;
in the step (2), the composition of the co-culture medium is as follows: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, acetosyringone AS 100. mu.M/L, plant gel 5g/L, pH 5.9;
in the step (3), the agrobacterium is GV3101, which contains expression vector pCAMBIA1301, and the antibiotic and its working concentration are: rifampicin (34mg/L), gentamicin (50mg/L) and kanamycin (50 mg/L);
in the step (4), the composition of the resuspension is as follows: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, 2, 4-D1.0 mg/L, KT 0.1mg/L, acetosyringone 100 μ M, pH 5.6;
in the step (6), the composition of the SIM differentiation medium is as follows: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, NAA 0.05mg/L, 6-BA 0.5mg/L, timentin 200mg/L, hygromycin B1.5mg/L, plant gel 5g/L, pH 5.9;
in the step (7), the SEM bud elongation growth medium comprises the following components: 4.43g/L of MS salt, 30g/L of cane sugar, 0.02mg/L of NAA, 0.05mg/L of 6-BA, 200mg/L of timentin, 1.5mg/L of hygromycin, 5g/L of agar and 5.9 of pH;
in the step (8), the RM rooting medium comprises the following components: MS salt 2.22g/L, sucrose 30g/L, NAA0.02mg/L, IBA 0.05mg/L, timentin 200mg/L, hygromycin B1.5mg/L, agar 5g/L, pH 5.9.
Example 2
The rest is the same as example 1 except that:
(3) activation and culture of bacterial species
Dipping agrobacterium strain preserved at ultralow temperature (-80 ℃) by using a sterile toothpick, streaking the agrobacterium strain on an LB solid culture medium containing antibiotics (kanamycin, rifampicin and gentamicin), and performing inverted culture at 28 ℃ for 3 days; picking a single colony, placing the single colony in a sterile test tube, adding 3mL of LB liquid culture medium containing antibiotics, and carrying out shake culture at 200rpm and 28 ℃ overnight; sucking 1.0mL of bacterial liquid, adding 50mL of LB liquid culture medium containing antibiotics, shaking and culturing at 200rpm and 28 ℃ to OD600＝0.4。
Example 3
The rest is the same as example 1 except that:
(3) activation and culture of bacterial species
Dipping Agrobacterium strain preserved at ultralow temperature (-80 deg.C) with sterile toothpick, and streaking on LB solid culture medium containing antibiotics (kanamycin, rifampicin and gentamicin)Performing inverted culture at 28 deg.C for 3 days; picking a single colony, placing the single colony in a sterile test tube, adding 3mL of LB liquid culture medium containing antibiotics, and carrying out shake culture at 200rpm and 28 ℃ overnight; sucking 1.0mL of bacterial liquid, adding 50mL of LB liquid culture medium containing antibiotics, shaking and culturing at 200rpm and 28 ℃ to OD600＝0.8。
Example 4
The rest is the same as example 1 except that:
(3) activation and culture of bacterial species
Dipping agrobacterium strain preserved at ultralow temperature (-80 ℃) by using a sterile toothpick, streaking the agrobacterium strain on an LB solid culture medium containing antibiotics (kanamycin, rifampicin and gentamicin), and performing inverted culture at 28 ℃ for 3 days; picking a single colony, placing the single colony in a sterile test tube, adding 3mL of LB liquid culture medium containing antibiotics, and carrying out shake culture at 200rpm and 28 ℃ overnight; sucking 1.0mL of bacterial liquid, adding 50mL of LB liquid culture medium containing antibiotics, shaking and culturing at 200rpm and 28 ℃ to OD600＝1.0。
Examples 5 to 7
The other steps are exactly the same as in example 1, with the difference that: the infestation times were 10 minutes (example 5), 20 minutes (example 6) and 25 minutes (example 7), respectively.
Examples 8 to 10
The other steps are exactly the same as in example 1, with the difference that: co-cultivation times were 2 days (example 8), 4 days (example 9) and 5 days (example 10), respectively.
Examples 11 to 13
The other steps are exactly the same as in example 1, with the difference that: the preculture time was 0 day (example 11), 2 days (example 12) and 4 days (example 13), respectively.
Examples 14 to 16
The other steps are exactly the same as in example 1, with the difference that: 0 (example 14), 50 (example 15) and 150. mu.M (example 16) acetosyringone were added at the time of infection, respectively.
And (4) analyzing results:
firstly, bacterial liquid OD600Influence of value on genetic transformation of populus argentea
The number of explants of the rooting-induced poplar prepared in examples 1 to 4 and the number of explants producing resistant buds were counted to calculate the transformation ratio, and the results are shown in Table 1.
OD600The values are different, the activity of Agrobacterium is also different, and the appropriate OD600The value is critical to the success of the transformation; selection of OD in the invention600Values of 0.4, 0.6, 0.8, 1.0, the results show that: OD600At 0.6, the conversion efficiency is highest, OD600Second order of conversion, OD, at 0.4600At 0.8 and 1.0, the conversion efficiency was lowest.
TABLE 1 OD600Effect on conversion efficiency
Secondly, influence of infection time on genetic transformation of populus argentea
In the example 1 of the present invention, the infection time in the step (4) was 15min, and under the condition that the other steps were completely the same, the infection times were 10 min, 20 min and 25 min, respectively; the number of explants and explants producing resistant shoots of the prepared induced differentiation poplar were observed, and the transformation rate was calculated, and the results are shown in table 2.
The infection time has an important influence on transformation efficiency, the genetic transformation is not facilitated due to too short time, and the browning and death of explants are easily caused due to too long time; the selected infection time of the invention is 10 minutes, 15 minutes, 20 minutes and 25 minutes, and the result shows that: the highest transformation efficiency was achieved at an infection time of 15 minutes and the lowest at 25 minutes.
TABLE 2 Effect of infection time on transformation efficiency
Influence of co-culture time on genetic transformation of populus argentifolia
The co-culture process after infection affects the transfer of T-DNA and the number of transformed cells, and thus, proper co-culture time plays an important role in improving transformation efficiency. In the embodiment 1 of the invention, the co-culture time is 3 days; the other steps are identical, except that the co-culture time is respectively 2 days, 4 days and 5 days; observing the number of explants of the prepared induced differentiation poplar and the number of explants producing resistant buds, and calculating the transformation rate, wherein the results are shown in Table 3; the results show that: when the co-culture time is 2 days to 3 days, the transformation efficiency is relatively high, and the frequency of the finally obtained hygromycin resistant buds is as high as 60 percent.
TABLE 3 Effect of Co-cultivation time on transformation efficiency
Fourth, the influence of the pre-culture time on the genetic transformation of the populus argentea
The pre-culture can enable the plant cells to integrate exogenous DNA more easily, and can promote acetosyringone in the culture medium to permeate into explants, so that the transformation efficiency is improved during infection; in the embodiment 1 of the invention, the pre-culture time is selected to be 6 days; the other steps are the same except that the pre-culture time is 0 day, 2 days and 4 days respectively; observing the number of explants of the prepared induced differentiation poplar and the number of explants producing resistant buds, and calculating the conversion rate, wherein the results are shown in Table 4; the experimental result shows that the transformation efficiency of the preculture for 6 days is up to 69% in a certain period of time, and the transformation efficiency difference of the preculture for 0 day, 2 days and 4 days is not obvious.
TABLE 4 Effect of preculture time on transformation efficiency
Influence of acetosyringone on genetic transformation of populus argentea
Phenolic substances such AS acetosyringone AS and the like are inducers necessary for agrobacterium to infect plant cells, and can activate vir genes and guide the transfer of T-DNA, thereby improving the transformation efficiency. During infection, Acetosyringone (AS) with a certain concentration is added, so that the genetic transformation efficiency can be effectively improved. In the embodiment 1 of the invention, 100 mu M acetosyringone is added during infection; the other is the same except that 0, 50 and 150 mu M acetosyringone is added at the time of infection; observing the number of explants of the prepared induced differentiation poplar and the number of explants producing resistant buds, and calculating the transformation rate, wherein the results are shown in Table 5; the results show that: when 100 mu M acetosyringone is added, the conversion efficiency is as high as 64 percent.
TABLE 5 Effect of Acetosyringone (AS) concentration on conversion efficiency
Compared with other genetic transformation systems, the method has relatively high transformation efficiency, can obtain a large number of transgenic seedlings by using fewer leaves of the tissue culture seedlings, saves a large amount of manpower and material resources, consumes less time in the whole process, and can provide convenience for constructing a mutant library or transgenes on a large scale.
The method has the advantages of high transformation efficiency, short period, high flux and the like, provides a feasible technical method for poplar transgene, and is a powerful technical means for genetic improvement of forest trees.
Claims (10)
1. A genetic transformation method for callus of populus silmuloides infected by agrobacterium comprises the following steps:
(1) callus induction
Taking the leaf of the tissue culture seedling of the silver glandular poplar, transversely cutting the leaf for 2-3 times along the vein direction by using an operation blade, paving the leaf on a callus induction culture medium, and carrying out induction culture for 30-45 days at 24 +/-1 ℃ under a dark condition;
(2) callus preculture
Cutting the callus obtained in the step (1) into small pieces, placing the small pieces in a CM co-culture medium, and pre-culturing for 2-6 days for later use;
(3) activation and culture of bacterial species
Dipping agrobacterium tumefaciens strain preserved at ultralow temperature by using a sterile toothpick, streaking the agrobacterium tumefaciens strain on an LB solid culture medium containing antibiotics, and carrying out inverted culture for 3 days at 28 ℃; picking a single colony, placing the single colony in a sterile test tube, adding 3mL of LB liquid culture medium containing antibiotics, and carrying out shake culture at 200rpm and 28 ℃ overnight; adding 1.0mL of bacterial liquid into 50mL of LB liquid culture medium containing antibiotics, performing shake culture at 200rpm and 28 ℃ to OD600＝0.4-1.0；
(4) Infection by infection
Placing the bacterial liquid obtained in the step (3) in a centrifugal tube, centrifuging for 15 minutes at 4 ℃ under 3500g, removing supernatant, and resuspending the bacteria by using heavy suspension to obtain the invaded liquid; placing the callus obtained in the step (2) into an invasion dye solution, and infecting for 10-25 minutes at room temperature;
(5) co-culturing:
taking out the callus, placing on sterile filter paper, sucking off residual infection liquid on the surface, placing on CM co-culture medium, dark culturing at 24 + -1 deg.C for 2-5 days;
(6) and (3) inducing adventitious buds:
taking out co-cultured callus, washing with sterile deionized water for 2-3 times, washing with 200mg/L sterile water solution of timentin-deionized water, soaking for 30-60min, drying surface water with sterile filter paper, transferring into SIM differentiation culture medium, culturing at 24 + -1 deg.C under sunlight irradiation for 16h, and growing adventitious bud from callus after 21 days;
(7) bud elongation growth culture:
transferring the callus with adventitious buds to an SEM bud elongation growth medium, and culturing for 2 weeks until the adventitious buds can grow to 1-2 cm;
(8) inducing rooting and detecting:
cutting off the bottom expanded part of the adventitious bud, transferring the cut-off part into an RM rooting culture medium to induce rooting, shearing leaves and extracting DNA, and carrying out PCR detection and GUS dyeing verification.
2. The genetic transformation method of Agrobacterium-infected Populus argenteus callus according to claim 1, wherein: in the step (1), the leaf blades of the tissue culture seedling of the silver adenophora populus are 3 weeks old.
3. The genetic transformation method of Agrobacterium-infected Populus argenteus callus according to claim 2, wherein: in the step (1), the callus induction culture medium comprises the following components: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, 2, 4-D1.0 mg/L, KT 0.1mg/L, plant gel 5g/L, pH 5.9.
4. The genetic transformation method of Agrobacterium-infected Populus argenteus callus according to claim 3, wherein: in the step (2), the pre-culture time is 6 days.
5. The genetic transformation method of Agrobacterium-infected Populus argenteus callus according to claim 4, wherein: in the step (2), the composition of the co-culture medium is as follows: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, acetosyringone 100 μ M/L, plant gel 5g/L, pH 5.9.
6. The method for genetic transformation of Agrobacterium-infected Populus argenteus callus according to claim 5, wherein the genetic transformation method comprises the steps of: in the step (3), the agrobacterium is GV3101, which contains expression vector pCAMBIA1301, and the working concentrations of antibiotics are respectively: rifampicin 34mg/L, gentamicin 50mg/L, kanamycin 50 mg/L.
7. The method for genetic transformation of Agrobacterium-infected Populus argenteus callus according to claim 6, wherein the genetic transformation method comprises: in step (3), OD600＝0.6。
8. The genetic transformation method of Agrobacterium-infected Populus argenteus callus according to claim 7, wherein: in the step (4), the composition of the resuspension is as follows: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, 2, 4-D1.0 mg/L, KT 0.1mg/L, acetosyringone 100 μ M, pH 5.6.
9. The method for genetic transformation of Agrobacterium-infected Populus argenteus callus according to claim 8, wherein the genetic transformation method comprises: in the step (4), infection is carried out for 15 min.
10. The method for genetic transformation of agrobacterium-infected populus argentea callus according to claim 9, wherein the genetic transformation method comprises the following steps: in the step (6), the composition of the SIM differentiation medium is as follows: WPM salt 2.41g/L, sucrose 20g/L, MES0.5g/L, NAA 0.05mg/L, 6-BA 0.5mg/L, timentin 200mg/L, hygromycin B1.5mg/L, plant gel 5g/L, pH 5.9;
in the step (7), the SEM bud elongation growth medium comprises the following components: 4.43g/L of MS salt, 30g/L of cane sugar, 0.02mg/L of NAA, 0.05mg/L of 6-BA, 200mg/L of timentin, 1.5mg/L of hygromycin, 5g/L of agar and 5.9 of pH;
in the step (8), the RM rooting medium comprises the following components: MS salt 2.22g/L, sucrose 30g/L, NAA0.02mg/L, IBA 0.05mg/L, timentin 200mg/L, hygromycin B1.5mg/L, agar 5g/L, pH 5.9.
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