阅读说明：本技术 一种农杆菌介导的沟叶结缕草遗传转化方法 (Agrobacterium-mediated genetic transformation method for zoysia matrella ) 是由 王凯 曲爱爱 刘建秀 李建建 郭海林 李晓慧 宗俊勤 于 2019-10-24 设计创作，主要内容包括：本发明公开了一种根癌农杆菌介导沟叶结缕草的遗传转化方法,包括菌液浓度、侵染时间、共培养时间、抗生素浓度、炼苗移栽和转基因植株的PCR检测等步骤。内容如下：最佳遗传转化体系为菌液OD<Sub>600</Sub>值为0.4,侵染30 min,共培养3 d后进行选择培养。特美汀在选择培养阶段最适抑菌浓度为250 mg/L,潮霉素愈伤组织筛选的最适选择压为40 mg/L,生根筛选的最适浓度为15 mg/L。通过GUS活性的组织化学分析和PCR鉴定,显示目的基因已成功转入沟叶结缕草基因组中。上述研究将为沟叶结缕草基因功能研究和分子育种提供重要技术支撑。该方法优点在于：获得了一种稳定、高效的沟叶结缕草遗传转化体系,为其遗传改良提供便利,同时为其功能基因的验证打下关键的前期基础。(The invention discloses a genetic transformation method of agrobacterium tumefaciens mediated zoysia japonica ditch, which comprises the steps of bacterial liquid concentration, infection time, co-culture time, antibiotic concentration, hardening seedling transplantation, PCR detection of transgenic plants and the like. The contents are as follows: the optimal genetic transformation system is bacterial liquid OD 600 The value is 0.4, infection is carried out for 30 min, and selective culture is carried out after co-culture for 3 d. The optimum bacteriostatic concentration of the timentin in the selective culture stage is 250 mg/L, the optimum selection pressure of the hygromycin callus screening is 40 mg/L, and the optimum concentration of the rooting screening is 15 mg/L. Histochemical analysis and PCR identification of GUS activity show that the target gene has been successfully transferred into the zoysia japonica genome. The above study will be zoysia matrella baseProvides important technical support for functional research and molecular breeding. The method has the advantages that: a stable and efficient zoysia japonica ditch genetic transformation system is obtained, convenience is provided for genetic improvement, and a key early-stage basis is laid for verification of functional genes of zoysia japonica ditch genetic transformation system.)

1. An agrobacterium-mediated genetic transformation method of zoysia matrella, which is characterized by comprising the following steps:

Preculture of the callus on a subculture medium for 7 d;

Preparation of agrobacterium infection liquid: performing plate-cutting culture on the agrobacterium liquid for 48 h, selecting a single clone, inoculating the single clone into 5 mL of agrobacterium liquid culture medium, performing shake culture, adding 800-₆₀₀And centrifuging to discard the supernatant, adding an equal volume of the infection solution for re-suspension to prepare an agrobacterium infection solution (containing 100 mu mol/ml AS (acetosyringone)) for later use.

2. And (3) transformation: first OD is applied₆₀₀Culturing for 1-2 h with a target agrobacterium infection solution (containing 100 mu mol/ml AS) of 0.4, transferring the embryogenic callus obtained by pre-culturing in the step (1) into the agrobacterium infection solution for infection for 30 min, transferring the infected callus onto sterilized filter paper, draining, transferring to a co-culture medium, and co-culturing for 3 d.

3. inducing differentiation, washing the co-cultured callus with sterile water containing Timentin for 1 time, washing with sterile water for 3 ~ 4 times, transferring to callus screening culture medium, and culturing for 20 d.

4. The surviving callus is transferred to a differentiation medium for growth and is cultured in light until the callus is differentiated.

5. Root selection culture and hardening seedling transplantation: and transferring the adventitious buds to a rooting screening culture medium for culturing for 20 d when the adventitious buds grow to 2-4 cm, transferring the resistant regenerated seedlings to the rooting culture medium after the resistant regenerated seedlings are obtained, opening a bottle cap to harden the seedlings for 3d when the test-tube seedlings grow to about 10 cm, and transplanting the test-tube seedlings to a plug tray.

6. The method according to claim 1, wherein the pre-incubation time in step (1) is 7 days.

7. The method of claim 1, wherein the OD of step (2)₆₀₀The value was 0.4.

8. The method according to claim 1, wherein before infection in step (3), 100 μmol/ml AS is added into the agrobacterium infection solution containing thalli, the culture temperature is 28 ℃, and the shaking speed is 100 r/min for 1-2 h; the culture medium of the agrobacterium infection liquid is MS + AS 100 mu mol/ml + casein 500 mg/L + sucrose (sucrose) 68.5 g/L + glucose 36g/L pH5.8, the infection time is 30 min, and the co-culture time is 3 d.

9. The method according to claim 1, wherein the callus selection medium in step (4) is MS + 6-benzylaminopurine (6-BA) 0.2 mg/L +2, 4-dichlorophenoxyacetic acid (2, 4-D) 2.0 mg/L + proline (pro) 1.15 g/L + sucrose 40 g/L + plant gel (phytagel) 3 g/L + Timentin 250 mg/L + hygromycin (Hyg) 40 mg/LpH5.8, the selection time is 20D, and the differentiation medium is MS + naphthylacetic acid (NAA) 0.1 mg/L + sucrose 40 g/L + agar (agar) 8 g/L + Timentin 250 mg/L pH5.8.

10. The method according to claim 1, wherein the rooting screening medium in step (5) is 1/2 MS + NAA 0.1 mg/L + sucrose 20 g/L + agar 8 g/L + Timentin 250 mg/L + Hyg 15 mg/L pH 5.8.

Technical Field

The invention relates to a genetic transformation method in the biological field, in particular to an agrobacterium tumefaciens-mediated genetic transformation method of zoysia matrella.

Background

Zoysia matrella (A. Merr.) (B. Merr.) (C. Merr.)Zoysia matrela) Is of the family Gramineae (Gramineae) Perennial warm-season turfgrass of the genus zoysia of the subfamily texatilis (choridoidea). The salt-resistant ecological composite fertilizer has the characteristics of slender leaves, dense grass layers, developed root systems, long green period and the like, is one of the most salt-resistant halophytes, is mostly used for urban greening, playground lawn and water and soil conservation, and is widely applied to tropical and subtropical regions of the world.

With the rapid development of biotechnology, transgenic technology is mature gradually, and specific target genes can be genetically transformed, so that the breeding period is shortened. Among the various plant transgenic methods, the Agrobacterium mediated method is the preferred method due to its advantages of high transformation efficiency, easy operation, low cost, etc. This method has also been successful in plants of the genus zoysia to date. It has been reported that Chaiminelian et al were first introduced by Agrobacterium-mediated methodgusThe gene is introduced into the embryogenic callus induced by Japanese zoysia japonica seeds to obtain a transgenic plant; the Wang Hefei et al and Ma Caiyun et al are respectively introduced by using agrobacterium-mediated methodDREBtransferring the gene into zoysia matsutake and zoysia japonica; hodgkin et al and Li Ruifen et al are each introduced by Agrobacterium mediated methodCBF1Transferring into Japanese and Chinese zoysia japonica.

However, few reports on genetic transformation of zoysia matrella are reported at present, and only in preliminary research on glyphosate-resistant genetic transformation of zoysia matrella, 7-age callus of zoysia matrella is taken as a material, after being infected by agrobacterium, 54 calli are obtained on a screening culture medium containing 2mmol/L, 3mmol/L and 5mmol/L glyphosate for 4 weeks respectively, and are regenerated for 16 weeks, and the regeneration seedlings are not identified, so that a genetic transformation system is incomplete. This greatly limits the development of molecular breeding. Therefore, on the basis of researching the influence of factors such as bacterial liquid concentration, infection time, co-culture time and the like on the zoysia matrella transgenic efficiency, a high-efficiency and stable zoysia matrella genetic transformation system is established, and the method has important significance for researching the gene function and breeding improvement.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a genetic transformation method for agrobacterium tumefaciens mediated channel zoysia japonica. The method is efficient and stable, and takes the embryonic callus induced by the stolons of zoysia japonica ditch as a transgenic receptor to establish a genetic transformation system of the embryonic callus.

The method for establishing zoysia matrella genetic transformation provided by the invention comprises the following steps:

(1) Preculture of callus (7 years old embryogenic callus induced from stoloniferous nods of zoysia virginiana): selecting faint yellow granular embryonic callus with a compact structure, and culturing the embryonic callus on a subculture medium for 7d to serve as a subsequent material; the culture conditions are as follows: 25 plus or minus 2 ℃ and 24 h darkness (humidity is 44%);

(2) Preparing agrobacterium infection liquid, namely unfreezing agrobacterium liquid preserved by 15 percent of glycerol at the temperature of minus 80 ℃, then performing plate-drawing culture on YEP solid culture medium containing kanamycin and rifampicin under the culture condition of 28 ℃ and 48 hours, selecting a single clone to inoculate the single clone into 5 mL of agrobacterium liquid culture medium for shake culture at the culture temperature of 28 ℃ and the shake speed of 200 ~ 220 r/min for 20 to 24 hours, adding 1000 mu l of the cultured agrobacterium liquid into 25 mL of agrobacterium liquid culture medium, and culturing the agrobacterium liquid at the temperature of 28 ℃ and the rpm of 220 for 5 to 6 hours until the agrobacterium grows to the length of 25 OD₆₀₀About 0.4, at room temperature 4000 rpm, centrifugation for 10 min. The supernatant was discarded, and an equal volume of the infection solution was added to resuspend it to prepare an Agrobacterium infection solution (containing 100. mu. mol/ml AS) for use.

(3) and (3) transformation: inoculating the embryogenic callus obtained by pre-culture in the step (1) into OD₆₀₀Infecting in the target agrobacterium suspension of =0.4 for 30 min to make agrobacterium sufficiently adsorbed on explant, transferring infected callus onto sterilized filter paper, draining residual bacterial liquid, adsorbing surface liquid, transferring onto co-culture medium, co-culturing for 2-3 d,

(4) And (3) induced differentiation, namely washing the co-cultured callus by sterile water containing Timentin for 1 time, washing by sterile water for 3 ~ 4 times, transferring to a callus screening culture medium for dark culture at 25 ℃ for 20 d, transferring the survival callus to a differentiation culture medium containing 250 mg/LTimentin for growth, and performing illumination culture (placing in a culture room for 24 h of illumination culture with the illumination intensity of 100 mu mol/(m ∙ s) at 25 ℃) until the callus is differentiated.

(5) Root selection culture and hardening seedling transplantation: transferring to rooting screening culture medium when adventitious bud grows to 2-4 cm, continuing culturing for 20 d to obtain resistance regenerated seedling, transferring to rooting culture medium containing 250 mg/L Timentin, hardening seedling for 3d (opening bottle cap and supplementing distilled water, 25 deg.C) when plantlet grows to about 10 cm, washing root agar with tap water, and transplanting to plug tray.

(6) GUS staining analysis, soaking the transformation material in GUS staining working solution, incubating at 37 ℃ for 1 ~ 24 h after wrapping with tinfoil paper, decolorizing with 70% ethanol for 1 ~ 3 h, and observing explant staining condition in a solid microscope.

(7) PCR detection of transgenic plants: extracting the genome DNA of the positive seedling obtained in the step (5), designing a primer aiming at the target gene, and identifying whether a target gene fragment is amplified by adopting a PCR technology, wherein if the identification is successful, the zoysia japonica ditch transgenic plant is obtained.

Compared with the prior art, the invention has the following advantages and effects:

The invention establishes a genetic transformation system method of zoysia japonica, which has the advantages that (1) the experimental process is simplified and the workload after transformation is greatly reduced by adopting a pressure screening culture medium to screen resistant callus and GUS identification to screen resistant plants. (2) A stable and efficient genetic transformation system method for zoysia matrella is obtained by utilizing the traditional monocotyledon genetic transformation method.

Drawings

FIG. 1 determination of inhibitory concentration of timentin

FIG. 2 determination of hygromycin screening concentration

FIG. 3 Effect of different Agrobacterium infection conditions on transformation efficiency

FIG. 4 GUS staining analysis of transformation Material

FIG. 5 Agrobacterium mediated genetic transformation of zoysia matrella and plant regeneration

FIG. 6 PCR detection of transgenic resistant plants

Detailed Description

The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer.

1.1 methods

1.1.1 Induction and subculture of zoysia japonica callus

The stolons of zoysia japonica furrows are induced to generate embryonic callus, and the callus induction and subculture are carried out by referring to methods such as chaimenhuang and the like.

TABLE 1 minimal Medium for plant regeneration and Agrobacterium culture

1.1.2 antibiotic susceptibility test

Determination of the inhibitory concentration of Timentin: and (3) respectively inoculating the callus soaked in the agrobacterium tumefaciens bacterial liquid for 30 min to callus subculture media containing 0, 100, 150, 200, 250 and 300 mg/L of Timentin, setting 3 times of each gradient, and observing the bacteriostasis condition. And after 30 d, counting the callus green seedling differentiation rate, and determining the optimal bacteriostatic concentration.

Determination of hygromycin (Hyg) screening concentration: and respectively inoculating the zoysia matrella embryonic callus on subculture media containing 0mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50mg/L Hyg, transferring to a callus differentiation culture medium after 20 days, counting the green seedling differentiation rate and seedling rate of the callus after 30 days, and preliminarily determining the hygromycin callus screening concentration. Respectively inoculating seedlings differentiated from the callus on rooting culture media containing 0mg/L, 5 mg/L, 10 mg/L, 15 mg/L and 20 mg/L of Hyg, observing the survival rate of green seedlings after 20 days, and primarily determining the screening concentration of hygromycin seedlings. On the basis, after the embryonic callus of the zoysia japonica is infected by agrobacterium, the zoysia japonica is inoculated on a subculture medium (containing Timentin) containing 0, 20, 30, 40 and 50mg/L Hyg, the zoysia japonica is transferred to a differentiation medium (containing Timentin) after 20 d, and the green seedling differentiation rate and the seedling survival rate of the callus are counted after 30 d. Respectively inoculating seedlings differentiated from the callus infected by the agrobacterium to rooting culture media containing 0, 5, 10, 15 and 20 mg/L Hyg, observing the survival rate of green seedlings after 20 days, and determining the hygromycin seedling screening concentration. Each of the above gradients was set to 3 replicates.

All the factors take the seedling rate of the callus and the survival rate of the regeneration seedlings as evaluation standards.

Seedling rate (%) = number of differentiated seedlings/total number of calluses × 100; survival rate of green seedlings (%) = number of green seedlings/number of total seedlings × 100.

1.1.3 preservation, culture and activation of Agrobacterium liquid

Thawing Agrobacterium tumefaciens liquid preserved at-80 ℃ with 15% glycerol at normal temperature, scratching and culturing in YEP + kanamycin (Kana) 50mg/L + rifampicin (Rif) 50mg/L solid culture medium under 28 ℃ for 48 h, selecting a single clone, inoculating the single clone into 5 mLYEP +50mg/L Kana +50mg/L Rif liquid culture medium, shaking and culturing at 28 ℃ for 20-24 h with shaking speed of 200 ~ 220 r/min, taking 1000 μ L of the cultured Agrobacterium tumefaciens 800 plus, adding the obtained 1000 μ L plus into 25 mL liquid culture medium containing YEP +50mg/L Kana +50mg/L Rif, culturing at 28 ℃ for 5-6 h at 220 rpm until the Agrobacterium grows to OD₆₀₀About 0.5, 25 mL of the above-mentioned bacterial suspension was put into 220 mL centrifuge tubes, and centrifuged at 4000 rpm at room temperature for 10 min. Abandoning the supernatant, adding a small amount of infection culture medium into the centrifuge tube to resuspend and precipitate, and then diluting the bacterial liquid to OD by using the infection culture medium₆₀₀About 0.4 (about 109/ml), 100 μmol/ml AS was added for infestation.

1.1.4 Agrobacterium-mediated genetic transformation, selection of resistant calli and plant regeneration

Refer to Zhang Fang et al, Zhang Li et al, and improve.

Before transformation, taking yellow granular embryogenic callusTissues were pre-cultured on subculture medium for 7 d. Placing the callus in Agrobacterium (bacterial liquid OD)₆₀₀0.4) soaking and dyeing the suspension for 30 min, culturing in the dark at 25 ℃ for 3d, washing the callus after co-culture for 1 time by using sterile water containing 250 mg/L Timentin, washing for 3 ~ 4 times by using the sterile water, transferring to a callus screening culture medium containing 250 mg/L Timentin and 40 mg/L Hyg for 20 d based on the dark culture at 25 ℃, transferring the survival callus to a differentiation culture medium containing 200 mg/L Timentin for growth, culturing by illumination until the callus is differentiated, cutting off seedlings with the length of about 2 cm, transferring to a screening culture medium containing 250 mg/L Timentin and 15 mg/L Hyg seedlings, continuing culturing for 20 d to obtain resistance regeneration seedlings, transferring to a rooting culture medium containing 250 mg/L Timentin, opening a bottle cap when the seedlings grow to about 10 cm, hardening the seedlings for 3d, washing agar on tap water roots, and transplanting into a hole tray.

1.1.5 improvement of the transformation conditions

Selection of Agrobacterium liquid concentration was performed by OD₆₀₀The callus was soaked for 30 min in 0.2, 0.4, 0.6 and 0.8 Agrobacterium suspensions and the residual bacteria liquid was drained with sterile filter paper. Co-culture, screening, differentiation and rooting culture were as above. The number of the positive transformation callus after screening is counted by GUS staining results, and 3 biological replicates are arranged in each gradient.

Selection of agrobacterium infection time: by OD₆₀₀The callus was soaked in 0.4 Agrobacterium suspension for 10, 20, 30 and 40min of infection time, respectively. Co-culture, screening, differentiation and rooting culture were as above. The number of the positive transformation callus after screening is counted by GUS staining results, and 3 biological replicates are arranged in each gradient.

Screening for co-cultivation time: by OD₆₀₀0.4 agrobacterium suspension is infected for 30 min, residual bacteria liquid is drained by sterile filter paper, the obtained product is placed on a callus subculture medium for dark culture at 25 ℃ for 1, 2, 3 and 4 days respectively, and screening, differentiation and rooting culture are carried out as above. The number of the positive transformation callus after screening is counted by GUS staining results, and 3 biological replicates are arranged in each gradient.

Transformation efficiency (%) = number of GUS staining positives/total number of calli after screening × 100

1.1.6 GUS staining analysis

the transformation material is soaked in GUS staining working solution, tin foil paper is wrapped and incubated at 37 ℃ for 1 ~ 24 h, 70% ethanol is used for decoloring for 1 ~ 3 h, and the explant staining condition is observed in a solid microscope.

1.1.7 PCR detection of resistant plants

Taking the tender leaves of hygromycin resistant plants and untransformed plants, and adopting a novel broad-spectrum plant genome DNA rapid extraction kit to extract genome DNA. Amplification by PCRHPTthe gene has the sequence as follows: HPT-F: 5'-GAAAAAGCCTGAACTCACCGC-3', respectively; HPT-R: 5'-TGCTCCATACAAGCCAACCAC-3', respectively; the expected fragment size was 729 bp. And (3) carrying out PCR amplification by taking the extracted zoysia sulcata genomic DNA as a template, taking the expression vector pCAMBIA1305.2 plasmid DNA as a positive control and taking the non-transgenic zoysia sulcata plant DNA as a negative control. The amplification system is as follows: mu.l of Premix Taq, 1. mu.l of DNA template, 1. mu.l each of HPT-F and HPT-R (20. mu. mol/L), and a 25. mu.l portion of deionized water. The amplification conditions were: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 1 min, and 35 cycles; extension at 72 ℃ for 10 min.

1.2 statistical analysis of data

All data were processed with Microsoft office excel 2016 and statistically analyzed using SPSS software (v 22.0).

2 results and analysis

2.1 determination of the inhibitory concentration of Timentin

As can be seen from FIG. 1, the bacteriostatic effect is gradually evident as the concentration increases. When the concentration is 300 mg/L, the agrobacterium can be completely inhibited from growing, but the differentiation rate of the callus is only 43.7 percent of that of the callus with the concentration of 250 mg/L; while the concentration of 200 mg/L has 59.7 percent of green seedling differentiation rate, but the bacteriostatic effect is not obvious. In order to effectively inhibit the growth of agrobacterium without influencing the regeneration of plants, 250 mg/L of Timentin is selected to inhibit the overgrowth of agrobacterium in the callus differentiation stage in the test.

2.2 determination of hygromycin screening concentration

As can be seen from FIG. 2, both the calli and seedlings of zoysia matrella were sensitive to hygromycin, the calli survival rate and the differentiated seedling survival rate were only 3.7% and 5.3% respectively when the hygromycin concentration was 50mg/L and 20 mg/L, and the calli differentiation and the seedling growth were severely inhibited, which is theoretically an ideal screening concentration. However, it can be seen from the figure that after Agrobacterium infection, the callus can not be normally differentiated under the hygromycin screening pressure of 50 mg/L; under the hygromycin screening pressure of 20 mg/L, differentiated seedlings can not survive, because the agrobacterium infection and the addition of timentin also cause certain damage to the growth and differentiation of callus tissues. When the hygromycin concentration is divided into 40 mg/L and 15 mg/L, the seedling rate of the callus is 6.3%, and the survival rate of the seedlings is 6.3%. Therefore, in this experiment, it was determined that the hygromycin selective concentration was 40 mg/L during the callus growth phase and 15 mg/L during the seedling growth phase.

2.3 Effect of transformation conditions on transformation efficiency

In agrobacterium-mediated genetic transformation, the concentration of agrobacterium liquid, infection time and co-culture time all have significant influence on transformation rate. As can be seen from FIG. 3A, the transformation efficiency of callus tends to increase with the increase of the concentration of Agrobacterium solution, when OD is higher₆₀₀When the conversion rate is 0.4, the conversion efficiency is highest and reaches 56 percent; however, as the concentration of the bacterial liquid increases, the conversion efficiency tends to decrease, and when OD is used₆₀₀0.6 ~ 0.8.8, the transformation efficiency was less than 50%, and the callus differentiation rate was decreased due to the browning and death of the callus caused by the vigorous growth of Agrobacterium, so the OD was₆₀₀An Agrobacterium strain solution impregnation concentration of 0.4 is most suitable.

As can be seen from FIG. 3B, the transformation efficiency of the callus tended to increase significantly as the agrobacteria dip-dyeing time was extended, with the highest transformation efficiency being 3.3 times the 10 min dip-dyeing and 1.4 times the 20min dip-dyeing when the dip-dyeing time was 30 min; this is due to the fact that the infection time is too short, and the agrobacterium attachment on the explants is insufficient, resulting in low callus transformation rate. However, with the continuous increase of the dip dyeing time, the transformation efficiency is remarkably reduced and the callus is damaged, and the transformation efficiency at the dip dyeing time of 40min is only 21% of that at the dip dyeing time of 30 min, and the difference is remarkable. This is because the infection time is too long, and the agrobacterium attached to the explant is too much, which causes excessive damage to the callus and is not favorable for obtaining the resistant callus. Therefore, the optimal agrobacteria staining time is 30 min.

The results in FIG. 3C show that the transformation efficiency of the callus tends to increase significantly with the increase of the co-culture time, when the co-culture time is 3d, the transformation efficiency of the callus is the highest, 70%, and the transformation efficiency is 2.6 times and 1.4 times of that of the co-culture time 1 d and 2 d, respectively; the reason is that the co-cultivation time is short, Agrobacterium infection is insufficient, and T-DNA cannot be efficiently transferred and expressed to plant cells. However, as the co-culture time is further prolonged, the transformation efficiency of the callus is reduced, and the transformation efficiency of the 4 d co-culture is only 57% of that of the 3d co-culture, because the co-culture time is prolonged, the quantity of agrobacterium adsorbed by the callus is increased, the damage to the callus is increased, and the later-stage callus growth is influenced. Therefore, the optimal co-cultivation time was 3 days.

2.4 GUS assay for transformation Material

According to the optimized agrobacterium transformation conditions, the zoysia japonica ditch callus is processed, and possible transgenic plants are obtained after co-culture, resistant callus screening, regenerated seedling growth and rooting, regenerated seedling screening, seedling hardening and transplanting (figure 5). GUS staining analysis is carried out on tissues in different stages, and the result shows that the callus in the screening stage presents blue after GUS staining, which indicates thatGUSThe gene was successfully expressed in callus (FIG. 4A), and after the resistant shoots were grown and acclimatized, GUS staining of transformed plants and leaves was blue, indicatingGUSThe gene was stably expressed in zoysia japonica plants (fig. 4B, C).

2.5 acquisition of transgenic resistant plants

And carrying out PCR detection on the regenerated plants after genetic transformation on the genome level. Randomly selecting 5 plants from 18 transplanted resistant plants, extracting leaf genome DNA, and amplifying by PCR reactionHPTA gene. The results showed that 5 transformed plants successfully amplified the 729 bp target fragment (FIG. 6), while the wild type zoysia japonica (F-) could not amplify the target band. The results further prove that the target gene is successfully integrated into the groove leavesZoysia genome.