Agrobacterium tumefaciens-mediated high-efficiency genetic transformation system of acacia melanoxylon and construction method thereof

Abstract

The application discloses a kind of Agrobacterium tumefaciens mediated efficient genetic transformation system of blackwood acacia and its construction method, with blackwood acacia excellent clone SR3 stem section as explant, and develops adventitious bud induction culture medium, using the infection liquid containing recombinant agrobacterium to infect blackwood acacia stem section without pre-culture, then carries out immersion and co-culture, and the explant is induced and screened culture to transgenic strain, obtain blackwood acacia resistant bud, and then screening obtains the blackwood acacia positive plant of genetic transformation completion.Therein, by optimizing experimental condition to improve the induction rate of adventitious bud and obtain efficient genetic transformation;The application provides technical support for gene introduction and knockout of original plant and the like, is beneficial to the molecular mechanism of parsing blackwood acacia heartwood formation, development etc., provides reference for the function analysis of important gene of blackwood acacia, and can further obtain transgenic and gene editing new variety on the basis of blackwood acacia excellent clone.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to an Agrobacterium tumefaciens-mediated high-efficiency genetic transformation system of Acacia melanoxylon and a construction method thereof. BACKGROUND

[0002] Acacia melanoxylon is a tall evergreen tree of the genus Acacia in the family Mimosaceae, which is originally from Australia. As a famous precious tree species in Australia, Acacia melanoxylon, together with walnut, redwood and teak, is one of the four precious wood species with large-scale output. Due to high wood density and good texture, Acacia melanoxylon is often used as excellent solid wood furniture, veneer and joinery, and is widely introduced in Oceania, Europe, Asia and Africa. In the 1990s, Acacia melanoxylon was introduced into China. Due to its fast growth, good wood quality, nitrogen-fixing nodules, rich litter and good soil improvement performance, etc., through breeding and improvement in the later period, an excellent clone with fast growth speed, straight stem shape and high heartwood rate has been selected, and the clone has been massively propagated and cultivated, and has become an ecological friendly precious tree species with medium and short periods in South China.

[0003] However, the current research on Acacia melanoxylon mainly focuses on clone selection, cultivation technology, tissue culture and propagation technology, and there is little research on callus induction and adventitious bud regeneration. There is no research on the establishment of a genetic transformation system at home and abroad, especially there is no research on the genetic transformation system for the cultivated excellent clone. In the present application, the stem segments of the tissue culture seedlings of the widely cultivated excellent clone SR3 of Acacia melanoxylon are used as the materials, a high-frequency regeneration system is established by developing a regeneration medium, and on this basis, an Agrobacterium tumefaciens-mediated genetic transformation system is optimized. Finally, through screening and identification, a stable transgenic bud seedling is obtained, and a high-efficiency genetic transformation system of the excellent clone of Acacia melanoxylon is established, which lays a foundation for further improvement and cultivation of transgenic new strains of Acacia melanoxylon. SUMMARY

[0004] In view of the above problems, the present application aims to provide an Agrobacterium tumefaciens-mediated high-efficiency genetic transformation system of Acacia melanoxylon and a construction method thereof.

[0005] The technical content of the present application is as follows:

[0006] The present application provides a construction method of an Agrobacterium tumefaciens-mediated high-efficiency genetic transformation system of Acacia melanoxylon, which comprises the following steps:

[0007] Step 1: Infection of Agrobacterium tumefaciens strain: pick the activated Agrobacterium single colony, transfer it to LB liquid medium, and cultivate it in a 28℃ and 200rpm constant temperature shaker for 16-24h until the OD600 value of the bacterial solution is 0.5, centrifuge at 4℃ for 10min, remove the supernatant, and add an equal volume of WPM+30g·L-1 sucrose resuspension solution to resuspend the precipitated bacterial cells;

[0008] The activation is carried out by streaking the Agrobacterium strain on a plate containing 30 mg·L -1 Rif and 50 mg·L -1 Kan;

[0009] The preparation of the Agrobacterium strain is as follows: the stored Agrobacterium tumefaciens GV3101 competence (purchased from Shanghai Weidi Biology) is taken out from a-80℃ refrigerator, inserted into ice, and when it is melted into an ice-water mixed state, 1 μL of pBI121 plasmid (Professor Lu Mengzhu of Zhejiang Agricultural University) is added to 100 μL of the competence for 5 min, and then it is quickly frozen in liquid nitrogen for 5 min, taken out from the liquid nitrogen, and smoothly placed in a 37℃ water bath for heat shock for 5 min, 700 μL of LB liquid medium is added, mixed uniformly by blowing, and then cultured at 28℃, 200 rpm for 2 h, and then the recovered bacterial solution is centrifuged at 6000 rpm for 1 min, the supernatant is discarded, and about 100 μL of the supernatant is mixed uniformly by blowing with a pipette, 50 μL of the bacterial solution is used to inoculate an LB plate containing 50 mg·L -1 Kan 20 mg·L -1 Rif;

[0010] Step 2: The cut explant stem section is immersed in the Agrobacterium liquid: the cut explant stem section is immersed in the resuspended Agrobacterium liquid for 20 min with slight shaking for 5-6 times, the surface bacterial liquid is taken out, and then transferred to a co-culture medium with a pH of 5.4 and sterile filter paper on the surface for dark culture for 48 h;

[0011] The pretreatment for obtaining the explant is as follows: the aseptic propagation seedlings of the excellent clone of Acacia melanoxylon are cultured on a propagation medium for 25-30 d, the plant illumination intensity is 100-300 μmol·m -2 ·s -1 , the time is 16 h, and the culture room temperature is 23±2℃;

[0012] The propagation medium comprises: mMS medium, 0.3 mg·L -1 BAP, 0.2 mg·L -1 IBA, 0.2 mg·L - 1 IAA, 30 g·L -1 sucrose, and 7.0 g·L -1 agar;

[0013] The cutting of the explant stem section is as follows: the Acacia melanoxylon bud seedlings with a seedling height of more than 3 cm are taken, the stem sections with axillary buds are removed, and 0.5-1 cm stem sections are cut as explants for standby use;

[0014] The co-culture medium comprises WPM, 0.10 mg·L -1 TDZ, 0.05 mg·L -1 IAA, 200.0 μM acetosyringone, 30.0 g·L -1 sucrose and 7.0 g·L -1 agar powder.

[0015] Step 3: screening culture of the explants for resistant shoots: the stems after co-culture are transferred to the callus induction medium added with 200 mg·L -1 Cef and 30 mg·L -1 Kan, and the medium is replaced every 2 weeks, and the browning and dead explants are removed to obtain explants with shoot points;

[0016] The callus induction medium has a pH of 5.8 and comprises WPM, 0.10 mg·L -1 TDZ, 0.05 mg·L - 1 IAA, 0.10 mg·L -1 AgNO3, 30.0 g·L -1 sucrose and 7.0 g·L -1 agar powder;

[0017] Step 4: screening culture and detection of the explants with shoot points;

[0018] The screening culture of the resistant plants is to transfer the explants with shoot points to the proliferation medium added with 200 mg·L -1 Cef and 30 mg·L -1 Kan, and the medium is replaced every 2 weeks, and the browning and dead explants are removed to obtain explants with shoot points;

[0019] The detection method is DNA extraction, PCR amplification and GUS staining: part of the leaves of the rooted seedlings are cut and labeled for DNA extraction on a clean bench, CTAB method is used for DNA extraction, NPTII and GUS gene specific primers are used for PCR amplification. The purpose of amplifying the band proves that the transgenic strain is stably integrated into the blackwood kernel genome, and GUS staining is used to further determine the detected strain to ensure the expression of GUS gene.

[0020] Beneficial effects:

[0021] The application provides a construction method of an Agrobacterium tumefaciens-mediated high-efficiency genetic transformation system of Acacia melanoxylon, and the method uses Acacia melanoxylon as a material, uses stem internodes of a proliferation tissue culture sprout seedling of a widely cultivated excellent clone SR3 of Acacia melanoxylon as a receptor, and develops an adventitious bud induction culture medium, and the regeneration system of the cluster adventitious buds induced from the stem segments is established by developing a formula of adding plant growth regulators, silver nitrate and the like, wherein the WPM medium added with TDZ has high inducibility to the cluster buds, and the addition of IAA and silver nitrate can greatly improve the regeneration rate of the Acacia melanoxylon adventitious buds, the highest induction rate of the adventitious buds reaches 75% by IAA, the induction regeneration rate of the adventitious buds reaches 85% by silver nitrate, and 30 mg / L of kanamycin is used as a screening pressure. -1 On the basis, under the mediation of the Agrobacterium tumefaciens, an exogenous gene is transferred into the Acacia melanoxylon, a transgenic strain is obtained through identification, it is confirmed that the T-DNA in the plasmid carried by the Agrobacterium tumefaciens has been inserted into the Acacia melanoxylon nuclear genome of the plant to be transformed, GUS staining detection of the transformed regeneration plant further proves that the transgenic strain has 35S promoter beta-galactosidase activity, and finally the stable transformation efficiency can reach 2.0%, and the genetic transformation system is successfully established.

[0022] The Agrobacterium tumefaciens-mediated high-efficiency genetic transformation system of the application solves the problem that there is no genetic transformation system for Acacia melanoxylon at present, and important gene function identification, genetic engineering improvement and gene editing technology cannot be applied, and provides a basis for carrying out important gene function identification and further improvement of Acacia melanoxylon. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a PCR detection and GUS staining diagram of the Acacia melanoxylon SR3 transgenic strain;

[0024] Figure 2 It is a fold line graph of the influence of the IAA concentration on the induction rate of the Acacia melanoxylon adventitious buds;

[0025] Figure 3 It is a fold line graph of the influence of AgNO3 on the induction rate of the Acacia melanoxylon adventitious buds;

[0026] Figure 4The process chart for the regeneration of Eucalyptus robusta adventitious buds;

[0027] Figure 5 The broken line chart for the influence of different concentrations of kanamycin on the induction rate of Eucalyptus robusta adventitious buds;

[0028] Figure 6 The broken line chart for the influence of different experimental conditions on the staining rate of GUS. DETAILED DESCRIPTION

[0029] The application will be described in further detail below with specific embodiments and drawings. It should be understood that these embodiments are only used to illustrate the application and are not used to limit the protection scope of the application. After reading the application, those skilled in the art can make various equivalent modifications of the application, which fall within the scope defined by the appended claims.

[0030] Unless otherwise specified, all raw materials and reagents of the application are conventional market raw materials and reagents.

[0031] Example 1

[0032] A method for constructing an Agrobacterium tumefaciens-mediated high-efficiency genetic transformation system of Eucalyptus robusta

[0033] Step 1: Take the stored Agrobacterium tumefaciens GV3101 competent cells (purchased from Shanghai Weidi Biology) from the-80℃ refrigerator, insert into ice, and when it is melted into ice water mixed state, add 1 μL of pBI121 plasmid (Professor Lu Mengzhu of Zhejiang Agricultural University) into 100 μL of competent cells, gently stir the bottom of the centrifugal tube with hands to mix, insert into ice for 5 min, then quickly freeze in liquid nitrogen for 5 min, take out from the liquid nitrogen, put into a 37℃ water bath for heat shock for 5 min, finally transfer to ice for ice bath for 5 min, and do not shake the centrifugal tube during the transfer process. Add 700 μL of LB liquid medium without antibiotics, mix well after blowing, and culture at 28℃, 200 rpm for 2 h. Centrifuge the recovered bacterial solution at 6000 rpm for 1 min, discard the supernatant, and mix about 100 μL of supernatant with a pipette gun. Take 50 μL of bacterial solution and spread it on LB solid medium containing 50 mg·L -1 Kan and 20 mg·L -1 Rif, and culture at 28℃ for 2 d.

[0034] Pick single colonies on LB solid medium containing 50 mg·L -1 Kan and 20 mg·L -1The LB liquid medium of Rif is cultured at 28℃ and 220rpm until the bacterial liquid becomes turbid, and the bacterial liquid of the transformed pBI121 plasmid is detected by PCR using the specific primers of NPTII and GUS genes for the transgenic plant PCR detection primers, wherein the transgenic plant PCR detection primers are shown in Table 1, the reaction system of PCR is shown in Table 2, the PCR reaction conditions of NPTII and GUS genes are shown in Table 3 and Table 4 respectively, and the bacterial liquid of the verification success is mixed with the glycerol preservation liquid in the proportion of 1:1, frozen by liquid nitrogen, and stored at-80℃. When used, activate, and store at-80℃. When used, activate, and store at-80℃. - 1 Rif and 50mg·L -1 Kan are performed on the plate, the activation of Agrobacterium is performed, a single colony is picked, and the LB liquid medium is transferred to the LB liquid medium, and cultured at 28℃ and 200rpm constant temperature shaker for 24h until the OD600 value of the bacterial liquid is 0.5, centrifuged at 4℃ for 10min, the supernatant is removed, and the same volume of resuspension liquid is added to resuspend the precipitated bacteria.

[0035] Table 1: Transgenic plant PCR detection primers

[0036]

[0037] The above sequences are SEQ ID NO.3-6, and the sequence of the GUS gene is shown in SEQ ID NO.1, and the sequence of the NPTII gene is shown in SEQ ID NO.2.

[0038] Table 2: PCR reaction system

[0039]

[0040] Table 3: NPTII gene PCR reaction conditions

[0041]

[0042] Table 4: GUS gene PCR reaction conditions

[0043]

[0044] Step 2: Prepare the material of the explant: the sterile propagation seedlings of the excellent clone of Acacia melanoxylon are cultured on the propagation medium for 30d, the light intensity of the plant is 300μmol·m -2 ·s -1 , the time is 16h, and the temperature of the culture room is 23℃;

[0045] The cut explant stem segments are immersed with Agrobacterium: take the blackwood eucalyptus seedlings with a height of more than 3 cm, remove the stem segments with axillary buds, cut 0.5 cm stem segments as explants, place the cut explant stem segments in resuspended Agrobacterium liquid for 20 min and shake gently 6 times, then take out the surface liquid, transfer to the co-culture medium with sterile filter paper on the surface and dark culture for 48 h;

[0046] The proliferation culture medium comprises: mMS medium, 0.3 mg·L -1 BAP, 0.2 mg·L -1 IBA, 0.2 mg·L - 1 IAA, 30 g·L -1 sucrose and 7.0 g·L -1 agar powder.

[0047] The co-culture medium comprises WPM, 0.10 mg·L -1 TDZ, 0.05 mg·L -1 IAA, 200.0 μM acetosyringone, 30.0 g·L -1 sucrose and 7.0 g·L -1 agar powder.

[0048] Step 3: screening culture of explants resistant buds: the stem segments after co-culture are transferred to the callus induction medium added with 200 mg·L -1 Cef and 30 mg·L -1 Kan, and the medium is replaced every 2 weeks, and the browned and dead explants are removed to obtain explants with bud points.

[0049] The callus induction medium has a pH of 5.8 and comprises WPM, 0.10 mg·L -1 TDZ, 0.05 mg·L - 1 IAA, 0.10 mg·L -1 AgNO3, 30.0 g·L -1 sucrose and 7.0 g·L -1 agar powder.

[0050] Step 4: screening of explants with bud points for resistant plants: the explants with bud points are transferred to the medium added with 200 mg·L -1 Cef and 30 mg·L -1Kan's proliferation medium for 20 days to the resistant bud elongation to 3-5 cm, and then detection, detection method for DNA extraction, PCR amplification and GUS staining: in the clean bench cut part of the leaf of the rooted seedlings and make a mark for DNA extraction, using CTAB method for DNA extraction, using NPTII and GUS gene specific primers for PCR amplification, PCR reaction system and conditions step 1, at the same time using GUS staining for further determination of the detected lines, to ensure the expression of GUS gene, the results as shown in Figure 1 , Figure 1 The lines amplified with the target band prove to be transgenic lines stably integrated into the blackwood genome, nptII (0.75k) and uidA (β-glucuronidase, GUS) (1.0k) genes have been successfully integrated into the nuclear genome of the transgenic lines, GUS staining of the transformed regeneration plants further proves that the transgenic lines have 35S promoter β-galactosidase activity, and the final stable transformation efficiency can reach 2.0%. A stable genetic transformation system of blackwood has been established.

[0051] Test example 1

[0052] Effect of different media on induction of blackwood SR3 adventitious buds

[0053] Select different TDZ concentrations (0.001, 0.005, 0.01, 0.05, 0.10 mg·L -1 ) and basic medium (WPM, MS) for testing. Each treatment has 12 plates of medium, and 20 explants are placed in each medium, a total of 240 explants for testing, and the statistics are made after 40 days of treatment. The test results are shown in Table 1.

[0054] Table 1 Effect of TDZ and basic medium on induction of blackwood adventitious buds

[0055]

[0056] As shown in Table 1, the effect of TDZ with a concentration of 0.05-0.1 mg·L -1 added to WPM medium is significant, and the best concentration of TDZ is 0.10 mg·L -1 , with the highest induction rate of adventitious buds of 28.36%, the callus induction rate of 100%, the callus being dense and white with green, and the formed buds being cluster buds.

[0057] Test example 2

[0058] Effect of IAA on adventitious bud regeneration

[0059] To further improve the induction rate of adventitious buds, different concentrations of IAA (0, 0.01, 0.05, 0.10, 0.20 mg·L -1 ) were added to the highest regeneration medium screened in the previous stage to induce callus and adventitious buds. At the same time, since it was observed that some adventitious buds were induced when the induced callus was transferred to the E. grandis proliferation medium, the callus induced for 40 days was transferred to the proliferation medium for 15 days, and the results are shown in Figure 2 . The results show that the highest induction rate of adventitious buds is 75.0% when 0.05 mg·L -1 IAA is added.

[0060] Test Example 3

[0061] Effect of AgNO3 on the regeneration of adventitious buds

[0062] To further improve the induction rate of adventitious buds, different concentrations of AgNO3 (0, 0.10, 1.0, 2.5, 5.0, 10.0 mg·L-1) were added to induce adventitious buds, and the results are shown in Figure 3 . The regeneration rate of adventitious buds reaches 85.0% when 0.10 mg·L-1 AgNO3 is added, which fully meets the further research on genetic transformation.

[0063] Test Example 4

[0064] Regeneration process of adventitious buds

[0065] Figure 4 A-E are pictures of stem segment explants, stem segment explants placed on adventitious bud induction medium for 40 days, callus transferred to proliferation medium for 1 day, adventitious buds transferred to proliferation medium for 25 days, and rooting induction for 25 days; after being cultured on the adventitious bud induction regeneration medium of the excellent clone SR3 of E. grandis for 40 days, white and dense callus was formed, and clear bud points and a small amount of adventitious buds were observed; after being transferred to the E. grandis proliferation medium for 15 days, a large amount of multiple shoots were formed, and the adventitious buds were elongated; the elongated adventitious buds were further transferred to the proliferation medium for propagation, and when the seedlings grew to more than 3 cm, the healthy bud seedlings were selected and transferred to the rooting medium for rooting induction to obtain complete plants.

[0066] Test Example 5

[0067] Determination of the screening concentration

[0068] To obtain suitable transgenic Acacia melanoxylon seedlings, it is necessary to use corresponding antibiotics for Acacia melanoxylon sensitivity test in order to obtain transgenic Acacia melanoxylon strains. The vector used to establish the system according to the present application is pBI121, which carries kanamycin resistance. Therefore, kanamycin is used for screening, and tests are carried out by using different concentrations of kanamycin (0, 10, 20, 30, 40, 50 mg·L -1 ) for testing, and the results are shown in Figure 5 , which shows that it is almost impossible to regenerate adventitious buds in the medium with 30 mg·L -1 kanamycin. The higher the concentration, the higher the mortality of adventitious buds. Therefore, 30 mg·L -1 kanamycin is selected as the screening pressure.

[0069] Test Example 6

[0070] Optimization of genetic transformation system

[0071] In order to obtain high-efficiency genetic transformation, the present application is based on the commonly used Agrobacterium strain GV3101 for dicotyledonous plants, and the transformation efficiency is optimized by optimizing the key pre-culture time (0, 3, 6, 9, 12 d) and co-culture time (48, 60, 72 h), and exogenous addition of different concentrations of acetosyringone (0, 10, 50, 100, 200 μM) and ultrasonic treatment time (0, 10, 20, 30, 40, 50, 60) in the co-culture medium; after co-culture of the explants, the explants are transferred to the selection medium for 7 days, and then subjected to GUS staining treatment, 3 repeats for each treatment, and at least 80 stem segment explants for each repeat; the GUS staining rate is calculated based on the staining of two segments of the stem, and the results are shown in Figure 6 .

[0072] As can be seen from Figure 6 , the pre-culture time significantly affects the GUS staining rate of Acacia melanoxylon stem segments, and the highest transformation efficiency (40.6%) can be obtained without pre-culture. At the same time, the co-culture time also significantly affects the transformation efficiency of Acacia melanoxylon, and the highest transformation efficiency is obtained after 48 h of co-culture, and with the increase of time, the transformation efficiency gradually decreases, which may be due to the excessive growth of the bacterial solution, which leads to the death of the cells at both ends of the stem, resulting in a decrease in the transformation efficiency. Short-term treatment of acetosyringone and ultrasonic wave can slightly improve the genetic transformation efficiency of Acacia melanoxylon, reaching 45.0% and 58.1%, respectively. After final analysis, it is determined that the genetic transformation of the excellent clone SR3 of Acacia melanoxylon is carried out without pre-culture, with 48 h of co-culture, 200 μM of acetosyringone added in the co-culture medium, and 20-30 s of ultrasonic treatment during the staining.

Claims

1. A method for constructing an Agrobacterium tumefaciens-mediated high efficiency genetic transformation system of Acacia melanoxylon, characterized by, The method comprises the following steps: Step 1: infecting Agrobacterium tumefaciens: picking single colony of activated Agrobacterium, transferring to LB liquid medium, culturing in constant temperature shaker for 16-24 hours until the OD600 value of the bacterial liquid is 0.5, centrifuging, removing supernatant, adding equal volume of sucrose resuspension of WPM+ to resuspend the precipitated bacteria; Step 2: dipping the cut explant stem in Agrobacterium: placing the cut explant stem in the resuspended Agrobacterium bacterial liquid for soaking and slight shaking, taking out the surface bacterial liquid, transferring to co-culture medium with sterile filter paper on the surface for dark culture for 48 hours; The co-culture medium comprises WPM, 0.10 mg·L -1 TDZ, 0.05 mg·L -1 IAA, 200.0 μM acetosyringone, 30.0 g·L -1 sucrose and 7.0 g·L -1 agar powder; Step 3: screening and culturing explant resistant buds: transferring the stem after co-culture to the callus induction medium added with Cef and Kan for screening and culturing, regularly replacing the medium, removing the browned and dead explants when replacing the medium, and obtaining explants with bud points; The PH of the callus induction medium is 5.8, containing WPM, 0.10 mg·L -1 TDZ, 0.05 mg·L -1 IAA, 0.10 mg·L -1 AgNO3, 30.0 g·L -1 Sucrose and 7.0 g·L -1 Agar powder; Step 4: Screening culture and detection of explants with bud points: explants with bud points were transferred to proliferation medium with 200 mg·L -1 Cef and 30 mg·L -1 Kan for 20 days for induction and elongation culture of adventitious buds until the resistant buds were elongated to 3-5 cm.

2. The method according to claim 1, wherein the method is characterized by, The Agrobacterium is GV3101 Agrobacterium strain carrying pBI121 plasmid with GUS gene.

3. The method for constructing an efficient genetic transformation system of *Agrobacterium tumefaciens* mediated by *Acacia confusa* according to claim 1, characterized in that, The activation of Agrobacterium described in step 1 was performed by incubating Agrobacterium in liquid medium containing 30 mg·L -1 Rifampicin and 50 mg·L -1 Kan on a plate with three streaks for activation.

4. The method according to claim 1, wherein the method is characterized by, The material for preparing explants in step 2 is: the aseptic propagation seedlings of the excellent clone of E. robusta are cultured on the propagation medium for 25-30 days, the light intensity for the plants is 100-300 μmol·m -2 ·s -1 , the time is 16 hours, and the temperature in the culture room is 23±2℃.

5. The method according to claim 4, wherein the method is characterized by, The proliferation medium comprises: mMS medium, 0.3 mg·L -1 BAP, 0.2 mg·L -1 IBA, 0.2 mg·L -1 IAA, 30 g·L -1 Sucrose and 7.0 g·L -1 Agar.

6. The method according to claim 1, wherein the method is characterized by: The cutting of the explant stem in step 2 is: taking blackwood acacia bud seedlings with a height of more than 3 cm, removing the stems with axillary buds, and cutting 0.5-1 cm stems as explants for standby.

7. The method for constructing an efficient genetic transformation system of *Agrobacterium tumefaciens* mediated by *Acacia confusa* according to claim 1, characterized in that, The detection method in step 4 is DNA extraction, PCR amplification and GUS staining.

Images