A genetic transformation method for hexaploid naked oats

Abstract

This application discloses a genetic transformation method for hexaploid naked oats, belonging to the field of plant genetic engineering technology. By optimizing the Agrobacterium-mediated oat genetic transformation system, the callus induction rate, shoot differentiation rate, and rooting rate of regenerated seedlings reached over 100%, 48.5%, and 80%, respectively. Furthermore, stable genetic transformation efficiency was obtained in hexaploid cultivated naked oat recipient materials with different genotypes, breaking the limitation that different genetic transformation systems need to be established for different genotype recipient materials.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to oat genetic transformation methods, and particularly to an optimized Agrobacterium-mediated genetic transformation method for hexaploid naked oats. Background Technology

[0002] With the advancement of the bio-breeding industry, establishing efficient genetic transformation systems for different crops is particularly important for obtaining transformants with excellent agronomic traits using transgenic, especially gene editing, technologies.

[0003] To date, research on identifying gene function in transgenic / gene-edited oats is almost nonexistent. The fundamental reason for this is the lack of a systematic and efficient oat genetic transformation system, which is the main limiting factor in identifying oat gene function. Existing reports indicate that researchers have successively conducted oat tissue culture studies using explants such as young spikelets (Fan Yinyan et al., 1996), young embryos (Zhao Yunyun et al., 2003), mature embryos (Cui Lin et al., 1998), inflorescence primordia (Jia Limin et al., 2009), ovaries (Liang Shouyi et al., 1984), and anthers (Wang et al., 2007). Transformed oat plants containing the Bar gene were obtained through gene gun transformation (Wan Shimei et al., 1998; Xu Chuanxiang et al., 2000), and GFP-marked transformant lines were obtained using Agrobacterium-mediated transformation (Zhang Yi et al., 2007; Wang Xunjing et al., 2012, 2015; Cho et al., 2003). However, in practice, the aforementioned studies have still not established an efficient and feasible oat genetic transformation system. Oats are monocotyledonous plants, and compared to dicotyledonous plants, Agrobacterium has a weaker ability to infect monocotyledonous plants, resulting in lower transformation rates, lower expression levels of the transformed exogenous genes, and genotype-dependent limitations. To improve the efficiency of Agrobacterium-mediated oat genetic transformation, this application used two hexaploid naked oat cultivars with different genotypes as recipient materials. Expression vectors carrying the target gene and marker protein were constructed using different vectors and promoters. By optimizing the culture medium formulation and key steps in the Agrobacterium-mediated genetic transformation system, transgenic lines with stable transformation efficiency were obtained. Summary of the Invention

[0004] All references cited herein are incorporated herein by reference. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise stated, the techniques used or mentioned herein are standard techniques known to one of ordinary skill in the art. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0005] This application provides an Agrobacterium-mediated genetic transformation method for oats, the method comprising:

[0006] S1. Place the peeled mature embryo shield face down into the callus induction medium and induce callus under dark culture conditions at 25°C.

[0007] S2, after immersing the induced callus tissue in the infection solution, place it on filter paper, absorb the excess bacterial solution, and then transfer it to a co-culture medium plate and co-culture at 28°C in the dark for 3 days.

[0008] S3, the co-cultured callus was transferred to differentiation medium and cultured under the conditions of 16h light / 8h dark culture, 4000LUX light intensity, and 25℃ until differentiated regenerated shoots appeared.

[0009] S4. Transfer the regenerated buds to the rooting medium for rooting culture to obtain regenerated seedlings;

[0010] The callus induction medium consisted of MS medium containing 30g maltose, 7g agar, 5.5mg / L 2,4-D, 0.8mg / L NAA, 0.2mg / L 6-BA, and 0.5g / L casein hydrolysate, with a pH of 5.6. On this medium, the callus induction rate reached 100%, and the induced callus tissue grew faster, exhibiting a pale yellow granular structure that facilitated the induction of new shoots.

[0011] Furthermore, before removing the mature embryo, the explants are disinfected. Specifically, mature oat seeds are disinfected with 75% alcohol and 10% sodium hypochlorite solution, and then rinsed with deionized water. After soaking the disinfected explants (oat seeds) in sterile distilled water for 10-15 hours, the mature embryo is removed by holding the seed with tweezers in one hand and using a scalpel in the other hand along the scutellum (including the scutellum).

[0012] Furthermore, the method for preparing the infection solution involves streaking Agrobacterium tumefaciens containing the expression vector, picking fresh single colonies grown on YEP plates and placing them into a YEP solution supplemented with 50 mg / L Kan and 50 mg / L rifampin, and incubating at 28°C and 200 rpm with shaking until the OD reaches 0.50. 600 Once the concentration reaches 0.8, centrifuge to collect the bacterial cells, wash the cells with Agrobacterium infection solution to remove residual YEP liquid culture medium, and then resuspend the cells in Agrobacterium infection solution containing 200 μM acetylsyl syringone. Shake the bacterial suspension at 140 rpm and 25℃ for 2-4 hours to obtain the infection solution.

[0013] Furthermore, the Agrobacterium infection solution is MS + 60g sucrose + 30g glucose + 5.5mg / L 2,4-D. Increasing the sucrose and glucose content in the Agrobacterium infection solution can increase the osmotic pressure of the infection solution, which is beneficial to improving the infection efficiency of Agrobacterium.

[0014] Furthermore, the infection process involves first immersing the callus tissue in 5 ml of Agrobacterium resuspension (without Agrobacterium) and then heat-shocking it in a 43°C water bath for 3 minutes, followed by the addition of OD. 600 = 0.6% Agrobacterium resuspension 20ml, vacuum for 1min, infect at 25℃ for 10min.

[0015] Furthermore, the co-culture medium consisted of MS medium containing 60 g maltose, 0.7 g / L proline, 0.125 g / L glutamine, 0.25 g / L cysteine, 5.5 mg / L 2,4-D, and 200 μM / L acetylsylgenone. The addition of proline and glutamine was beneficial in promoting the growth of embryogenic callus and reducing the impact of Agrobacterium infection on the differentiation ability of embryogenic callus.

[0016] Furthermore, the differentiation medium for inducing regenerated shoots was MS medium containing 30g maltose, 7g agar, 0.8mg / L NAA, 2.0mg / L 6-BA, 0.7g / L proline, and 0.125g / L glutamine, with a pH of 5.6. The positive rate of induced regenerated shoots reached a maximum of 48.5%.

[0017] Furthermore, the rooting medium is: MS medium + 30g maltose + 7g agar, pH=5.6. After shading the bottom of the culture flask from light, the rooting rate reaches over 80%.

[0018] Furthermore, the transformation expression vector used in this application is pCAMBIA- Pro PeNAC1 -P1:GFP-GUS and pBWA(V)BS-35S: AsMinpp 1, Agrobacterium is GV3101. More specifically, the promoter of the transformation expression vector used in this method is an inducible promoter. Pro PeNAC1 -P1 and the constitutive 35S promoter.

[0019] Furthermore, the method also includes GUS staining detection, in which a portion of the regenerated buds are selected and placed in GUS staining solution to detect whether the transformation is successful. Regenerated buds with a blue color reaction are considered positive buds that have been successfully transformed.

[0020] Furthermore, the method also includes obtaining transformed seedlings, transferring regenerated shoots to a rooting medium for rooting culture, taking leaves from regenerated seedlings that can root for PCR and GUS staining identification, and identifying those with correct PCR identification and blue color reaction as successfully transformed transgenic lines.

[0021] The beneficial technical effects of this application compared with the prior art are:

[0022] 1. This invention optimizes the Agrobacterium-mediated oat genetic transformation system, achieving callus induction rate, shoot differentiation rate, and rooting rate of regenerated seedlings exceeding 100%, 48.5%, and 80%, respectively. Furthermore, it achieves stable genetic transformation efficiency in hexaploid naked oat recipient materials with different genotypes. This system overcomes the limitation of requiring different genetic transformation systems for different genotype recipient materials.

[0023] 2. This invention detected and screened the expression of target genes / reporter genes driven by different promoters in transgenic plants. Promoter activity significantly affects the screening efficiency of positive plants; whether and how much the target gene is expressed depends on the promoter activity and properties. Existing oat genetic transformation systems have used the Nos promoter, rice actin 1 promoter, CaMV 35S promoter, and maize Ubi promoter to drive the expression of eukaryotic resistance genes / GFP / GUS proteins. Results showed that in oats, CaMV 35S or Ubi promoters also exhibited higher activity. In this invention, using the 35S promoter as a control, an inducible promoter isolated from Populus euphratica, possessing independent intellectual property rights, induced by hormones, drought, and high salt stress, was detected. Pro PeNAC1 -P1 drives the expression of the reporter gene (GUS) in transgenic oats. Detection showed that GUS was significantly expressed in floral organs such as young leaves, internodes of mature plants, and ovaries. The establishment of this genetic transformation system provides a novel plant-type promoter for obtaining oat transformants with breeding value. Attached Figure Description

[0024] Figure 1 The callus induced by mature oat embryos is shown in Figure A, where the callus induced by mature oat embryos of White Oat 2 is shown in Figure B, and the callus induced by White Oat 10 is shown in Figure B.

[0025] Figure 2 This is a diagram of the expression cassette structure of the expression plasmid used for oat transformation.

[0026] Figure 3 These are the results of experiments inducing shoot differentiation in hexaploid naked oats with different expression vectors and different genotypes. Figures A and B show the results of pBWA(V)BS-35S:AsMinpp 1 infecting white oat 2 and white oat 10, respectively; Figures C and D show the results of pCAMBIA- Pro PeNAC1 -P1:GFP-GUS was used to inoculate Baiyan 2 and Baiyan 10.

[0027] Figure 4 The positive rate of regenerated buds on the bud differentiation medium induced by Baiyan 10 was detected by GUS staining.

[0028] Figure 5It refers to the acquisition of transformed strains, where A is the regenerated bud in the rooting medium; and B is the transformed seedling transplanted into the soil after rooting.

[0029] Figure 6 These are the PCR identification results of the Baiyan 10 transformed strain.

[0030] Figure 7 The images show GUS staining to identify transformed lines and GUS expression sites. In the images, A represents GUS expression in differentiated buds; B represents GUS expression in young leaves; C represents GUS expression in floral organs; and D and E represent GUS expression in internodes. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0032] The oat varieties used in the following examples can be obtained from the "China Crop Germplasm Information Network" by obtaining relevant information and applying for the corresponding seeds.

[0033] Example 1: Screening of callus induction medium for mature oat embryos of different genotypes

[0034] Using two hexaploid cultivated naked oats, Baiyan 2 and Baiyan 10, as materials, plump and uniformly sized seeds (harvested within one year of maturity) were selected. After washing with distilled water, the seeds were first disinfected with 75% alcohol for 5 minutes in a clean bench, then rinsed three times with sterile distilled water. Next, they were disinfected with 10% sodium hypochlorite solution for 20 minutes, followed by five rinses with sterile distilled water until all residual disinfectant was removed. Finally, they were soaked in sterile distilled water for 10-15 hours (soaking facilitates embryo removal). Then, the mature embryo was removed along the scutellum (including the scutellum) using tweezers in one hand and a scalpel in the other.

[0035] The mature embryonic scutellaria, peeled off and placed face down, were placed in callus-inducing medium containing different hormone ratios (Table 2). After incubation at 25°C in the dark for 3-5 days, the emerging buds were cut off. Subculture was then performed every 7 days under the same 25°C dark incubation conditions, for a total of 3 subcultures, followed by subculture every 20 days. After 4-5 subcultures, pale yellow granular callus began to appear. The callus-inducing medium consisted of MS medium + 30g maltose + 7g agar + different hormone concentrations + 0.5g / L casein hydrolysate; pH = 5.6. The MS medium formulation is shown in Table 1 below.

[0036] Table 1 MS medium formulation

[0037] Trace elements mg / L μM <![CDATA[CoCI2·6H2O]]> 0.025 0.11 <![CDATA[CuSO4·5H20]]> 0.025 0.1 FeNaEDTA 36.7 100 <![CDATA[HBO3]]> 6.2 100.27 KI 0.83 5 <![CDATA[MnSO4·H2O]]> 16.9 100 <![CDATA[Na2MoO4·2H20]]> 0.25 1.03 <![CDATA[ZnSO4·7H20]]> 8.6 29.91 CaCl2 332.02 2.99 KH2PO4 170 1.25 KNO3 1990 18.79 MgSO4 180.54 1.5 NH4NO3 1650 20.61 Thiamine 0.1 0.3 Myo-inositol 100 554.94 Nicotinicacid 0.5 4.06 Pyridoxine HCl 0.5 2.43 Glycine 2 26.64

[0038] Table 2 Hormone Ratios in Callus Induction Culture Medium

[0039]

[0040] Experimental results showed that the callus induction rate of media No. 5 and No. 9 was 100%, higher than that of other media ratios; moreover, because the callus tissue grew faster in media No. 9, the callus diameter reached 5-6 mm after two weeks and 1-1.2 mm after four weeks, and the structure was light yellow, loose and granular, which was easy to differentiate and regenerate buds, media No. 9 was finally selected as the callus induction medium. Figure 1 AB). The formulation of medium No. 9 is MS + maltose 30g + agar 7g + 2,4-D 5.5mg / L + NAA 0.8mg / L + 6-BA 0.2mg / L + casein hydrolysate 0.5g / L; pH = 5.6.

[0041] This indicates that the hexaploid naked oat callus induction medium established in this invention has a more efficient induction rate compared to previously reported media.

[0042] Example 2: Construction of expression vector for oat transformation

[0043] The elements and structure of an expression vector have a significant impact on the transformation efficiency of plants. This invention uses pCAMBIA1304 and pBWA(V)BS (the aforementioned vectors are preserved in the laboratory of inventor Wang Junying) to construct a foreign gene expression vector. The full-length coding region of an oat multi-inositol-polyphosphate phosphatase gene, AsMinpp 1 (see Chinese Patent No.: 202211295369.0, whose nucleotide sequence is shown in SEQ ID NO: 1), is inserted downstream of the 35S promoter in the expression vector pBWA(V)BS. Figure 2 A). The PeNAC1 promoter (see Chinese Patent No.: CN200910158164.6) was truncated by 140 bp to obtain the sequence (P1) (as shown in SEQ ID NO: 2), which was then inserted into the pCAMBIA vector, replacing the 35S promoter upstream of the GUS gene in pCAMBIA. GUS is then expressed under the control of the PeNAC1-P1 promoter. Figure 2 B).

[0044] Example 3: Agrobacterium infection and co-culture

[0045] The constructed expression plasmid pCAMBIA- Pro PeNAC1-P1:GFP-GUS and pBWA(V)BS-35S:AsMinpp1s were introduced into Agrobacterium tumefaciens GV3101 using electroporation, targeting the promoter. Pro PeNAC1 After identification of the target genes -P1 and AsMinpp1 by bacterial PCR, the cells were streaked and fresh single colonies grown on YEP plates were picked and placed in YEP solution supplemented with 50 mg / L Kan and 50 mg / L rifampin. The cells were then incubated at 28°C with shaking at 200 rpm until OD500 reached the target value. 600 When the OD value reaches 0.8, centrifuge to collect the bacterial cells. Wash the cells with Agrobacterium infection solution to remove residual YEP liquid medium, and then resuspend the cells in Agrobacterium infection solution containing 200 μM acetylsuccinone. When the OD value of the bacterial suspension reaches 0.8, centrifuge to collect the bacterial cells. 600 At pH values ​​of 0.4, 0.6, and 0.8, the bacterial suspensions were shaken at 140 rpm and 25°C for 2-4 hours to obtain infection solutions. The Agrobacterium infection solution was: MS + 60 g sucrose + 30 g glucose + 5.5 mg / L 2,4-D, pH = 5.2. Increasing the sucrose and glucose content in the Agrobacterium infection solution can increase the osmotic pressure, which is beneficial for improving the infection efficiency of Agrobacterium.

[0046] Callus tissues from two well-grown naked oat varieties, Baiyan 2 and Baiyan 10, were immersed in infection solutions containing different expression vectors. The infection process involved four factors: infection solution concentration, infection time, water bath time, and vacuum time. Table 3 shows the statistical results of the effects of different combinations of these factors on the differentiation of callus tissue into positive regenerated shoots. After infection with different combinations, the callus tissues were placed on filter paper, excess bacterial solution was absorbed, and then transferred to co-culture plates at 28°C in the dark for 3 days. The co-culture medium formula, optimized by the inventors, was MS + 60g maltose + 0.7g / L proline + 0.125g / L glutamine + 0.25g / L cysteine ​​+ 5.5mg / L 2,4-D + 200μm / L acetylsylgenone, 9g agar, pH = 5.2. The addition of proline and glutamine is beneficial for improving the growth of embryogenic callus tissue and reducing the impact of Agrobacterium infection on the differentiation ability of embryogenic callus.

[0047] Example 4: Inducing Bud Regeneration

[0048] After co-culture, remove the plates from the incubator and select light yellow, fluffy callus tissue to transfer to differentiation medium. Under conditions of 16 hours of light / 8 hours of darkness, 4000 LUX light intensity, and 25°C, culture for about one week, green spots begin to appear on the callus tissue. Subculture once every two weeks until a large number of differentiated regenerated shoots are produced. Figure 3 Among them, the expression vector pCAMBIA- Pro PeNAC1-P1: GFP-GUS was used to infect 140 callus pieces of Baiyan No. 2 and 136 callus pieces of Baiyan No. 10, resulting in 61 and 66 differentiated buds respectively, with bud differentiation rates of 43.6% and 48.5%. Vector pBWA(V)BS-35S:AsMinpp 1 was used to infect 90 callus pieces of Baiyan No. 2 and 105 callus pieces of Baiyan No. 10, resulting in 35 and 45 differentiated buds respectively, with bud differentiation rates of 38.9% and 42.8%. The bud differentiation rate was calculated as the number of callus pieces with regenerated buds / the number of infected callus pieces.

[0049] The experimental results of this embodiment show that although the differentiation rates of the two vectors and two recipient materials differed on the same bud differentiation induction medium, there was no significant difference. This indicates that the Agrobacterium-infected bud-regenerating system optimized in this invention has a high bud differentiation rate in both naked oat varieties.

[0050] Whether Agrobacterium-mediated regeneration can differentiate into new shoots and the degree of differentiation are key aspects of establishing an Agrobacterium-mediated genetic transformation system. To improve the differentiation rate of new shoots after Agrobacterium infection, this invention optimized the Agrobacterium infection solution formulation, co-culture medium formulation, and shoot differentiation induction medium through a series of experiments. The optimized new shoot differentiation rate reached a maximum of 48.05%. According to the statistical results in Table 3, the optimal infection method is as follows: first, place the callus tissue in 5 ml of Agrobacterium resuspension (without Agrobacterium) and heat shock it in a 43°C water bath for 3 min, then add OD... 600 =0.6 Agrobacterium resuspension 20ml, vacuum for 1min, infect at 25℃ for 10min (Table 3). The culture medium for inducing shoot differentiation was: MS + maltose 30g + agar 7g + NAA 0.8mg / L + 6-BA 2.0mg / L + casein hydrolysate 0.5g / L, pH=5.6.

[0051] Example 5: GUS staining detection of induced bud regeneration

[0052] To determine the positive rate of regenerated shoots, some callus tissue that had differentiated into regenerated shoots was selected and placed in GUS staining solution. The tissue was then placed in a vacuum pump and stained for 14-18 hours in the dark. After destaining, the GUS staining was observed and photographed. The fresh GUS staining solution was prepared according to the kit instructions. The GUS staining results of the regenerated shoots showed that: of 77 samples of Baiyan 10, 37 were GUS positive, with a positive rate of 48.05%; of 82 samples of Baiyan 2, 31 were GUS positive, with a positive rate of 37.8%. Since only the expression vector pCAMBIA-Pro was available... PeNAC1 -P1:GFP-GUS carries the GUS selection marker, and the GUS staining rate, i.e., the positive rate of regenerated shoots, can reach up to 48.05% (Table 3). Figure 4 The positive rate of regenerated buds is calculated as GUS-staining positive buds / total number of infected regenerated buds.

[0053]

[0054] Example 6: Obtaining Transformed Lines

[0055] The regenerated shoots were transferred to a rooting medium for rooting culture. The rooting induction medium was MS medium + 30g maltose + 7g agar, pH = 5.6. When the transformed lines that could root normally developed 2-3 true leaves (i.e., 2 leaves and 1 bud) or 3 leaves and 1 bud, they were transferred to soil and hardened off in a greenhouse at 16h light / 8h dark, 22-25℃, and 60% humidity. The results showed that the rooting rate of the regenerated shoots from both naked oat varieties transformed using the two vectors was over 80%. Figure 5 A), the survival rate after transplanting was 100%. Figure 5 B).

[0056] Example 7: PCR detection and GUS staining identification of positive strains

[0057] DNA was extracted from leaves of regenerated seedlings of Baiyan 2 and Baiyan 10 transformed with the pBWA(V)BS expression vector without the marker protein. The 159bp sequence of the 35S promoter was detected by PCR using the following primers: forward: 5'-CTCTTACGACTCAATGACAAG-3' (SEQ ID NO:3), reverse: 5'-ATGGAATCCGAGGAGGTT-3' (SEQ ID NO:4). The results showed that amplification of the 35S promoter fragment using recipient material (WT) and Baiyan 10 transformants (OE1 to OE4) DNA as templates resulted in a 159bp band being amplified in both OE3 and OE4 transformants, indicating that the expression cassette in the expression vector was successfully integrated into the recipient genome. Figure 6 The positive results of the PCR-amplified 35S promoter sequence identification showed that the positive rates of Baiyan 2 and Baiyan 10 were 41.7% and 46.2%, respectively.

[0058] For expression vector pCAMBIA- Pro PeNAC1 -P1: Whole plants of Baiyan 2 and Baiyan 10, transformed with GFP-GUS, were subjected to GUS staining for identification. GUS staining results showed that Baiyan 2 had a positive rate of 46.7% with a blue reaction. Figure 7 D); Baiyan No. 10 has a 50% positive rate of blue reaction ( Figure 7 E), the positive rate is calculated as the GUS-stained positive strain coefficient / total transformant strain coefficient.

[0059] For identification Pro PeNAC1The expression of the target / marker gene driven by the -P1 promoter in transgenic lines was investigated, and the expression of GUS at various stages of the transgenic lines was examined. The results showed that GUS was expressed in regenerated buds (7A), young leaves (7B), and internodes (7C). Figure 7 It is expressed in DE and floral organs (pollen) (7C), especially in young tissues and flowers. Figure 7 AC).

[0060] Through the above embodiments, after optimizing the callus induction medium, Agrobacterium infection system, co-culture medium, bud differentiation induction medium, and rooting medium, the present invention established two hexaploid naked oat cultivation systems, Baiyan 2 and Baiyan 10, using two different types of promoters. The final transformation rates from callus infection to obtaining transgenic positive seedlings were 5.7%, 4.44%, and 6.6%, 5.7%, respectively (Table 4). The final transformation rate was calculated as the number of transgenic positive seedlings / the total number of infected callus pieces.

[0061] Table 4. Statistical data at different stages of the transformation of two hexaploid naked oats.

[0062]

Claims

1. A method for Agrobacterium-mediated genetic transformation of oats, characterized in that, The method includes: S1. Place the peeled mature embryo shield face down into the callus induction medium and induce callus under dark culture conditions at 25°C. S2, after immersing the induced callus tissue in the infection solution, place it on filter paper, absorb the excess bacterial solution, and then transfer it to a co-culture medium plate and co-culture at 28°C in the dark for 3 days. S3, the co-cultured callus was transferred to differentiation medium and cultured under the conditions of 16h light / 8h dark culture, 4000LUX light intensity, and 25℃ until differentiated regenerated shoots appeared. S4. Transfer the regenerated buds to the rooting medium for rooting culture to obtain regenerated seedlings; The callus induction medium consisted of MS medium containing 30 g maltose, 7 g agar, 5.5 mg / L 2,4-D, 0.8 mg / L NAA, 0.2 mg / L 6-BA, and 0.5 g / L casein hydrolysate. The infection process involved first placing the callus tissue in 5 ml of Agrobacterium-free resuspension, heat-shocking it at 43°C for 3 min, and then adding OD... 600 = 0.6% Agrobacterium resuspension 20ml, vacuum for 1min, infect at 25℃ for 10min, the expression vector in the infection solution contains Pro PeNAC1 -P1 promoter, Agrobacterium is GV3101.

2. The method according to claim 1, wherein the preparation method of the infection solution is to streak Agrobacterium tumefaciens containing the expression vector, pick fresh single colonies grown on YEP plates and place them in YEP solution containing 50 mg / L Kan and 50 mg / L rifampin, and culture at 28°C and 200 rpm with shaking until OD. 600 Once the concentration reaches 0.8, centrifuge to collect the bacterial cells, wash the cells with Agrobacterium infection solution to remove residual YEP liquid culture medium, and then resuspend the cells in Agrobacterium infection solution containing 200 μM acetylsyl syringone. Shake the bacterial suspension at 140 rpm and 25℃ for 2-4 hours to obtain the infection solution.

3. The method according to claim 2, wherein the Agrobacterium infection solution is MS + 60g sucrose + 30g glucose + 5.5mg / L 2,4-D.

4. The method according to claim 1, wherein the co-culture medium is MS + 60g maltose + 0.7g / L proline + 0.125g / L glutamine + 0.25g / L cysteine ​​+ 5.5mg / L 2,4-D + 200μm / L acetosyringone.

5. The method according to claim 1, wherein the differentiation medium for inducing regenerated shoots is MS + maltose 30g + agar 7g + NAA 0.8mg / L + 6-BA 2.0mg / L + proline 0.7g / L + glutamine 0.125g / L.

6. The method according to claim 1, wherein the rooting medium is MS + 30g maltose + 7g agar.

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