Non-tissue-culture-dependent pearl millet injection transformation method

Abstract

The invention discloses a non-tissue-culture-dependent pearl millet injection transformation method, and belongs to the technical field of plant genetic engineering. The method comprises the following steps: selecting germinated seeds of pearl foxtail millet with the primary bud length of 0.8-1.0 cm as receptors; preparing agrobacterium liquid, and adjusting the concentration to OD600 = 0.5; injecting the bacterial liquid into the embryo along the coleoptile direction of the seed until the bacterial liquid seeps from the end part of the coleoptile; co-culturing the transformed receptor for 24 hours under a dark condition after injection, and transplanting the transformed receptor into a seedling culture pot for cultivation; and finally, carrying out PCR (Polymerase Chain Reaction) screening. By optimizing key parameters such as the receptor development stage, the agrobacterium strain type and the bacterial liquid concentration, efficient genetic transformation of the pearl millet without tissue culture is achieved, and the T0 transformation positive rate reaches 63%. According to the invention, the transformation process is simplified, the transformation period is shortened, the cost is reduced, the transformation efficiency and stability are improved, and an effective technical support is provided for gene function research and molecular breeding of the pearl millet.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a non-tissue culture-dependent method for pearl millet injection transformation. Background Technology

[0002] Pearl millet ( Cenchrus americanus *Lysimachia morrhagica* (Morrone) is an important drought-resistant grain crop belonging to the Poaceae family, widely cultivated in Africa, South Asia, and arid and semi-arid regions. Due to its tolerance to poor soil, high temperatures, and strong drought resistance, *Lysimachia morrhagica* plays a vital role in ensuring food security in arid regions and in the development and utilization of feed and energy crops. With the deepening of molecular breeding and functional gene research, establishing a stable and efficient genetic transformation system for *Lysimachia morrhagica* has become an important technical foundation for advancing its gene function verification, molecular improvement, and stress-resistance breeding.

[0003] However, compared with model cereal crops such as rice and maize, pearl millet has obvious characteristics of being "difficult to tissue culture, difficult to regenerate, and difficult to transform." Existing pearl millet genetic transformation technologies mostly rely on tissue culture methods, such as embryogenic callus induction, regeneration culture, and selection propagation. These methods generally have the following problems: poor tissue culture response and low regeneration rate of pearl millet make it difficult to establish or stably replicate a complete transformation system; the transformation process is complex, including multiple steps such as explant treatment, callus induction, differentiation and regeneration, selection and transplanting, with a long cycle (often requiring several months) and high operational difficulty; due to the sensitivity of materials and culture conditions, low infection efficiency, and unstable regeneration process, the transformation efficiency is low and the reproducibility is poor; traditional methods rely on expensive equipment (such as gene guns) and a large amount of sterile culture consumables, resulting in high costs; the tissue culture system has high sterility requirements and a long operation cycle, making it extremely susceptible to bacterial or fungal contamination, resulting in material loss.

[0004] Currently, gene transformation methods for pearl millet mainly include tissue culture-dependent Agrobacterium-mediated transformation systems, gene gun / particle bombardment methods, and nanomaterial delivery methods. All of these methods suffer from the aforementioned problems to varying degrees. Although some studies have attempted to achieve exogenous gene transfer by directly injecting Agrobacterium-mediated transformation into meristematic tissues such as tubers and stem nodes, a systematic, reproducible, and stable process is still lacking in gramineous crops, especially pearl millet.

[0005] Therefore, developing a gene transformation method for pearl millet that does not require or depend on tissue culture is of great significance for improving the transformation efficiency of pearl millet, reducing the difficulty of operation, and promoting practical breeding applications. Summary of the Invention

[0006] The purpose of this invention is to provide a tissue culture-independent pearl millet injection transformation method to solve the problems of high dependence on tissue culture, cumbersome process, low efficiency, high cost and poor stability in the existing technology of pearl millet transformation.

[0007] To achieve the above objectives, the present invention provides a tissue culture-independent pearl millet injection transformation method, comprising the following steps: (1) Select pearl millet seeds with a primary bud length of 0.8–1.0 cm as transformation recipients; (2) Preparation of Agrobacterium tumefaciens bacterial culture containing the pCAMBIA1300 vector, wherein the pCAMBIA1300 vector contains a CaMV35S promoter-driven... eGFP Genes and the region located in T-DNA Hyg Resistance genes, and adjust the bacterial concentration to OD 600 =0.5; (3) Inject the Agrobacterium bacterial solution into the seed embryo along the direction of the seed coleoptile until the bacterial solution seeps out from the tip of the coleoptile; (4) Place the injected transformation recipient in a culture dish containing the Agrobacterium tumefaciens solution described in step (2) and culture it for 24 hours in the dark at room temperature. (5) After co-cultivation, transplant the seedlings into seedling pots for further cultivation; (6) Two to three weeks after transplanting, leaf samples were collected, genomic DNA was extracted, and marker genes were used as templates to extract the DNA. eGFP Genes and Hyg The genes were identified by PCR, and positive plants containing both of the above genes were obtained.

[0008] Further, the pearl millet germination seeds in step (1) are obtained by soaking the pearl millet seeds in 75% ethanol for 30 seconds, then disinfecting them with 4% sodium hypochlorite solution for 5 minutes, rinsing them with sterile water 4 to 6 times, and then culturing them for 48 hours under a photocycle of 28°C, 16 hours of light and 8 hours of darkness.

[0009] Furthermore, the Agrobacterium strain in step (2) is AGL1 or EHA105.

[0010] Further, in step (6), the eGFP The PCR amplification primers for the gene consist of an upstream primer having the nucleotide sequence shown in SEQ ID NO. 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO. 2; Hyg The PCR amplification primers for the gene consist of an upstream primer having the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer having the nucleotide sequence shown in SEQ ID NO.4.

[0011] The beneficial effects of this invention are as follows: (1) The present invention adopts the Agrobacterium injection transformation method that is not tissue culture dependent. By directly injecting the transformation gene vector into the meristematic region (growth point) of pearl millet seed embryo, the transformation operation no longer depends on the regeneration and culture of plant tissue, thus breaking through the limitations of tissue culture technology.

[0012] (2) This invention determined the optimal combination of technical parameters through systematic experiments: the optimal recipient material stage is a primary shoot length of 0.8–1.0 cm; strains AGL1 or EHA105 exhibited low toxicity in the live seed environment, with recipient survival rates reaching 48% and 41%, respectively; OD 600 = 0.5 bacterial concentration minimizes host damage while ensuring infection dose, and the synergistic effect of the three factors results in a T0 generation conversion positivity rate of 63%.

[0013] (3) This invention simplifies the transformation process by eliminating the complex operations and expensive culture media and equipment requirements of traditional tissue culture. No tissue culture is required during the transformation process, reducing reliance on sterile environments and differentiation media, thus significantly lowering costs. This reduced experimental cost makes large-scale application more feasible and economically efficient. Because this invention uses direct injection, the transformation cycle is significantly shortened compared to traditional tissue culture methods (T0 generation positive plants can be obtained in three weeks using this method; traditional tissue culture methods require approximately nine months or even longer). Traditional tissue culture methods typically require several months or even longer from tissue culture to plant regeneration, while the non-tissue culture injection transformation method of this invention can complete gene transformation in a shorter time, greatly improving the efficiency of research and application. Attached Figure Description

[0014] Figure 1 A flowchart of the overall process for the injection and transformation of pearl millet.

[0015] Figure 2 A standard schematic diagram of a healthy and complete pearl millet embryo.

[0016] Figure 3 The pCAMBIA1300 vector spectrum.

[0017] Figure 4 To investigate the effect of different primary shoot lengths on the positive conversion rate of injected receptors.

[0018] Figure 5 The effects of bacterial strain type and concentration on the survival rate and positivity rate of injected recipients.

[0019] Figure 6 A schematic diagram of the injection of pearl millet seed embryos.

[0020] Figure 7 For pearl millet seed embryo: primary bud.

[0021] Figure 8This is a PCR amplification diagram of marker genes in some positive plants; where 1-18 represent plants to be tested, and P represents pCAMBIA1300 (containing...). eGFP , Hyg The gene plasmid is a positive control, and "-" represents a wild-type plant as a negative control.

[0022] Figure 9 This is a fluorescence screening image of T0 generation positive strains.

[0023] Figure 10 Images of T0 generation positive plants and T1 generation positive plants. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0025] Example 1: Effect of different primary shoot lengths on the transformation of injected receptors To determine the optimal recipient material stage, 300 seed embryos each of *Pteris vittata* with initial bud lengths of 0.5 cm, 1.0 cm, and 1.5 cm were selected for Agrobacterium-mediated transformation. Specific procedures are as follows: Figure 1 As shown, the details are as follows: (1) Disinfection and germination of pearl millet seed embryos: Select healthy pearl millet seeds that are free of mold and of uniform size. Figure 2 Soak the seeds in 75% ethanol (Xilong, catalog number: 1280340101601, Jiuqu Technology) for 30 seconds, then disinfect them in 4% sodium hypochlorite solution (Maclean, catalog number: S828471, Jiuqu Technology) for 5 minutes. Rinse with sterile water 4-6 times and air dry thoroughly. Place the seeds on a petri dish moistened with double-layered filter paper, add water to almost cover the seeds, and incubate at 28℃ (16 hours light / 8 hours dark) for 48 hours to obtain germinated seeds with initial sprout lengths of 0.5 cm, 1.0 cm, and 1.5 cm, respectively. No additional treatment is required during germination; simply keep the seeds moist to meet germination conditions.

[0026] (2) Preparation of Agrobacterium tumefaciens culture: The culture contains Agrobacterium tumefaciens culture driven by the CaMV 35S promoter. eGFP Genes and the region located in T-DNA Hyg The pCAMBIA1300 plasmid vector for the resistance gene (the vector is deposited in the Grassland Research Center laboratory of the Institute of Tropical Crop Germplasm Resources, Chinese Academy of Tropical Agricultural Sciences; the vector and its construction method are derived from the academic paper "Adaptation Strategies and Molecular Mechanisms of Corolla Thermosensitive Movement in Yunnan Gentiana spp. for Alpine Breeding"; vector map as shown below) Figure 3 As shown; eGFP Genes and Hyg The resistance gene is present in the pCAMBIA1300 plasmid vector. eGFP Gene sequence: SEQ ID NO.5 Hyg Resistance gene sequence: SEQ ID NO.6) was transformed into Agrobacterium tumefaciens strain AGL1 (AGL1 competent cells, AC1020S, Shanghai Weidi Biotechnology Co., Ltd.), and then inoculated onto solid YEB medium and cultured at 28℃ for 48-72 hours. A single colony was picked and added to 5 mL of liquid YEB medium, and cultured at 28℃ and 200 rpm for 24 hours with shaking. Then, 100 μL of the culture was added to 100 mL of liquid YEB medium and cultured at 37℃ and 200 rpm until the desired concentration (OD) was reached. 600 (≈ 1.0), for future use. Each 1L of YEB medium (pH = 7.0±0.2) contains: 1 g yeast extract (OXOID, catalog number: LP0021B, Jiuqu Technology), 5 g peptone (OXOID, catalog number: LP0042B, Jiuqu Technology), 5 g beef extract (OXOID, catalog number: LP0029B, Jiuqu Technology), 5 g sucrose (Xilong Scientific, catalog number: 15700101, Jiuqu Technology), 0.24 g magnesium sulfate (Xilong Scientific, catalog number: 10500501, Jiuqu Technology), 15 g agar powder (White Shark, catalog number: BS195, Jiuqu Technology) (added when preparing solid medium), and water to 1L; the YEB medium contains 50 mg / L rifampicin (Rif; biosharp, BS296, Jiuqu Technology) and 100 mg / L kanamycin (Kan; biosharp, Item No.: BS152 (Jiuqu Technology).

[0027] Aliquot the cultured Agrobacterium tumefaciens into 50 mL centrifuge tubes, centrifuge at 4000 rpm for 10 minutes at 4℃, discard the supernatant, and resuspend the bacterial pellet in resuspending buffer until the OD value is reached. 600 = 0.5, for later use; the resuspension formulation is as follows: each 1 L of resuspension (pH= 5.7±0.1) contains: MS 4.42 g (Kulaibo, item number: PM1011, Beijing Kulaibo Technology Co., Ltd.), sucrose 30 g (Xilong Science, item number: 1570010101700, Jiuqu Technology).

[0028] (3) Injection transformation: Germinated seeds of different initial bud lengths were used as transformation recipients and dried on filter paper for 1 minute; 100 μL of Agrobacterium tumefaciens solution prepared in step (2) was drawn up with a 1 mL syringe and injected into the transformation recipients. The injection method of the bacterial solution was as follows: Figure 6): Place the primary bud and embryo of the transformation recipient upwards. The experimenter uses tweezers to fix the seed with one hand and injects Agrobacterium tumefaciens solution into the embryo along the direction of the coleoptile with the other hand until the solution seeps out from the tip of the coleoptile, thus obtaining the transformation recipient after injection. The transformation recipient after injection is then placed on filter paper to dry for 30 seconds.

[0029] (4) Co-cultivation and transplanting: After injection, the dried seeds are placed in a petri dish containing Agrobacterium tumefaciens solution and co-cultivated in the dark for 24 hours. Then, they are transplanted into seedling pots containing vermiculite and mixed substrate, thoroughly watered, and then cultivated at 28°C for 2-3 weeks.

[0030] The co-culture does not require additional culture medium; it simply refers to placing seeds that have been injected with Agrobacterium tumefaciens solution and then dried in a petri dish containing Agrobacterium tumefaciens solution and culturing them in the dark for 24 hours.

[0031] (5) Screening and identification of transformants: After 2-3 weeks of cultivation, leaf samples were collected, and genomic DNA was extracted using a modified TPS method: 0.1-0.2 g of fresh pearl millet leaves were placed in a 1.5 mL centrifuge tube, and 250 μL of TPS lysis buffer (Tris-HCl (100 mmol / L, pH 8.0; Merck, catalog number: V900312, Jiuqu Technology)), EDTA (10 mmol / L, pH 8.0; BioFroxx, catalog number: 1340GR500, Jiuqu Technology), KCl (1.0 mol / L), and deionized water were added to make up the difference. The mixture was thoroughly crushed and incubated at 75℃ for 20-30 minutes for high-temperature lysis. Afterward, the mixture was centrifuged at 12000 rpm for 10 minutes at room temperature, and the supernatant was collected in a new centrifuge tube. Then, an equal volume of isopropanol (Xilong Science, Product No.: 113249 (Jiuqu Technology) DNA was precipitated at -20℃ and washed twice with 1 mL of 70% ethanol, then dissolved in 30 μL of ddH2O to obtain genomic DNA.

[0032] Using the obtained DNA as a template, respectively... eGFP Gene (enhanced green fluorescent protein) and Hyg PCR detection was performed using specific primers (Table 1) for the gene (the gene encoding hygromycin in the T-DNA region of the pCAMBIA1300 plasmid). The PCR reaction system is shown in Table 2. The PCR reaction conditions were 95℃ for 5 min; 95℃ for 50 s, 52.5℃ for 1 min. eGFP ) / 57℃ 1min ( Hyg The PCR amplification products were detected by 1% agarose gel electrophoresis (BioFroxx, catalog number: 1110GR100, Jiuqu Technology). The amplification was performed at 72℃ for 1 min 30 s for 35 cycles, followed by 72℃ for 7 min.

[0033] Table 1 Primer Information

[0034] Table 2 PCR reaction system (10 µL system)

[0035] Experimental results are as follows Figure 4 As shown: the treatment group with a primary shoot length of 1.0 cm had the highest positive conversion rate (approximately 37%), significantly higher than the 0.5 cm group (approximately 24%) and the 1.5 cm group (approximately 16%). P < 0.05). Therefore, determining the initial shoot length as 0.8-1.0 cm as the optimal recipient material stage for this method can effectively improve the positive conversion rate of injection and enhance the stability and reproducibility of the method.

[0036] Example 2: Effects of different Agrobacterium strains on the survival rate of injected recipients To screen Agrobacterium strains suitable for the pearl millet injection transformation system, the following experiments were conducted: (1) Strains were selected: AGL1 (AGL1 competent cells, AC1020S, Shanghai Weidi Biotechnology Co., Ltd.), EHA105 (EHA105 competent cells, AC1010S, Shanghai Weidi Biotechnology Co., Ltd.) and GV3101 (GV3101 competent cells, AC1001S, Shanghai Weidi Biotechnology Co., Ltd.) were selected, each carrying the same pCAMBIA1300 expression vector (the vector was stored in the Grassland Research Center Laboratory of the Institute of Tropical Crop Germplasm Resources, Chinese Academy of Tropical Agricultural Sciences).

[0037] (2) Preparation of recipient materials: 300 pearl millet germinated embryos with a primary bud length of 0.8-1.0 cm were selected for each treatment group.

[0038] (3) Injection transformation: at the same bacterial concentration (OD) 600 = 0.5) and the treatment was carried out under the same injection conditions as in Example 1.

[0039] (4) Survival statistics: The survival rate of the injected recipients was statistically analyzed.

[0040] Experimental results are as follows Figure 5 As shown in A, the receptor survival rates in the AGL1 and EHA105 treatment groups were approximately 48% and 41%, respectively, with no significant difference between the two groups. P > 0.05), while the survival rate of the GV3101 treatment group was only about 20%, significantly lower than that of the AGL1 and EHA105 groups ( P< 0.05). Therefore, AGL1 or EHA105 are the preferred Agrobacterium strains for this method, which can effectively improve the survival rate of injected recipients and enhance the stability of the system.

[0041] Example 3: Effects of different bacterial suspension concentrations on receptor survival rate and positive conversion rate To optimize the bacterial concentration parameters in the Agrobacterium injection transformation system, the following experiments were conducted using Agrobacterium strain AGL1: (1) Setting the bacterial concentration: Set the OD 600 The bacterial culture gradients were 0.3, 0.5, 0.7, and 0.9.

[0042] (2) Preparation of recipient materials: 300 pearl millet germinated embryos with a primary bud length of 0.8-1.0 cm were selected for each treatment group.

[0043] (3) Injection conversion: Injection treatment was performed under the same conditions as in Example 1.

[0044] (4) Survival rate and positive transformation rate statistics: The survival rate of the injected recipients was statistically analyzed, and the positive transformation rate was statistically analyzed by PCR detection of the marker gene 2-3 weeks after transplanting (the operation of PCR detection of the marker gene is the same as in Example 1).

[0045] Experimental results show that: OD 600 = 0.3 and OD 600 The receptor survival rates in the 0.5 treatment group were approximately 54% and 47%, respectively, with no significant difference between the two groups. P > 0.05), all significantly higher than OD. 600 = 0.7 groups (approximately 37%) P < 0.05) and OD 600 = 0.9 groups (approximately 12%) P < 0.05) Figure 5 (B) in OD 600 The 0.5 treatment group had the highest conversion positivity rate (approximately 31%), significantly higher than the OD group. 600 = 0.3 groups (approximately 24%), OD 600 = 0.7 groups (approximately 15%) and OD 600 = 0.9 groups (approximately 6%) P < 0.05) Figure 5 (C in the middle).

[0046] Combining survival rate and conversion positivity rate results, OD 600 The 0.5 treatment group achieved the highest positive rate while maintaining a high receptor survival rate, thus confirming OD. 600 = 0.5 is the preferred concentration of the injection-transforming bacterial solution of the present invention.

[0047] Example 4: Verification of the complete conversion process Based on the above parameter optimization results, the complete conversion process was verified using optimal conditions. The overall operation process is as follows: Figure 1 As shown: Step 1: Preparation of Genetic Transformation Receptors (1) Disinfection of pearl millet embryo Select healthy, mold-free pearl millet seeds of uniform size. Figure 2 Rinse thoroughly with sterile water, then soak in 75% (v / v) ethanol (Xilong, product number: 1280340101601, Jiuqu Technology) for 30 seconds. After discarding the waste liquid, disinfect with 4% sodium hypochlorite solution (Maclean, product number: S828471, Jiuqu Technology) for 5 minutes. After discarding the waste liquid, rinse with sterile water 4-6 times until there is no disinfectant odor, and then air dry thoroughly.

[0048] (2) Germination of pearl millet embryo Place pearl millet seeds embryo-side up on a petri dish moistened with double-layered filter paper, add water until almost submerged, and incubate at 28℃ (16 hours light / 8 hours dark) for 48 hours to obtain germinated seeds. Germinated seeds with initial shoot lengths of 0.8–1.0 cm are used as transformation recipients. Figure 7 As shown.

[0049] Step 2: Preparation of Agrobacterium tumefaciens infection solution (1) Culture of Agrobacterium After transforming Agrobacterium tumefaciens strain AGL1 (AGL1 competent cells, AC1020S, Shanghai Weidi Biotechnology Co., Ltd.) with a vector containing the target marker gene, the cells were inoculated onto solid YEB medium and cultured at 28°C for 48–72 h. A single colony was picked and added to 5 mL of liquid YEB medium, and cultured at 28°C with shaking at 200 rpm for 24 hours. Then, 100 μL of the culture was added to 100 mL of liquid YEB medium and cultured at 37°C with shaking at 200 rpm until the desired concentration (OD) was reached. 600(≈ 1.0), for future use. Each 1L of YEB medium (pH = 7.0±0.2) contains: 1 g yeast extract (OXOID, catalog number: LP0021B, Jiuqu Technology), 5 g peptone (OXOID, catalog number: LP0042B, Jiuqu Technology), 5 g beef extract (OXOID, catalog number: LP0029B, Jiuqu Technology), 5 g sucrose (Xilong Scientific, catalog number: 15700101, Jiuqu Technology), 0.24 g magnesium sulfate (Xilong Scientific, catalog number: 10500501, Jiuqu Technology), 15 g agar powder (White Shark, catalog number: BS195, Jiuqu Technology) (added when preparing solid medium), and water to 1L; the YEB medium contains 50 mg / L rifampicin (Rif; biosharp, BS296, Jiuqu Technology) and 100 mg / L kanamycin (Kan; biosharp, Item No.: BS152 (Jiuqu Technology).

[0050] (2) Preparation of Agrobacterium resuspension Aliquot the cultured Agrobacterium tumefaciens into 50 mL centrifuge tubes (45 mL per tube), centrifuge at 4000 rpm for 10 min at 4℃, discard the supernatant, and resuspend the bacterial precipitate in resuspending buffer until the OD value is reached. 600 = 0.5, to be used for future reference; the resuspension formula is: each 1 L of resuspension (pH = 5.7±0.1) contains: MS 4.42 g, sucrose 30 g.

[0051] Step 3: Injecting germinated embryos with Agrobacterium for transformation (1) Germinated seeds with initial bud length reaching 0.8–1.0 cm were used as transformation recipients; (2) Place the conversion acceptor on filter paper and let it air dry for 1 minute; (3) Using a 1 mL syringe, draw 100 μL of Agrobacterium bacterial solution and inject it into the transformation recipient. The injection method is as follows: place the primary bud and embryo of the transformation recipient upwards, use tweezers to fix the seed with one hand, and use the other hand to inject Agrobacterium bacterial solution into the embryo along the direction of the coleoptile until the bacterial solution seeps out from the end of the coleoptile. Figure 6 As shown; (4) Place the transformed receptor after the bacterial solution is injected back onto filter paper and let it air dry for 30 seconds.

[0052] Step 4: Transformation and Recipient Co-culture and Transplantation After injection and drying, the transformation recipients were placed in a petri dish containing Agrobacterium tumefaciens solution and cultured in the dark for 24 hours. Then, they were transferred to seedling trays containing vermiculite and mixed substrate, thoroughly watered, and cultured at 28°C for another 2-3 weeks.

[0053] Step 5: Screening and Identification of Transformants (1) PCR detection was performed on the transformed contemporary (T0) seedlings obtained in step four. When the plants grew to the 4-leaf-1-heart stage, 2-3 leaves were collected from each plant, and leaf segments of about 2 cm were cut from each leaf and mixed for sampling.

[0054] (2) Genomic DNA was extracted from each transformed plant using the TPS rapid extraction method. Using the obtained DNA as a template, the DNA was then processed using… eGFP Gene (enhanced green fluorescent protein) and Hyg Specific primers (Table 1) and corresponding PCR systems (Table 2) were used to amplify the gene (the gene encoding hygromycin in the T-DNA region of the pCAMBIA1300 plasmid). Positive plants (those simultaneously containing...) were screened based on the PCR results. eGFP Genes and Hyg Gene, Figure 8 PCR-positive plants were then transplanted to the field to continue growing, resulting in T0 generation positive plants.

[0055] The ears of T0 generation positive plants were bagged and self-pollinated to obtain T1 generation seeds. Twenty T1 generation seeds were randomly selected from each main ear and germinated under indoor conditions. After the seedlings developed their fourth leaf, leaf samples were taken for genomic DNA extraction, and the process was carried out as described above. eGFP Genes and Hyg Gene PCR detection method is used to amplify and identify the target gene in the sample, thereby screening and obtaining T1 generation transformed plants.

[0056] This step involved PCR detection of the transformed T0 generation seedlings. Leaf samples were collected from 600 plants for testing, and 378 positive plants were ultimately selected, with a transformation rate of 63% for the T0 generation (Table 3). Ten T0 generation positive plants were randomly selected, and 20 seeds were randomly selected from each plant, for a total of 200 seeds (T1 generation). Germination and identification were performed, with a germination rate of approximately 70%. Seventy seedlings were randomly selected for identification, resulting in 48 positive T1 generation plants, with a positive rate of 68% for the T1 generation (Table 3). Compared with the wild-type control, leaves treated with Agrobacterium showed obvious GFP fluorescence, indicating that the exogenous plasmid (… eGFP The bacteria were successfully expressed in the leaves, and positive results were obtained by injecting Agrobacterium-mediated infection. Figure 9 Both the T0 and T1 generations of positive transformants showed normal heading and grain-filling abilities, and the T1 generation could still produce a certain amount of mature grains, indicating that the transformed material could stably inherit and form offspring seeds. Figure 10 This result verifies the advantages of the tissue culture-independent pearl millet injection transformation method of the present invention in improving gene transformation efficiency and stability.

[0057] Table 3. PCR detection results of T0 and T1 generation transgenic plants under optimal injection conditions.

[0058] This invention achieves efficient genetic transformation of *Polygonum chinense* without tissue culture by optimizing key parameters such as the developmental stage of the recipient material, the type of *Agrobacterium* strain, and the concentration of the bacterial culture. Compared with existing technologies, this invention has advantages such as simple process, short cycle, high efficiency, low cost, and good stability, providing effective technical support for gene function research and molecular breeding of *Polygonum chinense*.

Claims

1. A tissue culture-independent pearl millet injection transformation method, characterized in that, Includes the following steps: (1) Select pearl millet seeds with a primary bud length of 0.8–1.0 cm as transformation recipients; (2) Preparation of Agrobacterium tumefaciens bacterial culture containing the pCAMBIA1300 vector, wherein the pCAMBIA1300 vector contains a CaMV 35S promoter-driven... eGFP Genes and the region located in T-DNA Hyg Resistance genes, and adjust the bacterial concentration to OD 600 = 0.5; (3) Inject the Agrobacterium bacterial solution into the seed embryo along the direction of the seed coleoptile until the bacterial solution seeps out from the tip of the coleoptile; (4) Place the injected transformation recipient in a culture dish containing the Agrobacterium tumefaciens solution described in step (2) and culture it in the dark for 24 hours. (5) After co-cultivation, transplant the seedlings into seedling pots for further cultivation; (6) Two to three weeks after transplanting, leaf samples were collected, genomic DNA was extracted, and marker genes were used as templates to extract the DNA. eGFP Genes and Hyg The genes were identified by PCR, and positive plants containing both of the above genes were obtained.

2. The non-tissue culture-dependent pearl millet injection transformation method according to claim 1, characterized in that, In step (1), the germinated seeds of pearl millet are obtained by soaking the pearl millet seeds in 75% ethanol for 30 seconds, then disinfecting them with 4% sodium hypochlorite solution for 5 minutes, rinsing them with sterile water 4 to 6 times, and then culturing them for 48 hours under a photocycle of 28°C, 16 hours of light and 8 hours of darkness.

3. The non-tissue culture-dependent pearl millet injection transformation method according to claim 1, characterized in that, In step (2), the Agrobacterium strain is AGL1 or EHA105.

4. The non-tissue culture-dependent pearl millet injection transformation method according to claim 1, characterized in that, In step (6), the eGFP The PCR amplification primers for the gene consist of an upstream primer having the nucleotide sequence shown in SEQ ID NO. 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO. 2; Hyg The PCR amplification primers for the gene consist of an upstream primer having the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer having the nucleotide sequence shown in SEQ ID NO.4.

Images