GhWOX9 Application of genes in improving the ability of cotton embryogenic callus formation

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to GhWOX9 Application of a gene in improving the formation ability of cotton embryogenic callus GhWOX9 It is found in the application that after overexpression of the gene in cotton, the formation ability of cotton embryogenic callus is significantly improved. GhWOX9 The application creates a cotton transformant with the transgene, and the cotton transformant has a high embryogenic callus occurrence rate and somatic embryogenesis efficiency.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and particularly relates to... GhWOX9 Application of genes in improving the embryogenic callus formation ability of cotton varieties. Background Technology

[0002] Cotton is an important economic crop and textile raw material, but its genetic transformation efficiency is limited by its callus formation ability. Difficulty in cotton somatic embryogenesis and genotype restriction severely constrain cotton genetic transformation and transgenic breeding. The discovery and utilization of plant regeneration-related genes is an important way to overcome genotype dependence and promote plant somatic embryogenesis and genetic transformation. In recent years, some plant regeneration-related genes, such as Somatic Embryogenesis Receptor Kinase (… SERK ), Leafy Cotyledon1 ( LEC ), Leafy Cotyledon2 ( LEC ), BABY BOOM BBM ) and WUSCHEL ( WUS Genes such as [list of genes] have been found to promote somatic embryogenesis in plants. The application of some of these genes can effectively improve plant regeneration efficiency during tissue culture, enabling more efficient genetic transformation of genotypes that are difficult to regenerate. WOX Transcription factors are key regulators of plant cell totipotency, playing crucial roles in somatic embryogenesis, maintaining the state of meristematic stem cells, and organ development. Previous studies identified 40 transcription factors in cotton. WOX Genes, in transcriptome sequencing analysis of the entire process of cotton somatic embryogenesis, were found to contain only about half of the genes. WOX Genes are actively expressed at different stages of cotton somatic embryogenesis. Figure 1 It is worth noting that, although WOX The gene showed a significant promoting effect on somatic embryogenesis in crops such as Arabidopsis and maize, but its effect was not obvious in cotton. Transcriptome findings revealed... GhWUS Gene( WOXThe modern evolutionary branch of the GhWUS gene family shows only weak or no expression throughout the entire process of cotton somatic embryogenesis. Current techniques (Skoog F, Miller CO (1957) Chemical regulation of growth and organformation in plant tissue cultured in vitro. Symp Soc Exp Biol 11: 118–130.; Wang, K., Shi, L., Liang, X. et al. (2022) The gene TaWOX5 overcomes genotype dependence in wheat genetic transformation. Nat. Plants 8, 110–117.) suggest that different species may have different somatic embryogenesis mechanisms. Whether the involvement of the cotton GhWUS gene is essential for cotton somatic embryogenesis, or something else, remains to be seen. WOX Members may have similar roles and play an important compensatory role in cotton somatic embryogenesis, which warrants rethinking and further research. Summary of the Invention

[0003] To address the difficulty in embryogenic callus formation in existing non-regenerating cotton varieties, this invention provides... GhWOX9 Application of genes in improving the embryogenic callus formation ability of non-regenerating cotton varieties.

[0004] To solve the above problems, the present invention specifically adopts the following technical solution:

[0005] This invention discovered through WGCNA analysis GhWOX9 The gene is a representative gene in a module highly associated with the embryogenic acquisition process, a key stage of somatic embryogenesis in upland cotton. Its expression is sharply upregulated during embryogenic acquisition and remains highly expressed throughout subsequent embryogenesis and development. However, in materials that are difficult to regenerate... GhWOX9 He barely spoke throughout. (Implication) GhWOX9 Genes may play a crucial role in cotton somatic embryogenesis. The cotton variety TM-1, with high homozygosity, is a standard model variety for cotton gene function research, but it is difficult to genetically transform due to its difficulty in achieving somatic embryogenesis. Inducing non-embryonic callus into embryogenic callus is a bottleneck step in somatic embryogenesis, and TM-1 struggles to induce embryogenic callus. WOX9 The gene was transferred into the non-regenerating TM-1 gene and overexpressed, and it was found that it significantly promoted the formation of embryogenic callus.

[0006] Based on the above, the present invention providesGhWOX9 Genes, containing GhWOX9 Applications of gene-based biomaterials in one or more of the following:

[0007] (1) Application in improving the ability of cotton embryogenic callus formation,

[0008] (2) Application in the cultivation of genetically modified cotton.

[0009] The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell. GhWOX9 The nucleotide sequence of the gene is shown in SEQ ID NO:7.

[0010] The plasmid vector can be a pCAMBIA series vector. The host cell can be Agrobacterium.

[0011] In a further embodiment of the invention, the cotton is the TM-1 variety / genotype. This cotton variety is a non-renewable variety. GhWOX9 Overexpression of the gene in the non-regenerating TM-1 increased the embryogenic callus formation rate, indicating... GhWOX9 The gene significantly promotes the formation of embryonic callus.

[0012] On the other hand, the present invention provides a method for improving the ability of cotton embryogenic callus to form, comprising: by improving... GhWOX9 This is achieved through the expression level of the gene's nucleic acid.

[0013] In a further approach, overexpression was performed in receptor cotton. GhWOX9 Genes were used to obtain transgenic cotton; the embryogenic callus formation rate of the transgenic cotton was higher than that of the recipient cotton.

[0014] A further step in the plan will include GhWOX9 Agrobacterium, a plant expression vector for the gene, was transfected into the hypocotyl of cotton, and transgenic positive lines were obtained by screening.

[0015] In a further embodiment, the aforementioned GhWOX9 The gene can be used for plant transformation via a recombinant expression vector containing the gene.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) In this invention GhWOX9 Gene overexpression vectors via fragment recombination significantly improved the efficiency of callus formation in cotton.

[0018] (2) The present invention GhWOX9 Genes can enable efficient induction of embryogenic callus in non-regenerating cotton varieties (such as TM-1).

[0019] (3) This invention creates cotton (such as TM-1) transformants through genetic transformation, which improves the ability of callus formation and partially solves the industry problem of low efficiency of cotton genetic transformation.

[0020] (4) This invention shortens the breeding cycle, reduces the number of repeated experiments due to efficient callus induction, and has a high callus formation rate and somatic embryogenesis rate. Attached Figure Description

[0021] Figure 1 for GhWOX Expression heatmap of gene families at different somatic embryogenesis stages;

[0022] Figure 2 In Example 1 GhWOX9 Relative expression levels of the gene at different somatic embryogenesis stages in upland cotton (variety JH713) using qPCR; the two smaller graphs on the left and right represent the expression levels of the same gene (…). GhWOX9 Different expression levels of genes in cotton genome groups D and A.

[0023] Figure 3 In Example 2, pCAMBIA2300FLAG- GhWOX9 Schematic diagram of the recombinant carrier structure;

[0024] Figure 4 This is an image of embryogenic callus tissue of cotton hypocotyl cultured on tissue culture medium in Example 2 at 45 days.

[0025] Figure 5 This is a statistical chart showing the embryogenic callus formation rate of the three strains in Example 2.

[0026] Figure 6 For example, the three in Example 2 GhWOX9 Image of positive seedlings from overexpression strains. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] Example 1

[0029] This embodiment discusses WOXGene expression patterns during somatic embryogenesis in cotton. We performed RNA sequencing analysis on the entire process of somatic embryogenesis in upland cotton: the process begins with the hypocotyl dedifferentiating into non-embryonic callus (NEC), which then sequentially transforms into embryonic callus (EC), and further differentiates into whole embryo (GE), torpedo-shaped embryo (TE), and cotyledonary embryo (CE), thus obtaining the gene expression profiles at each stage of this development. We selected two... WOX Genes (targeting cotton genome groups A and D) GhWOX9 Gene GH_A05G1589 , GH_D05G1617 The expression profile obtained from RNA sequencing was verified using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Details are as follows:

[0030] (1) Samples were taken from upland cotton (variety JH713) at different somatic embryogenesis stages (T1-T9). After sampling, the samples were immediately flash-frozen in liquid nitrogen and stored at -80℃ to avoid RNA degradation. T1-T4 represent non-embryonic callus (NEC), T5 represents the non-embryonic to embryogenic callus transformation stage (NEC-EC), T6 represents the callus stage (EC), T7 represents the globular embryo stage (GE), T8 represents the torpedo embryo stage (TE), and T9 represents the cotyledonary embryo stage (CE).

[0031] (2) RNA was extracted using TRIzol or the plant-specific CTAB-LiCl method.

[0032] (3) cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.).

[0033] (4) Using the cDNA synthesized in step (3) as a template, design a target for cotton genome groups A and D. GhWOX9 Gene qPCR primers were selected, and qPCR amplification was performed. GhHis3 The gene (GenBank accession number: AF024716) was used as an internal reference gene.

[0034] Targeting cotton genome group A GhWOX9 Gene( GH_A05G1589 qPCR primers:

[0035] rt-WOX9-1-f: 5'-GCTCCTCCTATCACTAATATCTCCA-3' (SEQ ID NO: 1);

[0036] rt-WOX9-1-r: 5'-GAGAACTGGAAACGTCAACGAC-3' (SEQ ID NO: 2);

[0037] Targeting cotton genome group D GhWOX9 Gene( GH_D05G1617 qPCR primers:

[0038] rt-WOX9-8-f: 5'-TGCCAAAAAGCCCCTTACAC-3' (SEQ ID NO: 3);

[0039] rt-WOX9-8-r: 5'-GGTTAACCATCCCGGAGTTGA-3' (SEQ ID NO: 4).

[0040] The 20 μL qPCR reaction system contained 1 μL cDNA template, 10 μL 2×TransStart Top Green qPCRSuperMix (Beijing TransGen Biotech Co., Ltd.), 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, and 8 μL double-distilled water. The qPCR reaction program was as follows: pre-incubation at 95°C for 5 minutes, followed by 40 cycles: denaturation at 95°C for 10 seconds, annealing at 58°C for 10 seconds, and extension at 72°C for 10 seconds.

[0041] (5) The relative expression level was calculated using the 2^(-ΔΔCt) method. The relative quantification analysis was performed using the online tool “A shiny for the analysis of real-time PCR data” (https: / / ihope.shinyapps.io / qRT-PCR-Pipeline / ).

[0042] The results are as follows Figure 2 As shown: GhWOX9 The gene expression level was significantly upregulated during the T6-T9 period, indicating that the gene plays a role in the callus formation stage and the later stage of embryonic development during cotton somatic embryogenesis.

[0043] Example 2

[0044] Embryogenic callus from upland cotton variety TM-1: Provided by the Cotton Research Institute, Chinese Academy of Agricultural Sciences, obtained through plant tissue culture from TM-1 seeds. pCAMBIA2300FLAG vector: Provided by the Cotton Research Institute, Chinese Academy of Agricultural Sciences, which inserts a 3×FLAG tag (the target gene is inserted before this tag) into pCAMBIA2300. Agrobacterium LBA4404: Commercially available product. Upland cotton variety TM-1 seeds: Provided by the Cotton Research Institute, Chinese Academy of Agricultural Sciences.

[0045] 1. Total RNA extraction

[0046] Embryogenic callus tissue from the upland cotton variety TM-1 was used as material, and total RNA was extracted using the TRIZOL method, as follows:

[0047] (1) Take 100 mg of sample and grind it rapidly in liquid nitrogen. Add the powder to a 2.0 mL centrifuge tube containing 1.5 mL TRIZOL and let it stand at room temperature for 5 min.

[0048] (2) Add 0.4 mL of chloroform, invert to mix, and let stand at room temperature for 5 min;

[0049] (3) Centrifuge at 12000 rpm for 15 min, and transfer the upper aqueous phase to a new tube;

[0050] (4) Add an equal volume of pre-cooled isopropanol, mix well, and let stand at room temperature for 10 min;

[0051] (5) Centrifuge at 12000 rpm for 15 min and discard the supernatant;

[0052] (6) Add 1 mL of 75% ethanol and centrifuge at 7500 rpm for 5 min;

[0053] (7) Natural drying on the ultra-quiet table;

[0054] (8) Add 50 μL of LNase water to dissolve the RNA and store it for later use.

[0055] 2. GhWOX9 Gene cloning

[0056] RNA was reverse transcribed into cDNA using HISCRIPT III ALL-IN-ONE RT SUPERMIX PERFECT FOR PCR (Nanjing Novizan Biotechnology Co., Ltd.). Specific primers were designed using the cDNA as a template.

[0057] Upstream primer: 5'-GG GGTACC CCATGTCTTCATCAAACAGACACTGGC-3' (SEQ ID NO:5);

[0058] Downstream primer: 5'-CG GGATCC CGGATGGAAAAGGGGCTTAATGAGCG-3' (SEQ ID NO:6). The bases indicated by the horizontal lines are enzyme cleavage sites.

[0059] Using 2 × Phanta Flash Master Mix (Dye Plus) (Nanjing Novizan Biotechnology Co., Ltd.), the amplification was performed by PCR. GhWOX9The CDS of the gene, 50μL PCR reaction system: 25.0μL 2×PhantaFlash Master Mix, 2.0μL 10μM upstream primer, 2.0μL 10μM downstream primer, 2.0μL cDNA template, and double-distilled water to make up the volume.

[0060] PCR reaction procedure: pre-incubate at 95℃ for 5 minutes, followed by 32 cycles: denaturation at 95℃ for 20 seconds, annealing at 58℃ for 10 seconds, extension at 72℃ for 45 seconds, then 72℃ for 5 minutes and 25℃ for 2 minutes.

[0061] The amplified PCR products were subjected to agarose gel electrophoresis (2% concentration).

[0062] The FastPure Gel DNA Extraction Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.) was used to extract DNA from the DNA of the sample. GhWOX9 The method for recovering the target gene fragment is as follows:

[0063] (1) After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under UV light. It is recommended to use a paper towel to absorb all the liquid on the surface of the gel and cut it into pieces, and remove as much excess gel as possible. Weigh the gel (excluding the weight of the empty tube). 100 mg of gel is equivalent to 100 μL volume, which is taken as one gel volume.

[0064] (2) Add an equal volume of Buffer GDP. Incubate in a water bath at 50-55°C for 7-10 minutes, adjusting the time according to the size of the gel to ensure complete dissolution of the gel block. Invert the container twice during the water bath to accelerate the dissolution process.

[0065] (3) Briefly centrifuge to collect droplets on the tube wall. Place the FastPure DNA Mini Columns-G adsorption column in a 2 ml Collection Tube, transfer less than 700 µL of sol solution to the adsorption column, and centrifuge at 12000 rpm (13400 × g) for 30-60 s. If the sol volume is greater than 700 µL, return the adsorption column to the collection tube, transfer the remaining sol solution to the adsorption column, and centrifuge at 12000 rpm (13400 × g) for 30-60 s.

[0066] (4) Discard the filtrate and place the adsorption column in the collection tube. Add 300 µL of Buffer GDP to the adsorption column. Let stand for 1 min. Centrifuge at 12000 rpm (13400 × g) for 30-60 s.

[0067] (5) Discard the filtrate and place the adsorption column in the collection tube. Add 700µL of Buffer GW (with anhydrous ethanol added) to the adsorption column. Centrifuge at 12000 rpm (13400 × g) for 30-60 s.

[0068] (6) Repeat step (5).

[0069] (7) Discard the filtrate and place the adsorption column back into the collection tube. Centrifuge at 12000 rpm (13400 × g) for 2 min.

[0070] (8) Place the adsorption column in a 1.5 mL sterile centrifuge tube, add 20-30 µL of Elution Buffer to the center of the adsorption column, and let stand for 2 min. Centrifuge at 12000 rpm (13400 × g) for 1 min. Discard the adsorption column and store the DNA at -20℃.

[0071] The obtained DNA sequence was sequenced to obtain GhWOX9 The CDS sequence of the gene is as follows (SEQ ID NO:7):

[0072]

[0073] 3.pCAMBIA2300FLAG- GhWOX9 Carrier construction

[0074] Will GhWOX9 The gene CDS sequence was ligated into the laboratory pCAMBIA2300FLAG vector, specifically using... Kpn I and BamH I. The vector was linearized by enzyme digestion with two enzymes, and the gel was recovered and sequenced correctly. GhWOX9 Gene CDS (target gene fragment) is also used Kpn I and BamH I. Perform enzyme digestion. 50 μL digestion system: 1.0 μg target gene fragment / vector, 5.0 μL 10× Buffer, 1.0 μL... BamH I Enzyme, 1.0 μL Kpn Add I Enzyme and double-distilled water to a final volume of 50 μL.

[0075] The enzyme was digested at 37°C for 4 hours, and then the digestion products were subjected to agarose gel electrophoresis and gel recovery.

[0076] Add the target gene fragment at a concentration 3 to 10 times that of the vector (based on agarose gel electrophoresis results, if the sample volume is the same and the brightness is consistent, the concentration ratio of the two fragments is inversely proportional to the fragment size). Prepare a 10 μL ligation system (the following system is prepared with the target fragment concentration being 3 times that of the vector): 6.0 μL target gene fragment, 2.0 μL digested vector, 1.0 μL 10×Buffer (containing ATP) and 1.0 μL T4 DNA Ligase (200 U / μL).

[0077] The above system was ligated overnight at 16 °C. The ligation solution was then transformed into competent cells of *E. coli* Fast T1 (Nanjing Novizan Biotechnology Co., Ltd.) for amplification, as follows:

[0078] (1) Melt competent cells Fast T1 on ice;

[0079] (2) Add 5 μL of the ligation product to competent cells, gently aspirate and mix, place on ice for 25 min, and use 0.5 μL of pCAMBIA2300FLAG plasmid as a positive control;

[0080] (3) Heat shock the mixture in a 42°C water bath for 90 seconds, then place it on ice for 2 minutes;

[0081] (4) Add 800 μL LB and mix well. Incubate at 37°C and 150 r / min for 1 h.

[0082] (5) Centrifuge at 4000 r / min for 1 min, discard 600 μL of supernatant, use a pipette tip to aspirate the bacterial solution, spread the remaining 200 μL of bacterial solution evenly onto Kana-resistant LB solid medium, and air dry;

[0083] (6) Seal with sealing film and incubate overnight in a 37°C incubator;

[0084] (7) Select multiple single colonies and perform PCR identification using the primers shown in SEQ ID NO:5 and SEQ ID NO:6. After purification, send the colonies for sequencing. The colonies that are correctly sequenced are pCAMBIA2300FLAG- GhWOX9 Recombinant vector, schematic diagram of recombinant vector as shown below Figure 3 As shown.

[0085] 4. Transformation of Agrobacterium with recombinant plasmids

[0086] The correctly sequenced pCAMBIA2300FLAG- GhWOX9 The vector was transformed into Agrobacterium LBA4404 using the heat shock method for cotton genetic transformation. The method is as follows:

[0087] (1) Take a tube of Agrobacterium LBA4404 competent cells and place it on ice to dissolve. Add 10 μL of the plasmid to be transformed into the competent cells, gently aspirate and mix, and then incubate on ice for 25 min.

[0088] (2) Place the centrifuge tube containing the mixture into liquid nitrogen for 5 minutes, then place it in a 37°C water bath for 5 minutes, and then place it on ice for 2 minutes.

[0089] (3) In a clean bench, add 800 μL of LB culture medium into a centrifuge tube and incubate in a shaker at 200 r / min and 28 ℃ for 4 h;

[0090] (4) The transformed Escherichia coli culture was inoculated onto LB medium containing Kana (50 mg / L) and Rif (25 mg / L) double resistance, dried, and incubated in an incubator at 28°C for two days;

[0091] (5) Identification of Agrobacterium-positive single colonies, obtaining Agrobacterium-positive single colonies;

[0092] (6) Inoculate a single Agrobacterium-positive colony into an Erlenmeyer flask containing 10 mL of liquid LB medium with Kana and Rif double resistance, and incubate at 28°C and 200 r / min for 2 days.

[0093] 5. Agrobacterium infection of cotton hypocotyl

[0094] (1) Delinting cotton seeds: Take several TM-1 cotton seeds, put them in a large beaker, add a small amount of concentrated sulfuric acid, and immediately stir quickly with a glass rod until all the cotton seeds in the beaker turn black. Then immediately rinse with tap water, rinsing repeatedly to thoroughly remove the sulfuric acid (rinse at least 25 times). After that, spread the cotton seeds out on newspaper to dry.

[0095] (2) Cotton seed germination: Place about 300 delinted cotton seeds into a 250mL Erlenmeyer flask, add 150mL of 3%H2O solution, and shake to incubate (28℃ 120rpm). Replace with fresh 3%H2O2 solution every 8-12 hours. Sowing can be carried out after germination.

[0096] (3) Culture medium: After germination, the seed coat is removed under sterile conditions and the seeds are sown in sterile seedling culture medium (MS medium), sealed, and labeled (content, date). After 4 days of dark culture at 28℃, the seeds are cultured under light for 1 day (16h light + 8h dark). After 5 days, the seedlings will grow to more than 10cm, at which point the hypocotyl can be used for Agrobacterium infection or callus induction.

[0097] (4) Transfection with Agrobacterium: The bacteria transformed with pCAMBIA2300FLAG- GhWOX9 Agrobacterium plasmid was inoculated into 5 mL of LB medium containing Kana and Rif antibiotics. After shaking at 28°C until the culture turned orange and then brownish-yellow, a small amount of the culture was diluted 1:100 and added to LB medium containing AS (acetosyringone, final concentration 200 μM), Kana, and Rif antibiotics. The culture was then shaken at 28°C for 1 hour and ready for infection. Under aseptic conditions, the Agrobacterium culture was used to infect hypocotyl segments for 10 minutes.

[0098] (5) Callus induction: After blotting off excess bacterial solution from the hypocotyl with paper, place the hypocotyl on a co-culture medium (MS + 1 mg / L IAA + 1 mg / L 2,4-D + 1 mg / L KT) with 5-6 layers of sterile filter paper, seal and label it, and co-culture at 28℃ for 48 h; after co-culturing for 48 h, transfer the hypocotyl explants to a callus induction medium (B29, MS + 1 mg / L IAA + 1 mg / L 2,4-D + 1 mg / L KT) resistant to dexamethasone (DEX, 30 μM) for 20-30 days in the dark, followed by 24 h / day light culture for 20 days to induce callus.

[0099] 6. Induction of embryonic callus

[0100] The induced callus tissue was transferred to callus subculture medium (DB29, MS + 0.1 mg / L IAA + 0.1 mg / L 2,4-D + 0.1 mg / L KT) supplemented with DEX (30 μM) for 20 days. Fresh, differentiated callus tissue was then transferred to hormone-free callus subculture medium (DB29) supplemented with DEX (30 μM) for a second subculture. Subculture was repeated every 20 days, with fresh, differentiated callus tissue selected for each subculture. After approximately 2-3 subcultures, embryogenic callus tissue gradually emerged spontaneously. This embryogenic callus tissue was then transferred to embryoid induction medium (MSC medium, brand: Coollabo) to induce embryoid formation. The induced embryoids then grew into complete plants, which were then self-pollinated to obtain T3 generation seeds. The regeneration capacity of the T3 generation seeds was then assessed. Specifically as follows:

[0101] After germination of T3 generation seeds, the seed coat was removed under aseptic conditions, and the seeds were sown in aseptic seedling culture medium (MS medium), sealed, and labeled (content and date). After 4 days of dark culture at 28℃, the seeds were cultured under light for 1 day (16h light + 8h dark). After 5 days, the seedlings grew to over 10cm, at which point callus induction was performed on the hypocotyl. Callus induction: Hypocotyls were cut into 1cm segments and placed on co-culture medium (MS + 1mg / L IAA + 1mg / L 2,4-D + 1mg / L KT), sealed and labeled, and co-cultured at 28℃ for 48h; callus induction medium (B29, MS + 1mg / L IAA + 1mg / L 2,4-D + 1mg / L KT) was cultured in the dark for 20-30 days, followed by 20 days of light culture (24h / day); the induced callus was then transferred to callus subculture medium (DB29, MS + 0.1mg / L IAA + 0.1mg / L 2,4-D + 0.1mg / L KT). After subculturing for 20 days in KT (Kypertrophic Tissue), fresh, differentiated callus tissue was transferred to hormone-free callus subculture medium (DB29) for further subculturing. Subculturing was repeated every 20 days, with fresh, differentiated callus tissue selected for each subculturing. After approximately 2-3 subculturings, embryogenic callus tissue gradually began to spontaneously develop. This embryogenic callus tissue was then transferred to embryoid induction medium (MSC medium) to induce embryoid formation. The embryogenic callus development rate (EDR) was calculated at 25, 45, and 65 days of tissue culture on the callus subculture medium.

[0102] See results Figure 4-5 Statistical analysis revealed that the embryogenic callus formation rate of three lines was significantly higher than that of the control (pvalue <0.01).Figure 4 This demonstrates the transformation of cotton hypocotyls cultured on callus subculture medium at 45 days of tissue culture. GhWOX9 The transformed strains of the gene showed embryogenic callus (within the box), while the control strain did not form embryogenic callus. Figure 5 The embryogenic callus formation rate of the three strains was significantly higher than that of the control.

[0103] For these three GhWOX9 The overexpression lines were tested, with plasmids, H2O, and empty vectors used as controls. The results are as follows: Figure 6 As shown, three GhWOX9 All overexpression lines were positive.

[0104] The above results indicate that GhWOX9 Gene overexpression vectors via fragment recombination significantly improved the efficiency of cotton embryogenic callus formation. GhWOX9 The gene enables efficient induction of embryogenic callus in the non-regenerating cotton variety TM-1. Furthermore, a heritable TM-1 transformant was created, improving the formation capacity of embryogenic callus and partially solving the industry problem of low genetic transformation efficiency in cotton. Simultaneously, it shortens the breeding cycle, reduces the number of repeated experiments due to efficient callus induction, and exhibits a high rate of embryogenic callus formation and somatic embryogenesis.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. GhWOX9 Genes, containing GhWOX9 The application of gene-based biomaterials in one or more of the following, characterized by: (1) Application in improving the ability of cotton embryogenic callus formation, (2) Application in the cultivation of genetically modified cotton; The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell. GhWOX9 The nucleotide sequence of the gene is shown in SEQ ID NO:

7.

2. The application according to claim 1, characterized in that, The plasmid vector is a plant expression vector.

3. The application according to claim 1, characterized in that, The plasmid vector is a pCAMBIA series vector.

4. The application according to claim 1, characterized in that, The host cell is Agrobacterium.

5. The application according to claim 1, characterized in that, The cotton mentioned is of the TM-1 variety / genotype.

6. A method for improving the ability of cotton embryogenic callus formation, characterized in that, include: By improving GhWOX9 This is achieved through the expression level of the gene's nucleic acid, the stated GhWOX9 The nucleotide sequence of the gene is shown in SEQ ID NO:

7.

7. The method according to claim 6, characterized in that, Overexpression in receptor cotton GhWOX9 Genes were used to obtain transgenic cotton; the embryogenic callus formation rate of the transgenic cotton was higher than that of the recipient cotton.

8. The method according to claim 6, characterized in that, Will include GhWOX9 Agrobacterium, a plant expression vector for the gene, was transfected into the hypocotyl of cotton, and transgenic positive lines were obtained by screening.

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