Efficient genetic transformation method based on RUBY reporter gene and agrobacterium rhizogenes mediation

Abstract

The invention discloses a rubus cochinchinensis efficient genetic transformation method based on RUBY reporter gene and agrobacterium rhizogenes mediation, which realizes that the transformation period is shortened to be within 15 days and the transformation efficiency is improved to be more than 60% through direct explant treatment under a non-tissue culture condition. The method comprises the following specific steps: 1) preparing an explant material; (2) constructing an RUBY carrier and transforming agrobacterium rhizogenes; (3) infecting the explant by utilizing agrobacterium rhizogenes carrying a target gene; (4) co-culturing after infection and carrying out RUBY visual screening; 5) positive root molecular biology verification; (6) inducing positive roots into buds; according to the method, induction of the positive callus, the positive root and the positive bud of the rubus coreanus stem segment under the non-tissue culture condition is successfully achieved, a stable and efficient genetic transformation system is constructed, the method has the remarkable advantages of being easy and convenient to operate, low in cost, wide in applicability, high in reproducibility and the like, and a key technical support is provided for molecular breeding of the rubus coreanus.

Description

Technical Field

[0001] This invention belongs to the field of plant bioengineering, specifically relating to a highly efficient genetic transformation method for *Rhizopus rubra* mediated by the RUBY reporter gene and *Agrobacterium rhizogenes*. Background Technology

[0002] Wild raspberry (Rubus niveus Thunb.) is a small berry with a unique flavor and high nutritional, health, and economic value. It is rich in vitamins, pectin, and superoxide dismutase (SOD), as well as minerals, soluble fiber, and the anti-cancer substance ellagic acid. Widely loved in Europe and America, it has great development potential and is hailed as a third-generation small berry due to its unique and extremely high nutritional value. It has also been listed as one of the healthy small berries by the Food and Agriculture Organization of the United Nations. Currently, there are few reports of successful transgenic raspberry cultivation in China, and related genetic transformation mainly relies on Agrobacterium tumefaciens-mediated tissue culture regeneration systems. However, traditional Agrobacterium-mediated transformation methods are severely limited by genotype dependence, resulting in low transformation efficiency and requiring complex and time-consuming tissue culture processes. Especially for wild raspberry germplasm resources, due to genotype and other factors, it is difficult to establish a stable regeneration system, making it impossible to utilize its key genetic resources such as aroma, high yield, and stress resistance. Furthermore, existing raspberry transformation systems suffer from drawbacks such as delayed identification cycles, low efficiency (transformation frequency below 30% within 30-45 days), and long processing times. Therefore, establishing a non-tissue culture genetic transformation system can significantly improve genetic transformation efficiency and accelerate the regeneration cycle, which is crucial for advancing biological research, genetic breeding, and industrial development of raspberries and their wild relatives.

[0003] Agrobacterium rhizogenes, another highly efficient Agrobacterium species, can directly transform non-tissue cultured explants, establishing stable and efficient transformation systems in multiple species such as soybean and citrus. Compared to traditional methods, the Agrobacterium rhizogenes transformation system completes the entire transformation process in only 15-16 days, with a transformation frequency of 58-96%, significantly higher than traditional methods. Agrobacterium rhizogenes produces hairy roots with characteristics such as rapid growth, high differentiation degree, physiological, biochemical, and genetic stability, and simple operation control. Furthermore, non-tissue cultured explants can be directly transformed without aseptic treatment, greatly saving manpower and material costs.

[0004] Betaine is a type of plant natural product derived from the amino acid tyrosine. The bright red color of plants such as sugar beets, dragon fruit, and Swiss chard is the result of betaine accumulation. RUBY is a synthetic reporter gene that encodes three key enzymes in the betaine biosynthesis pathway. It converts tyrosine into bright red betaine pigment, which can be observed directly with the naked eye without special equipment or expensive reagents, offering significant advantages over traditional reporter genes such as β-glucuronidase (GUS) and luciferase.

[0005] Therefore, this invention, based on Agrobacterium rhizogenes and utilizing the RUBY reporter gene, selects non-tissue culture explants as transformation materials and systematically explores key factors such as different explant types, Agrobacterium strains, infection concentrations, and time. Ultimately, a genetic transformation system for *Rhizophora stylosa* was established, which not only obtained positive callus and positive roots but also positive buds induced by positive roots. This provides feasible technical support for domestic wild raspberry research in areas such as functional gene verification, genetic trait improvement, and new germplasm creation. Summary of the Invention

[0006] The purpose of this invention is to induce positive callus, positive roots, and positive buds in *Ardisia crenata*, and to provide a highly efficient genetic transformation method for *Ardisia crenata* based on the RUBY reporter gene and *Agrobacterium rhizogenes*. This method is a genetic transformation method for positive roots of *Ardisia crenata* mediated by *Agrobacterium rhizogenes* under non-tissue culture conditions, used for gene function verification studies. This system overcomes the problems of genetic transformation of *Ardisia crenata* under non-tissue culture conditions and can be used to verify the function of the visual reporter gene. This method is the first to combine the RUBY visual reporter gene with the *Agrobacterium rhizogenes* transformation system in the genetic transformation of *Ardisia crenata*, breaking through the technical bottlenecks of traditional transformation techniques, such as heavy reliance on tissue culture, strong genotype restriction, long transformation cycle (30-45 days), and low efficiency (<30%). Through direct explant treatment under non-tissue culture conditions, the transformation cycle is shortened to within 15 days, and the transformation efficiency is increased to over 60%. This invention successfully induced positive callus, positive roots and positive buds from stem segments of *Rhizophora stylosa* under non-tissue culture conditions, and constructed a stable and efficient genetic transformation system. It has significant advantages such as simple operation, low cost, wide applicability and strong reproducibility, and provides key technical support for molecular breeding of *Rhizophora stylosa*.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] A highly efficient genetic transformation method for *Rhizoctonia solani* based on the RUBY reporter gene and *Agrobacterium rhizogenes*-mediated transformation, which eliminates the need for tissue culture regeneration and achieves a transformation cycle of ≤15 days for positive roots, includes the following steps:

[0009] 1) Explant preparation: Stem segments of *Rhizophora stylosa* from the field were selected as explant materials;

[0010] 2) Vector construction: Construct a recombinant expression vector containing the target gene and the RUBY reporter gene, and transform it into Agrobacterium rhizogenes to obtain an engineered strain;

[0011] The RUBY reporter gene is a fusion gene containing three key enzyme genes for betaine synthesis: CYP76AD1, DODA, and glycosyltransferase, with each gene linked by a 2A self-cleaving peptide sequence; the Agrobacterium rhizogenes strains are K599, R1000, and C58C1; the recombinant expression vector is a binary vector containing T-DNA left and right boundary sequences, a replication origin, an antibiotic resistance marker, and a multiple cloning site;

[0012] 3) Infection treatment: Infect the cut site of the explant stem segment with the bacterial solution of the engineered strain;

[0013] 4) Co-culture: The infected explants were placed on moist vermiculite for co-culture;

[0014] 5) Visual screening: Identify positive hairy roots by observing the red betaine pigment produced by the RUBY reporter gene;

[0015] 6) Molecular verification: PCR and RT-qPCR were performed on the screened positive hairy roots to confirm the integration and expression of the target gene;

[0016] 7) Bud regeneration: Transfer the verified positive hairy roots to moist vermiculite and spray with gibberellin to induce the differentiation of positive buds;

[0017] 8) Verification of positive buds: Molecular biological tests are performed on the regenerated positive buds to confirm the stable inheritance of transgenes.

[0018] Furthermore, the stem segments selected are double-bud internode segments with a diameter of 0.6-0.8 cm and a length of 15 cm, with each stem segment retaining one leaf.

[0019] Furthermore, the conditions for the infection treatment are: bacterial suspension OD... 600 The value is 0.4-1.2, 100-200mM acetylsuccinone is added, the infection time is 10-20 minutes, and the infection temperature is 25-28℃; the co-culture conditions are: in a culture box containing moist vermiculite, dark culture at 25±2℃ for 3 days; the visual screening is carried out by visual observation, the hairy roots of positive transformation show obvious red or pink color, and the screening is carried out 7-14 days after infection.

[0020] Furthermore, in the molecular validation, PCR detection uses target gene-specific primers and RUBY gene-specific primers, and RT-qPCR uses an internal reference gene to standardize and analyze gene expression levels.

[0021] Furthermore, in the bud regeneration process, a 1:800 solution of carbendazim was sprayed, and a 2000 mg / L gibberellin solution was sprayed once a week. The culture conditions were 25±2℃, light intensity of 2000 LUX, and a photoperiod of 14h light / 10h darkness.

[0022] A second aspect of the present invention provides a transgenic *Rhododendron simsii* plant obtained by the above method, wherein the genome of the plant integrates an exogenous target gene and a RUBY reporter gene, and the root, stem or leaf tissues of the plant are red or pink, indicating that the RUBY reporter gene is continuously and stably expressed.

[0023] A third aspect of the present invention provides a recombinant expression vector for genetic transformation of *Rhizophora stylosa*, the vector containing a RUBY reporter gene and a cloning site, wherein the RUBY reporter gene comprises, in sequence, a CYP76AD1 gene, a first 2A peptide sequence, a DODA gene, a second 2A peptide sequence, and a glycosyltransferase gene.

[0024] Furthermore, the vector also includes a 35S promoter, a terminator sequence, T-DNA left and right boundary sequences, and a bacterial resistance selection marker; the 2A peptide sequence is selected from one or more of P2A, T2A, E2A, or F2A.

[0025] A fourth aspect of the present invention provides a recombinant Agrobacterium rhizogenes engineered strain containing the above-described vector.

[0026] The fifth aspect of this invention provides an application of the above-described method in the verification of gene function in *Rhizoma Scutellariae*.

[0027] The sixth aspect of this invention provides an application of the above-described method in molecular breeding of *Rhizophora stylosa*.

[0028] The seventh aspect of this invention provides an application of the above-described method in gene mining of wild raspberry germplasm resources.

[0029] The beneficial effects of this invention are:

[0030] 1. The genetic transformation system developed in this invention can successfully induce positive callus and positive roots of *Pteris vittata* by infecting stem segments. Positive buds were obtained by culturing positive roots. Based on RUBY transformation, the entire growth process can be continuously identified, and molecular detection shows that it is positive.

[0031] 2. This invention solves the problems of high cost, complex operation, long cycle and low transformation efficiency faced by the original genetic transformation of wild raspberries; the highest transformation rate of the existing genetic transformation system is 25%. After systematically screening the factors affecting transformation, the new transformation system can obtain positive roots in 14 days, with a positive rate of up to 83.7%.

[0032] 3. This invention can directly transform non-sterile materials of wild raspberries that are limited by genotype and cannot be tissue cultured, providing feasible theoretical and technical support for molecular breeding. Attached Figure Description

[0033] Figure 1 Plasmid map for modifying the RUBY reporter gene;

[0034] Figure 2 This is a flowchart illustrating the transformation of positive roots in *Rhizophora stylosa*. A represents explant branches; B represents prepared stem segments; C represents infected stem segments; D represents culture after transformation; E represents positive red callus formation at the base of the stem segment; and F represents positive red roots formed on the stem segment.

[0035] Figure 3 The results show the DNA detection results of the red positive roots of *Pteris vittata*. A represents the red positive root; B is the DNA identification electrophoresis image; Control represents the CK (control) leaves of *Pteris vittata*; 1-5 represent the red positive roots of four transformed plants.

[0036] Figure 4 The results show the effects of different Agrobacterium rhizogenes, different bacterial suspension concentrations, different infection times, and different stem segments with different internode numbers on the transformation rate of *Agrobacterium tumefaciens* explants. A represents different bacterial suspension concentrations; B represents different Agrobacterium rhizogenes; C represents different infection times; and D represents different stem segments with different internode numbers.

[0037] Figure 5 The images show the DNA detection results for the red positive buds of *Pteris vittata*. A represents positive buds induced by positive roots; B is the DNA identification electrophoresis image; C is the RNA identification electrophoresis image. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the embodiments do not limit the scope of protection of the present invention.

[0039] This embodiment provides a highly efficient genetic transformation method for *Rhizoctonia solani* mediated by the RUBY reporter gene and *Agrobacterium rhizogenes*, including the following steps:

[0040] 1. Preparation of explant materials from *Rhizophora stylosa*

[0041] Material selection: Healthy, semi-lignified one-year-old branches of *Pteris vittata*, with a branch diameter of 0.6-0.8 cm.

[0042] Branch treatment: Wash the branches, rinse off the dust with clean water, and use filter paper to absorb any residual liquid on the surface of the branches.

[0043] Stem segment preparation: Use pruning shears to cut the washed branches into 15cm stem segments, keeping one leaf in each stem segment. Soak the stem segments in an 800-fold carbendazim solution for 30 minutes, then remove and air dry for later use.

[0044] Internode number conditions: Stem segments with different internode numbers were prepared: first, single-bud internode segment; second, double-bud internode segment; third, triple-bud internode segment. The second internode number condition was preferred for infection.

[0045] 2. Expression vector construction and Agrobacterium rhizogenes transformation

[0046] The RUBY reporter gene was constructed using pDR5-RUBY as the backbone, amplified with specific primers containing the 35S promoter, and expressed as a gene-editing vector driven by the 35S promoter. Recombinant ligation primers highly homologous to the pDR5-RUBY vector were designed using SanpGene 6.0.2 bioinformatics software. Subsequently, the pDR5:RUBY vector was digested with Sma I and Nco I restriction endonucleases. Figure 1 Following previous transformation experimental protocols, when using the reporter gene as the target vector, no blank vector control was set up. Wild-type Rubus was used as a negative control, and molecular detection was combined to verify the specificity of positive roots.

[0047] Transformation of Agrobacterium rhizogenes: Using chemical transformation, the recombinant vector was transformed into different Agrobacterium rhizogenes competent cells, including K599, R1000, and C58C1. Strains containing the RUBY vector were cultured on YEP solid medium (50 mg / L kanamycin, 50 mg / L streptomycin) and TY medium (50 mg / L kanamycin, 50 mg / L streptomycin) at 28°C for 2 days, and single colonies were picked for detection.

[0048] 3. Agrobacterium rhizogenes carrying the target gene infects explants.

[0049] Agrobacterium preparation: Positive strains were streaked onto TY solid medium containing antibiotics and incubated at 28°C for 2 days. Single colonies were picked and added to 800 μL of TY liquid medium containing antibiotics and activated on a shaker (28°C, 200 rpm) for 24 h. 10 μL of the activated bacterial culture was added to 10 mL of TY liquid medium containing antibiotics and shaken on a shaker (28°C, 200 rpm) for 20 h. The OD of the bacterial culture was measured. 600 To determine the concentration, the bacterial suspension was centrifuged (5000 rpm, 5 min), the supernatant was discarded, and the suspension was resuspended in MES infection solution. Based on the previously determined concentration, the suspension was resuspended to an OD value using calculations. 600 =0.4, 0.8, 1.2, preferred OD 600=0.8. The resuspension was allowed to stand in the dark for 2 hours. MES infection solution components: 1M MgCl2 10mL / L, 1M MES (2-(N-morpholino)ethanesulfonic acid) 10mL / L, 100mM AS (acetyl eugenone) 5mL / L, Silwet L-77 (99%) 5mL / L, dd H2O Up to 100mL, pH = 5.8.

[0050] Explant contamination: Without requiring a laminar flow hood, under non-sterile conditions, prepare the explant material. Wearing gloves, use tweezers to gently immerse the explants in three different bacterial solutions for 10 min, 20 min, and 30 min, preferably 10 min. Gently shake the beaker containing the explant and bacterial solution mixture every 2 minutes. After the countdown, remove the explant material and blot dry any remaining bacterial solution on the surface with sterile filter paper. Transfer the contaminated explants to a culture box containing moist vermiculite.

[0051] 4. Co-culture and maintenance of explants after infection

[0052] The infected explants were co-cultured at 24℃ in the dark for 3 days. Then, the co-cultured explants were transferred to a culture rack, and the humidity was maintained at 80%-90%. The culture conditions were 25±2℃, light intensity of about 2000LUX, and photocycle of 14h light / 10h dark. A 1:800 Hogland nutrient solution was sprayed every 3 days.

[0053] 5. Positive root sampling and detection

[0054] After 14 days of cultivation, the explants were removed from the substrate, and data related to the red positive roots were collected. The positive root samples were frozen in liquid nitrogen and stored at -80℃. DNA was extracted from the stored samples, and real-time quantitative PCR was performed to confirm that gene expression was consistent with the red phenotype. To verify the DNA of the transgenic *Aralia elata*, DNA samples were extracted from the roots of identified transgenic and wild-type *Aralia elata*. These samples were then used as templates for PCR amplification using 35S-F and RUBYseq-R primers. Primers 35S_F and RUBYseq_R were designed, and the amplified band length was 493 bp. The primers were synthesized by Qingke Biotechnology (Beijing).

[0055] 35S_F:Taagggactgaccacccgggtgagacttttcaacaaagggtaatatcgg(SmaⅠ)

[0056] RUBYseq_R:Gggtcgcatgatccatggtcagcgtgtcctctccaaatg(NcoⅠ)

[0057] 6. Positive roots induce positive buds

[0058] Rinse the obtained positive roots with clean water, remove the lateral roots, keep only the main root, and cut it into 2cm long root segments. Then place them in a culture box containing moist vermiculite, and partially bury them in the vermiculite. The culture conditions are 25±2℃, light intensity of about 2000LUX, photocycle of 14h light / 10h dark, spray with carbendazim 1:800 solution, and spray with 2000mg / L gibberellin solution (GA3) once a week. Positive buds can be obtained after 25 days.

[0059] Example 1: Overexpression of 35S:RUBY promotes betaine synthesis in *Agrobacterium rhizogenes* explants infected with different *Agrobacterium* species under non-tissue culture conditions.

[0060] (I) Experimental Methods

[0061] 1. Experimental Materials

[0062] Select healthy, one-year-old semi-lignified branches from the current year, with a branch diameter of 0.6-0.8cm. Figure 2 ).

[0063] 2. Stem segment preparation

[0064] Wash the branches thoroughly, rinse off any dust with clean water, and blot dry any remaining liquid with filter paper. Use pruning shears to cut the washed branches into single-bud internode stem segments, retaining one leaf on each segment. Immerse the stem segments in an 800-fold dilution of carbendazim solution for 30 minutes, then remove and air dry before use.

[0065] 3. Construction of the carrier and preparation of bacterial culture

[0066] Primers for homologous recombination ligation with the pDR5:RUBY vector were designed, and the pDR5:RUBY vector was digested with BamHI and PstI restriction endonucleases. The CaMV35S promoter sequence was inserted between the sticky ends to construct the 35S:RUBY vector. Figure 1Using chemical transformation, the correctly sequenced recombinant vector was transformed into different Agrobacterium rhizogenes competent cells, including K599, R1000, and C58C1. The bacterial culture was streaked onto solid YEP medium containing antibiotics (50 mg / L kanamycin, 50 mg / L streptomycin) and incubated at 28°C for 2 days. Single colonies were picked and transferred to 800 μL of liquid TY medium containing antibiotics (50 mg / L kanamycin, 50 mg / L streptomycin) and activated on a shaker (28°C, 200 rpm) for 24 h. 20 μL of the activated bacterial culture was transferred to a 50 mL centrifuge tube containing 20 mL of liquid TY medium (50 mg / L kanamycin, 50 mg / L streptomycin) and shaken on a shaker (28°C, 200 rpm) for 20 h. The bacterial concentration was measured, and the bacterial suspension was centrifuged (4000 rpm, 10 min). The supernatant was discarded, and the bacterial suspension was resuspended in liquid MES infection buffer. Based on the previously measured concentration, the bacterial suspension was resuspended to a concentration of OD100 using calculations. 600 =1.0. Place the resuspension in the dark for 2 hours. MES infection solution components: 1M MgCl2 10mL / L, 1M MES (2-(N-morpholino)ethanesulfonic acid) 10mL / L, 100mM AS (acetyl eugenone) 5mL / L, Silwet L-77 (99%) 5mL / L, ddH2O Up to 100mL, pH=5.8.

[0067] 4. Explant infection

[0068] Without needing to be in a laminar flow hood or under non-sterile conditions, the explant material was divided into three groups, and the explants were placed into the bacterial solution using tweezers. Figure 2 The explant material and bacterial solution were soaked for 10, 20, and 30 minutes respectively. During this time, the beaker containing the explant material and bacterial solution mixture was gently shaken every 2 minutes. Figure 1 After the countdown ends, the explants are removed, and any residual bacterial solution on the surface of the explant material is blotted dry with sterile filter paper. The infected explants are then transferred to a culture box containing moist vermiculite. The infected explants are then cultured in the dark at 24°C for 3 days. Subsequently, the co-cultured explants are transferred to a culture rack, where the humidity is maintained at 80%-90%, the culture conditions are 25±2°C, the light intensity is approximately 2000 LUX, the photocycle is 14h light / 10h dark, and 1:800 Hoagland nutrient solution is sprayed every 3 days.

[0069] 5. DNA extraction and real-time quantitative PCR analysis

[0070] Adopting rapid DNA was extracted from transgenic positive roots using a column-based nucleic acid extraction kit. DNA was also extracted from transgenic roots and untransformed roots. PCR verification was performed using the 35S:RUBY gene as the target gene. Plasmid DNA served as a positive control, and DNA from plants not transformed with the RUBY gene served as a negative control. A 20 μL RT-qPCR reaction system included: 2 μL forward and reverse primers (10 μM), 7 μL H2O, 1 μL DNA, and 10 μL Taq Mix. The RT-qPCR reaction was performed using a Bio-Rad CFX 96 instrument. RUBY was used as an internal reference gene in the qPCR, with primer sequences 35S_F and RUBYseq_R.

[0071] (II) Experimental Results

[0072] DNA testing of the red positive roots of *Prickly ash* amplified the target fragment. Figure 3 This indicates that the RUBY gene is present in the transformed plants, and the positive root phenotype after expression is a bright red color visible to the naked eye. All three Agrobacterium rhizogenes can achieve genetic transformation of *Rabdosia rubescens*, with K599 showing the highest transformation efficiency at 54.17%. When using K599 for *Rabdosia rubescens* transformation, the positive rates of other Agrobacterium rhizogenes such as C58C1 and R1000 were significantly lower than K599. K599 Agrobacterium rhizogenes showed the highest efficiency in transforming *Rabdosia rubescens* explants into RUBY. Figure 4 ).

[0073] Example 2: Overexpression of 35S:RUBY promotes betaine synthesis in *Agrobacterium rhizogenes* explants infected with different concentrations and infection times under non-tissue culture conditions.

[0074] (I) Experimental Methods

[0075] 1. Experimental Materials

[0076] The method is the same as in Example 1.

[0077] 2. Stem segment preparation

[0078] The method is the same as in Example 1.

[0079] 3. Construction of the carrier and preparation of bacterial culture

[0080] The method is the same as in Example 1, and the bacterial suspension is resuspended to a concentration of OD100 by calculation. 600 =0.4, 0.8 and 1.2. The resuspension was placed in the dark for 2 hours.

[0081] 4. Explant infection

[0082] Without the need for a clean bench or under non-sterile conditions, the explant material was divided into three groups, and the explants were placed into the bacterial solution with tweezers and soaked for 10 min, 20 min and 30 min respectively.

[0083] 5. DNA extraction and real-time quantitative PCR analysis (qPCR)

[0084] The method is the same as in Example 1.

[0085] (II) Experimental Results

[0086] In experiments with different bacterial suspension concentrations, stem segments of *Rhizophora stylosa* were uniformly soaked for 30 minutes at a bacterial suspension infection concentration of OD0.05. 600 The conversion efficiency was highest at a ratio of 0.8, reaching 46.67%, with a positive rate significantly higher than that of OD. 600 =0.4 and 1.2 ( Figure 4 In experiments with different infection times, a concentration of OD was used. 600 When the bacterial suspension with an OD value of 0.8 was used to uniformly infect explants of *Rhizophora stylosa*, the highest transformation efficiency (63.89%) was observed at an infection time of 10 min. At an infection time of 10 min, the positive rate was significantly higher than the control at infection times of 20 min and 30 min. When the bacterial suspension concentration was OD... 600 The highest efficiency was observed in the transformation experiment of *Rhizophora stylosa* explants at 0.8 μg / mL and 10 min of infection.

[0087] Example 3: Overexpression of 35S:RUBY in explants of *Agrobacterium rhizogenes* with different internode numbers promoted betaine synthesis using the Agrobacterium rhizogenes genetic transformation system.

[0088] (I) Experimental Methods

[0089] 1. Experimental Materials

[0090] The method is the same as in Example 1.

[0091] 2. Stem segment preparation

[0092] The method is the same as in Example 1. Stem segments with different numbers of internodes were prepared: the first type was a single-bud stem segment; the second type was a double-bud stem segment; and the third type was a triple-bud stem segment.

[0093] 3. Construction of the carrier and preparation of bacterial culture

[0094] The method is the same as in Example 1. K599 Agrobacterium was selected, and the bacterial suspension was resuspended to a concentration of OD100 by calculation. 600 =0.8.

[0095] 4. Explant Infection and Maintenance

[0096] Without needing to be in a laminar flow hood or under non-sterile conditions, explant materials with different numbers of intersegments were divided into three groups. The explants were then placed into the bacterial solution using forceps and soaked for 10 minutes. The method is the same as in Example 1.

[0097] 5. DNA extraction and real-time quantitative PCR analysis (qPCR)

[0098] The method is the same as in Example 1.

[0099] (II) Experimental Results

[0100] The positive rates of *Agrobacterium rhizogenes* explants with different numbers of internodes showed significant differences after transformation with the *Agrobacterium rhizogenes* system, with the highest positive rate observed in stem segments with two buds and internodes. Figure 4 The positive rate of single-bud and three-bud internode stem segments was significantly lower than that of double-bud internode segments, at 68.15% and 62.96% respectively, indicating that double-bud internode stem segments were the most efficient in the experiment of converting betaine substances into compounds.

[0101] Example 4: Induction of positive buds from red-positive roots of *Pteris vittata*

[0102] 1. Experimental Materials

[0103] Red positive roots of *Rhizophora stylosa* that have passed molecular verification were selected, and the induced root system was cultured until browning.

[0104] 2. Stem segment preparation

[0105] Wash the roots thoroughly, rinse off any dust with clean water, and blot dry with filter paper. Remove any lateral roots, leaving only the main root. Use scissors to cut the washed, sun-loving roots into 2cm long sections.

[0106] 3. Cultivation environment and conditions

[0107] Then place it in a culture box containing moist vermiculite, partially bury it in the vermiculite, spray with a 1:800 solution of carbendazim, and spray with a 2000 mg / L gibberellin solution (GA3) once a week. Positive buds can be obtained after 25 days.

[0108] 4. DNA extraction and real-time quantitative PCR analysis (qPCR)

[0109] The method is the same as in Example 1.

[0110] (II) Experimental Results

[0111] Positive shoots induced by positive roots were verified by molecular methods. Figure 5 The plant was identified as a positive case.

[0112] The above description provides examples of the preferred embodiments of the present invention. Parts not detailed herein are common knowledge to those skilled in the art. The scope of protection of the present invention is determined by the claims. Any equivalent modifications based on the technical teachings of the present invention are also within the scope of protection of the present invention.

Claims

1. A highly efficient genetic transformation method for *Rabbittia rubra* mediated by the RUBY reporter gene and *Agrobacterium rhizogenes*, characterized in that, Includes the following steps: 1) Explant preparation: Stem segments of *Rhizophora stylosa* from the field were selected as explant materials; 2) Vector construction: Construct a recombinant expression vector containing the target gene and the RUBY reporter gene, and transform it into Agrobacterium rhizogenes to obtain an engineered strain; The RUBY reporter gene is a fusion gene containing three key enzyme genes for betaine synthesis: CYP76AD1, DODA, and glycosyltransferase, with each gene linked by a 2A self-cleaving peptide sequence; the Agrobacterium rhizogenes strains are K599, R1000, and C58C1; the recombinant expression vector is a binary vector containing T-DNA left and right boundary sequences, a replication origin, an antibiotic resistance marker, and a multiple cloning site; 3) Infection treatment: Infect the cut site of the explant stem segment with the bacterial solution of the engineered strain; 4) Co-culture: The infected explants were placed on moist vermiculite for co-culture; 5) Visual screening: Identify positive hairy roots by observing the red betaine pigment produced by the RUBY reporter gene; 6) Molecular verification: PCR and RT-qPCR were performed on the screened positive hairy roots to confirm the integration and expression of the target gene; 7) Bud regeneration: Transfer the verified positive hairy roots to moist vermiculite and spray with gibberellin to induce the differentiation of positive buds; 8) Verification of positive buds: Molecular biological tests are performed on the regenerated positive buds to confirm the stable inheritance of transgenes.

2. The method according to claim 1, characterized in that: The stem segments selected are double-bud internode segments with a diameter of 0.6-0.8 cm and a length of 15 cm, with each segment retaining one leaf.

3. The method according to claim 1, characterized in that: The conditions for the infection treatment are: bacterial suspension OD 600 The value is 0.4-1.2, 100-200mM acetylsuccinone is added, the infection time is 10-20 minutes, and the infection temperature is 25-28℃; the co-culture conditions are: in a culture box containing moist vermiculite, dark culture at 25±2℃ for 3 days; the visual screening is carried out by visual observation, the hairy roots of positive transformation show obvious red or pink color, and the screening is carried out 7-14 days after infection.

4. The method according to claim 1, characterized in that: In the molecular validation, PCR detection used target gene-specific primers and RUBY gene-specific primers, and RT-qPCR used internal reference genes to standardize and analyze gene expression levels.

5. The method according to claim 1, characterized in that: In the bud regeneration process, a 1:800 solution of carbendazim was sprayed, and a 2000 mg / L gibberellin solution was sprayed once a week. The culture conditions were 25±2℃, light intensity of 2000 LUX, and photoperiod of 14h light / 10h darkness.

6. A transgenic *Pteris vittata* plant obtained by the method according to any one of claims 1-5, characterized in that: The plant's genome integrates a foreign target gene and a RUBY reporter gene. The root, stem, or leaf tissues of the plant are red or pink, indicating the continuous and stable expression of the RUBY reporter gene.

7. A recombinant expression vector for genetic transformation of *Rhizophora stylosa*, characterized in that: The vector contains a RUBY reporter gene and a cloning site. The RUBY reporter gene comprises, in sequence, the CYP76AD1 gene, the first 2A peptide sequence, the DODA gene, the second 2A peptide sequence, and the glycosyltransferase gene.

8. The carrier according to claim 7, characterized in that: The vector also includes a 35S promoter, a terminator sequence, T-DNA left and right boundary sequences, and a bacterial resistance selection marker; the 2A peptide sequence is selected from one or more of P2A, T2A, E2A, or F2A.

9. A recombinant Agrobacterium rhizogenes engineered strain containing the vector described in any one of claims 7-8.

10. The application of the method according to any one of claims 1-5 in the functional verification of the red raspberry gene, molecular breeding, or gene mining of other wild raspberry germplasm resources.

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