A rhododendron leaf transient transformation method based on minimally invasive pretreatment and semi-liquid culture and application

Abstract

This invention discloses a method for transient transformation of rhododendron leaves based on minimally invasive pretreatment and semi-liquid culture, and its application. Addressing the problems of insufficient Agrobacterium penetration and poor post-infection culture conditions caused by the thick cuticle, non-planar nature, and dense rough hairs on the underside of Hainan rhododendron leaves, this invention employs a microneedle roller with a needle length of 0.5–1.0 mm for minimally invasive pretreatment of the leaves. After minimally invasive pretreatment, the leaves are immersed in Agrobacterium culture and subjected to negative pressure infection at -0.10–-0.09 MPa for 1.5–2.5 h, followed by a slow restoration to normal pressure over 15–25 min. After infection, the leaves are placed in a semi-liquid system with an agar concentration of 2–3 g / L and cultured at 20°C in the dark for 3 days. The transient transformation method for rhododendron leaves provided by this invention can promote uniform infection of Agrobacterium in rhododendron leaves and maintain the tissue state of the infected leaves, and can be used for transient overexpression, silencing, and functional verification studies of genes in Hainan rhododendrons.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method and application of instantaneous transformation of rhododendron leaves based on minimally invasive pretreatment and semi-liquid culture. Background Technology

[0002] Transient gene expression technology in plants is characterized by its short cycle time and relatively simple operation, making it an important means of rapidly verifying gene function. Stable genetic transformation of woody plants typically involves a long cycle, high technical difficulty, and limited transformation efficiency. Therefore, establishing a transient expression system suitable for woody plants can help shorten the gene function verification cycle.

[0003] Rhododendron genus ( Rhododendron Rhododendron plants are important woody ornamental plants with abundant germplasm resources. Compared with herbaceous model plants, the genetic transformation and gene function verification of Rhododendron species are still limited by the efficiency and stability of the transformation system. Hainan Rhododendron ( Rhododendron hainanense It has a typical woody leaf structure and can be used as research material for establishing a method for instantaneous transformation of Rhododendron leaves.

[0004] When using Hainan Rhododendron leaves as material, the conventional instantaneous leaf transformation method mainly encounters the following technical problems: 1. Physical barriers are difficult to overcome: The leaves of Hainan Rhododendron have a high degree of leatheriness and a thick cuticle, making it difficult for Agrobacterium tumefaciens to fully enter the leaf tissue. When using a needleless injector for local forced injection, due to the dense woody leaf tissue and the high resistance to liquid entry, the bacterial solution is prone to splashing or overflowing at the injection site, resulting in limited effective penetration and uneven infection. It may also cause local tissue mechanical damage and water staining.

[0005] For example, invention application CN118272429A discloses a method for Agrobacterium infiltration of transient gene expression in Phoebe zhennan. This Agrobacterium infiltration method includes the following steps: (1) preparing Agrobacterium infection solution; (2) creating wounds on the back of non-exposed Phoebe zhennan leaves using a microneedle roller, applying the infection solution described in step (1) to the wounds so that all wounds are in contact with the infection solution, cleaning the infection solution with sterile water after 5-10 minutes, spraying an appropriate amount of water to keep the plant adequately moist and humid, and then dark-culturing the infected plants to allow Agrobacterium to penetrate into the plant leaf cells. This invention application uses a microneedle roller to create wounds, which is beneficial for subsequent Agrobacterium infiltration into the leaves, but the method of applying the infection solution in step (2) is to draw the infection solution with a needleless syringe, press it against the wound, and inject it under local pressure. For leaves of woody plants with a high degree of leatheriness and dense tissue, the resistance to the entry of bacterial solution into the tissue is significant when applying local pressure with a needle-free injector. This can easily lead to splashing or overflow at the injection site, limiting the effective amount of bacterial solution entering the leaf tissue and affecting the uniformity of penetration. Furthermore, continuous local pressure may cause mechanical damage to the tissue. Therefore, this method does not address the problems of bacterial solution splashing, impeded penetration, and mechanical damage to the tissue during local injection of Rhododendron hainanense leaves.

[0006] 2. Special leaf morphology affects the culture state after infection: Hainan rhododendron leaves are non-planar (slightly curled), and the underside of the leaves is densely covered with rough, appressed hairs. In solid or high agar concentration culture systems, the effective contact between the leaves and the aqueous phase is limited, and detached leaves are prone to dehydration and shriveling; in pure liquid systems, complete submersion of the leaves may cause insufficient gas exchange and induce tissue browning.

[0007] 3. Pressure changes can easily cause tissue damage: During the rapid release of pressure after vacuum infiltration, the leaves of Hainan Rhododendron may suffer mechanical damage due to sudden pressure changes, which will affect the subsequent cultivation status.

[0008] For example, invention application CN112458109A discloses a highly efficient transient plant transgenic method based on negative pressure and temporary immersion. This highly efficient transient plant transgenic method introduces exogenous genes into plant explants efficiently and transiently through negative pressure and short-term repeated infection, which is time-saving, highly efficient, and easy to operate. However, in this highly efficient transient plant transgenic method, the explant leaves are immersed in Agrobacterium infection solution and placed in a vacuum pump to maintain a pressure of 0.1-0.4 MPa, and the pressure is rapidly released after 2-4 minutes. This method does not address the problem of mechanical damage caused by sudden pressure changes in Rhododendron hainanense leaves.

[0009] Therefore, it is necessary to establish a method for instantaneous transformation of Rhododendron hainanense leaves that can take into account leaf epidermal barrier breakthrough, Agrobacterium infiltration, post-infection culture state, and tissue integrity. Summary of the Invention

[0010] The purpose of this invention is to address the problems of insufficient bacterial penetration, poor culture status after infection, and easy tissue damage caused by the thick cuticle of Hainan Rhododendron leaves, non-planar leaves, and dense rough hairs on the underside of the leaves. This invention provides a method for instantaneous transformation of Hainan Rhododendron leaves based on minimally invasive pretreatment and semi-liquid phase culture, and its application.

[0011] This method improves the uniformity of Agrobacterium penetration through microneedle pretreatment and reduces damage caused by sudden pressure changes by combining prolonged negative pressure with slow recovery to normal pressure. Meanwhile, since the infected detached leaves need to be cultured in a system that maintains tissue hydration, and if exposed directly to air after infection, the leaves are prone to dehydration and shriveling, suitable semi-liquid phase culture conditions after infection were determined by screening using agar concentration gradients based on the floating and sinking characteristics and hydration status of Hainan Rhododendron leaves.

[0012] This invention first provides a method for transient transformation of rhododendron leaves based on minimally invasive pretreatment and semi-liquid phase culture, comprising the following steps: Step 1: Construct an engineered Agrobacterium containing a target gene expression vector, and prepare a bacterial culture of the engineered Agrobacterium; Step 2: Use a microneedle roller to perform a minimally invasive pretreatment on the back of the rhododendron leaf to obtain the pretreated leaf; Step 3: Use the bacterial solution described in Step 1 to infect the pretreated leaves described in Step 2, and obtain the infected leaves. Step 4: Place the infected leaves from Step 3 into an agar semi-liquid culture system for post-infection culture to obtain leaves expressing the target gene; the concentration of agar in the agar semi-liquid culture system is 2~3 g / L.

[0013] The rhododendron leaves described in this invention are the tender leaves of plants in the genus Rhododendron; these tender leaves are those that have basically unfolded, are light green in color, have a relatively soft texture, and have not yet undergone obvious leathery transformation. The epidermal barrier of these tender leaves is relatively weak, which is conducive to Agrobacterium infection, and the accumulation of polysaccharides and polyphenols is relatively low, which is beneficial for RNA extraction and molecular detection.

[0014] Preferably, the tender leaves of the rhododendron species are tender leaves of Rhododendron hainanense.

[0015] Since changing the diameter of the microneedle does not significantly affect the effect of microneedle pretreatment, the main focus was on comparing the effects of different microneedle lengths on the minimally invasive pretreatment of rhododendron leaves.

[0016] Preferably, the microneedle diameter of the microneedle roller in step 2 is 0.1~0.3 mm, and the microneedle length is 0.5~1.0 mm.

[0017] More preferably, the microneedle of the microneedle roller in step 2 has a needle body diameter of 0.2 mm and a needle length of 0.5 mm.

[0018] Preferably, the microneedle roller in step 2 is a 540-needle steel sheet dense microneedle roller with a steel sheet diameter of 20mm and a total of 540 needles.

[0019] Preferably, the Agrobacterium engineered strain mentioned in step 1 is Agrobacterium GV3101.

[0020] Preferably, the minimally invasive pretreatment in step 2 involves rolling a microneedle roller twice on the back of the rhododendron leaf, ensuring that the leaf remains intact without any visible continuous cracks.

[0021] Preferably, the infection step described in step 3 includes negative pressure osmosis and restoration to normal pressure; The negative pressure infiltration is performed under pressure conditions of -0.10 to -0.09 MPa for a time of 1.5 to 2.5 h.

[0022] Preferably, the negative pressure infiltration is performed under the following conditions: -0.095 MPa for 2 hours.

[0023] The step of restoring normal pressure is to allow the gas pressure inside the device to slowly return to normal pressure within 15 to 25 minutes after negative pressure permeation.

[0024] Preferably, the step of restoring normal pressure involves allowing the air pressure inside the device to slowly return to normal pressure within 20 minutes after negative pressure permeation.

[0025] This invention also provides the application of the aforementioned method for transient transformation of rhododendron leaves based on minimally invasive pretreatment and semi-liquid culture, for the transient expression of exogenous genes, transient silencing of genes, or application in gene function research in rhododendron leaves.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. Improving the culture state of detached leaves after infection: Considering the non-planar curled morphology and densely covered rough hairs on the underside of Hainan Rhododendron leaves, a gradient screening using 0, 1, 2, 3, 4, and 5 g / L agar concentrations was conducted, ultimately determining 2–3 g / L as the optimal concentration range. At 0–1 g / L, leaves easily sank and turned brown; at 4–5 g / L, leaves showed dehydration and shriveling; while at 2–3 g / L, leaves maintained their normal morphology and green color without significant browning. This indicates that this concentration range is more suitable for maintaining the water content and tissue state of detached Hainan Rhododendron leaves after infection.

[0027] 2. Promoting Agrobacterium penetration and reducing tissue damage: A combination of "microneedle minimally invasive treatment + prolonged negative pressure + slow recovery to normal pressure" is employed. 0.5~1.0 mm microneedle rollers can create relatively uniformly distributed micropores on the leaf surface; combined with negative pressure penetration, this promotes the entry of the bacterial solution into the leaf tissue. Compared to local pressure injection with a needle-free injector, the slow recovery to normal pressure avoids bacterial solution splashing or overflow, limited effective penetration, and uneven infection caused by greater resistance to liquid entry during local injection into woody leaves, and reduces the risk of local mechanical damage.

[0028] 3. Establish a rapid gene function verification method: without the need for a complete tissue culture and regeneration process, it can be used for transient expression or silencing of exogenous genes in Rhododendron hainanense leaves, and combined with fluorescence observation, qRT-PCR, and analysis of phenotypic, cell death and reactive oxygen species under stress conditions to carry out gene function verification. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the dense microneedle roller structure used for minimally invasive pretreatment of explants in Example 2 (1 is the needle tip, 2 is the roller, 3 is the shaft, and 4 is the handle).

[0030] Figure 2 This is a comparison chart of different minimally invasive explant pretreatment conditions and different agar concentrations in Example 2. Among them, Figure 2 A in the figure is a comparison of the state of young leaves of Hainan Rhododendron after different minimally invasive pretreatment methods (scribing method, manual punching, and treatment with 0.2 mm, 0.5 mm and 1.0 mm microneedle rollers) followed by negative pressure infection. Figure 2 B in the figure shows the state of infected young leaves of Hainan Rhododendron after 3 days of culture at 20℃ in the dark in culture systems with different agar concentrations of 0, 1, 2, 3, 4 and 5 g / L.

[0031] Figure 3 The images shown are laser confocal microscopy images of young leaves of Rhododendron hainanense after infection with TRV-empty mixed bacterial solution in Example 3 (Bright represents bright field image, GFP represents green fluorescence image, and Merge represents superimposed image of bright field and green fluorescence).

[0032] Figure 4 This is an agarose gel electrophoresis image of total RNA from tender leaves of Rhododendron hainanense in Example 3.

[0033] Figure 5 The bar chart shows the transcriptional level qRT-PCR detection results in Example 3 (** indicates a difference of P<0.01 compared to the corresponding control group). Figure 5 In the text, A represents the concentration of YFP-RCA1 in the tender leaves of Rhododendron hainanense. RCA1 Image of gene expression detection results; Figure 5B in the text refers to the concentration of B in the young leaves of Rhododendron hainanense after treatment with TRV-RCA1 mixed bacterial solution. RCA1 Image showing the results of gene expression detection.

[0034] Figure 6 Transient overexpression and transient silencing in Example 4 RCA1 Phenotypic observation of young leaves of Hainan Rhododendron after genetic modification and treatment at 42℃ for 12 h.

[0035] Figure 7 Transient overexpression and transient silencing in Example 4 RCA1 Histochemical staining images of physiological indicators of young leaves of *Rhododendron hainanense* after treatment at 42℃ for 12 h following gene modification. Figure 7 In the diagram, A represents trypan blue staining. Figure 7 B in the image represents the DAB staining pattern; Figure 7 C in the diagram represents the NBT staining pattern. Detailed Implementation

[0036] The Hainan Rhododendron material used in the examples was obtained from Hainan Rhododendron cuttings from the Ornamental Plant Germplasm Resource Garden of Zhejiang University.

[0037] The Hainan rhododendron material selected in this embodiment was young leaves of Hainan rhododendron that were in a uniform growth state, had basically unfolded, had a tender green color, a relatively soft texture, and had not yet shown obvious leatheriness. Young leaves are more susceptible to Agrobacterium infection than older leaves, and at the same time, they accumulate relatively less polysaccharides and polyphenols, which is beneficial for total RNA extraction and molecular detection.

[0038] In the embodiments RCA1 Transient gene silencing was achieved using the TRV vector system, specifically a two-component vector system consisting of TRV1 and TRV2. TRV2-empty was an empty vector carrying a GFP tag, which was used for construction. RCA1 The vector backbone of the transient gene silencing vector TRV2-RCA1; both TRV2-empty and TRV2-RCA1 need to be used in conjunction with the helper vector TRV1 component during actual leaf infection.

[0039] In Example 1, "TRV2-empty" and "TRV2-RCA1" were used to represent the corresponding TRV2 component carriers, respectively. In Examples 2-4, Agrobacterium bacterial suspension carrying the auxiliary carrier TRV1 component was mixed with Agrobacterium bacterial suspension carrying the TRV2-empty empty carrier and used as a control, with "TRV-empty" indicating the bacterial suspension or the leaves treated with the bacterial suspension; Agrobacterium bacterial suspension carrying the auxiliary carrier TRV1 component was mixed with Agrobacterium bacterial suspension carrying the TRV2-RCA1 carrier and used for silencing. RCA1 The gene is indicated by "TRV-RCA1" to represent the bacterial solution or the leaf treated with the bacterial solution.

[0040] Example 1: Construction of transient expression vector from Rhododendron hainanense leaves and preparation of Agrobacterium tumefaciens engineered strain This embodiment details the construction process of the transient overexpression vector YFP-RCA1 and the transient silencing vector TRV2-RCA1: using TRV2-empty as the backbone for insertion... RCA1 TRV2-RCA1 was obtained by gene reconstruction; both TRV2-empty and TRV2-RCA1 are TRV2 components of the TRV system and need to be used in conjunction with the auxiliary vector TRV1 component during actual leaf infection.

[0041] 1. Construction of recombinant plasmids containing the target gene (1) Primer design and target fragment amplification: Based on Hainan Rhododendron ( Rhododendron hainanense ) RCA1 Gene sequences (as shown in SEQ ID NO:1) and multiple cloning site sequences of selected vectors (transient overexpression vector backbone pHB-YFP and transient silencing empty vector backbone TRV2-empty carrying GFP tag) were used to design specific amplification primers, and homologous arm sequences consistent with the sequences on both sides of the vector restriction site were added to the 5' end of the primers.

[0042] The primer sequences for the YFP-RCA1 vector are shown in SEQ ID NO:2 and SEQ ID NO:3; the primer sequences for the TRV2-RCA1 vector are shown in SEQ ID NO:4 and SEQ ID NO:5.

[0043] Young leaves of *Rhododendron hainanense* were collected, and total RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (purchased from Tiangen Biotech Co., Ltd.). Using the extracted total RNA as a template, first-strand cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (catalog number RR047A, purchased from TaKaRa). The obtained *Rhododendron hainanense* cDNA was used as a template for PCR amplification, and the resulting cDNA with homologous arms was recovered and purified. RCA1 Target fragment of the gene.

[0044] (2) Vector linearization: Select restriction endonuclease Pst I and Spe The transient overexpression vector backbone pHB-YFP was subjected to double enzyme digestion, and restriction endonucleases were selected simultaneously. Eco RI and Bam The transiently silenced empty vector backbone TRV2-empty was subjected to double enzyme digestion with HI, and the linearized vector fragment was recovered by gel excision.

[0045] (3) Homologous recombination ligation: Using the homologous recombinase Exnase II (purchased from Novizan), the homologous recombination enzyme obtained in step (1) of this embodiment was ligated. RCA1 The target gene fragment is ligated with the linearized vector fragment obtained in step (2) of this embodiment to construct a structure containing the target gene. RCA1 Recombinant plasmids of genes.

[0046] (4) Transformation and identification of Escherichia coli: The two recombinant plasmids were transformed separately into Escherichia coli DH5α competent cells. After screening with kanamycin and sequencing verification, the correct recombinant plasmids were extracted using the TIANprep Mini Plasmid Kit (purchased from Tiangen Biotech Co., Ltd.).

[0047] 2. Preparation of engineered Agrobacterium bacteria The verified recombinant plasmids (YFP-RCA1 and TRV2-RCA1) were transformed into Agrobacterium GV3101 competent cells using a freeze-thaw method. Simultaneously, YFP-empty, TRV2-empty with a GFP tag, and the helper vector TRV1 were transformed into Agrobacterium GV3101 competent cells. Positive clones were screened on LB agar plates containing kanamycin (50 mg / L) and rifampin (50 mg / L), identified by PCR, and stored for later use. Note that the Agrobacterium strains corresponding to TRV2-empty or TRV2-RCA1 must be used in conjunction with the Agrobacterium strain corresponding to TRV1 for subsequent leaf infection.

[0048] The PCR identification primer sequences for recombinant plasmid YFP-RCA1 are shown in SEQ ID NO:6 and SEQ ID NO:7; the PCR identification primer sequences for recombinant plasmid TRV2-RCA1 are shown in SEQ ID NO:8 and SEQ ID NO:9.

[0049] Example 2: Establishment of a transient transformation system for Rhododendron hainanense leaves 1. Strain activation: Agrobacterium GV3101 strains containing overexpression vectors YFP-RCA1 and YFP-empty, as well as various Agrobacterium GV3101 strains used in the TRV system for preparing TRV-RCA1 and TRV-empty infection solutions, were streaked separately. Single colonies were picked and inoculated into LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampin) and cultured at 28°C in a shaker until OD500. 600 It reaches 1.0~1.2.

[0050] 2. Cell Collection and Resuspension: Centrifuge the bacterial suspension at 6000 rpm for 10 min and discard the supernatant. Resuspend the cells in Agrobacterium infection medium (basal components include MgCl2 and MES (2-morpholinoethanesulfonic acid)). Just before use, add acetylsyleugenone (AS) to a final concentration of 150 μM to adjust the bacterial concentration to OD. 600 The concentration is 1.0~1.2, and the mixture is left to stand at room temperature for 1~2 hours.

[0051] For the TRV system, after the bacterial suspensions of each component were collected, resuspended and induced, before leaf infection, the Agrobacterium suspension carrying the auxiliary carrier TRV1 component was mixed with the Agrobacterium suspension carrying the TRV2-empty empty carrier and recorded as "TRV-empty"; the Agrobacterium suspension carrying the auxiliary carrier TRV1 component was mixed with the Agrobacterium suspension carrying the TRV2-RCA1 carrier and recorded as "TRV-RCA1".

[0052] 3. Minimally invasive pretreatment and selection of explants: Pick tender leaves of Rhododendron hainanense and wipe them clean with water.

[0053] Treatment groups were set up using a scribing method, a manual perforation method, and dense microneedle rollers with needle lengths of 0.2 mm, 0.5 mm, and 1.0 mm, respectively, to treat the back of tender leaves of *Rhododendron hainanense* and compare the treatment effects. The microneedle roller used was a 540-needle steel plate dense microneedle roller (purchased from Jiangsu Great Wall Medical Device Co., Ltd., product registration certificate number: Su Xie Zhu Zhun 20222201004), with a steel plate diameter of 20 mm and a total of 540 needles, each with a needle diameter of 0.2 mm. Only the needle length was varied for comparison. The structure of the dense microneedle roller is as follows: Figure 1 As shown, 1 is the needle tip, 2 is the roller, 3 is the spool, and 4 is the handle.

[0054] Figure 2 In Figure A, the leaf state of each minimally invasive pretreatment group after negative pressure infection is shown, with different degrees of water-soaked infiltration observed in the leaves after different treatments. Although the streak method and manual perforation method can create local wounds, the wound location and depth are uneven, which can easily lead to excessive local damage and patchy infection. The pores formed by the 0.2 mm microneedle roller are relatively shallow. Both the 0.5 mm and 1.0 mm microneedle rollers can form more uniformly distributed micropores on the leaf surface, which is conducive to the entry of bacterial solution into the leaf tissue under negative pressure.

[0055] Considering the uniformity of micropores, the effectiveness of infection, and the integrity of the leaf, a 0.5 mm microneedle roller is preferred as the standard treatment condition, while a 1.0 mm microneedle roller can be used for leaves with a relatively thick cuticle. In this embodiment, during specific operation, a sterilized 0.5 mm dense microneedle roller is used to gently press and roll twice on the back of the leaf, ideally until the leaf has no visible continuous cracks and remains intact.

[0056] 4. Prolonged Negative Pressure Infiltration and Slow Recovery to Normal Pressure: The leaves, pretreated with 0.5 mm microneedles, were completely immersed in the Agrobacterium infection solution and placed in a vacuum desiccator. The vacuum pump was turned on, and the air pressure was reduced to -0.095 MPa and maintained at negative pressure for 2 hours to promote the penetration of the bacterial solution into the leaf tissue. After treatment, the air intake rate was controlled to allow the air pressure inside the device to slowly recover to normal pressure within 20 minutes to reduce the risk of mechanical damage caused by sudden pressure changes. After 0.5 mm microneedle pretreatment combined with the above negative pressure and slow recovery procedure, the leaves exhibited a relatively uniform water-soaked state.

[0057] 5. Agar concentration screening and post-infection semi-liquid culture: Culture system preparation: The basal liquid phase used in this embodiment is sterile water, without adding any other basal culture medium components. Agar powder was weighed and added to sterile water to prepare culture systems with agar concentrations of 0, 1, 2, 3, 4, and 5 g / L, respectively, where 0 g / L was the liquid control without added agar. Each concentration culture system was sterilized by high-temperature autoclaving and then cooled to room temperature for use. Infected detached leaves need to continue culturing in a culture system that can maintain tissue hydration; if directly exposed to air, the leaves are prone to water loss and shriveling. Therefore, this embodiment screened for suitable post-infection culture conditions by adjusting the agar concentration.

[0058] Post-infection culture condition screening and operation: Leaves pretreated with 0.5 mm microneedles and subjected to negative pressure infection were taken out. After negative pressure infection, the bacterial solution had entered or attached to the leaf tissue, but further culture was still required to complete the subsequent Agrobacterium T-DNA transfer and transient expression of the target gene. Excess bacterial solution was washed off the surface and excess water was absorbed. The leaves were then placed in agar concentration systems of 0, 1, 2, 3, 4, and 5 g / L, respectively, and cultured at 20℃ in the dark for 3 days before comparing the leaf condition.

[0059] like Figure 2 As shown in Figure B, when the agar concentration was 0 g / L and 1 g / L, the leaves sank to the bottom of the system and showed obvious browning; when the agar concentration was 2 g / L and 3 g / L, the leaves maintained normal shape and green color, without obvious browning, and the culture condition was good; when the agar concentration was 4 g / L and 5 g / L, the system fluidity decreased, and the leaves showed obvious water loss and shriveling, and the culture condition was poor. Considering the sinking and floating position of the leaves, the degree of browning, and the appearance of the tissue, an agar concentration of 2-3 g / L was finally determined as the optimal agar concentration range for Rhododendron hainanense leaves for semi-liquid phase culture after infection.

[0060] It should be noted that different plants or different types of leaves may have different floating and sinking positions and tissue states in the culture system due to differences in individual leaf weight, thickness, degree of curling, and surface hairs, and the suitable agar concentration range may vary accordingly. In this example, the 2-3 g / L agar concentration is the parameter range obtained by screening young leaves of Hainan Rhododendron through the above-mentioned 0-5 g / L agar concentration gradient experiment.

[0061] After determining the conditions, the subsequent transformation effect verification in this embodiment adopted a 2 g / L agar semi-liquid culture system, and the infected leaves were cultured at 20℃ in the dark for 3 days.

[0062] Example 3: Molecular-level verification of conversion effect 1. GFP fluorescence observation: Leaves of *Rhododendron hainanense* infected with TRV-empty mixed bacterial solution and cultured for 3 days were observed using a laser confocal microscope. Since the empty vector component in the TRV-empty infection system carries a GFP tag, the results are as follows... Figure 3 As shown, compared with the sterile water control group, the leaves of the TRV-empty treatment group showed obvious green fluorescence (GFP), proving that the TRV system can introduce exogenous genes into the leaf cells of Rhododendron hainanense and achieve transient expression.

[0063] 2. Transcription level detection: After wiping the surface moisture of the cultured leaves, total RNA was extracted from the leaves of Rhododendron hainanense in the YFP-RCA1 treatment group, YFP-empty control group, TRV-RCA1 mixed bacterial solution treatment group and TRV-empty control group using the RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit (purchased from Tiangen Biotech Co., Ltd.).

[0064] Agarose gel electrophoresis results showed clear total RNA bands in the leaves of four types of Hainan Rhododendron (e.g.) Figure 4 (As shown), the integrity was good. RNA concentration was determined using NanoDrop, and 1 μg of RNA was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (catalog number RR047A, purchased from TaKaRa). The diluted cDNA was then used as a template for qRT-PCR. qRT-PCR experiments were performed using TB Green dye (purchased from TaKaRa). The instrument used was a BIO-RAD CFX Connect Real-Time System, with 18S rRNA as an internal control. -ΔΔCt The relative expression levels of genes were analyzed and statistical significance was tested.

[0065] Detection RCA1The gene-specific primer sequences are shown in SEQ ID NO:10 and SEQ ID NO:11; the internal reference primer sequences are shown in SEQ ID NO:12 and SEQ ID NO:13.

[0066] The qRT-PCR reaction system consisted of 4 μL cDNA template, 0.5 μL each of forward and reverse primers, and 5 μL of TB Green dye mixture. The qRT-PCR reaction program was as follows: 95℃ for 2 min; 95℃ for 5 s, 58℃ for 30 s, 39 cycles; 95℃ for 5 s; melting curve analysis at 65~95℃.

[0067] like Figure 5 The results showed that, compared with the YFP-empty control group, the YFP-RCA1 bacterial solution treatment group had significantly higher levels of bacteria in the leaves. RCA1 Increased gene expression; compared with the TRV-empty control group, the TRV-RCA1 mixed bacterial solution treatment group showed increased gene expression in leaves. RCA1 The decrease in gene expression indicates that the transient transformation method of the present invention can achieve transient regulation of the expression of the target gene.

[0068] Example 4: RCA1 Verification of the heat resistance function of genes To verify the application of the transient transformation method of the present invention in gene function research, the transient overexpression obtained in Example 2 was analyzed. RCA1 Genes and transient silencing RCA1 Transformed young leaves of *Rhododendron hainanense* were subjected to high-temperature stress treatment. The detached leaves were placed at 42℃ for 12 h, followed by phenotypic observation and staining of physiological indicators. 1. Phenotypic observation: such as Figure 6 As shown, after high-temperature treatment, overexpression RCA1 The gene retains better leaf green color and exhibits less wilting; silencing... RCA1 The gene showed more pronounced leaf browning and wilting, and the phenotype was more severe than that of the corresponding control group.

[0069] 2. Cell death and reactive oxygen species detection: such as Figure 7 As shown, the degree of leaf damage was detected using histochemical staining. All reagents used were purchased from Beijing Coollab Technology Co., Ltd. The specific methods and results are as follows: (1) Trypan blue staining (indicating cell death): Trypan blue phytoscopy was used for detection. Leaves of *Rhododendron hainanense* treated with high temperature were washed with distilled water and dried. The leaves were then completely immersed in trypan blue staining solution and allowed to stand at room temperature for 3 hours. After staining, the leaves were removed and washed with 75% (v / v) ethanol for 30 minutes, repeated three times. Finally, the leaves were immersed in 95% (v / v) ethanol for about 2 days until the chlorophyll was completely removed. It was observed that dead cells were stained blue, such as... Figure 7 The overexpression shown in A is... RCA1 The blue coloration of the gene-treated leaves was significantly lighter than that of the control, indicating less cell necrosis; while the silent leaves... RCA1 The leaves with the most gene-derived coloration indicate severe cell damage.

[0070] (2) DAB staining (indicating H2O2 accumulation): The plant tissue reactive oxygen species detection kit (DAB method) was used for detection. DAB was dissolved in DAB diluent to prepare the staining working solution according to the kit instructions. Leaves were immersed in the working solution and subjected to negative pressure filtration at -0.1 MPa for 30 min. Afterward, the leaves were removed and stained at room temperature in the dark for 12 h until the positive areas (H2O2 accumulation areas) turned reddish-brown. The leaves were then rinsed with distilled water and immersed in 95% (v / v) ethanol in a water bath at 70-80℃ for decolorization until all the green color of the leaves faded. Results are as follows: Figure 7 As shown in B, the H2O2 accumulation site is brownish-red, indicating overexpression. RCA1 The gene-implanted leaves showed less brown accumulation than the control, while the silenced gene... RCA1 The leaves of the gene accumulate more brown color.

[0071] (3) NBT staining (indicating O2) - (Accumulation): The reactive oxygen species (ROS) assay kit for plant tissues (NBT method) was used for detection. Following the kit instructions, NBT was dissolved in NBT diluent to prepare the staining working solution. Leaves were immersed in the working solution and subjected to negative pressure filtration at -0.1 MPa for 30 min. Subsequently, the leaves were removed and stained at room temperature in the dark for 12 h until the positive areas turned deep blue. The leaves were then removed and immersed in 95% (v / v) ethanol and destained in a water bath at 70–80 °C until the green color completely disappeared. Results are as follows: Figure 7 As shown in C, O2 - The accumulation sites appear blue, a trend consistent with DAB staining results, indicating overexpression. RCA1 Genome O2 - Little accumulation, silence RCA1 Genome O2 - A considerable amount has been accumulated.

[0072] The above results indicate that RCA1Transient overexpression and silencing of genes can produce different high-temperature stress responses, indicating that the transient transformation system established in this invention can be used for rapid verification of gene function in Hainan rhododendron.

Claims

1. A method for instantaneous transformation of rhododendron leaves based on minimally invasive pretreatment and semi-liquid phase culture, characterized in that, Includes the following steps: Step 1: Construct an engineered Agrobacterium containing a target gene expression vector, and prepare a bacterial culture of the engineered Agrobacterium; Step 2: Use a microneedle roller to perform a minimally invasive pretreatment on the back of the rhododendron leaf to obtain the pretreated leaf; Step 3: Use the bacterial solution described in Step 1 to infect the pretreated leaves described in Step 2, and obtain the infected leaves. Step 4: Place the infected leaves from Step 3 into an agar semi-liquid culture system for post-infection culture to obtain leaves expressing the target gene; the concentration of agar in the agar semi-liquid culture system is 2~3 g / L.

2. The method for instantaneous transformation of rhododendron leaves according to claim 1, characterized in that, The azalea leaves mentioned are the tender leaves of plants in the genus Rhododendron.

3. The method for instantaneous transformation of rhododendron leaves according to claim 2, characterized in that, The tender leaves of the rhododendron species mentioned are tender leaves of Rhododendron hainanense.

4. The method for instantaneous transformation of rhododendron leaves according to claim 1, characterized in that, The microneedles of the microneedle roller have a needle diameter of 0.1~0.3 mm and a needle length of 0.5~1.0 mm.

5. The method for instantaneous transformation of rhododendron leaves according to claim 1, characterized in that, The Agrobacterium engineered strain mentioned in step 1 is Agrobacterium GV3101.

6. The method for instantaneous transformation of rhododendron leaves according to claim 1, characterized in that, The minimally invasive pretreatment described in step 2 involves rolling a microneedle roller on the back of a rhododendron leaf 1 to 3 times.

7. The method for instantaneous transformation of rhododendron leaves according to claim 1, characterized in that, The infection process described in step 3 includes negative pressure osmosis and restoration to normal pressure.

8. The method for instantaneous transformation of rhododendron leaves according to claim 7, characterized in that, The negative pressure infiltration is performed under pressure conditions of -0.10 to -0.09 MPa for a duration of 1.5 to 2.5 h. The step of restoring normal pressure involves allowing the gas pressure inside the device to return to normal pressure within 15-25 minutes after negative pressure permeation.

9. The application of the method for instantaneous transformation of rhododendron leaves based on minimally invasive pretreatment and semi-liquid phase culture as described in any one of claims 1 to 8, characterized in that, The applications are transient expression of exogenous genes or transient silencing of genes in rhododendron leaves, or applications in gene function research.

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