A genetic transformation method of Eucalyptus grandis mediated by Agrobacterium rhizogenes and its application

Abstract

The invention discloses a genetic transformation method of Eucalyptus grandis mediated by Agrobacterium rhizogenes and its application. The Eucalyptus grandis superior clone DH32-29 of the invention is used as an explant material, and the transformation system is further optimized by screening important factors such as the selection of explants, the type of Agrobacterium strain, the concentration of bacterial solution, the immersion time and the treatment method, and an efficient and stable genetic transformation system of Eucalyptus grandis mediated by Agrobacterium rhizogenes is established, and regenerated plants are successfully obtained, laying a foundation for the identification of specific gene functions of eucalyptus and the genetic improvement of eucalyptus in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic transformation, and particularly relates to a genetic transformation method of Eucalyptus urophylla mediated by Agrobacterium rhizogenes and an application thereof. Background Art

[0002] Eucalyptus is the collective name for plants in the genera Eucalyptus, Corymbia, and Angophora of the Myrtaceae family. Eucalyptus wood is of excellent quality and serves as a primary raw material for the construction, furniture, and paper industries, making significant contributions to my country's timber security and ecological progress. Eucalyptus urophylla × E. grandis, a hybrid of E. urophylla and E. grandis, exhibits significant hybrid vigor, boasts uniform, straight trunks, a short rotation cycle, and a high yield, making it the primary eucalyptus plantation variety in my country. With the rapid development of my country's timber processing industry, the demand for timber resources has also increased. Limited resources have led to an imbalance between supply and demand. To meet this growing demand, researchers have prioritized the development of fast-growing, high-quality, and high-yielding eucalyptus varieties.

[0003] Traditional breeding methods primarily include selection, hybridization, and polyploidy. These methods often suffer from limitations such as long breeding cycles, low success rates for improvement, difficulty in stably inheriting desirable traits, and poor control precision, making them difficult to meet the requirements for rapid, targeted cultivation of new eucalyptus varieties. In the 1980s, Parson et al. first used genetic engineering techniques to transfer exogenous genes into poplar (Populus L.) for expression, successfully producing herbicide-resistant poplars, thus marking the beginning of forest genetic engineering. Compared to traditional breeding, transgenic technology not only circumvents limitations such as reproductive isolation caused by interspecific hybrid sterility, enabling gene exchange and thus achieving stable expression of exogenous desirable genes, but also maintains the desirable traits of the original variety or strain. In recent years, with the vigorous development of genomics and molecular biology, genetic engineering has received increasing attention in optimizing forest variety improvement and accelerating genetic breeding processes, and holds great promise for application. Currently, genetic engineering of eucalyptus primarily focuses on genetic engineering for disease and insect resistance, stress tolerance, herbicide resistance, and wood quality improvement.

[0004] Plant genetic modification techniques primarily include Agrobacterium-mediated transfection, gene gun techniques, polyethylene glycol-mediated transfection, pollen tube transfection, and electroporation. Agrobacterium-mediated transfection is widely used due to its ease of use, low cost, low copy number, minimal gene silencing, and ability to transform larger DNA fragments. Agrobacterium-mediated transfection is divided into Agrobacterium tumefaciens-mediated and Agrobacterium rhizogenes-mediated methods, which contain the tumorigenic Ti plasmid and the rhizogenic Ri plasmid, respectively, and have a wide host range. Under natural conditions, Agrobacterium enters plant cells by infecting wounded parts of the plant and inserts the T-DNA fragments contained in the Ti or Ri plasmid into the plant genome, thereby transferring and integrating foreign genes into the host plant cells. Subsequently, through established plant tissue culture techniques, complete transgenic plants are ultimately obtained, earning them the reputation of being a natural and highly efficient transgenic vector. However, the prerequisite for using Agrobacterium tumefaciens-mediated transformation of plants to obtain transgenic plants is the establishment of an efficient regeneration system, which is also the main reason why only a small number of plants can currently be subjected to transgenic breeding. Forest trees, especially eucalyptus, have high heterozygosity, different varieties and clones have different genetic backgrounds, and they also have a large number of secondary metabolites. Therefore, establishing an efficient regeneration system is difficult, resulting in difficulty in establishing a genetic transformation system for eucalyptus, which in turn affects the identification of functional genes and the development of transgenic breeding. Currently, there are few reports on Agrobacterium rhizogenes-mediated hairy root transformation systems in eucalyptus. Moreover, due to the inconsistent genetic backgrounds of different eucalyptus varieties and clones, the genetic methods also vary. There are no reports on the genetic transformation system of Eucalyptus grandis, a cultivated variety widely planted in South my country. Therefore, the establishment of an efficient and stable Agrobacterium rhizogenes-mediated genetic transformation system for Eucalyptus grandis can provide a powerful tool for further identification of gene functions related to eucalyptus lignin synthesis, secondary metabolite synthesis, etc. Summary of the Invention

[0005] This study aims to utilize plant transgenic technology to establish a simple, rapid, efficient, and stable Agrobacterium rhizogenes-mediated genetic transformation system for Eucalyptus urophylla. This system can achieve stable root transformation in a relatively short period of time. By optimizing key influencing factors in the transformation system, the transformation efficiency can be further improved, resulting in a larger number of transgenic plants and providing stable experimental material for subsequent studies of gene function. Furthermore, preliminary exploration of adventitious bud induction experiments based on eucalyptus hairy roots will provide a new approach and method for the subsequent breeding of new eucalyptus varieties.

[0006] The present invention aims to provide a method for genetic transformation of Eucalyptus urophylla mediated by Agrobacterium rhizogenes, comprising the following steps:

[0007] S1. Take the leaves of the proliferating seedlings cultured in the proliferation medium for 20-25 days as explants, and place the proliferating seedling leaf explants with 1-2 wounds on the surface in a suspension of Agrobacterium rhizogenes MSU440 at OD600 = 0.3 for 30 minutes;

[0008] The soaking process is performed by soaking and ultrasonic treatment for 5 seconds, and shaking is performed every 5 minutes during the soaking process.

[0009] S2. After the infection, the suspension on the surface of the explant was blotted dry with sterile filter paper, and then placed on co-cultivation medium and co-cultivated in the dark for 3 days;

[0010] The co-cultivation medium was composed of 20.0 g / L sucrose, 6.0 g / L agar powder, 50 μM AS, and the balance was WPM;

[0011] S3. After the co-culture, the explants were transferred to hairy root induction medium and cultured for 2-4 weeks to obtain hairy roots;

[0012] The hairy root induction medium is composed of: 20.0 g / L sucrose, 2.5 mg / L PVP, 2.0 g / L PVPP, 0.8 g / L hydrolyzed casein, 200 mg / L Cef, 6.0 g / L agar powder, and the balance is WPM;

[0013] S4. Cut the hairy roots into small segments, inoculate them into a bud induction medium, and culture them until small green buds appear. Then, transfer the small green buds into a proliferation medium for proliferation culture.

[0014] The bud induction medium is composed of the following: 0.2 mg / L TDZ, 0.2-0.5 mg / L IBA, 20.0 g / L sucrose, 6.0 g / L agar powder, and the balance is WPM;

[0015] S5. When the green buds grow to 2-3 cm, transfer them to rooting medium for rooting culture to obtain rooted seedlings.

[0016] Preferably, the proliferation medium is composed of: 0.5 mg / L 6-BA, 0.1 mg / L NAA, 20.0 g / L sucrose, 6.0 g / L agar powder, and the remainder is MS medium.

[0017] Preferably, the proliferated seedlings are robust seedlings with a height exceeding 2 cm.

[0018] Preferably, the frequency of the ultrasonic wave is 50 Hz.

[0019] Preferably, the culture conditions are a light exposure time of 16 h / d and a light intensity of 100 μmol / m 2·s, culture temperature 25±2℃.

[0020] Preferably, the composition of the bud induction medium can also be as follows: 0.2 mg / L TDZ, 0.1 mg / L IAA, and the balance is WPM.

[0021] Preferably, the cutting of the hairy roots into small segments is cutting the hairy roots into small segments with a length of 0.5-1.0 cm.

[0022] Preferably, the rooting medium is composed of: 0.5 mg / L IBA, 20 g / L sucrose, 6 g / L carrageenan, and the balance is 1 / 2 MS.

[0023] Beneficial effects of the present invention:

[0024] The present invention uses the superior asexual line DH32-29 of Eucalyptus urophylla, which is widely planted in South China, as the explant material to establish a simple, rapid, efficient and stable Agrobacterium rhizogenes-mediated genetic transformation system for Eucalyptus urophylla. Stable root transformation can be achieved in a relatively short period of time. The transformation efficiency is improved by optimizing relevant factors during the transformation system, thereby obtaining more transgenic plants. This provides a new approach and method for the future identification of the functions of specific eucalyptus genes (such as genes related to lignin synthesis), and also lays a foundation for the genetic improvement of eucalyptus and the cultivation of new varieties.

[0025] The system established by the present invention successfully obtained a small number of regenerated plants, bringing hope for inducing transgenic regeneration plants based on the hairy roots of Eucalyptus urophylla, providing a new path and method for the rapid cultivation of transgenic plants, and also providing new ideas for other non-model tree species. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effects of different strains and explants on hairy root induction; AF are the hairy root induction of leaves after transformation with control (culture medium without strain), Ar1193, ArQual, ArA4, MSU440, and K599 Agrobacterium; GL are the hairy root induction of stem segments after transformation with control, Ar1193, ArQual, ArA4, MSU440, and K599 Agrobacterium.

[0027] Figure 2 The effects of different strains and explants on the induction rate of hairy roots; A is the induction rate of hairy roots after leaves and stem segments were induced by different Agrobacterium, and B is the length of hairy roots after leaves and stem segments were induced by different Agrobacterium.

[0028] Figure 3Figure 2 is a state diagram of the hairy root growth process; AD is the state of the leaf on the 1st, 7th, 14th and 28th day after infection with Agrobacterium rhizogenes MSU440 in culture on hairy root induction medium; EH is the state of a single leaf on the 1st, 7th, 14th and 28th day after infection with Agrobacterium rhizogenes MSU440 in culture on hairy root induction medium.

[0029] Figure 4 is the effect of MSU440 bacterial solution concentration on hairy root induction; AE are the effects of MSU440 Agrobacterium bacterial solution on hairy root induction when the absorbance OD600 is 0.1, 0.3, 0.5, 0.7, and 0.9, respectively.

[0030] Figure 5 This is the effect of MSU440 bacterial solution concentration on the induction rate of hairy roots and the induction rate of transgenic hairy roots.

[0031] Figure 6 is the effect of immersion time on the induction of hairy roots; AE are the effects of immersion time of 10, 30, 50, 70 and 90 min on the induction of hairy roots.

[0032] Figure 7 The effect of immersion time on the induction rate of hairy roots and the induction rate of transgenic hairy roots.

[0033] Figure 8 D is the effect of different co-culture time on hairy root induction; AD is the effect of co-culture on hairy root induction after 1, 2, 3 and 4 days, respectively.

[0034] Figure 9 The figure shows the effect of co-culture time on the induction rate of hairy roots and the induction rate of transgenic hairy roots.

[0035] Figure 10 is the effect of acetosyringone concentration on hairy root induction; AE is the effect of adding 0, 50, 100, 150, and 200 μM AS to the co-culture medium on hairy root induction.

[0036] Figure 11 The effect of adding different concentrations of acetosyringone to the co-culture medium on the induction rate of hairy roots and the induction rate of transgenic hairy roots.

[0037] Figure 12 AD represents the effects of different treatments on hairy root induction; AD represents the effects of soaking, co-shaking, ultrasonic-assisted treatment for 5s and ultrasonic-assisted treatment for 10s on hairy root induction.

[0038] Figure 13 The results show the effects of different treatments on the induction rate of hairy roots and the induction rate of transgenic hairy roots.

[0039] Figure 14 Figure 3 is the effect of different concentrations of Cef on the induction rate of hairy roots and the length of hairy roots; Figure 3 is the effect of different concentrations of Cef on the induction rate of hairy roots, and Figure 3 is the effect of different concentrations of Cef on the induction rate of hairy roots.

[0040] Figure 15 Figure 5. Effects of different concentrations of kanamycin on the induction rate and length of hairy roots. ...

[0041] Figure 16 The GUS staining results of hairy roots; AC are non-transgenic hairy roots, and DF are successfully transgenic hairy roots.

[0042] Figure 17 PCR identification of hairy roots; from left to right: DNA2000 marker (M), positive control: roots infected with Agrobacterium rhizogenes MSU440 (+), ddH2O (W), negative control: roots not infected with Agrobacterium (-), and hairy roots induced on hairy root induction medium containing 20 mg / L kanamycin (L1-L7).

[0043] Figure 18 Diagram of the regeneration process of adventitious buds induced by hairy roots; AF are the cases of inducing adventitious buds on the bud induction medium for 1d, 7d, 15d, 30d, 45d, and 60d, respectively.

[0044] Figure 19 PCR identification of adventitious buds; from left to right: DNA2000 marker (M), positive control: buds induced by roots infected with Agrobacterium rhizogenes MSU440 (+), negative control: buds induced by roots not infected with Agrobacterium (-), ddH2O (W) and regenerated bud DNA (L1-L4).

[0045] Figure 20 A is a morphological comparison of the regenerated buds and the wild type; A is the wild type proliferated seedling, B is the regenerated bud proliferated seedling, C is the wild type rooted seedling, and D is the regenerated bud rooted seedling.

[0046] Figure 21 This is the flow chart of the impregnation process. DETAILED DESCRIPTION

[0047] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0048] Example 1

[0049] 1. Materials and Methods

[0050] 1.1 Plant materials

[0051] In the following examples, sterile seedlings of the superior clone DH32-29 of Eucalyptus grandis were used as materials. Leaves and stem segments of robust, budding plants exceeding 2 cm in height were selected as explant materials. The formation of hairy roots of eucalyptus was induced by two methods. The transformation system was further optimized by screening important factors such as the type of Agrobacterium strain, bacterial solution concentration, infection time, and treatment method, in order to establish an efficient and stable Agrobacterium rhizogenes-mediated genetic transformation system of Eucalyptus grandis, thereby laying the foundation for the identification of specific gene functions of eucalyptus and genetic improvement of eucalyptus in the future. The seedlings were subcultured for 20-25 days in a proliferation medium (i.e., MS minimal medium (Murashige and Skoog) supplemented with 0.5 mg / L 6-benzylaminopurine (6-BA), 0.1 mg / L α-naphthaleneacetic acid (NAA), 20.0 g / L sucrose, and 6.0 g / L agar powder) under the conditions of 16 h / d of light intensity and 100 μmol / m 2 ·s, culture temperature 25±2° C. The materials used in the following examples were preserved and cultured at the Tropical Forestry Research Institute, Chinese Academy of Forestry.

[0052] 1.2 Strains and vectors

[0053] The plant expression vector used in this experiment is pBI121, which contains the 35S promoter, NOS terminator, NPTⅡ gene, and GUS gene. This vector is maintained in our laboratory. The five strains of Agrobacterium rhizogenes used in this experiment (MSU440, Ar1193, ArA4, ArQual, and K599) were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0054] 1.3 Reagents and culture media

[0055] (1) LB medium: NaCl 10.0 g, Tryptone 10.0 g, Yeast extract 5.0 g, agar powder 15.0 g (add to solid medium), fully dissolve, adjust pH to about 7.0, add water to 1000 mL, and then sterilize by autoclaving at 121°C for 20 min.

[0056] (2) Resuspension medium: Dissolve 2.5 g of WPM (purchased from Duchefa, catalog number M0219) and 20.0 g of sucrose. Adjust the pH to approximately 5.8 and add water to a volume of 1000 mL. Sterilize by autoclaving at 121°C for 20 min. This is used to prepare the bacterial suspension.

[0057] (3) Co-culture medium: Dissolve 2.5 g of WPM, 20.0 g of sucrose, and 6.0 g of agar powder. Adjust the pH to approximately 5.8 and add water to a volume of 1000 mL. Sterilize by autoclaving at 121°C for 20 min.

[0058] (4) Hairy root induction medium: 2.5 g WPM, 20.0 g sucrose, 2.5 mg PVP, 2.0 g PVPP, 0.8 g casein hydrolyzate, 200.0 mg Cef, and 6.0 g agar powder. After fully dissolving, adjust the pH to approximately 5.8 and add water to a volume of 1000 mL. Subsequently, sterilize by autoclaving at 121°C for 20 min.

[0059] (5) Acetosyringone (AS) stock solution: Weigh 0.196 g of acetosyringone powder, add a small amount of DMSO and stir thoroughly to dissolve, then add water to make up to the volume in a 10 mL volumetric flask. Filter and sterilize using a 0.22 μm filter membrane in a clean bench, then aliquot and store.

[0060] (6) Kanamycin (Kan) stock solution: Weigh 1.0 g of kanamycin powder, add water and stir thoroughly to dissolve, then add purified water to a 10 mL volumetric flask, filter sterilize using a 0.22 μm filter membrane in a clean bench, and then aliquot and store.

[0061] (7) Cef storage solution: Weigh 10.0 g of Cef powder, add a small amount of sterile water to fully dissolve it, add purified water to a 50 mL volumetric flask, filter and sterilize using a 0.22 μm filter membrane in a clean bench, and then store in aliquots.

[0062] (8) Thidiazuron (TDZ) stock solution: Weigh 0.01 g of thiazuron powder, add a small amount of 1 M NaOH to fully dissolve it, add pure water to a 100 mL volumetric flask, filter and sterilize using a 0.22 μm filter membrane in a clean bench, and then divide and store.

[0063] (9) Indoleacetic acid (IAA) stock solution: Weigh 0.05 g of indoleacetic acid powder, add a small amount of anhydrous ethanol to fully dissolve it, add pure water to a 100 mL volumetric flask, filter and sterilize using a 0.22 μm filter membrane in a clean bench, and then divide and store.

[0064] (10) 2,4-Dichlorophenoxyacetic acid (2,4-D) stock solution: Weigh 0.1 g of 2,4-D powder, add a small amount of 1 M NaOH to fully dissolve it, add pure water to a 100 mL volumetric flask, filter and sterilize using a 0.22 μm filter membrane in a clean bench, and then divide and store.

[0065] (11) Kinetin (KT) stock solution: Weigh 0.05 g of KT powder, add a small amount of 1 M HCl to fully dissolve it, add pure water to a 100 mL volumetric flask, filter and sterilize using a 0.22 μm filter membrane in a clean bench, and then store in aliquots.

[0066] (12) Indolebutyric acid (IBA) stock solution: Weigh 0.01 g of IBA powder, add a small amount of anhydrous ethanol to fully dissolve it, add pure water to a 100 mL volumetric flask, filter and sterilize using a 0.22 μm filter membrane in a clean bench, and then divide and store.

[0067] (13) Benzyl adenine (6-BA) stock solution: Weigh 0.01 g of thiadiazole powder, add a small amount of 1 M HCl to fully dissolve it, add pure water to a 100 mL volumetric flask, filter and sterilize using a 0.22 μm filter membrane in a clean bench, and then aliquot and store.

[0068] (14) α-Naphthylacetic acid (NAA) stock solution: Weigh 0.05 g of naphthaleneacetic acid powder, add a small amount of 95% ethanol aqueous solution to fully dissolve it, add pure water to a 100 mL volumetric flask, filter and sterilize using a 0.22 μm filter membrane in a clean bench, and then store in aliquots.

[0069] 1.3 Experimental methods

[0070] 1.3.1 Selection and transformation of Agrobacterium rhizogenes strains

[0071] Five different Agrobacterium rhizogenes strains (MSU440, Ar1193, ArA4, ArQual, and K599) were used, and the transformation plasmid was pBI121. The transformation method was chemical transformation.

[0072] 1.3.2 Effects of different treatments on the transformation efficiency of eucalyptus hairy roots

[0073] (1) Optimization of Agrobacterium strains and explants

[0074] Prepare one tube of suspension (OD600 = 0.5) of each of five different Agrobacterium rhizogenes carrying the pBI121 plasmid. Take leaves and stem segments of proliferating seedlings cultured for 20-25 days as explants. Gently incise one or two wounds on the surface of the explants with a scalpel. Place them in suspensions containing different Agrobacterium strains for 30 minutes, mark them, and shake them every 5 minutes. Then, remove the infected explants, blot the surface of the explants with sterile filter paper, place them on co-cultivation medium, and co-cultivate them in the dark for 3 days. Then, transfer them to hairy root induction medium and continue culturing for another 4 weeks. The hairy root induction rate and conversion rate are calculated. Five replicates are performed for each treatment, with 20-25 explants per replicate.

[0075] (2) Optimization of bacterial solution concentration

[0076] After strain and explant optimization, the selected strain was shake-cultured to OD600 values ​​of 0.1, 0.3, 0.5, 0.7, and 0.9, respectively. Equal volumes of suspensions were prepared using resuspension medium, and the prepared explants were placed in each suspension for 30 minutes, with shaking every 5 minutes. The infected explants were then removed, the bacterial suspension on the surface blotted with sterile filter paper, and placed on co-cultivation medium for 3 days in the dark. The explants were then transferred to hairy root induction medium and cultured for another 4 weeks. Hairy root induction and conversion rates were then measured. Five replicates were performed for each treatment, with 20-25 explants per replicate.

[0077] (3) Optimization of soaking time

[0078] After determining the strain, explants, and bacterial solution concentration, we continued to explore the optimal infection time. We placed the prepared explants in the same five tubes of suspension for 10, 30, 50, 70, and 90 minutes. The bacterial solution on the surface of the infected explants was blotted with sterile filter paper. The explants were then placed on co-cultivation medium and incubated in the dark for 3 days. They were then transferred to hairy root induction medium and cultured for another 4 weeks. The hairy root induction rate and conversion rate were calculated. Each treatment was replicated five times, with 20-25 explants per replicate.

[0079] (4) Addition of acetosyringone to the resuspension medium

[0080] Under the specified conditions of bacterial strain, explant, bacterial concentration, and infection time, the suspension was inoculated with 0, 50, 100, 150, and 200 μM acetosyringone. The inoculated explants were blotted dry with sterile filter paper, placed on co-cultivation medium, and incubated in the dark for 3 days. The explants were then transferred to hairy root induction medium and cultured for another 4 weeks. The hairy root induction and conversion rates were then calculated. Five replicates were performed for each treatment, with 20-25 explants per replicate.

[0081] (5) Optimization of co-culture time

[0082] Under defined conditions for bacterial strain, explant, bacterial concentration, infection time, and the amount of acetosyringone added to the suspension, the infected explants were blotted dry with sterile filter paper, placed on co-cultivation medium, and cultured in the dark for 1, 2, 3, and 4 days. The explants were then transferred to hairy root induction medium and cultured for another 4 weeks. The hairy root induction and conversion rates were then calculated. Each treatment was replicated five times, with 20-25 explants per replicate.

[0083] (6) Optimization of processing methods

[0084] Four different infiltration methods were used: immersion for 30 minutes, shaking for 30 minutes, immersion combined with ultrasonic treatment for 5 seconds, and immersion combined with ultrasonic treatment for 10 seconds. Based on the optimized factors of strain type, bacterial solution concentration, infiltration time, and co-incubation time, these treatments were optimized as optimal transformation conditions. Each treatment was replicated five times, with 20-25 explants per replicate. The ultrasonic frequency used was 50 Hz, and the shaking frequency was 100 rpm.

[0085] 1.3.3 Screening of antibiotic concentration

[0086] (1) Determination of Cef antibacterial agent concentration

[0087] Cef was added to the hairy root induction medium at concentrations of 100, 200, 300, 400, and 500 mg / L, respectively. Four weeks later, the hairy root induction rate and root length of the leaves were measured to determine the effect of the Cef inhibitor on hairy root induction. Each treatment was replicated five times, with 20-25 explants per replicate.

[0088] (2) Determination of Kan screening concentration

[0089] Kan was added to the hairy root induction medium at concentrations of 0, 10, 15, 20, 25, 30, and 35 mg / L. After four weeks, the hairy root induction rate and hairy root length were calculated to determine the appropriate screening concentration. Each treatment was replicated five times, with 20-25 explants per replicate.

[0090] 1.3.4 Detection of transgenic hairy roots

[0091] (1) GUS histochemical staining

[0092] The hairy roots obtained by infecting the wild-type strain MSU440 were used as negative controls. The hairy roots on the resistance plate were selected, and all the hairy roots were rinsed with pure water. The surface moisture of the hairy roots was dried with clean filter paper and quickly placed in a 15 mL centrifuge tube with 5-bromo-4-chloro-3-indolyl glucoside (X-gluc) dye solution so that the hairy roots were completely immersed in the dye solution. The centrifuge tube was protected from light with tin foil to prevent the active ingredients in the GUS dye solution from inactivating. It was then placed in a vacuum filtration pump for 30 minutes and transferred to a 37°C incubator in the dark for 12-24 hours. It was decolorized with 70% alcohol by volume until the alcohol was colorless. The 70% alcohol by volume was replaced every 3 hours. The staining was observed with the naked eye and then observed with a stereo microscope and photographed for storage. The staining rate was statistically analyzed.

[0093] (2) DNA extraction (CTAB method)

[0094] a. Use hairy roots infected with wild-type strain MSU440 as a negative control. Take a single hairy root from a resistance plate as a single DNA sample. Take two samples from each resistance plate, repeating the process twice for a total of 10 tubes. Rinse the hairy roots with pure water and dry the surface moisture with clean filter paper. Slightly chop the roots (wipe the scissors clean after each sample) and place them in a 2 mL EP tube. Add 6-8 3-5 mm diameter steel balls and quickly freeze in liquid nitrogen for 1 minute to ensure adequate grinding.

[0095] b. Place the sample rack containing the sample tube in liquid nitrogen for 1 minute to fully freeze the sample rack. Then, place the sample rack on a ball mill as soon as possible, set the frequency to 25-30 Hz, and the oscillation time to 3 minutes.

[0096] c. Add 1 mL of CTAB extract and 10 μL of mercaptoethanol to the sample, incubate in a 60-65°C water bath for 1 hour, and shake every 5 minutes.

[0097] d. Add an equal volume of a mixture of chloroform and isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1), seal the tube, shake well, centrifuge at 12,000 rpm for 10 min, and pipette as much supernatant as possible into a new 1.5 mL EP tube.

[0098] e. Add 2 / 3 volume of refrigerated anhydrous ethanol to the supernatant, shake gently to mix, and place in a -20°C refrigerator for more than 1 hour (or overnight).

[0099] f. Immediately remove the refrigerated sample and centrifuge it at 12,000 rpm for 10 minutes. Slowly discard the supernatant. Add 1 mL of 70% alcohol by volume and centrifuge it at 12,000 rpm for 5 minutes. Slowly discard the supernatant. Add 1 mL of 95% alcohol by volume and centrifuge it at 12,000 rpm for 5 minutes. Slowly discard the supernatant. Invert the tube onto clean toilet paper to absorb the remaining alcohol. Be careful not to let the precipitate flow out. Place the tube upright to dry.

[0100] g. Add 50 μL of 1×TE or ddH2O, let it soak for 5-10 minutes, and then fully dissolve the DNA by bouncing or oscillating.

[0101] h. Add RNase A (final concentration 50.0 μg / mL) to each tube and keep warm in a 37°C water bath for 30 min.

[0102] i. Measure the concentration of each DNA sample, label it, and store it in a -80°C freezer for long-term storage.

[0103] (3) PCR molecular identification

[0104] The rola and rolb gene-specific primers (rola-F / rola-R and rolb-F / rolb-R) listed in Table 1 were used to confirm the hairy root identity of the induced roots. The GUS and NPTII gene-specific primers (GUS-F / GUS-R and NPTII-F / NPTII-R) were used to confirm the stable integration of the reporter gene GUS into the eucalyptus hairy roots. The specific PCR amplification reaction system is shown in Table 2. The PCR reaction conditions were: 94°C for 5 min; 35 cycles of 94°C for 30 s, 58°C / 56°C / 57°C / 55°C (corresponding to the annealing temperatures of rola / rolb / GUS / NPTII, respectively) for 45 s, and 72°C for 45 s; 72°C for 7 min; and a 4°C incubation period. PCR products were analyzed by electrophoresis on a 1.2% (M / V) agarose gel at 180 V.

[0105] Table 1 Primer list

[0106]

[0107]

[0108] Table 2 PCR amplification reaction system

[0109] Ingredients Sample load Ex Taq (5 μmol / μL) 8μL Upstream primer (10 μM) 0.5μL Downstream primer (10 μM) 0.5μL DNA template 1 μL <![CDATA[ddH2O]]> Add water to a total volume of 20 μL

[0110] 1.3.5 Adventitious bud induction based on hairy roots

[0111] One to two weeks after the explants were inoculated, white or red callus appeared at the incision and gradually differentiated into numerous hairy roots densely covered with white root hairs. After culturing on the hairy root induction medium for another one to two weeks (i.e., culturing on the hairy root induction medium for 2 to 4 weeks), the hairy roots reached a length of approximately 2 to 10 cm. Subsequently, rapidly growing, relatively robust hairy roots in the hairy root induction medium were selected, their root tips excised to prevent further elongation, and the hairy roots were cut into 0.5 to 1.0 cm long segments. These were then transferred to bud induction medium containing various plant hormones (as shown in Table 3 below). After two months, the callus induction status and callus condition were recorded and regularly observed. Once the calli on the hairy roots developed small green buds, they were transferred to a proliferation medium for adventitious bud elongation. Six to eight plates were used for each treatment, with 20 to 25 explants per plate.

[0112] The proliferation culture, hairy root induction, bud induction, and rooting culture processes were all carried out in the culture room under the following conditions: a light intensity of 100 μmol / m 2 ·s, culture temperature 25±2℃.

[0113] Table 3 Bud induction medium

[0114]

[0115]

[0116] Note: The basal culture medium used was WPM medium. Different exogenous hormones were added to the WPM medium according to Table 3 to prepare different bud induction media (20.0 g / L sucrose and 6.0 g / L agar powder needed to be added).

[0117] 1.3.6 Data Statistical Analysis

[0118] Hairy root induction rate = number of explants producing hairy roots / total number of explants × 100%;

[0119] Positive transformation rate = number of blue-stained hairy roots / total number of stained roots × 100%;

[0120] Excel and GraphPad Prism 8 were used to record the data of the relevant experiments and draw the bar graphs. SPSS 22.0 analysis software was used for one-way analysis of variance (ANOVA) and Duncan's multiple comparisons (p = 0.05). All percentages were square root arcsine processed before data analysis.

[0121] The calculation formula for the square root arcsine transform is: ASIN(SQRT(X)).

[0122] 2. Results and Analysis

[0123] 2.1 Optimization of hairy root induction and transformation system

[0124] 2.1.1 Effects of different strains and explants on hairy root induction in eucalyptus

[0125] Different strains of Agrobacterium rhizogenes have different abilities to induce hairy roots in plants. In this experiment, five different types of Agrobacterium rhizogenes were selected, including ArA4, Ar1193, K599, MSU440, and ArQual (all five strains are wild-type strains without expression vectors), and the experiments were conducted using leaves and stem segments as explants. Figure 1 As shown in the figure, the five Agrobacterium rhizogenes strains showed obvious differences in the induction of hairy roots of eucalyptus, among which the agrobacterium type ArA4 ( Figure 1 D) and MSU440( Figure 1 The E) strain can induce a large number of hairy roots, while the cucumber base type K599 has almost no hairy roots ( Figure 1F in the figure). It was also found that all five strains were able to produce more hairy roots when using leaves as explants compared to stem segments. MSU440 and ArA4 had the highest hairy root induction rates, at 81.0% and 79.1%, respectively. Slightly different from leaves, ArA4 had the highest hairy root induction rate of 59.2% when using stem segments as explants, followed by MSU440, with a hairy root induction rate of 25.4%. Figure 2 A in the figure). By measuring the length of hairy roots, it was found that the average length of hairy roots induced by Ar1193 was the longest, at 5.2 cm, followed by MSU440, which had an average length of 3.2 cm. Figure 2 Furthermore, MSU440 required a shorter time to induce hairy roots ( Figure 3 ), after 7 days of induction, it can be seen that obvious hairy roots have been induced ( Figure 3 B and Figure 3 F), and then the hairy roots were cultured for 3 weeks to allow them to elongate. After 28 days of induction, the average length of the hairy roots was more than 3 cm ( Figure 3 (D and H in Figures 1 and 2). A comprehensive comparison of hairy root induction rate, root length, and morphology showed that using MSU440 on leaves as explants yielded the best results. Therefore, in subsequent experiments, we optimized the transformation system using Agrobacterium rhizogenes MSU440 as the infection strain and leaves as the recipient material.

[0126] 2.1.2 Effects of different bacterial concentrations on hairy root induction in eucalyptus

[0127] The concentration of Agrobacterium tumefaciens affects the infection efficiency of Agrobacterium rhizogenes to a certain extent. In this experiment, five different concentration gradients of Agrobacterium rhizogenes MSU440 suspensions transformed with expression vector pBI121 were set up. The results are as follows: Figure 4 and Figure 5 As shown in the figure, the concentration of bacterial solution significantly affected the induction rate and conversion rate of hairy roots in eucalyptus leaves. Figure 5As can be seen, both the hairy root induction rate and the conversion rate showed an overall trend of increasing first and then decreasing within the bacterial concentration range of OD600 = 0.1-0.9. At an OD600 of 0.3, the hairy root induction rate peaked at 24.6%, significantly higher than in the other treatments. The hairy root conversion rate also peaked at an OD600 of 0.3, and the transgenic hairy root induction rate (GUS conversion rate) was 20.5%, also significantly different from the other concentration treatments. Compared to wild-type Agrobacterium MSU440, the plasmid-containing Agrobacterium exhibited significantly reduced hairy root induction ability, likely due to the insertion of the exogenous gene and the addition of antibiotics to the hairy root induction medium. Therefore, based on the analysis of the hairy root induction rate and GUS conversion efficiency, an OD600 of 0.3 was selected as the optimal infection concentration for Agrobacterium rhizogenes MSU440.

[0128] 2.1.3 Effects of different immersion times on hairy root induction in eucalyptus

[0129] Based on 2.1.2, this experiment set up 5 different treatments for immersion time. The results are as follows: Figure 6 As shown in the figure, it is more intuitive to show that the immersion time affects the induction results of eucalyptus leaves to a certain extent. Further data statistics and GUS staining analysis show that: within the immersion time range of 10min-90min, the hairy root induction rate and GUS conversion rate both show an overall trend of first increasing and then decreasing ( Figure 7 When the explants were immersed in the immersion solution for 30 minutes, the hairy root induction rate and conversion rate reached their peak values ​​of 23.2% and 20.2%, respectively, and were significantly different from those in the other treatment groups. Therefore, 30 minutes was selected as the optimal immersion time.

[0130] 2.1.4 Effect of co-cultivation time on hairy root induction in Eucalyptus

[0131] If the co-cultivation time is too long, it will lead to excessive reproduction of Agrobacterium, making it difficult to sterilize in the later stage and causing the explants to brown and die. If the co-cultivation time is too short, the number of Agrobacterium attached to the surface of the explants will be small, resulting in a low conversion rate. This experiment set up 4 different co-cultivation times, namely 1, 2, 3, and 4 days. Figure 8 As shown in , different co-cultivation times have a certain degree of influence on the induction rate of hairy roots in eucalyptus leaves. The statistical analysis of the results shows that ( Figure 9), after 2-4 days of co-cultivation, the hairy root induction rate and transformation efficiency of leaf explants were significantly improved compared with the control (co-cultivation time was 1 day). Among them, the hairy root induction rate and transgenic hairy root transformation efficiency of leaf explants reached peak values ​​after 3 days of co-cultivation treatment, with the hairy root induction rate being 28.1% and the transgenic hairy root transformation efficiency (GUS transformation efficiency) reaching 23.9%. Therefore, the co-cultivation time was selected as 3 days for subsequent experiments.

[0132] 2.1.5 Effect of acetosyringone addition on hairy root induction in eucalyptus

[0133] The addition of phenolic substances can induce the activation and expression of Vir genes on the Ri plasmid T-DNA, promoting the entry of Agrobacterium T-DNA into the plant genome. Acetosyringone (AS) is a commonly used phenolic compound. In this experiment, 0, 50, 100, 150, and 200 μM of AS were added to the co-culture medium. The results are shown in Figure 2. Figure 10 and Figure 11 As shown, adding an appropriate amount of AS can improve the transformation efficiency of hairy roots in eucalyptus leaves, but excessive AS can cause leaf browning and death, thereby reducing the transformation effect. When 50 μM acetosyringone was added to the co-culture medium, the hairy root induction rate and the transgenic hairy root induction rate reached their highest peaks, at 44.7% and 32.8%, respectively, representing increases of 18.6% and 15.0%, respectively, compared to the control (without acetosyringone). Therefore, 50 μM acetosyringone was selected for subsequent experiments.

[0134] 2.1.6 Effects of different treatments on hairy root induction in eucalyptus

[0135] This experiment set up four different treatment methods, namely soaking, shaking, ultrasonic assisted treatment for 5 seconds and ultrasonic assisted treatment for 10 seconds, and the soaking time was 30 minutes. Figure 12 and 13 As shown, the ultrasound-assisted treatment group significantly improved the hairy root induction and transformation efficiency of eucalyptus leaves compared to the immersion and co-shaking treatment groups. Both the hairy root induction and transformation efficiency reached their highest values ​​after 5 seconds of ultrasound treatment, with a hairy root induction rate of 52.5% and a transgenic hairy root induction rate of 43.7%. The hairy root transformation efficiency of eucalyptus leaves decreased with increasing ultrasound treatment time. Furthermore, ultrasound-treated explants were found to be mostly reddish-brown in color, with a dense surface of numerous hairy roots, indicating healthy growth. Therefore, 5 seconds of ultrasound-assisted treatment combined with immersion is the optimal treatment for inducing transformation in eucalyptus leaves.

[0136] 2.2 Determination of antibiotic concentration

[0137] (1) Determination of Cef antibacterial agent concentration

[0138] In order to inhibit the growth of Agrobacterium, an appropriate amount of antibacterial growth factor should be added. At the same time, the addition of antibacterial agents may affect the induction and growth of hairy roots. In this experiment, different concentrations of Cef were added to the hairy root induction medium to determine the appropriate antibacterial agent concentration. The Cef concentration was set at 100, 200, 300, 400, and 500 mg / L. The results are shown in Figure 2. Figure 14 As shown in the results, within a certain concentration range, different Cef concentrations had no significant effect on the induction rate and length of hairy roots in Eucalyptus grandis leaves. At a 200 mg / L addition, Agrobacterium in the culture medium was largely inhibited within a certain timeframe. Considering the hairy root induction rate, phenotype, and economic benefits, the optimal Cef inhibitory concentration was determined to be 200 mg / L.

[0139] (2) Determination of Kan screening concentration

[0140] On the basis of adding Cef at an antibacterial concentration of 200 mg / L, untransformed eucalyptus leaves were inoculated into hairy root induction medium containing different concentrations of kanamycin to determine the appropriate Kan screening pressure. Figure 15 As shown, Kan concentration significantly affected the induction rate and length of hairy roots. Compared with the control (no kanamycin), increasing kanamycin concentration significantly inhibited hairy root growth and shortened hairy root length. At a concentration of 20 mg / L, hairy root growth was completely inhibited. Therefore, 20 mg / L of Kan was selected as the optimal concentration for screening transgenic hairy roots.

[0141] 2.3 Detection of transgenic hairy roots

[0142] 2.3.1 GUS staining of hairy roots

[0143] Agrobacterium rhizogenes MSU440 was selected, leaves were used as explants, the OD600 of the Agrobacterium suspension was 0.3, the immersion time was 30 min and ultrasonic treatment was 5 s, and the explants were co-cultured in a co-culture medium with an addition of 50 μM AS for 3 days. The explants were transferred to a hairy root induction medium supplemented with 20 mg / L Kan and continued to be cultured for 20-35 days. The hairy roots reached 2-10 cm in length, and the hairy roots on the resistance plate were selected for GUS histochemical staining analysis ( Figure 16 Results showed that the GUS gene had successfully integrated into the genome of eucalyptus hairy roots induced by Agrobacterium rhizogenes. Kan screening revealed a hairy root induction rate of 52.5%, of which 83.2% showed a color reaction, resulting in a conversion rate (transgenic hairy root induction rate) of 43.7%. (The color reaction indicated that the GUS gene had integrated into the genome.)

[0144] 2.3.2 Molecular detection of hairy roots

[0145] The results are as follows Figure 17 : Specific fragments were successfully amplified, and their band sizes were consistent with the target gene sizes, namely 496bp (GUS gene), 742bp (NPTII gene), 519bp (rola gene), and 423bp (rolb gene), respectively. Among them, L1-L7 successfully amplified rola and rolb genes, confirming that eucalyptus hairy roots had been successfully obtained, and L1-L3 and L6-L7 successfully amplified GUS and NPTⅡ genes, confirming that transgenic hairy roots had been successfully obtained.

[0146] 2.4 Adventitious bud induction based on hairy roots

[0147] 2.4.1 Induction of adventitious buds

[0148] Previous studies have shown that hairy roots induced by wild-type MSU440 exhibit stable integration, a higher hairy root induction rate, and faster growth, making them more suitable for subsequent experimental material requirements than plasmid-transformed strains. Therefore, the hairy roots used in this experiment were all derived from the Eucalyptus grandis hairy root induction system and wild-type MSU440 strains, using the optimal combination of conditions screened above. Six different exogenous hormones (TDZ, IBA, 6-BA, IAA, 2,4-D, and KT) were added to WPM basal medium, resulting in 14 different combinations to identify the exogenous hormone combination that best induced hairy root calli of eucalyptus.

[0149] Table 4 shows that the three shoot induction media (WPM + 0.2 mg / L TDZ + 0.1 mg / L IAA (combination 5), WPM + 0.2 mg / L TDZ + 0.2 mg / L IBA (combination 2), and WPM + 0.2 mg / L TDZ + 0.5 mg / L IBA (combination 3)) achieved high callus induction rates of 19.5%, 37.4%, and 45.9%, respectively. The induced calli were pink in color and had a loose structure. In contrast, the calli in the TDZ and 2,4-D combination all showed yellowish-white color, loose structure, and lack of differentiation ability, eventually turning brown and dying. Therefore, based on these results, further development of shoot induction media for higher-quality calli is warranted.

[0150] Table 4 Addition of external hormones in bud induction medium and its effects

[0151]

[0152]

[0153] 2.4.2 Obtaining hairy root regenerated plants

[0154] Hairy roots were cultured on different bud induction media as shown in Table 4 for 1-2 months ( Figure 18 ), a total of 4 regenerated buds were obtained, of which 2 regenerated buds were obtained with WPM+TDZ 0.2mg / L+IBA0.2mg / L (combination 2), 1 regenerated bud was obtained with WPM+TDZ 0.2mg / L+IAA0.1mg / L (combination 5), and 1 regenerated bud was obtained with WPM+TDZ 0.2mg / L+KT 0.5mg / L (combination 13). When the buds grew to about 1cm, they were transferred to a proliferation medium for proliferation culture to form a large number of proliferation buds. 2-3cm long proliferation buds were cut and transferred to a rooting medium (the rooting medium composition is: 1 / 2MS medium + 0.5mg / L IBA + 20g / L sucrose + 6g / L carrageenan) to induce rooting. Figure 20 It can be found that compared with the wild-type propagated seedlings and rooted seedlings, the propagated seedlings and rooted seedlings of regenerated buds phenotypically exhibit dwarf plants, compact plant shape, shortened internodes, wrinkled leaves, curled leaf edges and dark green leaf color. They also show strong rooting ability, with thick roots and densely covered root hairs on the surface.

[0155] The CTAB method was used to extract the leaf DNA of the regenerated plants of eucalyptus hairy roots, and PCR amplification was performed using the specific primers of the rola and rolb genes of Agrobacterium rhizogenes (Table 1) to confirm that the regenerated plants had integrated the hairy root-inducing genes from the Ri plasmid of Agrobacterium rhizogenes. Figure 19 As shown, the four regenerated shoots all integrated the rola and rolb genes, confirming that the approach of inducing adventitious shoots based on the hairy roots of Eucalyptus urophylla to obtain transgenic plants is feasible.

[0156] 3. Conclusion

[0157] (1) The genetic transformation system mediated by Agrobacterium rhizogenes was established with leaves as explants. The seven important influencing factors, including Agrobacterium rhizogenes strain type, explant type, bacterial solution concentration, immersion time, co-cultivation time, the amount of acetosyringone added to the immersion solution, and treatment method, were comprehensively analyzed. The final conclusion was that the best genetic transformation system was selected, with Agrobacterium rhizogenes MSU440, leaves as explants, OD600 of 0.3, immersion time of 30 min (immersion combined with ultrasonic-assisted treatment for 5 s), co-cultivation in a co-cultivation medium with an addition of 50 μM AS for 3 days, and ultrasonic treatment for 5 s. After screening and cultivation with 20 mg / L kanamycin, PCR molecular identification and GUS staining confirmed that the exogenous gene was stably integrated into the genome of the eucalyptus hairy roots, and the transformation rate reached 43.7%. The genetic transformation system of Eucalyptus grandis DH32-29 leaves mediated by Agrobacterium rhizogenes was preliminarily established (for the specific process, see Figure 21 ).

[0158] (2) Based on the induction of adventitious buds from eucalyptus hairy roots, the combination of 0.2 mg / L TDZ with a lower concentration of IAA (0.1 mg / L) and the combination of 0.2 mg / L TDZ with higher concentrations of IBA (0.2 mg / L and 0.5 mg / L) resulted in higher callus induction rates and the differentiation of a small number of regenerated buds. Leaf DNA from regenerated plants of eucalyptus hairy roots was extracted using the CTAB method, and PCR amplification was performed using primers specific for the rola and rolb genes of Agrobacterium rhizogenes. This confirmed that the regenerated plants had integrated the hairy root-inducing genes from the Ri plasmid of Agrobacterium rhizogenes.

Claims

1. A method for genetic transformation of Eucalyptus grandis mediated by Agrobacterium rhizogenes, characterized in that: The following steps are involved: S1. Take the leaves of the proliferating seedlings cultured in the proliferation medium for 20-25 days as explants, and place the proliferating seedling leaf explants with 1-2 wounds on the surface in the MSU440 Agrobacterium rhizogenes suspension with OD600 = 0.3 for 30 minutes; The dyeing is performed by immersion combined with ultrasonic treatment at a frequency of 50 Hz for 5 seconds, and shaking once every 5 minutes during the immersion period; S2. After the infection, the suspension on the surface of the explant was dried with sterile filter paper, and then placed on the co-culture medium and co-cultured in the dark for 3 days; The co-culture medium was composed of: 20.0 g / L sucrose, 6.0 g / L agar powder, 50 μM AS, and the balance was WPM; S3. After the co-culture, the explants are transferred to a hairy root induction medium and cultured for 2-4 weeks to obtain hairy roots; The composition of the hairy root induction medium is as follows: 20.0 g / L sucrose, 2.5 mg / L PVP, 2.0 g / L PVPP, 0.8 g / L hydrolyzed casein, 200 mg / L Cef, 6.0 g / L agar powder, and the balance is WPM; S4. cutting the hairy roots into small segments, inoculating them into a bud induction medium and culturing them until small green buds grow out, and then transferring the small green buds into a proliferation medium for adventitious bud elongation culture; The bud induction medium is composed of: 0.2 mg / L TDZ, 0.2-0.5 mg / L IBA, 20.0 g / L sucrose, 6.0 g / L agar powder, and the balance is WPM; The proliferation medium is composed of: 0.5 mg / L 6-BA, 0.1 mg / L NAA, 20.0 g / L sucrose, 6.0 g / L agar powder, and the remainder is MS medium; S5. When the green buds grow to 2-3 cm, transfer them to rooting medium for rooting culture to obtain rooted seedlings; The rooting medium is composed of: 0.5 mg / L IBA, 20 g / L sucrose, 6 g / L carrageenan, and the balance is 1 / 2 MS.

2. The method according to claim 1, characterized in that The proliferated seedlings are robust seedlings with a height exceeding 2 cm.

3. The method according to claim 1, characterized in that The culture conditions are as follows: the illumination time is 16 h / d and the illumination intensity is 100 μmol / m 2 ·s, culture temperature 25±2℃.

4. The method according to claim 1, characterized in that: The composition of the bud induction medium can also be as follows: 0.2 mg / LTDZ, 0.1 mg / L IAA, 20.0 g / L sucrose, 6.0 g / L agar powder, and the balance is WPM.

5. The method according to claim 1, characterized in that The method of cutting the hairy roots into small segments is to cut the hairy roots into small segments with a length of 0.5-1.0 cm.

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