Agrobacterium rhizogenes-mediated genetic transformation method for olea europaea

Abstract

The invention discloses an agrobacterium rhizogenes-mediated olive genetic transformation method, and belongs to the field of plant genetic engineering. According to the agrobacterium rhizogenes-mediated olea europaea genetic transformation method provided by the invention, rooted olea europaea seedlings are taken as explants, and are subjected to rooting culture after being infected by agrobacterium rhizogenes, so that olea europaea plants with roots are obtained. According to the method, the hairy roots are directly used as research vectors, the steps of callus induction, plant regeneration and the like are omitted, the transgenic efficiency of the olea europaea can be improved to 20% or above, even 35% or above, the hairy roots can be rapidly formed within 3-6 weeks, and the experimental period is greatly shortened.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a method for genetic transformation of olive trees mediated by Agrobacterium rhizogenes. Background Technology

[0002] Olive (Olea europaea L.) is an important woody oilseed plant widely distributed in the Mediterranean region. Olive oil, extracted from the fruit of the olive tree, is considered a nutrient-rich edible oil. It not only plays a vital role in the Mediterranean diet but also enjoys a high reputation in the international market. As an important economic forest species, olive's genetic transformation system has long suffered from low efficiency and regeneration difficulties, severely limiting the progress of gene function research and variety improvement. Traditional Agrobacterium-mediated transformation methods have low success rates in olive, and the regeneration rate of transformed plants is insufficient, making it difficult to obtain stable transgenic lines. Furthermore, olive tissue culture has a long cycle and is prone to browning, further increasing the difficulty of transgenic operations. Therefore, developing an efficient and stable olive genetic transformation system is of great significance for overcoming the bottlenecks in gene function research and accelerating the breeding of superior varieties.

[0003] *Agrobacterium rhizogenes* is a Gram-negative soil bacterium belonging to the Rhizobium family. It transfers T-DNA fragments into plant cell genomes via its Ri plasmid, a process regulated by vir genes on the Ri plasmid. The proteins encoded by these vir genes recognize signaling molecules (such as phenolic compounds) produced at the site of plant injury and initiate the cleavage, transfer, and integration of T-DNA. After being cleaved from the Ri plasmid, the T-DNA fragment enters the plant cell nucleus through the bacterial-plant cell channel and randomly integrates into the plant genome. The T-DNA region of the Ri plasmid contains several key genes, such as rolA, rolB, rolC, and rolD. Expression of these genes in plant cells significantly alters the plant's hormone metabolism and cell division regulation mechanisms, promoting the growth of hairy roots. Hairy roots are characterized by rapid growth, genetic stability, and ease of in vitro culture. They can efficiently synthesize plant secondary metabolites, thus holding significant application value in plant genetic engineering, metabolic engineering, and drug development. Summary of the Invention

[0004] In view of one or more problems existing in the prior art, the present invention aims to solve the technical difficulties of olive transgenic technology, and provide a transgenic method suitable for olive, providing technical support for molecular breeding and functional genomics research of olive.

[0005] One aspect of the present invention provides a method for genetic transformation of olive trees mediated by Agrobacterium rhizogenes, which uses rootless olive seedlings as explants, and after infection with Agrobacterium rhizogenes, carries out root culture to obtain olive trees carrying transgenic root plants.

[0006] In some embodiments, the Agrobacterium rhizogenes is selected from Agrobacterium rhizogenes K599, Agrobacterium rhizogenes MSU440 and Agrobacterium rhizogenes C58C1, preferably Agrobacterium rhizogenes K599.

[0007] In some embodiments, the method for preparing the explant includes the following steps: partially removing the root segment of a sterile olive seedling to obtain a rootless olive seedling as the explant.

[0008] In some embodiments, the sterile olive seedlings are obtained by the following steps: sterilized olive seeds are inoculated into a kernel growth medium and cultured for 1-2 months under a sterile environment with a culture temperature of 23±2 ℃, a light intensity of 2250-2750 lux, and a photoperiod of 16±1 h to obtain the sterile olive seedlings; wherein the kernel growth medium consists of: MS basal medium + 1.4-1.6 mg / L gibberellin + 25-35 g / L sucrose + 2.5-3.5 g / L plant gel.

[0009] In some embodiments, the olive genetic transformation method includes the following operations:

[0010] (1) Explant preparation: The root segment of the sterile olive seedling was partially removed, and the remaining part was used as the explant;

[0011] (2) Infection and co-culture: The cut of the explant is immersed in Agrobacterium rhizogenes infection solution for infection. After infection, the infection solution at the cut of the explant is dried and inserted into the seed kernel growth medium for co-culture to obtain co-cultured explants;

[0012] (3) Insert the co-cultured explants obtained in step (2) into the rooting medium for rooting culture to obtain olive trees carrying transgenic root plants.

[0013] In some embodiments, the co-culture conditions in step (2) are: a culture temperature of 23±2 ℃ and dark culture for 3-4 days; the composition of the seed kernel growth medium is: MS basal medium + 1.4-1.6 mg / L gibberellin + 25-35 g / L sucrose + 2.5-3.5 g / L plant gel.

[0014] In some embodiments, the rooting medium in step (3) consists of: MS basal medium + 1.4-1.6 mg / L gibberellin + 25-35 g / L sucrose + 2.5-3.5 g / L plant gel + 45-55 mg / L kanamycin + 100-150 mg / L termethin.

[0015] In some implementations, the conditions for root cultivation in step (3) include: 23±2 ℃, light cultivation for 16 h / d, dark cultivation for 8 h / d, light intensity of 2250-2750 lux, and optional cultivation for 2-3 weeks.

[0016] In some embodiments, the rooting efficiency of the olive genetic transformation method after two months of rooting culture is over 70%, and the transgenic efficiency is over 20%.

[0017] In some embodiments, the rooting efficiency of the olive genetic transformation method after two months of rooting culture is above 80%, preferably above 90%, and the transgenic efficiency is above 25%, preferably above 35%, and even more preferably above 40%.

[0018] Another aspect of the present invention provides a method for cultivating transgenic olive plants, which includes obtaining olive plants carrying transgenic roots according to the above method, and performing molecular detection on the transgenic olive roots to obtain transgenic olive plants infused with the target gene.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. Traditional transgenic technologies (such as Agrobacterium-mediated leaf disc method and gene gun method) require the regeneration of complete plants through plant tissue, which easily leads to chimerism due to the mixing of transformed and non-transformed cells, making subsequent screening difficult and resulting in poor genetic stability. In contrast, this invention directly induces the differentiation of hairy roots of olive through Agrobacterium rhizogenes. The hairy roots develop from a single transformed cell, resulting in completely homogeneous genotypes, which fundamentally eliminates the risk of chimerism and significantly improves the reliability of transgenic materials.

[0021] 2. Due to its high degree of lignification and imperfect regeneration system, the efficiency of traditional transgenic methods (such as callus transformation) for olive trees is usually less than 5%, and the regeneration cycle is as long as 6-12 months. This invention, through infection with Agrobacterium rhizogenes, increases the transgenic efficiency of olive trees to over 20%, and even over 40%, and hairy roots can form rapidly within 3-6 weeks, significantly shortening the experimental cycle.

[0022] 3. Traditional methods rely on complex plant regeneration systems, while this invention directly utilizes hairy roots as a research vehicle, eliminating steps such as callus induction and plant regeneration, making it particularly suitable for woody plants like olive that are difficult to regenerate. Furthermore, the hairy root system can be directly used for the synthesis of secondary metabolites or the verification of gene function, without waiting for the development of a complete plant, thus accelerating the research process. Attached Figure Description

[0023] Figure 1 These are photographs of the hair roots of the CK control group and the eGFP fluorescent tag group, as detected by the LUYOR-3410RB fluorescent flashlight in Example 1.

[0024] Figure 2 Comparison of PCR detection of the target gene after DNA extraction from wild-type olive roots and transgenic positive roots obtained in Example 1. Detailed Implementation

[0025] This invention aims to provide a method for genetic transformation of olive trees mediated by Agrobacterium rhizogenes using rootless olive seedlings as explants, mainly including the following steps:

[0026] 1) Preparation of sterile olive seedlings: Sterilized olive seeds were inoculated into kernel growth medium and cultured for 1-2 months under sterile conditions with a culture temperature of 23±2 ℃, a light intensity of 2250-2750 lux, and a photoperiod of 16±1 h to obtain sterile olive seedlings; wherein the kernel growth medium may consist of: MS basal medium + 1.4-1.6 mg / L gibberellin + 25-35 g / L sucrose + 2.5-3.5 g / L plant gel;

[0027] 2) Explant preparation: The root segment of the sterile olive seedling is partially removed (for example, the root is removed at the junction of the hypocotyl and the root segment, or the part with part of the hypocotyl is removed above the junction of the hypocotyl and the root segment), and the remaining part is used as the explant.

[0028] 3) Infection and co-culture: The cut ends of the explants are immersed in Agrobacterium rhizogenes infection solution (e.g., prepared by chemical transformation of plasmids into competent Agrobacterium rhizogenes cells). After infection, the infection solution at the cut ends of the explants is aspirated, and the explants are inserted into seed growth medium for co-culture to obtain co-cultured explants. The co-culture conditions are: culture temperature of 23±2 ℃, dark culture for 3-4 days. The composition of the seed growth medium is: MS basal medium + 1.4-1.6 mg / L gibberellin + 25-35 g / L sucrose + 2.5-3.5 g / L plant gel.

[0029] 4) Insert the co-cultured explants obtained in step 3) into the rooting medium for rooting culture to obtain olive trees carrying transgenic roots. The composition of the rooting medium can be: MS basal medium + 1.4-1.6 mg / L gibberellin + 25-35 g / L sucrose + 2-3 g / L plant gel + 40-55 mg / L kanamycin + 100-150 mg / L termethin. The conditions for rooting culture include: 25±2 ℃, light culture for 16 h / d, dark culture for 8 h / d, light intensity of 2250-2750 lux, and culture for 2-3 weeks.

[0030] Based on the olive genetic transformation method provided by this invention, the rooting efficiency after two months of rooting culture is above 70%, preferably above 80%, more preferably above 90%, and the transgenic efficiency is above 20%, preferably above 25%, more preferably above 35%, and even more preferably above 40%.

[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments are only used to further illustrate the present invention and are not intended to limit the scope of the present invention.

[0032] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0033] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials in this invention. In fact, the sources of the biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted according to the instructions in the embodiments. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0034] Example 1: Agrobacterium rhizogenes-mediated genetic transformation of olive trees

[0035] 1) Preparation of sterile olive seedlings

[0036] 1.1) Obtaining olive seeds: Collect mature olives, remove the seeds with shells from the fruit, thoroughly wash away any remaining fruit residue with detergent, rinse thoroughly with clean water, and air dry naturally in a well-ventilated environment for 3 months. After air drying, use a hammer to crack the shells of the seeds to obtain the shell-free kernels.

[0037] 1.2) Disinfection of olive seeds: Collect a quantity of shelled olive kernels from step 1.1), soak them in tap water for 3 days, then drain and transfer them to a clean bench. First, soak them in 75% alcohol for 30 seconds, then rinse them three times with sterile water, each rinse lasting 5 minutes. Next, immerse the seeds in a 10% sodium hypochlorite solution for 15 minutes for disinfection, then rinse them five times with sterile water, each rinse lasting 5 minutes. Finally, soak the disinfected seeds in sterile water for later use.

[0038] 1.3) Cultivation of olive seeds: After disinfection in step 1.2), the seeds were placed on sterile filter paper to absorb surface moisture, and then inoculated into kernel growth medium. The seeds were cultivated for 2 months at a temperature of 23±2 ℃, a light intensity of 2500 lux, and a photoperiod of 16 h. The seeds germinated and developed into sterile seedlings. The kernel growth medium was MS basal medium + 1.5 mg / L gibberellin + 30 g / L sucrose + 2.5 g / L plant gel, and the pH of the medium was 5.7.

[0039] 2) Explant preparation

[0040] Remove the radicle portion of the sterile olive seedling obtained in step 1) (for example, remove the radicle at the junction of the hypocotyl and radicle of the seedling, or remove the portion with the hypocotyl above the junction of the hypocotyl and radicle of the seedling), and use the remaining portion as an explant.

[0041] 3) Infection and co-cultivation

[0042] 3.1) Preparation of Agrobacterium rhizogenes K599 bacterial culture: The pROK2 empty vector plasmid with eGFP enhanced green fluorescent tag was transformed into competent cells of Agrobacterium rhizogenes K599 using chemical transformation.

[0043] 3.1.1) The specific operation of the chemical transformation method of Agrobacterium rhizogenes is as follows: Take K599 competent Agrobacterium rhizogenes (purchased from Shanghai Weidi Biotechnology Co., Ltd.) stored at -80 ℃ and let it partially melt at room temperature. When it is in an ice-water mixed state, insert it into ice. Add 1 μg of pROK2-eGFP plasmid DNA (long-term storage in our laboratory) to every 100 μL of competent cells, mix thoroughly by pipetting, and then place in a series of incubation methods: ice for 5 min, liquid nitrogen for 5 min, water bath at 37 ℃ for 5 min, and ice bath for 5 min. After the ice bath, remove the cells and place them in a clean bench. Add 700 μL of antibiotic-free TY liquid medium (formulation: add 5 g of peptone and 3 g of yeast extract to an Erlenmeyer flask, add water to a volume of 1 L, dissolve completely, and autoclave at 121 ℃ for 20 min; prepare a 1 M calcium chloride aqueous solution and autoclave at 121 ℃ for 20 min; add 10 mL of sterile 1 M calcium chloride aqueous solution to every 1 L of sterilized TY liquid medium. If preparing TY solid medium, add 15 g of agar powder). Incubate at 28 ℃ with shaking for 2 h. Centrifuge at 6000 rpm for 1 min to collect the bacteria. Take about 100 μL of supernatant, gently pipette to resuspend the bacterial block, and spread it on TY solid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin. Invert the medium and incubate at 28 ℃ for 48 h.

[0044] 3.1.2) Select Agrobacterium rhizogenes containing the target plasmid and perform primary culture at 28 °C in TY medium containing 50 mg / L streptomycin and 50 mg / L kanamycin. Then, perform liquid shaking culture at a ratio of 1:50 to expand the culture until the bacterial concentration reaches OD200. 600 =0.4, bacterial cells were collected, and finally the bacterial cells were resuspended in seed kernel growth liquid medium until OD200 reached. 600 =0.4, to be used in subsequent infection experiments.

[0045] 3.2) Infection and Co-culture: The hypocotyls of the explants obtained in step 2) with cuts were immersed in the K599 Agrobacterium rhizogenes infection solution from step 3.1) for 10 min. After infection, the seedlings were removed, and the bacterial solution at the hypocotyl was blotted dry with sterile filter paper. The seedlings were then placed on seed kernel growth medium for co-culture. The culture conditions were 25 ℃ and in the dark for 3 days.

[0046] 4) Culture of transgenic roots

[0047] Remove the seedlings from the co-culture medium after 3 days of co-culture in step 3.2), wash off any remaining Agrobacterium rhizogenes on the surface with sterile water containing 100 mg / L Timentin (CAS No.: 86482-18-0), blot dry on sterile filter paper, and place them in a seed growth medium containing 50 mg / L kanamycin and 125 mg / L Timentin for root development culture. The culture conditions are: 25 ℃, light culture for 16 h / d, dark culture for 8 h / d, and light intensity of 2500 lux.

[0048] 5) Detection of genetically modified roots

[0049] The roots obtained in step 4) were tested for green fluorescence using a Luyang fluorescent flashlight, and the results are as follows: Figure 1 The images shown are photographs of the hair roots of the CK control group (CK being explants with the hypocotyl removed but without infection) and the eGFP fluorescent tag group, respectively, as detected by Lu Yang fluorescent flashlight.

[0050] DNA was extracted from the roots of the control (CK) and transgenic roots separately (using the Jinsha Biotechnology FlaPure Plant DNA Extraction Kit (DE711-50)). The results of PCR cloning of the target eGFP gene are shown below. Figure 2 In the figure, lines 1-5 represent five different transgenic root systems. It can be seen that the CK control group did not have the eGFP target gene band, while the transgenic root systems contained the eGFP target gene band.

[0051] According to the test results in this embodiment, the K599 Agrobacterium rhizogenes infection of the hypocotyl of olive seedlings showed that the seedlings began to grow basal roots after 2 weeks, and the rooting efficiency (represented by the number of seedlings with roots / the number of infected explants) was 95.24% after 2 months, and the transgenic efficiency (represented by the number of positive transgenic plants in the seedlings with roots / the number of infected explants) was 42.86%.

[0052] Example 2: Agrobacterium rhizogenes-mediated genetic transformation of olive.

[0053] Example 2 was performed in accordance with the procedure of Example 1, except that the composition of the seed growth medium and the rooting medium used was different. Specifically, the seed growth medium used in Example 2 consisted of: MS basal medium + 1.4 mg / L gibberellin + 25 g / L sucrose + 2 g / L plant gel; the rooting medium used consisted of: MS basal medium + 1.4 mg / L gibberellin + 25 g / L sucrose + 2 g / L plant gel + 40 mg / L kanamycin + 100 mg / L termethin.

[0054] According to the test results in this embodiment, the K599 Agrobacterium rhizogenes infection of the hypocotyl of olive seedlings showed that the seedlings began to grow dermal roots after 2 weeks, and the rooting efficiency was 91.30% after 2 months, with a transgenic efficiency of 39.13%.

[0055] Example 3: Agrobacterium rhizogenes-mediated genetic transformation of olive trees

[0056] Example 3 was performed in accordance with the procedure of Example 1, except that the composition of the seed growth medium and the rooting medium used was different. Specifically, the seed growth medium used in Example 3 consisted of: MS basal medium + 1.6 mg / L gibberellin + 35 g / L sucrose + 3 g / L plant gel; the rooting medium consisted of: MS basal medium + 1.6 mg / L gibberellin + 35 g / L sucrose + 3 g / L plant gel + 55 mg / L kanamycin + 150 mg / L termethin.

[0057] According to the test results in this embodiment, the K599 Agrobacterium rhizogenes infection of the hypocotyl of olive seedlings showed that the seedlings began to grow dermal roots after 2 weeks, and the rooting efficiency was 88.46% after 2 months, with a transgenic efficiency of 38.46%.

[0058] Example 4: Agrobacterium rhizogenes-mediated genetic transformation of olive.

[0059] This embodiment 4 is performed in accordance with the operation of embodiment 1, with the only difference being:

[0060] In step 3) infection and co-culture, Example 4 prepared a bacterial suspension of *Agrobacterium rhizogenes* MSU440. The pROK2 empty vector carrying an eGFP-enhanced green fluorescent tag was chemically transformed into competent *Agrobacterium rhizogenes* cells of MSU440. Subsequently, the hypocotyls of the explants obtained in step 2) with cuts were immersed in the *Agrobacterium rhizogenes* infection solution of MSU440 for 10 min. After infection, the seedlings were removed, the bacterial suspension at the hypocotyl was blotted dry with sterile filter paper, and they were placed in seed growth medium for co-culture. The culture conditions were 25 ℃, dark culture for 3 days.

[0061] The specific procedure for the chemical transformation method of Agrobacterium rhizogenes is as follows: MSU440 competent cells of Agrobacterium rhizogenes stored at -80 ℃ were partially thawed at room temperature and then inserted into ice while in an ice-water mixture. 1 μg of pROK2-eGFP plasmid DNA was added to every 100 μL of competent cells, and the mixture was pipetted and incubated sequentially in ice for 5 min, liquid nitrogen for 5 min, a 37 ℃ water bath for 5 min, and an ice bath for 5 min. After the ice bath, the cells were removed and placed in a clean bench, and 700 μL of antibiotic-free TY liquid medium was added. The mixture was incubated at 28 ℃ with shaking for 2 h. The cells were collected by centrifugation at 6000 rpm for 1 min. Approximately 100 μL of the supernatant was collected, gently resuspended by pipetting, and spread onto TY solid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin. The medium was then inverted and incubated at 28 ℃ for 48 h.

[0062] Agrobacterium rhizogenes containing the target plasmid was selected and cultured in TY medium containing 50 mg / L streptomycin and 50 mg / L kanamycin at 28 °C for primary inoculation. Then, a liquid-liquid agitation culture was performed at a 1:50 ratio until the bacterial concentration reached OD200. 600 =0.4, bacterial cells were collected, and finally the bacterial cells were resuspended in seed kernel growth liquid medium until OD200 reached. 600 =0.4, to be used in subsequent infection experiments.

[0063] According to the test results in this embodiment, the infection of olive seedling hypocotyl with MSU440 Agrobacterium rhizogenes showed that the seedlings began to grow rhizogenes after 3 weeks, the rooting efficiency was 88% after 2 months, and the transgenic efficiency was 20%.

[0064] Example 5: Agrobacterium rhizogenes-mediated genetic transformation of olive trees

[0065] This embodiment 5 is performed in accordance with the operation of embodiment 1, with the only difference being:

[0066] In step 3) infection and co-culture, Example 5 prepared a bacterial suspension of *Agrobacterium rhizogenes* C58C1. The pROK2 empty vector carrying an eGFP-enhanced green fluorescent tag was chemically transformed into competent *Agrobacterium rhizogenes* C58C1 cells. Subsequently, the hypocotyls of the explants obtained in step 2) with cuts were immersed in the *Agrobacterium rhizogenes* C58C1 infection solution for 10 min. After infection, the seedlings were removed, the bacterial suspension at the hypocotyl was blotted dry with sterile filter paper, and the seedlings were placed on seed growth medium for co-culture. The culture conditions were 25 °C and in the dark for 3 days.

[0067] The specific procedure for the chemical transformation method of Agrobacterium rhizogenes is as follows: C58C1 competent cells of Agrobacterium rhizogenes stored at -80 ℃ are partially thawed at room temperature and then inserted into ice while in an ice-water mixture. 1 μg of pROK2-eGFP plasmid DNA is added to every 100 μL of competent cells, and the mixture is pipetted and incubated sequentially in ice for 5 min, liquid nitrogen for 5 min, a 37 ℃ water bath for 5 min, and an ice bath for 5 min. After the ice bath, the cells are removed and placed in a clean bench, and 700 μL of antibiotic-free TY liquid medium is added. The mixture is then incubated at 28 ℃ with shaking for 2 h. The cells are collected by centrifugation at 6000 rpm for 1 min. Approximately 100 μL of the supernatant is collected, gently resuspended by pipetting, and spread onto TY solid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin. The medium is then inverted and incubated at 28 ℃ for 48 h.

[0068] Agrobacterium rhizogenes containing the target plasmid was selected and cultured in TY medium containing 50 mg / L streptomycin and 50 mg / L kanamycin at 28 °C for primary inoculation. Then, a liquid-liquid agitation culture was performed at a 1:50 ratio until the bacterial concentration reached OD200. 600 =0.4, bacterial cells were collected, and finally the bacterial cells were resuspended in seed kernel growth liquid medium until OD200 reached. 600 =0.4, to be used in subsequent infection experiments.

[0069] According to the test results in this embodiment, the C58C1 Agrobacterium rhizogenes infection of the hypocotyl of olive seedlings showed that the seedlings began to grow rhizogenes after 3 weeks, and the rooting efficiency was 79.17% after 2 months, with a transgenic efficiency of 20.83%.

[0070] Comparative Example 1: Agrobacterium rhizogenes-mediated genetic transformation of olive.

[0071] Comparative Example 1 was conducted according to the procedures of Example 1, except that the composition of the kernel growth medium and the rooting medium used was different. Specifically, the kernel growth medium used in Comparative Example 1 consisted of: MS basal medium + 1.5 mg / L 2,4-D + 0.2 mg / L 6-BA + 30 g / L sucrose + 2.5 g / L plant gel; the rooting medium used consisted of: MS basal medium + 1.5 mg / L 2,4-D + 0.2 mg / L 6-BA + 30 g / L sucrose + 2.5 g / L plant gel + 50 mg / L kanamycin + 125 mg / L termethin.

[0072] The results of testing in this comparative example showed that K599 Agrobacterium rhizogenes infected the hypocotyl of olive seedlings as follows: the seedlings began to grow basal roots after 4 weeks, the rooting efficiency was 47.62% after 2 months, and the transgenic efficiency was 9.52%.

[0073] Comparative Example 2: Agrobacterium rhizogenes-mediated genetic transformation of olive

[0074] Comparative Example 2 was performed following the procedures of Example 1, except that the composition of the rooting medium used was different. Specifically, the rooting medium used in Comparative Example 2 consisted of: MS basal medium + 2 mg / L 2,4-D + 0.5 mg / L 6-BA + 30 g / L sucrose + 2.5 g / L plant gel + 50 mg / L kanamycin + 125 mg / L termethin.

[0075] According to the test results, in this comparative example, the K599 Agrobacterium rhizogenes infection of the hypocotyl of olive seedlings showed that the seedlings began to grow dermal roots after 4 weeks, and the rooting efficiency was 52.63% after 2 months, with a transgenic efficiency of 10.53%.

[0076] Comparative Example 3: Agrobacterium rhizogenes-mediated genetic transformation of olive.

[0077] Comparative Example 3 was performed following the procedures of Example 1, except that the composition of the seed growth medium used was different. Specifically, the seed growth medium used in Comparative Example 3 consisted of: MS basal medium + 2.5 mg / L 2,4-D + 0.5 mg / L 6-BA + 30 g / L sucrose + 2.5 g / L plant gel.

[0078] The results of K599 Agrobacterium rhizogenes infection of the hypocotyl of olive seedlings in this comparative example were as follows: the seedlings began to grow dermal roots after 3 weeks, and the rooting efficiency was 77.27% after 2 months, with a transgenic efficiency of 18.18%.

[0079] Comparative Example 4: Agrobacterium rhizogenes-mediated genetic transformation of olive.

[0080] Comparative Example 4 was conducted following the procedures of Example 1, except that the compositions of the seed growth medium and rooting medium used were different. Specifically, the seed growth medium used in Comparative Example 4 consisted of: MS basal medium + 2.0 mg / L gibberellin + 40 g / L sucrose + 4.0 g / L plant gel; the rooting medium used consisted of: MS basal medium + 2.0 mg / L gibberellin + 40 g / L sucrose + 4.0 g / L plant gel + 60 mg / L kanamycin + 200 mg / L termethin.

[0081] According to the test results, in this comparative example, the K599 Agrobacterium rhizogenes infection of the hypocotyl of olive seedlings showed that the seedlings began to grow dermal roots after 4 weeks, and the rooting efficiency was 45% after 2 months, with a transgenic efficiency of 15%.

[0082] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for genetic transformation of Olea europaea by Agrobacterium rhizogenes, characterized by, The oil olive genetic transformation method uses a de-rooted oil olive seedling as an explant, and after infection by Agrobacterium rhizogenes, the explant is subjected to rooting culture to obtain an oil olive plant with roots.

2. The method for genetic transformation of Olea europaea according to claim 1, characterized by, The Agrobacterium rhizogenes is selected from Agrobacterium rhizogenes K599, Agrobacterium rhizogenes MSU440, and Agrobacterium rhizogenes C58C1, and is preferably Agrobacterium rhizogenes K599.

3. The method for genetic transformation of Olea europaea according to claim 1 or 2, characterized in that, The preparation method of the explant comprises the following operation: a root section of a sterile oil olive seedling is cut off, and the obtained de-rooted oil olive seedling is used as the explant. Optionally, the sterile oil olive seedling is obtained by the following operation: after disinfection, the oil olive seed is inoculated into a kernel growth medium, and is cultured in a sterile environment at a culture temperature of 23±2 ℃, a light intensity of 2250-2750 lux, and a light period of 16±1 h for 1 month to obtain the sterile oil olive seedling. The kernel growth medium comprises the following components: MS basic medium+1.4-1.6 mg / L gibberellin+25-35 g / L sucrose+2.5-3.5 g / L plant gel.

4. The method for genetic transformation of Olea europaea according to any one of claims 1-3, characterized in that, The oil olive genetic transformation method comprises the following operations: (1) Explant preparation: a root section of a sterile oil olive seedling is cut off, and the remaining part is used as the explant; (2) Infection and co-culture: the cut of the explant is immersed in an Agrobacterium rhizogenes infection solution for infection, the infection solution is absorbed after the infection is completed, and the explant is inserted into a kernel growth medium for co-culture to obtain a co-cultured explant; (3) The co-cultured explant obtained in step (2) is inserted into a rooting culture medium for rooting culture to obtain an oil olive plant with roots.

5. The method for genetic transformation of Olea europaea according to claim 4, characterized by, The co-culture in step (2) is performed at a culture temperature of 23±2 ℃ and in darkness for 3-4 days. The kernel growth medium comprises the following components: MS basic medium+1.4-1.6 mg / L gibberellin+25-35 g / L sucrose+2.5-3.5 g / L plant gel.

6. The method for genetic transformation of Olea europaea according to claim 4 or 5, characterized in that, The rooting culture medium in step (3) comprises the following components: MS basic medium+1.4-1.6 mg / L gibberellin+25-35 g / L sucrose+2.5-3.5 g / L plant gel+45-55 mg / L kanamycin+100-150 mg / L timentin.

7. The method for genetic transformation of Olea europaea according to any one of claims 4-6, characterized in that, The rooting culture in step (3) is performed at 23±2 ℃, under light culture for 16 h / d and dark culture for 8 h / d, and at a light intensity of 2250-2750 lux, and is optionally performed for 4-8 weeks.

8. The method for genetic transformation of Olea europaea according to any one of claims 1-7, characterized in that, The oil olive genetic transformation method has a rooting efficiency of 70% or more and a transgenic efficiency of 20% or more after 2 months of rooting culture.

9. The method of genetic transformation of Olea europaea according to claim 8, characterized in that, The oil olive genetic transformation method has a rooting efficiency of 80% or more, preferably 90% or more, and a transgenic efficiency of 25% or more, preferably 35% or more, and further preferably 40% or more after 2 months of rooting culture.

10. A method of breeding a transgenic olea europaea plant, characterized in that, The method comprises obtaining an oil olive plant with transgenic roots by the method in any one of claims 1-9, and performing molecular detection on the transgenic roots of the oil olive to obtain an oil olive plant into which a target gene is introduced.

Images