An agrobacterium-mediated method for transient transformation of oil palm zygotic embryos

Abstract

This invention provides a method for transient transformation of oil palm zygote embryos based on Agrobacterium-mediated transformation, which employs the Agrobacterium-mediated approach and is based on the optimal Agrobacterium concentration (OD) for stable transformation of oil palm. 600 The optimal Agrobacterium infection time for zygotic embryos was determined by measuring the concentration of acetylsuccinone and co-culture time. The transient GUS expression staining rate was compared between zygotic embryos of different oil palm varieties and at different growth stages. The method described in this invention optimizes infection conditions based on stable transformation parameters, clarifies the optimal explant materials and treatment process, and effectively improves transient transformation efficiency and experimental repeatability. This study establishes for the first time an Agrobacterium-mediated transient transformation system for oil palm zygotic embryos and obtains zygotic embryos most suitable for transient transformation, achieving efficient transient expression of exogenous genes in oil palm zygotic embryos. This provides a rapid and low-cost technical means for studying oil palm gene function.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and more specifically, relates to a method for transient transformation of oil palm zygote embryos based on Agrobacterium-mediated transformation. Background Technology

[0002] Oil palm (Elaeis guineensis Jacq.) is the world's highest-yielding oilseed crop and also a distinctive and advantageous economic crop in Hainan, my country.

[0003] Genetic transformation technology is a core tool for oil palm variety improvement, gene function research, and molecular breeding, and suitable recipient materials are a fundamental prerequisite for achieving efficient genetic transformation. In oil palm genetic transformation, zygotic embryos (ZE) are the most promising recipient materials, including immature embryos (IE) and mature embryos (ME). Compared with other explants, oil palm zygotic embryos are more sensitive to the culture medium, have extremely strong regeneration capabilities, and a shorter cycle for regenerating complete plants, making them one of the commonly used recipient selections in oil palm genetic transformation.

[0004] Transient transformation is a key technology in plant gene function research, rapid expression of exogenous proteins, and metabolic engineering regulation. Its core is to rapidly and temporarily introduce exogenous genes or molecules into plant cells without integrating them into the plant genome. It has the advantages of simple operation, short cycle, and rapid experimental results. It can be widely used for rapid screening and functional verification of exogenous genes, providing important technical references and preliminary foundations for subsequent stable genetic transformation.

[0005] Agrobacterium-mediated transient expression technology has been widely used in transient transformation studies of various plants due to its high transformation efficiency, stable expression of exogenous genes, and minimal damage to plant cells. The key factors affecting the efficiency of Agrobacterium-mediated transient expression are similar to those for stable genetic transformation, mainly including Agrobacterium suspension density, infection time, co-culture time, and acetylsylgenone (AS) concentration. However, the process of obtaining oil palm zygotic embryos is more complex, and their transient transformation efficiency is not only affected by the aforementioned general factors but also by specific factors such as variety (i.e., recipient genotype) and post-pollination development stage. Furthermore, due to the small size of oil palm zygotic embryos, their optimal infection time differs significantly from that of other plant recipient tissues, making it difficult to directly apply existing transient transformation parameters from other plants.

[0006] To date, there are no reports in China on the transient transformation of oil palm zygotic embryos of different varieties and at different post-pollination developmental stages mediated by Agrobacterium. Current technologies cannot meet the demand for efficient transient transformation of oil palm zygotic embryos and are insufficient to provide reliable technical support for oil palm gene function research, molecular breeding, and genetic improvement. Therefore, developing an efficient and stable Agrobacterium-mediated transient transformation method adapted to the characteristics of oil palm zygotic embryos has significant practical implications and application value. Summary of the Invention

[0007] To address the aforementioned problems in the prior art, this invention develops an efficient and stable Agrobacterium-mediated transient transformation method that is adapted to the characteristics of oil palm zygote embryos.

[0008] Specifically, the present invention adopts the following technical solution:

[0009] This invention provides a method for transient transformation of oil palm zygote embryos based on Agrobacterium-mediated transformation, comprising the following steps:

[0010] (1) Obtaining the zygote: Disinfect the oil palm seeds; cut the seed coat to keep the kernel containing the embryo and disinfect it, and then pick out the complete zygote under sterile conditions for transformation;

[0011] (2) Preparation of Agrobacterium infection solution: Agrobacterium containing recombinant plasmid was streaked onto a plate and incubated for propagation; the propagated Agrobacterium was scraped into EgzeSCM liquid culture medium, the bacterial cells were suspended, and the mixture was shaken for 10-30 minutes. The OD of the bacterial solution was then measured. 600 Adjust the value to 0.5-0.6 and keep it in reserve;

[0012] The liquid culture medium EgzeSCM is composed of: Y3 medium + 100~350ml coconut water + 20~45g sucrose + 50~250μM acetylsyleugenone, with a pH of 5.5;

[0013] (3) Infection and co-culture: The zygote was placed in Agrobacterium infection solution for 1-60 minutes, and then the bacterial solution on the surface of the zygote was removed and spread evenly on the co-culture medium EgzeCoM. It was co-cultured at 19-22℃ in the dark for 2-4 days.

[0014] The co-culture medium EgzeCoM consists of: Y3 medium + 100-350 ml coconut water + 20-45 g sucrose + 50-250 μM acetylsylgenone + 0.05-0.5 g / L Cysteine, with a pH of 5.5;

[0015] (4) De-sterilization and cleaning: Use sterile water to clean the co-cultured zygote multiple times until the sterile water is clear; prepare an antibacterial antibiotic solution, transfer the zygote into it and soak for 10 minutes; after pouring out the solution, spread the zygote flat on sterile filter paper and blow dry its surface under sterile conditions to complete the de-sterilization and cleaning process.

[0016] The antibacterial antibiotic solution is a 350-600 mg / L cefotaxime sodium solution or a 350-600 mg / L termethin solution.

[0017] (5) Subculture: The sterilized and cleaned zygotes were transferred to EgzeIM medium and subcultured at a constant temperature of 28°C in the dark. The medium was changed periodically.

[0018] The composition of the culture medium EgzeIM is: Y3 medium + 300~500 mg / L cefotaxime sodium solution or termethin solution + 5~30 mg / L cyproconazole + 100~300 ml coconut water + 25~50 g / L sucrose + 1.5~4 g / L plant gel + 0.5~5 g / L activated carbon, pH 5.5;

[0019] (6) GUS staining identification: The transformation results are identified by GUS staining.

[0020] It should be understood that the composition of the Y3 culture medium described in this invention is: Y3 macro-+Y3 micro-+Y3 iron salt+Y3 organic matter (refer to Eeuwens C. Mineral requirements for growth and callus initiation of tissue explants excised from mature coconut palms (Cocos nucifera) and cultured in vitro [J]. Physiologia Plantarum, 1976, 36(1): 23-8.), which is common knowledge in the field.

[0021] In one or more embodiments, the oil palm seeds in step (1) are selected from: thin-shelled variety seeds after 3 months (3 MAP) of pollination, thin-shelled variety seeds after 6 months (6 MAP) of pollination, thick-shelled variety seeds after 3 months (3 MAP) of pollination, thick-shelled variety seeds after 4 months (4 MAP) of pollination, and thick-shelled variety seeds after 5 months (5 MAP) of pollination; the length of the zygote is 3 mm to 6 mm.

[0022] In one or more embodiments, the step of disinfecting oil palm seeds in step (1) is to soak them in 5%~25% sodium hypochlorite for 5~30 minutes.

[0023] In one or more embodiments, the step of disinfecting the kernel containing the embryo in step (1) is as follows: the kernel is soaked in sodium hypochlorite at a concentration of 15-30%, placed on a shaker, rotated at 120-140 rpm, shaken for 15-30 min, the sodium hypochlorite is discarded, and sterile water is added to wash 5-10 times until it is clear.

[0024] In one or more embodiments, the Agrobacterium in step (2) is Agrobacterium tumefaciens EHA105 or LBA4404 or GV3101.

[0025] In one or more embodiments, the recombinant plasmid in step (2) is pCEiEPSPS-GUS, whose T-DNA nucleotide sequence is shown in SEQ ID NO. 1.

[0026] In one or more embodiments, the infiltration step (3) is as follows: Agrobacterium tumefaciens solution is drawn into a centrifuge tube containing a zygote embryo, the centrifuge tube is gently shaken 20 to 30 times, and then static infiltration is performed.

[0027] In one or more embodiments, the zygote is placed in Agrobacterium infection solution for 5 to 15 minutes in step (3).

[0028] In one or more embodiments, during step (4), the zygote can be gently shaken up and down manually or placed on a shaker during soaking in the antibacterial antibiotic solution.

[0029] In one or more embodiments, the culture medium is changed once every 1 to 3 months during step (5) of subculture.

[0030] In one or more embodiments, in step (6), GUS staining is performed for identification 7 to 210 days after the zygote is infected with Agrobacterium.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] This invention provides a method for transient transformation of oil palm zygote embryos mediated by Agrobacterium. This method employs Agrobacterium-mediated transformation and is based on the optimal Agrobacterium concentration (OD) for stable transformation of oil palm. 600The optimal Agrobacterium infection time for zygotic embryos was determined by measuring the concentration of acetylsuccinone and co-culture time. The transient GUS expression staining rate was compared between zygotic embryos of different oil palm varieties and at different growth stages. The method described in this invention optimizes infection conditions based on stable transformation parameters, clarifies the optimal explant materials and treatment process, and effectively improves transient transformation efficiency and experimental repeatability. This study establishes for the first time an Agrobacterium-mediated transient transformation system for oil palm zygotic embryos and obtains zygotic embryos most suitable for transient transformation, achieving efficient transient expression of exogenous genes in oil palm zygotic embryos. This provides a rapid and low-cost technical means for oil palm gene function research. The establishment of this system overcomes the limitations of traditional oil palm genetic transformation, which involves long cycles and cumbersome operations, and has significant theoretical and applied value for promoting molecular biology research in oil palm and accelerating the molecular breeding process of tropical oil crops. Attached Figure Description

[0033] Figure 1 The image shows the GUS recombinant vector pCEiEPSPS-GUS in Example 1 of this invention; where GUS: β-glucuronidase gene; OsUbip: rice ubiquitin promoter; NOSTer: NOS terminator; EPSPS: glyphosate resistance gene; Ubipro: maize ubiquitin promoter; OsUbiT: rice ubiquitin terminator.

[0034] Figure 2 The following are examples of the effects of different immersion times on the GUS staining rate of 6MAP ​​zygotes of the thin-shelled oil palm in Example 2 of the present invention (a), the effects of different immersion times on the GUS staining rate of 5MAP zygotes of the thick-shelled oil palm (b), the effects of different culture times after immersion of zygotes at different stages of the thin-shelled species on the GUS staining rate (c), and the effects of different culture times after immersion of zygotes at different stages of the thick-shelled species on the GUS staining rate (d); error bars represent standard errors.

[0035] Figure 3This is a comparison of GUS staining at different culture times after zygotic embryos of thin-shelled and thick-shelled oil palms were infected with Agrobacterium in Example 3 of the present invention; where ae represents GUS staining images of 3MAP zygotic embryos of thin-shelled oil palms after 7d, 40d, 80d, 120d, and 210d of induction culture after infection with Agrobacterium; fj represents GUS staining images of 6MAP ​​zygotic embryos of thin-shelled oil palms after 7d, 40d, 80d, 120d, and 210d of induction culture after infection with Agrobacterium; ko represents GUS staining images of 3MAP zygotic embryos of thick-shelled oil palms after 7d, 40d, 80d, and 120d of induction culture after infection with Agrobacterium. GUS staining images of 210d and 210d; pt are GUS staining images of 4MAP zygote embryos of thick-shelled species after incubation with Agrobacterium, induced culture for 7d, 40d, 80d, 120d and 210d respectively; uy are GUS staining images of 5MAP zygote embryos of thick-shelled species after incubation with Agrobacterium, induced culture for 7d, 40d, 80d, 120d and 210d respectively; bar=2mm. Detailed Implementation

[0036] An Agrobacterium-mediated, highly efficient genetic transformation method for oil palm embryogenic callus, comprising the following steps:

[0037] (1) Obtaining the zygotic embryo: Oil palm seeds (thin-shelled varieties 3 months after pollination (3 MAP) and 6 months after pollination (6 MAP), and thick-shelled varieties 3 months after pollination (3 MAP), 4 months after pollination (4 MAP), and 5 months after pollination (5 MAP)) were collected. The seeds were first soaked in 5%–25% sodium hypochlorite solution for 5–30 min. The seed coat was cut off, and the kernel containing the embryo was extracted. This kernel was then soaked in 15–30% sodium hypochlorite solution on a shaker at 120–140 rpm for 15–30 min. The sodium hypochlorite was discarded, and the kernel was washed 5–10 times with sterile water until it became clear. The sterilized kernel containing the embryo was carefully removed from the embryo (approximately 3 mm–6 mm) using the sharp end of a sterile scalpel on a clean bench and placed in a 2 ml centrifuge tube suspension for transformation.

[0038] (2) Preparation of Agrobacterium infection solution: Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105, LBA4404, or GV3101 containing recombinant plasmids was streaked with an inoculation loop. After 36-48 h of colony growth, the single colony was streaked to continue propagation for another 36-48 h, and cultured at a constant temperature of 28℃. The propagated bacteria were scraped into 50 ml of EgzeSCM liquid medium containing 50-250 μm acetylsyringone (AS), the bacterial cells were suspended, and the mixture was shaken for 10-30 min, and the OD was adjusted. 600 =0.5~0.6, for backup;

[0039] The liquid culture medium EgzeSCM consists of: Y3 medium + 100~350ml coconut water + 20~45g sucrose + 50~250μM acetylsyleugenone, pH 5.5;

[0040] The recombinant plasmid is pCEiEPSPS-GUS, and its T-DNA nucleotide sequence is shown in SEQ ID NO. 1.

[0041] (3) Infection and co-culture: Aspirate the suspended bacterial solution into a centrifuge tube containing the immature embryo, gently shake the centrifuge tube 20-30 times and incubate (room temperature, dark) for 0 min, 3 min, 5 min, 15 min, 30 min and 60 min; pour the infected complete zygote into the co-culture medium EgzeCoM, aspirate the bacterial solution, spread it evenly on the medium, and co-culture at 19-22℃ in the dark for 2-4 days;

[0042] The co-culture medium EgzeCoM consists of: Y3 medium + 100~350ml coconut water + 20~45g sucrose + 50~250μM acetylsylgenone + 0.05~0.5 g / L Cysteine, pH 5.5.

[0043] (4) Sterilization and cleaning: After co-culturing for 2-4 days, the zygote embryos are washed multiple times with sterile water until the sterile water is clear; prepare a solution containing antibacterial antibiotics, transfer the zygote embryos into it and soak for 10 minutes, during which time it can be gently shaken up and down manually; after the liquid is poured out, the above zygote embryos are spread flat on sterile filter paper, blown in an ultra-clean workbench for 30 min to 90 min, and turned over several times to dry until dry, remove excess liquid, and let the surface dry;

[0044] The co-culture medium EgzeIM consists of: Y3 medium + 300-500 mg / L cephalosporin or termethin solution + 5-30 mg / L buprofen + 100-300 ml coconut water + 25-50 g / L sucrose + 1.5-4 g / L plant gel + 0.5-5 g / L activated charcoal, pH 5.5; the antibacterial antibiotic solution is 350-600 mg / L cefotaxime sodium solution or 350-600 mg / L termethin solution.

[0045] (5) Subculture: Transfer the surface-dried zygotes to EgzeIM medium and culture them at a constant temperature of 28°C in the dark. Replace the medium once every 1 to 3 months during subculture.

[0046] (6) GUS staining identification: The transient expression effect was identified by histochemical GUS staining, and the staining rate of zygotes was finally counted. Identification was carried out at five time points after immersion (7 d, 40 d, 80 d, 120 d and 210 d).

[0047] GUS staining rate = (Number of GUS-stained calluses / Total number of calluses) × 100%.

[0048] Example

[0049] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0050] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0051] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0053] The oil palm seeds used in the following examples were all from the Coconut Research Institute of the Chinese Academy of Tropical Agricultural Sciences.

[0054] Example 1: Construction of a transient conversion vector for oil palm zygotic embryos

[0055] Using the plant binary expression vector pCEiEPSPS containing the TIPS-EiEPSPS selection gene from previous studies as the backbone vector (Ouyang C, Liu W, Chen S, et al. The Naturally Evolved EPSPS From Goosegrass Confers High Glyphosate Resistance to Rice [J]. Frontiers in PlantScience, 2021, 12: 756116.), the GUS gene expression cassette was ligated into this vector, and the recombinant vector was named pCEiEPSPS-GUS. Figure 1The nucleotide sequence of its T-DNA is shown in SEQ ID NO. 1. This vector plasmid was transformed into Agrobacterium competent cells, and transiently transformed into zygote embryos.

[0056] Example 2: Effect of different immersion times on the GUS staining rate of transient transformation of oil palm zygote embryos

[0057] Appropriate immersion time is a key condition for the successful transient transformation of zygotes.

[0058] This embodiment compares the GUS staining rate of zygote embryos from two oil palm varieties at different developmental stages (6 months after pollination of thin-shelled oil palm (6 MAP) and 5 months after pollination of thick-shelled oil palm (5 MAP)) at different immersion times. The Agrobacterium tumefaciens immersion time was set at six time intervals: 0 min, 3 min, 5 min, 15 min, 30 min, and 60 min. The oil palm zygote embryos were immersed in a solution containing the recombinant plasmid pCEiEPSPS-GUS, 50–250 μM AS, and OD. 600 The embryos were cultured in Agrobacterium solutions with a viscosity of 0.5–0.6. After 7 days of induction culture, the transient GUS staining rate of zygotes was calculated. The GUS staining rate was calculated as: (Number of GUS-stained calluses / Total number of calluses) × 100%.

[0059] The results showed that, for zygotes of the thin-shelled oil palm variety, 6 months after pollination (6 MAP), the GUS staining rate was 100% after 5 min of staining and 7 days of culture; the GUS staining rate was 97.78% after 15 min of staining and 7 days of culture; there was no significant difference between the two. For zygotes of the thick-shelled oil palm variety, 5 months after pollination (5 MAP), the GUS staining rate was 82.22% after 5 min of staining and 7 days of culture, significantly higher than the 68.89% after 15 min of staining and 7 days of culture. Figure 2 (a, b). Based on the above results, the optimal immersion time for zygotic embryos is preferably 5 min.

[0060] Example 3: Comparison of GUS staining rates during transient transformation of zygotic embryos from different varieties and at different growth stages

[0061] To further compare the effects of oil palm variety and zygotic embryo growth period on transient transformation GUS staining, zygotic embryos of thin-shelled varieties 3 months (3 MAP) and 6 months (6 MAP) after pollination, and zygotic embryos of thick-shelled varieties 3 months (3 MAP), 4 months (4 MAP) and 5 months (5 MAP) after pollination, were selected for 5 min staining and GUS staining rate statistics at 5 culture time periods (7 d, 40 d, 80 d, 120 d and 210 d) (the calculation formula is the same as in Example 2).

[0062] Observation and statistics showed that after 7 days of MAP zygote induction culture of the thin-shelled oil palm variety 3, a portion of the embryos were randomly selected for GUS staining. The conical embryos showed no obvious changes, the embryos were relatively soft and collapsed, and no staining was observed. Figure 3 (a); then at 40 d and 80 d, the size of the zygote did not change, and there was no GUS staining (a). Figure 3 (b, c); at 120 d and 210 d, a few embryos were swelling, but most embryos did not change in size and did not show GUS staining ( Figure 3 (d, e). After 7 days of MAP induction culture of zygote embryos of the thin-shelled oil palm variety 6, the randomly selected zygotes showed the highest GUS staining rate, at 100%. As the culture time increased, the zygotes gradually swelled, forming primary callus tissue, and slowly developed into granular embryogenic callus tissue. The amount of granular callus tissue increased, but the GUS staining rate generally showed a decreasing trend. Figure 2 c). For the three stages of zygotic embryos in the thick-shelled oil palm variety, the development of the zygotic embryos was similar to that of the thin-shelled zygotic embryos with increasing culture time, and the GUS staining rate generally showed a decreasing trend; the highest GUS staining rate in the 3MAP zygotic embryos was obtained after 7 days of culture following staining, at 51.11% ( Figure 2 d; Figure 3 The highest GUS staining rate in MAP zygote embryos was obtained after 7 days of culture following infiltration, reaching 82.22%. Figure 2 d; Figure 3 (u~y); however, the highest GUS staining rate of 4MAP zygotes was 97.78% after 40 days of culture following staining, and then showed a downward trend.

[0063] Based on the above results, zygotic embryos of thin-shelled oil palm varieties with 6 MAP and zygotic embryos of thick-shelled oil palm varieties with 4 MAP or 5 MAP are suitable as recipient materials for Agrobacterium-mediated transient transformation; while zygotic embryos with 3 MAP are unstable and not very suitable as recipient materials.

[0064] The above results indicate that the present invention has successfully established an Agrobacterium-mediated transient transformation system for oil palm zygotic embryos and obtained zygotic embryos most suitable for transient transformation, enabling efficient transient expression of exogenous genes in oil palm zygotic embryos, providing a rapid and low-cost technical means for the study of oil palm gene function.

[0065] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for transient transformation of oil palm zygote embryos based on Agrobacterium-mediated transformation, characterized in that, Includes the following steps: (1) Obtaining the zygote: Disinfect the oil palm seeds; cut off the seed coat to keep the kernel containing the embryo and disinfect it, and then pick out the complete zygote under sterile conditions for transformation; (2) Preparation of Agrobacterium infection solution: Agrobacterium containing recombinant plasmid was streaked onto a plate and incubated for propagation; the propagated Agrobacterium was scraped into EgzeSCM liquid culture medium, the bacterial cells were suspended, and the mixture was shaken for 10-30 minutes. The OD of the bacterial solution was then measured. 600 Adjust the value to 0.5-0.6 and keep it in reserve; The liquid culture medium EgzeSCM consists of: Y3 medium + 100-350 ml coconut water + 20-45 g sucrose + 50-250 μM acetylsyleugenone, with a pH of 5.

5. (3) Infection and co-culture: The zygote was placed in Agrobacterium infection solution for 1-60 minutes, and then the bacterial solution on the surface of the zygote was removed and spread evenly on the co-culture medium EgzeCoM. It was co-cultured at 19-22℃ in the dark for 2-4 days. The co-culture medium EgzeCoM consists of: Y3 medium + 100-350 ml coconut water + 20-45 g sucrose + 50-250 μM acetylsylgenone + 0.05-0.5 g / L Cysteine, with a pH of 5.

5. (4) De-sterilization and cleaning: Use sterile water to clean the co-cultured zygote multiple times until the sterile water is clear; prepare an antibacterial antibiotic solution, transfer the zygote into it and soak for 10 minutes; after pouring out the solution, spread the zygote flat on sterile filter paper and blow dry its surface under sterile conditions to complete the de-sterilization and cleaning process. The antibacterial antibiotic solution is a 350-600 mg / L cefotaxime sodium solution or a 350-600 mg / L termethin solution. (5) Subculture: The sterilized and cleaned zygotes were transferred to EgzeIM medium and subcultured at a constant temperature of 28°C in the dark. The medium was changed periodically. The composition of the culture medium EgzeIM is: Y3 medium + 300~500 mg / L cefotaxime sodium solution or termethin solution + 5~30 mg / L cyproconazole + 100~300 ml coconut water + 25~50 g / L sucrose + 1.5~4 g / L plant gel + 0.5~5 g / L activated carbon, pH 5.5; (6) GUS staining identification: The transformation results are identified by GUS staining.

2. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, The oil palm seeds mentioned in step (1) are selected from: thin-shelled variety seeds after 3 months of pollination, thin-shelled variety seeds after 6 months of pollination, thick-shelled variety seeds after 3 months of pollination, thick-shelled variety seeds after 4 months of pollination, and thick-shelled variety seeds after 5 months of pollination; the length of the zygote is 3 mm to 6 mm.

3. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, The steps for disinfecting oil palm seeds in step (1) are as follows: soak them in 5%~25% sodium hypochlorite for 5~30 minutes; the steps for disinfecting the kernel containing the embryo are as follows: place the kernel in a 15~30% sodium hypochlorite solution, place it on a shaker, rotate at 120~140 rpm, shake for 15~30 minutes, discard the sodium hypochlorite, and wash with sterile water 5~10 times until it is clear.

4. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, The Agrobacterium mentioned in step (2) is Agrobacterium tumefaciens EHA105, LBA4404, or GV3101.

5. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, The recombinant plasmid mentioned in step (2) is pCEiEPSPS-GUS, and its T-DNA nucleotide sequence is shown in SEQ ID NO.

1.

6. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, The infiltration step (3) is as follows: aspirate the Agrobacterium tumefaciens solution into a centrifuge tube containing a zygote embryo, gently shake the centrifuge tube 20-30 times, and then allow it to stand for infiltration.

7. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, In step (3), the zygote embryo is placed in the Agrobacterium infection solution for 5 to 15 minutes.

8. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, In step (4), the zygote can be gently shaken up and down manually or placed on a shaker during the soaking in the antibacterial antibiotic solution.

9. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, In step (5), the culture medium should be changed once every 1 to 3 months during the subculture.

10. The method for transient transformation of oil palm zygotic embryos based on Agrobacterium-mediated transformation as described in claim 1, characterized in that, In step (6), GUS staining is performed after the zygote is infected with Agrobacterium and cultured for 7 to 210 days.

Images