Agrobacterium rhizogenes-mediated efficient genetic transformation method for citrus

Abstract

The invention relates to the technical field of plant genetic transformation, and particularly discloses an agrobacterium rhizogenes-mediated efficient genetic transformation method for citrus. The composition is mainly an infection solution and comprises an MS liquid culture medium, agrobacterium rhizogenes K599 containing target genes and acetosyringone. The preparation method comprises the following steps: culturing citrus seedlings, preparing an agrobacterium infection solution containing acetosyringone, trimming the roots of the seedlings, making wounds, wrapping the wounds with cotton slivers soaked with the infection solution, inducing hairy roots through dark culture and light culture, and finally extracting hairy root nucleic acid to identify transgenic plants. The method can be used for citrus genetic improvement, functional gene verification and metabolic engineering research, and a transgenic hairy root material can be efficiently obtained; in addition, the preparation method is easy and convenient to operate, acceptor materials are good in adaptability, the agrobacterium infection stability can be guaranteed, the problems that traditional transformation is low in efficiency and long in period are solved, and the quality of transgenic materials can be accurately confirmed in the identification link.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic transformation, more particularly, it relates to a high-efficiency genetic transformation method of citrus mediated by Agrobacterium rhizogenes. BACKGROUND

[0002] As a global important economic fruit tree, the genetic improvement of citrus is of great significance to enhance disease resistance, improve fruit quality and enhance environmental adaptability. In the research of citrus genetic improvement, Agrobacterium-mediated genetic transformation technology is the core means to realize the targeted introduction of target genes and cultivate excellent varieties. Among them, Agrobacterium rhizogenes, as a gram-negative soil bacterium of Rhizobiaceae Agrobacterium, can infect most dicotyledonous plants including citrus. Since the hairy root genetic transformation system mediated by Agrobacterium rhizogenes was discovered in 1907, it has been widely used in plant functional biology, metabolic engineering and root-rhizosphere interaction research due to its simple operation, rapid transformation and high efficiency. In recent years, it has also been applied in the genetic improvement and disease resistance research of citrus and other fruit trees, providing a reliable technical basis for the optimization of citrus genetic transformation technology.

[0003] However, the current citrus genetic transformation field still relies on Agrobacterium tumefaciens-mediated transformation system, which has significant technical shortcomings. Not only is the transformation efficiency of citrus generally low, only 0.1%-11%, which cannot meet the demand of efficient acquisition of transgenic materials, but also the recovery period of transgenic plants is as long as 6-12 months, and an additional 3-6 months is needed to propagate enough clones, with a total period of 9-18 months. The long period seriously restricts the efficiency of citrus genetic improvement research, and cannot meet the needs of rapid functional gene verification and variety improvement test in actual research. SUMMARY

[0004] In order to solve the problems of low transformation efficiency and long period of Agrobacterium tumefaciens-mediated citrus transformation in the prior art, the present application provides a high-efficiency genetic transformation method of citrus mediated by Agrobacterium rhizogenes.

[0005] A high-efficiency genetic transformation method of citrus mediated by Agrobacterium rhizogenes, comprising the following steps:

[0006] S1, citrus seedling culture: citrus seeds are sown in substrate soil and cultured under light and temperature conditions to obtain seedling with hypocotyl diameter of 1.5-2mm;

[0007] S2, preparation of infection solution: Agrobacterium rhizogenes containing target genes is activated, the concentration of the bacterial solution is adjusted with MS liquid medium, and acetyl cinnamone is added to obtain the infection solution;

[0008] S3, seedling infection treatment: trimming the roots of the seedlings, making wounds on the hypocotyls, and wrapping the wounds with cotton soaked with the infection solution;

[0009] S4, hairy root induction culture: placing the infected seedlings in a high humidity environment for dark culture, and then transferring to light culture conditions to induce hairy roots;

[0010] S5, transgenic plant identification: extracting DNA and RNA from the induced hairy roots for transgenic positive screening and target gene expression level detection.

[0011] By using the above technical scheme, the citrus seeds are sown in the substrate soil and cultured under specific light and temperature conditions to obtain seedlings with a hypocotyl diameter of 1.5-2 mm, which plays a role in providing transformation receptor materials with suitable physiological state and uniform specifications, thereby laying a foundation for subsequent infection operations; the Agrobacterium rhizogenes K599 containing the target gene is activated and a bacterial solution is prepared, and acetosyringone is added, which plays a role in constructing an infection system with transformation ability and promoting T-DNA transfer, thereby providing a carrier for introducing the target gene into citrus cells, wherein the infection solution comprises Agrobacterium rhizogenes K599 bacterial bodies, MS liquid medium, and acetosyringone, wherein the bacterial body concentration is OD600 value 0.65-0.75, and the final concentration of acetosyringone is 50 mg / L; this component range can synergistically optimize the activity of the bacterial bodies and the efficiency of T-DNA transfer, avoiding the failure of infection caused by too low concentration or the toxicity to plants caused by too high concentration; by trimming the roots of the seedlings, making wounds on the hypocotyls, and wrapping the wounds with cotton soaked with the infection solution, the contact area between the Agrobacterium and the receptor cells is increased, and a microenvironment conducive to the attachment and infection of the Agrobacterium is created, thereby improving the introduction efficiency of the target gene; by placing the infected seedlings in a high humidity environment for dark culture and then transferring to light culture conditions, the physiological environment suitable for the occurrence of hairy roots is simulated, and the induction of signal transduction and the differentiation of hairy roots by the Agrobacterium is promoted, thereby realizing the effective induction of hairy roots; by extracting DNA and RNA from the hairy roots and performing transgenic positive screening and target gene expression level detection, respectively, the role of accurately identifying transgenic positive materials and verifying the expression state of the target gene is played, thereby ensuring that the transgenic plants meet the expectations.

[0012] Preferably, in step S1, the culture temperature is 25-27°C, the culture time is 3 or 4 weeks, and healthy seedlings are selected for subsequent experiments.

[0013] By adopting the technical scheme, the seedling is provided with a suitable growth environment to ensure stable development to a specification of 1.5-2 mm in diameter of the hypocotyl, and a receptor material with high activity and physiological state is screened out, thereby providing a uniform and high-activity receptor basis for precise operation of a wound, effective infection of Agrobacterium in a subsequent step, and avoiding influence of inconsistent growth state or insufficient activity of the seedling on stability of a subsequent transformation process.

[0014] Preferably, in step S1, the light cycle of the culture is 16 hours of light and 8 hours of darkness.

[0015] By adopting the technical scheme, a natural light environment suitable for growth of the citrus seedling is simulated, and accumulation of photosynthetic organic matter and physiological metabolism of respiration of the seedling are balanced, thereby ensuring stable development of the seedling to a target specification of 1.5-2 mm in diameter of the hypocotyl, and maintaining a healthy physiological state of the seedling, and providing a physiological-activity-consistent receptor material basis for root pruning, wound making of the hypocotyl, and infection of Agrobacterium in a subsequent step.

[0016] Preferably, in step S2, the activated Agrobacterium rhizogenes K599 is cultured at 28°C for 24-48 hours, and OD 600 of the bacterial solution is determined by using a UV spectrophotometer, and the OD 600 value of the MS liquid medium is adjusted to 0.65-0.75.

[0017] By adopting the technical scheme, a suitable growth temperature is provided for the Agrobacterium rhizogenes K599 to promote its large-scale reproduction and maintain high activity, and the concentration of the bacterial solution is precisely controlled to keep the number and activity of the bacterial solution of each batch consistent, thereby providing the Agrobacterium rhizogenes K599 solution with stable concentration and sufficient activity for a subsequent infection step, avoiding problems of rotting of a wound of the seedling due to too high concentration of the bacterial solution or affecting target gene introduction efficiency due to too low concentration, and ensuring stability of the infection system.

[0018] Preferably, in step S2, the acetyl cinnamone is added to make the final concentration thereof in the infection solution 50 mg / L.

[0019] By adopting the technical scheme, expression of a Vir region gene of the Ri plasmid in the Agrobacterium rhizogenes K599 is activated, and a key signal is provided for T-DNA transfer from the Agrobacterium to the citrus cell, and on the basis of ensuring effective activation of the Vir region gene, the concentration range can avoid problems of insufficient signal due to too low concentration or adverse effects on activity of the Agrobacterium and the wound of the citrus seedling due to too high concentration, thereby providing stable signal support for efficient introduction of the target gene into the citrus cell, and ensuring efficiency and stability of T-DNA transfer in a subsequent infection process.

[0020] Preferably, in step S3, the length of the roots after pruning is 4-5 cm; and the wounds are specifically 3 wounds on each side of the hypocotyl near the root, with a depth of 0.5-1 mm.

[0021] By adopting the above technical solutions, the basic absorption function of the seedling root system is maintained to ensure normal physiological metabolism after infection, the contact area between the hairy root Agrobacterium and the hypocotyl cells is increased to provide more infection sites, and the depth of the wounds is controlled to avoid damaging the core tissue of the hypocotyl and causing the seedling to wither or be infected, thereby creating favorable conditions for the cotton strip soaked in the infection solution to fully contact the wounds, the hairy root Agrobacterium to successfully attach and enter the citrus cells, ensuring the survival ability of the seedling after infection, and providing sufficient recipient cell sites for the introduction of target genes, avoiding problems such as affecting the vitality of the seedling due to improper root length, or causing low infection efficiency or damage to the seedling due to improper wound quantity / depth.

[0022] Preferably, the size of the cotton strip is 2-3 cm in length and 0.8-1.2 cm in width.

[0023] By adopting the above technical solutions, the wounds on both sides of the hypocotyl are precisely covered, and the cotton strip is ensured to fully adhere to the wounds. At the same time, this size can absorb a sufficient amount of infection solution to maintain the effective concentration of the infection solution at the wounds, avoiding rapid loss of the infection solution due to a too small cotton strip, or causing abnormal humidity around the wounds due to a too large cotton strip, thereby providing suitable support for the hairy root Agrobacterium to continuously contact the wound cells and stably complete the infection process, ensuring that each wound has sufficient opportunity to contact the bacterial solution, and avoiding problems such as affecting the infection efficiency or causing damage to the seedling due to improper cotton strip size.

[0024] Preferably, in step S4, the environmental humidity during dark culture is 70%-85%, and the dark culture time is 1-3 days.

[0025] By adopting the above technical solutions, the humidity of the wound environment of the citrus seedling is maintained to prevent the seedling from dehydrating, and suitable humidity conditions are provided for the hairy root Agrobacterium to attach to the wound cells and activate infection-related genes. At the same time, a dark environment is adopted to avoid interference from light on the early infection process of the Agrobacterium, and to ensure that the Agrobacterium has sufficient time to complete the preparation for T-DNA transfer, thereby creating a stable initial environment for hairy root induction on the basis of ensuring the physiological activity of the seedling after infection, avoiding problems such as affecting the attachment of the Agrobacterium due to too low humidity causing the wounds to dry out, or causing the wounds to rot due to too high humidity, or causing insufficient infection of the Agrobacterium due to too short dark culture time, or causing the seedling to grow excessively due to too long dark culture time, thereby providing preliminary protection for the subsequent differentiation of the hairy roots after transfer to light culture.

[0026] Preferably, in step S4, the light intensity of the light condition is 100-150 μmol / m² / s.

[0027] By adopting the above technical solution, suitable photosynthetic energy is provided for citrus seedlings that are transitioned from dark culture to light, balancing their photosynthetic organic matter synthesis and transpiration water consumption. This intensity can meet the accumulation of photosynthetic products required for seedlings to maintain normal physiological metabolism, providing a material basis for hairy root differentiation, while avoiding problems such as excessive light causing seedling leaves to transpire too quickly and wounds to dry out, or insufficient light causing insufficient photosynthetic efficiency and decreased seedling vitality. This ensures a smooth transition of seedlings from dark culture to light environment, providing stable energy and physiological environment support for the continuous differentiation and growth of hairy roots, and ensuring the continuity and stability of the hairy root induction process.

[0028] Preferably, in step S5, the transgene positive screening is performed by PCR identification, and the target gene expression level detection is performed by qRT-PCR analysis.

[0029] By adopting the above technical solutions, PCR identification can amplify the target gene fragment through specific primers, accurately identify positive hairy roots containing the target gene, and exclude negative materials that have not been transformed with the target gene. qRT-PCR analysis can quantify the RNA expression level of the target gene by monitoring fluorescence signals in real time and clearly determine the expression status of the target gene in positive hairy roots. The two work together to complete the identification of transgenic materials from two core dimensions: whether the target gene has been introduced and whether the target gene is effectively expressed. This avoids subsequent experimental biases caused by screening only positive results without confirming the expression status, and provides a reliable identification basis for accurately obtaining chimeric plants with effective expression of the target gene in the roots, ensuring the quality of the final transgenic materials and the accuracy of subsequent functional studies.

[0030] In summary, this application has the following beneficial effects:

[0031] 1. Because this application uses the Agrobacterium rhizogenes K599-mediated genetic transformation system, by optimizing the combination of bacterial concentration and adding acetylsuccinone, and by innovatively using cotton strips to wrap the wound to achieve continuous infection, it has achieved significant results in increasing the hairy root induction rate of multiple citrus varieties to 52.8%-84.6% and shortening the transformation cycle to several weeks.

[0032] 2. In this application, robust seedlings with a hypocotyl diameter of 1.5-2 mm cultivated under specific light and temperature conditions are preferred as recipient materials. As this standardized procedure ensures the consistency of the physiological state of the recipient materials, it achieves a stable effect of improving the success rate and reproducibility of genetic transformation operations.

[0033] 3、The method of the application provides a feasible technical path for obtaining genetically transformed materials under non-tissue culture conditions. By precisely regulating the concentration of Agrobacterium rhizogenes K599 infection liquid to an OD600 value of 0.65-0.75 using an MS liquid culture medium, and by cooperating with the use of 50 mg / L acetyl syringone as a high-efficiency inducer, the transfer and integration of T-DNA are effectively promoted in the whole non-sterile substrate soil culture system, so that high-efficiency induction and stable genetic transformation of citrus hairy roots are realized without the premise of complex tissue culture, and a simpler and faster experimental material obtaining scheme is provided for citrus functional gene research. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a flow chart of a method for preparing Agrobacterium rhizogenes-mediated high-efficiency genetic transformation of citrus provided by the application;

[0035] Figure 2 is a schematic diagram of the infection process and induced hairy roots of the Agrobacterium rhizogenes-mediated genetic transformation method of citrus provided by the application;

[0036] Figure 2 F in the figure is a schematic diagram of the infection process and induced hairy roots of the Agrobacterium rhizogenes-mediated genetic transformation method of citrus provided by the application at the 4th week after treatment;

[0037] Figure 2 G in the figure is a schematic diagram of the infection process and induced hairy roots of the Agrobacterium rhizogenes-mediated genetic transformation method of citrus provided by the application at the 8th week after treatment;

[0038] Figure 3 is a diagram of the occurrence and transgenic root expression identification of 35s-CsCIPK7 transgenic hairy roots of P. trifoliatum provided by the application;

[0039] Figure 4 is a diagram of the occurrence and transgenic root expression identification of 35s-CsCIPK7 transgenic hairy roots of P. trifoliatum provided by the application. DETAILED DESCRIPTION

[0040] The application will be further described in detail below in combination with examples and comparative examples.

[0041] Technical ideas:

[0042] The existing Agrobacterium tumefaciens-mediated citrus genetic transformation technology has the problems of low transformation efficiency and long cycle, the main reasons are as follows: first, the concentration and activity of bacterial liquid lack precise control, the T-DNA transfer signal of Agrobacterium is insufficient, resulting in low target gene introduction efficiency; second, the traditional infection method, the contact between Agrobacterium and receptor cells is insufficient and the continuous action time is short, it is difficult to form stable infection; third, the culture and treatment conditions of the receptor material are not optimized, the size of the seedlings is not uniform, the wound state is not suitable, which further reduces the stability of the transformation; fourth, the culture and identification link after transformation lacks systematic design, which leads to slow hairy root differentiation and insufficient accuracy of transgenic material screening.

[0043] The technical scheme aims at the above problems, adopts Agrobacterium rhizogenes K599 mediated system, optimizes the culture conditions of bacterial liquid, cultures Agrobacterium rhizogenes at 28°C and adjusts the OD600 value of bacterial liquid to 0.65-0.75, and adds acetosyringone to activate the T-DNA transfer signal; innovatively uses a cotton strip of appropriate size to wrap the wound to achieve continuous infection, and combines with the treatment method of accurately trimming the seedling roots to reserve 4-5cm root length and cutting 3 wounds on each side of the hypocotyl to increase the contact area; systematically regulates the temperature, light cycle of seedling culture, humidity, time and light intensity of dark culture, and guarantees the activity of the receptor material and the differentiation of the hairy roots; finally, through the double-dimensional identification method of PCR identification of transgenic positive and qRT-PCR detection of target gene expression, high-quality transgenic materials are efficiently and quickly obtained.

[0044] Preparation Example 1

[0045] The method for constructing Agrobacterium rhizogenes K599 engineering strain containing target gene is as follows:

[0046] Firstly, the coding sequence of the target gene (such as CsCIPK7 gene) is obtained by PCR amplification or gene synthesis; the target gene fragment is subjected to ligation reaction with a plant expression vector subjected to the same enzyme digestion, the plant expression vector is preferably pCAMBIA1300 series or pBI121 series, and carries 35S promoter and terminator beneficial to expression in plants, and antibiotic resistance marker for bacterial screening; the ligation product is transformed into E. coli DH5α competent cells, cultured on LB solid plate containing corresponding antibiotic and subjected to colony PCR screening, and the plasmid of positive clone is extracted for sequencing verification; the recombinant plasmid correctly sequenced is transformed into Agrobacterium rhizogenes K599 competent cells by heat shock or electroporation, and subjected to resistance screening on YEB solid plate containing corresponding antibiotic at 28°C, and finally identified by colony PCR to obtain Agrobacterium rhizogenes K599 engineering strain containing correct recombinant plasmid, which is preserved in glycerol-containing medium at-80°C for subsequent preparation of infection liquid.

[0047] The following are the main raw materials and reagents used in the preparation examples, examples and comparative examples, their sources and specifications are as follows; the reagent not specified is the commercially available analytical pure and above grade product:

[0048] 1, Acetyl eugenitol is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., and the item number is S30264.

[0049] Example 1

[0050] The application provides a kind of Agrobacterium-mediated citrus efficient genetic transformation method provided in the embodiment, comprising the following steps:

[0051] S1, citrus seedling culture: seed of small leaf apricot is sown in matrix soil, and is cultured under light and temperature conditions, to obtain seedling with 1.5mm diameter of hypocotyl;

[0052] Wherein, the culture temperature is 26 DEG C, the culture time is 3 weeks, and the healthy seedling is selected for subsequent test;

[0053] Wherein, the culture light cycle is 16h light, 8h dark.

[0054] S2, preparation of infection solution: activate Agrobacterium rhizogenes K599 containing target gene, adjust OD 600 Value to 0.75 with MS liquid medium;

[0055] The activated Agrobacterium rhizogenes K599 is cultured at 28 DEG C for 36 hours, and the OD 600 Value of bacterial solution is adjusted to 0.75 with MS liquid medium; 600

[0056] Wherein, acetyl eugenitol is added to make its final concentration in the infection solution 50 mg / L.

[0057] S3, seedling infection treatment: wherein, the root of seedling is trimmed, and the wound is made on the hypocotyl, and the wound is wrapped with cotton soaked with infection solution;

[0058] Wherein, the root length is 4.5cm after trimming the root;

[0059] Wherein, the wound is made by cutting 3 wounds on both sides of the hypocotyl near the root, and the wound depth is 0.5mm;

[0060] Wherein, the size of cotton is 2.5cm long and 1.0cm wide.

[0061] S4, hairy root induction culture: the infected seedling is placed in high humidity environment for dark culture, and then transferred to light culture to induce hairy roots; ​

[0062] wherein the environmental humidity during the dark culture is 77%, and the dark culture time is 2 days;

[0063] wherein the light intensity of the light condition is 125 μmol / m2 / s.

[0064] S5, transgenic plant identification: extracting DNA and RNA from the induced hairy roots, and performing transgenic positive screening and target gene expression level detection;

[0065] wherein the transgenic positive screening is PCR identification, and the target gene expression level detection is qRT-PCR analysis.

[0066] Example 2

[0067] The embodiment of the present application provides a high-efficiency genetic transformation method of citrus mediated by Agrobacterium rhizogenes, comprising the following steps:

[0068] S1, citrus seedling culture: sowing small leaf orange seeds in substrate soil, and culturing under light and temperature conditions to obtain seedlings with hypocotyl diameter of 1.8 mm;

[0069] wherein the culture temperature is 25°C, the culture time is 3 weeks, and the healthy seedlings are selected for subsequent experiments;

[0070] wherein the culture light cycle is 16 h light and 8 h darkness.

[0071] S2, preparation of infection solution: activating Agrobacterium rhizogenes K599 containing a target gene, adjusting OD 600 of the bacterial solution to 0.65 by using MS liquid medium, and adding acetosyringone to obtain the infection solution;

[0072] The activated Agrobacterium rhizogenes K599 is cultured at 28°C for 24 hours, and the OD 600 of the bacterial solution is adjusted to 0.65 by using MS liquid medium; 600

[0073] wherein the final concentration of acetosyringone in the infection solution is 50 mg / L.

[0074] S3, seedling infection treatment: trimming the roots of the seedlings, making wounds on the hypocotyls, and wrapping the wounds with cotton soaked with the infection solution;

[0075] wherein the length of the trimmed roots is 4 cm;

[0076] wherein the wound making specifically comprises cutting 3 wounds on the left and right sides of the hypocotyls close to the roots, and the depth of the wounds is 0.75 mm;

[0077] ​The size of the cotton strip is 2 cm in length and 0.8 cm in width.

[0078] S4, hairy root induction culture: the infected seedlings are placed in a high humidity environment for dark culture, and then transferred to a light culture condition to induce hairy roots;

[0079] The environmental humidity during dark culture is 70%, and the dark culture time is 1 day.

[0080] The light intensity of the light culture condition is 100 μmol / m 2 / s.

[0081] S5, transgenic plant identification: DNA and RNA are extracted from the induced hairy roots, and transgenic positive screening and target gene expression level detection are performed;

[0082] The transgenic positive screening is PCR identification, and the target gene expression level detection is qRT-PCR analysis.

[0083] Example 3

[0084] The embodiment of the application provides a high-efficiency genetic transformation method of citrus mediated by Agrobacterium rhizogenes, comprising the following steps:

[0085] S1, citrus seedling culture: the seeds of small leaf orange are sown in the substrate soil, and the seedlings with a hypocotyl diameter of 2 mm are obtained under light and temperature conditions;

[0086] The culture temperature is 27℃, the culture time is 4 weeks, and the healthy seedlings are selected for subsequent experiments;

[0087] The culture light cycle is 16h light and 8h darkness.

[0088] S2, preparation of infection solution: the activated Agrobacterium rhizogenes K599 containing the target gene is adjusted to OD 600 , and acetyl vanillin is added to obtain the infection solution;

[0089] The activated Agrobacterium rhizogenes K599 is cultured at 28℃ for 48 hours, the OD 600 value of the bacterial solution is adjusted by using a UV spectrophotometer, and the OD 600 value is adjusted to 0.70 by using MS liquid medium;

[0090] The acetyl vanillin is added to make the final concentration in the infection solution 50 mg / L.

[0091] S3, seedling infection treatment: the roots of the seedlings are trimmed, wounds are made on the hypocotyls, and the wounds are wrapped with cotton strips soaked with the infection solution;

[0092] The root length is 5 cm after pruning;

[0093] The manufacturing wounds are three wounds with a depth of 1 mm on each of the left and right sides of the hypocotyl near the root;

[0094] The size of the cotton strip is 3 cm in length and 1.2 cm in width.

[0095] S4, hairy root induction culture: the infected seedlings are placed in a high humidity environment for dark culture, and then transferred to a light culture condition for culture to induce hairy roots;

[0096] The environmental humidity during dark culture is 85%, and the dark culture time is 3 days.

[0097] The light intensity of the light culture condition is 150 μmol / m² / s.

[0098] S5, transgenic plant identification: DNA and RNA are extracted from the induced hairy roots for transgenic positive screening and target gene expression level detection;

[0099] The transgenic positive screening is PCR identification, and the target gene expression level detection is qRT-PCR analysis.

[0100] Example 4

[0101] The difference from Example 1 is only that in step S1, Shatian pomelo seeds are used, and the remaining steps are the same as those in Example 1.

[0102] Example 5

[0103] The difference from Example 1 is only that in step S1, sugar orange seeds are used, and the remaining steps are the same as those in Example 1.

[0104] Example 6

[0105] The difference from Example 1 is only that in step S1, local lime seeds are used, and the remaining steps are the same as those in Example 1.

[0106] Example 7

[0107] The difference from Example 1 is only that in step S1, Feilong Citrus seeds are used, and the remaining steps are the same as those in Example 1.

[0108] Example 8

[0109] The difference from Example 1 is only that in step S1, Anjiang sour pomelo seeds are used, and the remaining steps are the same as those in Example 1.

[0110] The hairy root induction rates and transformation rates of different citrus varieties are shown in Table 1.

[0111] Table 1:

[0112] Citrus varieties Hairy root induction rate Hairy root transformation rate Small leaf trifoliate orange 84.6±9.6 56.7±6.7 Flying dragon trifoliate orange 71.1±10.8 63.3±3.3 Ice sugar orange 52.8±12.6 60.0±5.8 Local lime 68.0±8.7 43.3±6.7 Anjiang sour pummelo 82.0±9.7 83.3±8.8 Shatian pummelo 59.3±7.9 66.7±8.8

[0113] Through various embodiments, the present study evaluates the hairy root induction ability and induction rate and transformation rate of a total of 6 citrus varieties including citrus rootstock trifoliate orange, flying dragon, and Hunan local varieties of Bingtang orange, local lime, Anjiang sour pummelo, and Shatian pummelo. The results show that the hairy root induction rate is between 52.8% and 84.6%, with the highest induction rate of trifoliate orange; the hairy root transformation rate is between 43.3% and 83.3%, with the highest transformation rate of Shatian pummelo.

[0114] Comparative Example 1

[0115] The present comparative example adopts the traditional genetic transformation method of Agrobacterium tumefaciens EHA105.

[0116] The present comparative example adopts the conventional Agrobacterium tumefaciens EHA105-mediated citrus genetic transformation method in the art to compare the advantages of the hairy root Agrobacterium K599 system used in the present application in terms of transformation efficiency and cycle. The specific steps are as follows:

[0117] 1. Receptor preparation: The trifoliate orange seedling (hypocotyl diameter 1.5 mm) obtained by culture in Example 1 is used as the receptor material, and the operation is the same as S1 in Example 1.

[0118] 2. Infection and co-culture: Activate Agrobacterium tumefaciens EHA105 containing the same target gene (CsCIPK7), and adjust the OD value of the bacterial solution to 0.75 with MS liquid medium. 600

[0119] 3. Infection treatment: Trim the roots of the seedling, leaving a root length of 4.5 cm; cut 3 wounds on both sides of the hypocotyl near the root, with a wound depth of 0.75 mm; use a cotton strip (length 2.5 cm, width 1.0 cm) soaked with the infection solution to wrap the wounds. The whole process is operated under non-tissue culture conditions without using any antibiotic selection medium.

[0120] 4. Culture and observation: After treatment, the seedling is planted back into the substrate soil, cultured in darkness for 2 days at a humidity of 77%, and then cultured under light intensity of 125 μmol / m² / s, and the hairy root induction is observed regularly.

[0121] 5. Identification: After 8 weeks of culture, the tissue is extracted from the base incision, and the DNA is extracted for PCR identification.

[0122] The test results of the present comparative example are as follows: ​

[0123] Hairy root induction rate: 0% (Agrobacterium tumefaciens EHA105 failed to induce hairy roots under non-tissue culture conditions);

[0124] Transformation rate: no transgenic positive material was obtained by PCR detection;

[0125] Cycle: no effective transformed material was obtained in the 8-week observation period;

[0126] The comparison conclusion of the example is as follows:

[0127] The comparative example proves that under the same non-tissue culture conditions, using the traditional Agrobacterium tumefaciens EHA105 system and using the same receptor material, target gene and physical wounding treatment method as the present application, hairy roots cannot be induced, and no transgenic signal is detected. The results show that under non-tissue culture conditions, the Agrobacterium tumefaciens EHA105 system does not have the ability to induce citrus hairy roots and achieve genetic transformation, while the Agrobacterium rhizogenes K599 system used in the present application can successfully achieve genetic transformation under the same conditions, which reflects the essential difference between the two transformation systems in mechanism and application conditions.

[0128] The specific embodiment is only an explanation of the present application, and is not a limitation of the present application. Those skilled in the art can make modifications to the present embodiment without creative contribution after reading the present specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes, characterized in that: Includes the following steps: S1. Citrus seedling cultivation: Citrus seeds are sown in substrate soil and cultivated under light and temperature conditions to obtain seedlings with a hypocotyl diameter of 1.5-2 mm. S2. Preparation of infection solution: Activate Agrobacterium rhizogenes K599 containing the target gene, adjust the bacterial concentration with MS liquid medium, and add acetylsuccinone to obtain the infection solution; S3. Seedling infection treatment: The roots of the seedlings are pruned, wounds are made on the hypocotyls, and the wounds are wrapped with cotton strips soaked in the infection solution. S4. Hairy root induction culture: The infected seedlings were placed in a high humidity environment for dark culture, and then transferred to light conditions for culture to induce the production of hairy roots. S5. Identification of transgenic plants: DNA and RNA were extracted from the induced hairy roots for transgenic positive screening and detection of the expression level of the target gene.

2. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S1, the culture temperature is 25-27°C, the culture time is 3 or 4 weeks, and robust seedlings are selected for subsequent experiments.

3. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S1, the light cycle for cultivation is 16 hours of light followed by 8 hours of darkness.

4. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S2, the activated Agrobacterium rhizogenes K599 was cultured at 28°C for 24-48 hours, and the OD of the bacterial culture was measured using a UV spectrophotometer. 600 The OD value was adjusted using MS liquid medium. 600 The value is between 0.65 and 0.

75.

5. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S2, acetylsuccinone is added to achieve a final concentration of 50 mg / L in the infiltration solution.

6. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S3, after pruning the roots, the remaining root length is 4-5cm; the creation of the wounds specifically involves making 3 wounds on each side of the hypocotyl near the root, with a wound depth of 0.5-1mm.

7. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The tampons are 2-3cm long and 0.8-1.2cm wide.

8. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S4, the ambient humidity during the dark culture is 70%-85%, and the dark culture time is 1-3 days.

9. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S4, the light intensity of the illumination condition is 100-150 μmol / m² / s.

10. The method for efficient genetic transformation of citrus mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S5, the positive screening for transgenes is performed by PCR identification, and the detection of the target gene expression level is performed by qRT-PCR analysis.

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