Tissue culture method for establishing agrobacterium K599 mediated honeysuckle genetic transformation system

Abstract

The invention discloses a tissue culture method for establishing an agrobacterium K599 mediated honeysuckle genetic transformation system. The method comprises the following steps: by taking a tender honeysuckle stem as an explant, thoroughly disinfecting, infecting a K599 agrobacterium solution carrying a GUS (glucuronidase) reporter gene, carrying out tissue culture for a certain period, and inducing the honeysuckle stem to generate adventitious roots or adventitious buds after removing bacteria by the agrobacterium. Furthermore, after antibiotic positive screening, the adventitious roots are transferred into a rooting culture medium for expanding propagation, or the adventitious buds are cut and then transplanted and cultured into new honeysuckle plants. The K599 agrobacterium disclosed by the invention can be introduced into plasmids for editing genes with required phenotypes, and then the plasmids are cultured by the method, so that a honeysuckle genetic transformation system with the required phenotypes is expected to be obtained.

Description

Technical Field

[0001] The invention belongs to the research field of traditional Chinese medicine genetic transformation system, and aims to provide a specific method for establishing a stable honeysuckle genetic transformation system mediated by Agrobacterium rhizogenes K599. Background Art

[0002] Honeysuckle (Lonicera japonica Thunb.) is a traditional Chinese medicine with the functions of clearing away heat and detoxifying, dispersing wind and dissipating heat. It is widely used in clinical prescriptions of traditional Chinese medicine and Chinese patent medicine. Artificial introduction and cultivation of honeysuckle has a long history, and there are large planting areas in Shandong, Henan, Hebei and other provinces in my country. At present, the cultivation of honeysuckle is based on different planting purposes such as medicinal or ornamental. The varieties selected include big hairy flower, tree four-season flower, golden flower No. 3, Beihua No. 1, etc. Compared with the increasingly widespread application of honeysuckle in medicinal-grade horticultural ornamental applications, its demand for excellent varieties far exceeds its breeding process. Moreover, under the current planting model, the phenomenon of germplasm mixing and degradation is also very serious. The lack of a stable heritage transformation system in the selection and breeding process is one of the main reasons for the very slow progress of the selection and breeding of new honeysuckle varieties.

[0003] Agrobacterium rhizogenes is a Gram-negative soil bacterium of the genus Agrobacterium in the family Rhizobium. It has the excellent characteristics of strong infection ability and wide infection range. K599 Agrobacterium rhizogenes contains pRi2659 agrobacterium-type Ri plasmid, which has a wide host range. Most dicotyledons, a few monocotyledons and individual gymnosperms can become its hosts, and it also has streptomycin resistance. Agrobacterium rhizogenes can induce transgenic hairy adventitious roots when infecting plant explants. The obtained hairy roots originate from a single cell without chimeras and can grow rapidly and autonomously on a culture medium without plant hormones. It can be used as a bioreactor to produce protein products expressed by exogenous genes and active ingredients of medicinal plants.

[0004] The present invention is based on the deficiencies of the prior art and provides a tissue culture method for preparing a honeysuckle genetic transformation system with stable transformation performance using Agrobacterium K599 as a mediating vector. The invention uses young honeysuckle stems as explants, and infects them with K599 Agrobacterium bacterial solution carrying the target gene to induce the production of an adventitious root genetic transformation system carrying the target gene or a new honeysuckle plant carrying the target gene. The established genetic transformation system can provide an important technical means for breeding new excellent varieties of honeysuckle and improving their quality. Summary of the invention

[0005] The purpose of the present invention is to use honeysuckle stem segments as explants to be infected by Agrobacterium carrying target genes to produce adventitious roots through tissue culture, thereby providing a method for genetic transformation of honeysuckle based on tissue culture. The present invention induces honeysuckle to produce adventitious roots by using rhizogenes Agrobacterium K599, laying a good technical support for optimizing the construction of a genetic transformation system for honeysuckle.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a tissue culture method for establishing a honeysuckle genetic transformation system mediated by Agrobacterium K599, the method comprising the following steps:

[0008] S1. Spread the Agrobacterium K599 strain carrying the GUS reporter gene plasmid evenly on the TY solid medium and invert it for activation culture; then pick a single colony and inoculate it in the TY liquid medium for shaking culture activation, and then further inoculate the activated bacterial liquid into the TY liquid medium and culture it until the OD of the bacterial liquid reaches 0. 600 The value is between 0.6 and 0.8, and then the bacterial solution is resuspended in MES buffer to obtain MES infection solution;

[0009] S2, cutting the stem of the honeysuckle plant as an explant, cleaning and fully disinfecting it, immersing the lower end of the incision in the MES infection solution for infection, and then transferring it to a growth medium for dark culture;

[0010] S3. Transferring the stem segments after dark culture to a culture medium containing antibiotics and culturing them until adventitious roots grow out, and then cutting the adventitious roots and transferring them to a growth culture medium for expanded culture.

[0011] Preferably, if the stem segment does not grow adventitious roots in the medium containing antibiotics, it is transferred to a rooting medium to accelerate rooting induction.

[0012] Preferably, the explant is a healthy tender stem with leaf buds on a honeysuckle plant.

[0013] Preferably, the GUS reporter gene plasmid is pre-connected with an exogenous target gene to be introduced into the honeysuckle genome.

[0014] Preferably, the explants are disinfected by alternately immersing them in 75% ethanol solution and 15% sodium hypochlorite solution.

[0015] Preferably, the formula of the TY liquid culture medium is: 5 g / L Tryptone + 3 g / L Yeast extract + 10 ml / L 1M calcium chloride.

[0016] Preferably, the formula of the TY solid culture medium is: 5 g / L Tryptone + 3 g / L Yeast extract + 10 ml / L 1M calcium chloride + 15 g / L agar powder.

[0017] Preferably, the formula of the MES buffer is: MS culture medium 4.47 g / L + 1M MES 10 ml / L + 1M Mgcl2 10 ml / L + 200 mM AS 100 ul / L.

[0018] Preferably, the formula of the growth medium is: 1 / 2MS+sucrose 20.0 g / L+agar 9.3 g / L.

[0019] Preferably, the formula of the culture medium containing antibiotics is: 1 / 2MS+sucrose 20.0 g / L+agar 9.3 g / L+antibiotic 400 mg / L; the antibiotic is preferably ceftriaxone sodium.

[0020] Preferably, the formula of the rooting medium is: 1 / 2MS+NAA1.5mg / L+sucrose 20g / L+agar 9.3g / L+antibiotic 400mg / L.

[0021] Compared with the prior art, the beneficial effects that may be brought about by the implementation of the present invention are as follows:

[0022] Based on the characteristics of plant active regeneration ability and asexual reproduction, the method of the present invention is expected to be applied to a wider range of plant species, achieving trait improvement and germplasm innovation of more characteristic resource plants and economic crops, so as to promote the development of basic research and applied research in related fields, and provide theoretical and methodological support for the efficient genetic improvement of characteristic plants and modern agricultural breeding applications.

[0023] Compared with the prior art method of generally using mercuric chloride combined with sodium hypochlorite solution to disinfect explants, the present invention, based on multiple experimental verifications, uses a method of soaking and rinsing in sodium hypochlorite and 75% ethanol in sequence, which can more fully disinfect the honeysuckle explants, thereby avoiding the contamination of the explants and subsequent tissue culture links by external pathogens.

[0024] Compared with the process of forming callus tissue and then dedifferentiating to generate adventitious roots, the present invention greatly shortens the cultivation time and provides an important technical means for quickly improving honeysuckle varieties and enhancing the quality of medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 Flow chart of the tissue culture method established for the Agrobacterium K599-mediated genetic transformation system of Honeysuckle;

[0027] Figure 2 The adventitious roots grown from the honeysuckle stem segment in the embodiment of the present invention;

[0028] Figure 3 This is the GUS staining verification result of the positive plants in the examples of the present invention. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0030] The invention provides a tissue culture method of a honeysuckle genetic transformation system mediated by Agrobacterium rhizogenes K599, which can quickly cultivate honeysuckle plants containing exogenous target genes, shorten the time required in the excellent gene screening process, and improve the efficiency and accuracy of genetic material screening.

[0031] like Figure 1 As shown, in an embodiment of the present invention, a tissue culture method of a honeysuckle genetic transformation system mediated by Agrobacterium rhizogenes K599 is provided, and its basic steps are as follows:

[0032] S1. Spread the Agrobacterium K599 strain carrying the GUS reporter gene plasmid evenly on the TY solid medium and invert it for activation culture; then pick a single colony and inoculate it in the TY liquid medium for shaking culture activation, and then further inoculate the activated bacterial liquid into the TY liquid medium and culture it until the OD of the bacterial liquid reaches 0. 600 The value is between 0.6 and 0.8, and then the bacterial solution is resuspended in MES buffer to obtain MES infection solution for use.

[0033] S2. Cut the stem of the honeysuckle plant as an explant, clean and fully disinfect it, and immerse the lower end of the cut in the MES infection solution for infection, so that the explant stem segment is infected with K599 Agrobacterium carrying the GUS reporter gene, and then transfer it to a growth medium for dark culture.

[0034] S3. Transfer the stem segments after dark culture to a culture medium containing antibiotics and culture them until adventitious roots grow. Cut the adventitious roots and transfer them to a growth culture medium for expanded culture.

[0035] In addition, if the stem segment does not grow adventitious roots or roots too slowly in the culture medium containing antibiotics, the stem segment can also be transferred to a rooting medium to accelerate rooting induction. The obtained adventitious roots can be cut and transferred to a growth medium for expanded culture.

[0036] It should be noted that the GUS reporter gene plasmid carried in the above-mentioned Agrobacterium K599 can be used to connect the exogenous target gene that needs to be introduced into the honeysuckle genome, and the honeysuckle genetic transformation system of the desired phenotype can be obtained by this culture method. The exogenous target gene can be recombined onto the GUS reporter gene plasmid by enzyme cutting and connection, and then the recombinant plasmid is introduced into the Agrobacterium cell according to the conventional operation of Agrobacterium-mediated infection. The exogenous target gene can be selected according to the actual transformation requirements, and this is not limited. The GUS reporter gene plasmid can facilitate the GUS staining verification of the target gene.

[0037] In the above culture method, the specific formulas of the five culture media can be adjusted according to actual needs, and the tissue culture methods and operation details at different stages can also be optimized according to actual needs.

[0038] The following further provides a specific implementation method of the tissue culture method of the honeysuckle genetic transformation system mediated by Agrobacterium rhizogenes K599, and the steps are as follows:

[0039] 1) Preparation of TY medium: Preparation of medium required for the growth of Agrobacterium K599, including TY liquid medium and TY solid medium;

[0040] 2) Prepare MES infection solution: Spread Agrobacterium K599 evenly on TY solid medium, invert and activate for 2 days; then pick a single colony and inoculate it into 1 mL TY liquid medium, and incubate at 28°C and 200 r / min. -1 The activated bacteria were inoculated into 30 mL TY liquid medium and cultured until the OD of the bacteria reached 0. 600 The value falls between 0.6 and 0.8, and the bacterial solution is resuspended in MES buffer to obtain MES infection solution for use;

[0041] 3) Explant selection and disinfection: Select healthy, disease-free and insect-free tender stems from the honeysuckle plant as explants, cut off the mature part, remove the terminal buds and young leaves, and use sterile water in an ultra-clean workbench to rinse off the dust and garbage attached to the surface of the explant; then rinse with 15% sodium hypochlorite for 5 minutes 1-3 times, rinse with sterile water 1-3 times, and finally soak in 75% ethanol for 30 seconds, rinse with sterile water 1-3 times, and place on sterile filter paper for natural ventilation to dry.

[0042] 4) Explant infection and dark culture: Place the sterilized explant with the upper end facing upwards, ensure that the lower cut is immersed in MES infection solution for 30 minutes, and then transfer to growth medium for dark culture for 48 hours. Then transfer the explant after 48 hours of dark culture to a medium containing the antibiotic ceftriaxone sodium to inhibit the growth of Agrobacterium.

[0043] 5) Removal of Agrobacterium: After 48 hours of dark culture, the explants were transferred to a medium containing sodium ceftriaxone to inhibit the growth of Agrobacterium, and the culture was continued to induce the explants to grow a small amount of adventitious roots.

[0044] 6) Accelerating rooting: After the Agrobacterium stops growing, observe for 5-10 days and transfer the explants that have not grown adventitious roots to rooting medium to accelerate the induction of rooting of the stem segments as needed.

[0045] 7) The obtained adventitious roots are cut and transferred to a growth medium for expansion culture.

[0046] The culture medium and MES buffer involved in the above tissue culture method can be formulated as follows:

[0047] I. TY liquid culture medium: 5 g / L tryptone + 3 g / L yeast extract + 10 ml / L 1M calcium chloride;

[0048] II. TY solid medium: Tryptone 5g / L + Yeast extract 3g / L + 1M calcium chloride 10ml / L + agar powder 15g / L;

[0049] III. Growth medium: 1 / 2MS + sucrose 20.0g / L + agar 9.3g / L;

[0050] IV. Culture medium containing antibiotic ceftriaxone sodium: 1 / 2MS + sucrose 20.0 g / L + agar 9.3 g / L + antibiotic 400 mg / L;

[0051] Ⅴ. The formula of rooting medium is: 1 / 2MS + NAA 1.5 mg / L + sucrose 20 g / L + agar 9.3 g / L + antibiotics 400 mg / L.

[0052] VI. The formula of MES buffer is: MS medium 4.47 g / L + 1M MES 10 ml / L + 1M Mgcl2 10 ml / L + 200 mM AS 100 ul / L.

[0053] The method provided in the present invention can effectively shorten the time required in the excellent gene screening process and improve the efficiency and accuracy of genetic material screening.

[0054] The above method is applied to a specific example below to demonstrate its specific technical effect in combination with data.

[0055] Example

[0056] (I) Prepare different functional culture media. The specific methods are as follows:

[0057] 1. Prepare TY medium

[0058] According to the formula of TY liquid culture medium: 5g / L of tryptone + 3g / L of yeast extract + 10ml / L of 1M calcium chloride, prepare TY culture medium: weigh 5g of Tryptone and 3g of Yeast extract, stir and melt in water, dilute the mixed solution to 1L, and dispense 30ml into a conical flask. Then weigh 55.5g of calcium chloride and melt in water, dilute to 500ml, sterilize at 121℃ for 20 minutes to obtain 1M calcium chloride solution. After sterilization, add 300uL of 1M calcium chloride solution to the 30ml mixed solution in the conical flask in the clean bench to obtain TY liquid culture medium.

[0059] In addition, according to the formula of TY solid culture medium: Tryptone 5g / L + yeast extract 3g / L + 1M calcium chloride 10ml / L + agar powder 15g / L, TY solid culture medium was prepared: on the basis of TY liquid culture medium, agar powder with a final concentration of 15g / L was added to obtain TY solid culture medium.

[0060] 2. Prepare growth medium

[0061] According to the formula: 1 / 2MS + sucrose 20.0g / L + agar 9.3g / L, prepare the growth medium: weigh 2.3g 1 / 2MS and 20g sucrose, stir and melt in water, adjust the pH to 5.8, dilute the solution to 1L, pour it into a sterilized bottle containing 9.3g agar, and sterilize at 121℃ for 20 minutes. After sterilization, dispense 100ml into the culture bottle in the clean bench to obtain the growth medium.

[0062] 3. Prepare culture medium containing antibiotics

[0063] According to the formula: 1 / 2MS + sucrose 20.0g / L + agar 9.3g / L + antibiotic 400mg / L, prepare the culture medium containing antibiotics: weigh 2.3g 1 / 2MS and 20g sucrose, stir and melt in water, adjust the pH to 5.8, dilute the solution to 1L and pour it into a sterile bottle containing 9.3g agar, sterilize at 121℃ for 20 minutes. After sterilization, add 1ml 400mg / mL ceftriaxone sodium in the clean bench and dispense 100ml into the culture bottle to obtain the antibiotic culture medium.

[0064] 4. Prepare rooting medium

[0065] Prepare the rooting medium according to the formula: 1 / 2MS + NAA 1.5mg / L + sucrose 20g / L + agar 9.3g / L + antibiotics 400mg / L: weigh 2.3g 1 / 2MS and 20g sucrose and stir to melt in water, add 1.5mg NAA, adjust the pH to 5.8, dilute the solution to 1L and pour it into a sterilized bottle containing 9.3g agar, sterilize at 121℃ for 20 minutes. After sterilization, dispense 100ml into the culture bottle in the clean bench to obtain the rooting medium.

[0066] 5. Prepare MES infection solution: evenly spread Agrobacterium K599 carrying GUS reporter gene plasmid (which can be recombined with exogenous target gene as needed) on TY solid medium, invert and activate culture for 2 days; then pick a single colony and inoculate it in 1 mL TY liquid medium, incubate at 28°C, 200 r·min -1 The activated bacteria were inoculated into 30 mL TY liquid medium and cultured until the OD of the bacteria reached 0. 600 The value falls between 0.6-0.8, and then the bacterial solution is resuspended in MES buffer to obtain MES infection solution for use. Among them, the formula of MES buffer is: MS culture medium 4.47g / L + 1M MES 10ml / L + 1MM MgCl2 10ml / L + 200mM AS 100ul / L.

[0067] (II) Disinfect the honeysuckle explants. The specific method is as follows:

[0068] Use plants grown indoors as the selection object, select healthy, disease-free and insect-free tender stems with leaf buds as explants, separate the leaves and stem segments, and rinse the dust and garbage on the surface of the explants with running water. Further rinse the dust and garbage with sterile water in the clean bench, then rinse with 15% sodium hypochlorite for 5 minutes 1-3 times, rinse with sterile water 1-3 times, and finally soak in 75% ethanol for 30 seconds, rinse with sterile water 1-3 times, and place on sterile filter paper for natural ventilation and drying.

[0069] (III) The sterilized explants are infected and then cultured in the culture medium obtained in (I) above to induce rooting. The specific method is as follows:

[0070] After the prepared stem segment is disinfected, the morphological upper end is facing upward, and the lower cut is immersed in the MES infection solution containing Agrobacterium K599 for infection for 30 minutes, and then further inoculated in the pre-sterilized growth medium for dark culture for 48 hours. After 48 hours, the Agrobacterium attached to the explant stem segment is rinsed with sterile water and dried with sterile filter paper. The stem segment is then transferred to the antibiotic culture medium to inhibit the growth of Agrobacterium and induce rooting. After the experiment, the rooting of the honeysuckle stem segment infected with Agrobacterium K599 is shown in Table 1 and Figure 2 shown.

[0071] Table 1 Infection and growth of Agrobacterium K599

[0072]

[0073] In addition, in order to quickly induce rooting of explants, explants that root slowly or cannot root in antibiotic culture medium can be transferred to rooting medium to induce stem segments to accelerate rooting.

[0074] (IV) Positive plant verification, the specific method is as follows:

[0075] When adventitious roots grow out of the honeysuckle stem segment, cut off the newly grown adventitious roots and verify them with GUS staining. Add an appropriate amount of prepared GUS staining working solution until the material is completely covered; wrap it with tin foil (try to avoid light during the whole process) and incubate it at 37℃ for 1-24h. If necessary, 70% alcohol can be used to remove the chlorophyll in the sample. The blue on the white background is the GUS expression site (see Figure 3 ), which can be observed with the naked eye or under a microscope.

[0076] After positive antibiotic selection, the adventitious roots are transferred to a growth medium for propagation, or the adventitious buds are divided and transplanted to grow new honeysuckle plants.

[0077] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A tissue culture method for establishing a honeysuckle genetic transformation system mediated by Agrobacterium K599, characterized in that: The method comprises the following steps: S1. Spread the Agrobacterium K599 strain carrying the GUS reporter gene plasmid evenly on the TY solid medium and invert it for activation culture; then pick a single colony and inoculate it in the TY liquid medium for shaking culture activation, and then further inoculate the activated bacterial liquid into the TY liquid medium and culture it until the OD of the bacterial liquid reaches 0. 600 The value is between 0.6 and 0.8, and then the bacterial solution is resuspended in MES buffer to obtain MES infection solution; S2, cutting the stem of the honeysuckle plant as an explant, cleaning and fully disinfecting it, immersing the lower end of the incision in the MES infection solution for infection, and then transferring it to a growth medium for dark culture; S3. Transferring the stem segments after dark culture to a culture medium containing antibiotics and culturing them until adventitious roots grow out, and then cutting the adventitious roots and transferring them to a growth culture medium for expanded culture.

2. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 1, characterized in that: If the stem segment does not grow adventitious roots in the medium containing antibiotics, it is transferred to a rooting medium to accelerate root induction.

3. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 1 or 2, characterized in that: The GUS reporter gene plasmid is pre-connected with an exogenous target gene to be introduced into the honeysuckle genome.

4. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 1 or 2, characterized in that: The explants are sterilized by alternately soaking in 75% ethanol solution and 15% sodium hypochlorite solution.

5. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 1 or 2, characterized in that: The formula of the TY liquid culture medium is: 5 g / L of tryptone + 3 g / L of yeast extract + 10 ml / L of 1M calcium chloride.

6. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 1 or 2, characterized in that: The formula of the TY solid culture medium is: 5 g / L of tryptone + 3 g / L of yeast extract + 10 ml / L of 1M calcium chloride + 15 g / L of agar powder.

7. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 1 or 2, characterized in that: The formula of the MES buffer is: MS culture medium 4.47 g / L+1M MES 10 ml / L+1M Mgcl2 10 ml / L+200 mM AS 100 ul / L.

8. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 1 or 2, characterized in that: The formula of the growth medium is: 1 / 2MS+sucrose 20.0 g / L+agar 9.3 g / L.

9. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 1 or 2, characterized in that: The formula of the culture medium containing antibiotics is: 1 / 2MS+sucrose 20.0 g / L+agar 9.3 g / L+antibiotic 400 mg / L; the antibiotic is preferably ceftriaxone sodium.

10. The tissue culture method for establishing the honeysuckle genetic transformation system mediated by Agrobacterium K599 as claimed in claim 2, characterized in that: The formula of the rooting medium is: 1 / 2MS+NAA1.5mg / L+sucrose 20g / L+agar 9.3g / L+antibiotic 400mg / L.

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