Agrobacterium rhizogenes mediated sunflower hairy root transformation system

Abstract

The invention discloses an agrobacterium rhizogenes mediated sunflower hairy root transformation system, and belongs to the technical field of genetic transformation. The transformation system comprises the following steps: accelerating germination of sunflowers, transferring germinated sunflower seeds into a seedling tray, wrapping with a preservative film for moisturizing, and culturing in an illumination incubator; the method comprises the following steps: culturing an agrobacterium rhizogenes Ar. Qual strain containing a pBI121-GUS (glucuronidase) plasmid; preparing a bacterial liquid; the method comprises the following steps of: beveling sunflower seedlings along a position 3cm below cotyledons, and immersing cuts into a bacterium solution containing pBI121-GUS plasmid agrobacterium rhizogenes Ar.qual; the sunflower seedlings are transferred to a seedling raising plate to be cultured; culturing for 5-6 days, and transferring the seedlings into a filter paper system of a culture dish to promote root growth. According to the agrobacterium rhizogenes-mediated sunflower hairy root transformation system, the genetic transformation efficiency of sunflowers is improved, the transformation period is greatly shortened, and obtained hairy root plants are good in growth consistency, robust in seedlings and capable of being used in molecular breeding research.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic transformation, and in particular relates to a sunflower hairy root transformation system mediated by Agrobacterium rhizogenes. Background Art

[0002] In the field of modern agricultural biotechnology, improving plant traits through genetic transformation technology, such as increasing yield, enhancing nutritional quality, improving stress resistance, insect resistance and disease resistance, is an important way to achieve crop improvement. Although transgenic technology has made significant progress, for some crops, such as sunflower, it is still challenging to achieve stable genetic transformation due to its limited in vitro regeneration ability. As an important oil crop, the genetic improvement of sunflower is of great significance for improving oil yield and quality.

[0003] Agrobacterium tumefaciens is a natural genetic transformation tool that can introduce foreign genes into plant genomes through the T-DNA fragment of its Ti plasmid, which has become a key technology in plant genetic engineering. However, for crops that are difficult to transform, such as sunflower, the traditional Agrobacterium tumefaciens-mediated transformation method is inefficient and difficult to obtain stable transgenic plants. Agrobacterium rhizogenes is another Agrobacterium bacterium that can induce plants to form hairy roots, and its Ri plasmid (root-inducing plasmid) is another effective tool for plant genetic transformation. Similar to the Ti plasmid of Agrobacterium tumefaciens, the Ri plasmid can also introduce foreign genes into plants through T-DNA, but the hairy roots formed during the transformation process provide a unique system for studying plant roots. Since the mid-20th century, the application of Ri plasmids in plant genetic transformation has gradually been developed, especially in crops that are difficult to transform, such as sunflower, showing its unique advantages.

[0004] The structure of Ri plasmid is similar to that of Ti plasmid, containing T-DNA region, toxicity region (Vir), functional metabolic region (OPCA) and replication origin region (Ori). Functionally, Ri plasmid can mediate the transfer of T-DNA, allowing foreign genes to be expressed in plant cells. According to the type of opine, Ri plasmid can be divided into mannopine type, agropine type, cucumine type and isocucumine type. These different types of plasmids differ in transformation efficiency and plant response. Summary of the invention

[0005] The present invention aims to provide a sunflower hairy root transformation system mediated by Agrobacterium rhizogenes, which improves the genetic transformation efficiency of sunflower and greatly shortens the transformation cycle. The obtained hairy root plants have good growth consistency and strong seedlings, and can be used in molecular breeding research.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A sunflower hairy root transformation system mediated by Agrobacterium rhizogenes comprises the following steps:

[0008] S1. Sunflower germination: Select full sunflower seeds and place them evenly on a seedling tray covered with filter paper, cover the filter paper, soak the filter paper and seeds with distilled water to fully moisten them, and place them in a constant temperature incubator at 25°C for germination for 2-3 days. During this period, rinse the filter paper and seeds with running water every day;

[0009] S2. Sunflower cultivation: transfer the germinated sunflower seeds into seedling trays, wrap with plastic wrap to keep moisture, and place in a light incubator for 72-96 hours;

[0010] S3, culture of Agrobacterium rhizogenes Ar.qual strain containing pBI121-GUS plasmid;

[0011] S4, preparation of the bacterial solution of Agrobacterium rhizogenes Ar.qual containing pBI121-GUS plasmid;

[0012] S5. Cut the sunflower seedlings obliquely 3 cm below the cotyledons, immerse the cut surface in the Agrobacterium rhizogenes Ar.qual bacterial solution containing the pBI121-GUS plasmid obtained in step S4, place 10 seedlings in each conical flask, and place the conical flask in a shaker at 100 rpm and 28°C for 5-6 hours;

[0013] S6. Transfer the sunflower seedlings to a seedling tray, wherein the lower layer of the seedling tray is covered with two layers of toilet paper and the upper layer is covered with a layer of filter paper. A layer of filter paper is added to the seedling incision to keep moisture, and the seedling incision is covered and cultured. The filter paper at the seedling incision is moistened with distilled water every day to always maintain a moist environment;

[0014] S7. After culturing for 5-6 days at 60% humidity and 25°C, the seedlings were transferred to a culture dish filter paper system to promote root growth.

[0015] Preferably, in step S2, the seedling tray is placed with a matrix with a humidity of 70%, and the matrix consists of peat, vermiculite and perlite.

[0016] Preferably, the matrix consists of peat, vermiculite and perlite in a weight ratio of 2:1:1.

[0017] Preferably, in step S2, the culture conditions of the light incubator are: light culture temperature is 25°C, culture time is 16h, dark culture temperature is 23°C, culture time is 23°C, and relative air humidity is 60%.

[0018] Preferably, in step S3, the culturing of the strain comprises the following steps:

[0019] A1. Thaw the competent cells of Agrobacterium rhizogenes Ar.qual stored at -80°C at room temperature and place them in ice when they are in an ice-water mixture state;

[0020] A2. Add pBI121-GUS plasmid, mix cells and plasmid thoroughly, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0021] A3. Add 700 μL of TY liquid medium without antibiotics, culture in a shaker at 28°C, 220 rpm for 2 h, centrifuge at 6000 rpm for 1 min, discard part of the supernatant, keep 100 μL of liquid to resuspend the precipitate, mix evenly, spread on a TY solid medium containing kanamycin at a final concentration of 50 μg / mL, and culture at 28°C for 24-96 h to obtain the rhizogenes Agrobacterium Ar.qual strain containing the pBI121-GUS plasmid.

[0022] Preferably, the TY liquid culture medium comprises the following components by weight: 5 g of tryptone, 3 g of yeast extract, and 1.11 g of calcium chloride. The components are dissolved in distilled water and the volume is fixed to 1 L.

[0023] Preferably, the TY solid medium comprises the following components by weight: 5 g of tryptone, 3 g of yeast extract, 1.11 g of calcium chloride, and 15 g of agar. The components are dissolved in distilled water and the volume is adjusted to 1 L.

[0024] Preferably, in step S4, the preparation of the bacterial solution comprises the following steps:

[0025] B1. Pick the Agrobacterium rhizogenes Ar.qual strain containing the pBI121-GUS plasmid, transfer it to TY liquid medium containing kanamycin and streptomycin with a final concentration of 50 μg / mL, culture it on a shaker at 220 rpm at 28°C for 12 h, add glycerol to obtain a bacterial solution, and freeze it at -80°C for later use;

[0026] B2, take 100 μL of the bacterial solution obtained in step B1, add it to TY liquid medium containing kanamycin and streptomycin with a final concentration of 50 μg / mL, and culture overnight at 220 rpm and 28°C to obtain culture A;

[0027] B3, to a TY liquid medium containing kanamycin and streptomycin at a final concentration of 50 μg / mL, acetosyringone, MES solution, and then culture A obtained in step B2 were added, and cultured overnight at 220 rpm and 28° C. to obtain culture B;

[0028] B4. Centrifuge culture B at 6000 rpm for 5 min at room temperature, discard the supernatant, resuspend in infection solution, mix evenly, measure OD600 value, dilute to OD600=1, and obtain Agrobacterium rhizogenes Ar.qual bacterial solution containing pBI121-GUS plasmid, and pour into a sterilized conical flask in an amount of 50 mL.

[0029] Preferably, the TY liquid culture medium comprises the following components by weight: 5 g of tryptone, 3 g of yeast extract, and 1.11 g of calcium chloride. The components are dissolved in distilled water and the volume is fixed to 1 L.

[0030] Preferably, the MES solution comprises the following components by weight: MES 19.52 g, ddH2O fixed to 100 mL, and the MES solution is sterilized by filtering with a filter membrane with a pore size of 0.22 μm.

[0031] Preferably, in step B4, the method for preparing the infection solution comprises the following steps:

[0032] (1) Weigh 1.95 g MSE and 2.21 g MgCl2 into a volumetric flask;

[0033] (2) Add 1 mL of 200 mM AS solution and adjust the volume to 1 L;

[0034] (3) Place the above solution in a high pressure sterilizer and sterilize at 121°C for 20 min to obtain the infection solution.

[0035] Compared with the prior art, the present invention has the following advantages and technical effects:

[0036] The present invention discloses a sunflower hairy root transformation system mediated by Agrobacterium rhizogenes. The hairy roots induced by the system provide a model system that is easy to operate and observe for studying the sunflower root system, and is convenient for studying the root system structure, metabolism and signal transduction. Compared with traditional transformation methods, the system reduces physical damage to plant cells, improves the safety of transgenic plants, and has good environmental adaptability, and is easy to apply under different laboratory and field conditions.

[0037] The present invention not only improves the genetic transformation efficiency of sunflower, but also provides an efficient, stable and safe new method for the study of plant functional genes and crop improvement. It has important scientific significance and broad application prospects and can be extended to other crops, especially those that are difficult to transform by traditional methods, thereby broadening the application scope of genetic transformation technology.

[0038] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a specific implementation process of the sunflower hairy root transformation system induced by Agrobacterium rhizogenes of the present invention, wherein: Figure 1 A in the figure is a model diagram. Figure 1 B in the figure is a specific experimental flow chart;

[0040] Figure 2 The results of the induced rooting of sunflower in Example 1;

[0041] Figure 3 The growth conditions of the rooted sunflower seedlings in the culture dish filter paper system in Example 1;

[0042] Figure 4 The staining results of GUS transformation in sunflower hairy roots, where: Figure 4 A in the figure is the dyeing result of comparative example 1, Figure 4 B in the figure is the dyeing result of Example 1. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0044] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0045] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0046] The plasmid used in Example 1 was pBI121-GUS, which was purchased from Changsha Abiwei Biotechnology Co., Ltd.

[0047] The Agrobacterium rhizogenes strain was Ar.qual, purchased from Puint Biotechnology Co., Ltd.

[0048] TY liquid medium includes the following components by weight: 5 g of tryptone, 3 g of yeast extract, and 1.11 g of calcium chloride. The components are dissolved in distilled water and the volume is adjusted to 1 L.

[0049] TY solid medium includes the following components by weight: 5 g of tryptone, 3 g of yeast extract, 1.11 g of calcium chloride, and 15 g of agar. The components are dissolved in distilled water and the volume is adjusted to 1 L.

[0050] The MES solution includes the following components by weight: MES 19.52 g, ddH2O is fixed to 100 mL, and the MES solution is sterilized by filtering with a filter membrane with a pore size of 0.22 μm.

[0051] The preparation method of the infection solution comprises the following steps:

[0052] (1) Weigh 1.95 g MSE and 2.21 g MgCl2 into a volumetric flask;

[0053] (2) Add 1 mL of 200 mM AS solution and adjust the volume to 1 L;

[0054] (3) Place the above solution in a high pressure sterilizer and sterilize at 121°C for 20 min to obtain the infection solution.

[0055] Example 1 This example provides a sunflower hairy root transformation system mediated by Agrobacterium rhizogenes, comprising the following steps:

[0056] S1. Sunflower germination: Select full sunflower seeds and place them evenly on a seedling tray covered with filter paper, cover the filter paper, soak the filter paper and seeds with distilled water, cover to keep moisture, and place them in a 25°C constant temperature incubator for germination for 3 days. During this period, rinse the filter paper and seeds with running water every day;

[0057] S2. Sunflower cultivation: The germinated sunflower seeds are transferred into seedling trays, wrapped with plastic wrap to keep moisture, and placed in a light incubator for 96 hours. When the two cotyledons are unfolded, the seedling trays are placed with a substrate with a humidity of 70%, and the substrate is composed of peat, vermiculite and perlite in a mass ratio of 2:1:1. The light incubation temperature in the light incubator is 25°C, the incubation time is 16 hours, the dark incubation temperature is 23°C, the incubation time is 23°C, and the relative humidity of the air is 60%;

[0058] S3, culturing the Agrobacterium rhizogenes Ar.qual strain containing the pBI121-GUS plasmid, comprising the following steps:

[0059] A1. Thaw the competent cells of Agrobacterium rhizogenes Ar.qual stored at -80°C at room temperature and place them in ice when they are in an ice-water mixture state;

[0060] A2. Take 100 μL of Agrobacterium rhizogenes Ar.qual competent cells, add 1 μL of pBI121-GUS plasmid, gently flick the bottom of the centrifuge tube to fully mix the cells and plasmid, and then place on ice for 5 min, place in liquid nitrogen for 5 min, place in a 37°C water bath for 5 min, and place in an ice bath for 5 min.

[0061] A3. Add 700 μL of TY liquid medium without antibiotics, culture in a shaker at 28°C and 220 rpm for 2 h, centrifuge at 6000 rpm for 1 min, discard part of the supernatant, keep 100 μL of liquid to resuspend the precipitate, mix evenly, spread on a TY solid medium containing kanamycin at a final concentration of 50 μg / mL, and culture at 28°C for 72 h to obtain the rhizogenes Agrobacterium Ar.qual strain containing the pBI121-GUS plasmid.

[0062] S4, the preparation of the Agrobacterium rhizogenes Ar.qual bacterial solution containing the pBI121-GUS plasmid comprises the following steps:

[0063] B1. Pick the Agrobacterium rhizogenes Ar.qual strain containing the pBI121-GUS plasmid, transfer it to 3 mL of TY liquid medium containing kanamycin and streptomycin at a final concentration of 50 μg / mL, shake and culture at 220 rpm at 28°C for 12 h, add glycerol to obtain a bacterial solution, and freeze it at -80°C for later use;

[0064] B2, take 100 μL of the bacterial solution obtained in step B1, add it to 3 mL of TY liquid culture medium containing kanamycin and streptomycin at a final concentration of 50 μg / mL, and culture overnight at 220 rpm and 28°C to obtain culture A;

[0065] B3, take 10 mL of TY liquid medium containing kanamycin and streptomycin at a final concentration of 50 μg / mL, add 10 μL of 200 mM / L acetosyringone, 100 μL of 1 mol / L MES solution, and then add 300 μL of culture A obtained in step B2, and culture at 220 rpm and 28°C overnight to obtain culture B;

[0066] B4. Centrifuge culture B at 6000 rpm for 5 min at room temperature, discard the supernatant, resuspend in 10 mL of infection solution, mix evenly, measure the OD600 value, dilute to OD600=1, and obtain the Agrobacterium rhizogenes Ar.qual bacterial solution containing the pBI121-GUS plasmid. Pour into a sterilized conical flask in an amount of 50 mL.

[0067] S5. Cut the sunflower seedlings obliquely 3 cm below the cotyledons, immerse the cut surface in the Agrobacterium rhizogenes Ar.qual bacterial solution containing the pBI121-GUS plasmid obtained in step S4, place 10 seedlings in each conical flask, and place the conical flask in a shaker at 100 rpm and 28°C for 6 h;

[0068] S6. Transfer the sunflower seedlings to a seedling tray, wherein the lower layer of the seedling tray is covered with two layers of toilet paper and the upper layer is covered with a layer of filter paper. A layer of filter paper is added to the seedling incision to keep moisture, and the seedling incision is covered and cultured. The filter paper at the seedling incision is moistened with distilled water every day to always maintain a moist environment;

[0069] S7. Cultivate at 60% humidity and 25°C for 6 days, and transfer the seedlings to the culture dish filter paper system to promote root growth.

[0070] Comparative Example 1 This comparative example provides a sunflower hairy root transformation system mediated by Agrobacterium rhizogenes. The specific steps are the same as those in Example 1, except that the pBI121-GUS plasmid is not transferred into the Agrobacterium rhizogenes Ar.qual strain.

[0071] The test results are as follows Figure 2-Figure 3 .

[0072] Depend on Figure 2 It can be seen that hairy roots can grow at the incision position of the hypocotyls 6 days after the sunflower hypocotyls were treated with the rhizogenes Agrobacterium strain Ar.qual.

[0073] Depend on Figure 3 It can be seen that after the sunflower seedlings that have grown hairy roots are transferred into the culture dish, the sunflower can survive normally and produce a large number of roots.

[0074] The sunflower hairy roots of Example 1 and Comparative Example 1 were dyed, and the test method was as follows:

[0075] (1) Place the GUS staining working solution in a 100 mL conical flask, take out the sunflower roots grown in the culture dish filter paper system for 3 days and place them in the conical flask for staining. Each flask can hold 10 to 30 roots and stain overnight in a 28°C incubator.

[0076] (2) Take out the overnight stained material and observe the root coloration under a stereomicroscope. Figure 4 .

[0077] Depend on Figure 4 It can be seen that after GUS staining, it can be observed that the sunflower roots are dyed blue, indicating that the gus gene has been successfully transformed into the sunflower roots.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A sunflower hairy root transformation system mediated by Agrobacterium rhizogenes, characterized in that: The steps include: S1. Sunflower germination: Select full sunflower seeds and place them evenly on a seedling tray covered with filter paper, cover the filter paper, soak the filter paper and seeds with distilled water to fully moisten them, and place them in a constant temperature incubator at 25°C for germination for 2-3 days. During this period, rinse the filter paper and seeds with running water every day; S2. Sunflower cultivation: transfer the germinated sunflower seeds into seedling trays, wrap with plastic wrap to keep moisture, and place in a light incubator for 72-96 hours; S3, culture of Agrobacterium rhizogenes Ar.qual strain containing pBI121-GUS plasmid; S4, preparation of the bacterial solution of Agrobacterium rhizogenes Ar.qual containing pBI121-GUS plasmid; S5. Cut the sunflower seedlings obliquely 3 cm below the cotyledons, immerse the cut surface in the Agrobacterium rhizogenes Ar.qual bacterial solution containing the pBI121-GUS plasmid obtained in step S4, place 10 seedlings in each conical flask, and place the conical flask in a shaker at 100 rpm and 28°C for 5-6 hours; S6. Transfer the sunflower seedlings to a seedling tray, wherein the lower layer of the seedling tray is covered with two layers of toilet paper and the upper layer is covered with a layer of filter paper. A layer of filter paper is added to the seedling incision to keep moisture, and the seedling incision is covered and cultured. The filter paper at the seedling incision is moistened with distilled water every day to always maintain a moist environment; S7. After culturing for 5-6 days at 60% humidity and 25°C, the seedlings were transferred to a culture dish filter paper system to promote root growth.

2. The Agrobacterium rhizogenes-mediated sunflower hairy root transformation system according to claim 1, characterized in that: In step S2, a matrix with a humidity of 70% is placed in the seedling tray, and the matrix consists of peat, vermiculite and perlite.

3. The Agrobacterium rhizogenes-mediated sunflower hairy root transformation system according to claim 1, characterized in that: In step S2, the culture conditions of the light incubator are: light culture temperature is 25°C, culture time is 16h, dark culture temperature is 23°C, culture time is 23°C, and relative air humidity is 60%.

4. The Agrobacterium rhizogenes-mediated sunflower hairy root transformation system according to claim 1, characterized in that: In step S3, the culturing of the strain comprises the following steps: A1. Thaw the competent cells of Agrobacterium rhizogenes Ar.qual stored at -80°C at room temperature and place them in ice when they are in an ice-water mixture state; A2. Add pBI121-GUS plasmid, mix cells and plasmid thoroughly, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. A3. Add 700 μL of TY liquid medium without antibiotics, culture in a shaker at 28°C, 220 rpm for 2 h, centrifuge at 6000 rpm for 1 min, discard part of the supernatant, keep 100 μL of liquid to resuspend the precipitate, mix evenly, spread on a TY solid medium containing kanamycin at a final concentration of 50 μg / mL, and culture at 28°C for 24-96 h to obtain the rhizogenes Agrobacterium Ar.qual strain containing the pBI121-GUS plasmid.

5. The Agrobacterium rhizogenes-mediated sunflower hairy root transformation system according to claim 1, characterized in that: In step S4, the preparation of the bacterial solution includes the following steps: B1. Pick the Agrobacterium rhizogenes Ar.qual strain containing the pBI121-GUS plasmid, transfer it to TY liquid medium containing kanamycin and streptomycin with a final concentration of 50 μg / mL, culture it on a shaker at 220 rpm at 28°C for 12 h, add glycerol to obtain a bacterial solution, and freeze it at -80°C for later use; B2, take 100 μL of the bacterial solution obtained in step B1, add it to TY liquid medium containing kanamycin and streptomycin with a final concentration of 50 μg / mL, and culture overnight at 220 rpm and 28°C to obtain culture A; B3, to a TY liquid medium containing kanamycin and streptomycin at a final concentration of 50 μg / mL, acetosyringone, MES solution, and then culture A obtained in step B2 were added, and cultured overnight at 220 rpm and 28° C. to obtain culture B; B4. Centrifuge culture B at 6000 rpm for 5 min at room temperature, discard the supernatant, resuspend in infection solution, mix evenly, measure OD600 value, dilute to OD600=1, and obtain Agrobacterium rhizogenes Ar.qual bacterial solution containing pBI121-GUS plasmid, and pour into a sterilized conical flask in an amount of 50 mL.

6. The Agrobacterium rhizogenes-mediated sunflower hairy root transformation system according to claim 5, characterized in that: In step B4, the method for preparing the infection solution comprises the following steps: (1) Weigh 1.95 g MSE and 2.21 g MgCl2 into a volumetric flask; (2) Add 1 mL of 200 mM AS solution and adjust the volume to 1 L; (3) Place the above solution in a high pressure sterilizer and sterilize at 121°C for 20 min to obtain the infection solution.

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