A genetic transformation method for Elymus nutans Griseb. var. nutans

Abstract

The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a genetic transformation method for perennial Elymus nutans. The present invention provides a genetic transformation method for Elymus nutans. Using mature embryos as explants for inducing callus formation has the characteristics of convenient material collection and simple and easy operation, avoiding the supply problem of explant material collection. At the same time, the consistent physiological state of mature embryos results in good repeatability of experiments. Additionally, the infection solution contains acetosyringone, and acetosyringone (AS, Acetosyringone) can promote the growth of Agrobacterium and enhance its Agrobacterium activity, thereby improving the transformation efficiency. The bactericidal treatment effectively inhibits the browning phenomenon of callus, enabling the callus to resume normal growth. Transgenic Elymus nutans plants are obtained using the technical solution of the present invention, which can greatly promote the research of researchers on the gene functions of Elymus nutans.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a genetic transformation method for perennial Elymus nutans Background Art

[0002] Elymus nutans Elymus nutans ), belonging to the genus Elymus of the tribe Triticeae in the subfamily Pooideae of the family Poaceae, is a perennial tufted herb. Elymus nutans is extremely widely distributed in China and adapts to most of the grassland ecosystems in China. Elymus nutans has strong drought and cold tolerance, good forage value, and plays an irreplaceable role in soil and water conservation, grassland establishment, ecological restoration, etc. At the same time, as a Triticeae plant, Elymus nutans retains many primitive traits and gene characteristics of this tribe. Therefore, the establishment of the genetic transformation system of Elymus nutans lays a foundation for the functional genomics research of forage grasses in the family Poaceae

[0003] In recent years, researchers have successfully established a tissue culture system for Elymus nutans using young spikes as explants. However, it is difficult to obtain young spikes as explants, and their developmental status is inconsistent under different planting conditions and different sampling times, which easily leads to poor experimental repeatability Summary of the Invention

[0004] The present invention provides a genetic transformation method for perennial Elymus nutans. Using the method of the present invention for genetic transformation, mature embryos are used as explants, which are convenient to obtain and have a consistent physiological state, with good repeatability, and the genetic transformation method is simple and easy to operate

[0005] To solve the above technical problems, the present invention proposes the following technical solutions

[0006] The present invention provides a genetic transformation method for Elymus nutans, comprising the following steps

[0007] Inducing callus culture of mature embryos of Elymus nutans to obtain callus

[0008] Infecting and co-culturing the callus with an Agrobacterium infection solution to obtain infected callus; the OD of the Agrobacterium infection solution 600 is 0.6 - 0.8; the infection solution contains acetosyringone, and the concentration of acetosyringone is 0.2 - 0.25 mM

[0009] Performing sterilization treatment and culture on the infected callus to obtain Elymus nutans plants

[0010] Preferably, the culture includes sequentially performing differentiation culture, screening, and rooting culture to obtain Elymus nutans plants

[0011] Preferably, the reagent used for the sterilization treatment includes a cephalosporin antibacterial solution at a concentration of 0.4 - 0.6 g / L.

[0012] Preferably, the time for each sterilization treatment is 8 - 10 min, and the number of sterilization treatments is 3 - 5 times.

[0013] Preferably, the Agrobacterium infection time is 8 - 12 min.

[0014] Preferably, the co - culture time is 2 - 3 d; the co - culture is carried out in the dark; the temperature for the co - culture is 25 ± 2°C.

[0015] Preferably, the medium used for callus induction culture includes an induction medium, and the composition of the induction medium includes: 4 - 4.43 g / L MS, 4 - 6 mg / L 2,4 - D, 40 - 60 mg / L CH, 1 - 3 mg / L ABA, 0.04 - 0.07 mg / L KT, 28 - 30 g / L sucrose, and 7 - 9 g / L agar.

[0016] Preferably, the medium used for differentiation culture includes a differentiation and regeneration medium, and the composition of the differentiation and regeneration medium includes: 2 - 2.2 g / L MS, 0.01 - 0.03 mg / L KT, 0.3 - 0.5 mg / L NAA, 100 - 200 mg / L TMT, 12.5 - 15 g / L sucrose, and 7 - 9 g / L agar; the differentiation and regeneration medium is replaced every 14 d during the differentiation culture process; the differentiation culture is carried out until the adventitious buds reach a length of 5 - 8 cm.

[0017] The medium used for rooting culture includes a rooting medium, and the composition of the rooting medium includes: 4 - 4.43 g / L MS, and also includes 0.3 - 0.5 g / L NAA, 100 - 200 mg / L TMT, 28 - 30 g / L sucrose, and 7 - 9 g / L agar.

[0018] Preferably, the time for callus induction culture is 30 - 45 d; the induction culture is carried out in the dark; the temperature for the induction culture is 25 ± 2°C.

[0019] Preferably, the medium used for screening includes a screening medium, and the types of antibiotics in the screening medium include hygromycin; the composition of the screening medium includes: 4 - 4.43 g / L MS, 30 mg / L hygromycin, 28 - 30 g / L sucrose, and 7 - 9 g / L agar.

[0020] Advantages of the present invention: The present invention provides a genetic transformation method for Elymus nutans. In the method provided by the present invention, the mature seeds of Elymus nutans with plump grains are dehulled to obtain mature embryos. Using the mature embryos as explants for inducing callus formation has the characteristics of convenient material collection and simple and easy operation. Mature embryos are easily obtained, avoiding the supply problem of explant material collection. At the same time, the consistent physiological state of mature embryos makes the experiment highly repeatable. Additionally, the infection solution contains acetosyringone, which can promote the growth of Agrobacterium and enhance its activity, thereby improving the transformation efficiency. The bactericidal treatment effectively inhibits the browning of callus, enabling the callus to resume normal growth. Transgenic Elymus nutans plants are obtained using the technical solution of the present invention, which can greatly promote the research on the gene function of Elymus nutans by researchers.

[0021] The present invention provides a genetic transformation method mediated by Agrobacterium tumefaciens based on mature embryos of Elymus nutans. The established Agrobacterium genetic transformation system based on mature embryos of Elymus nutans provides certain technical guidance for exploring the gene function of Elymus nutans, laying a foundation for the transfer of target genes, the creation and cultivation of new materials, and the research on gene function of perennial Elymus nutans. At the same time, it also lays a foundation for the directional molecular design and improvement of Elymus nutans varieties. Description of the Drawings

[0022] Figure 1 It is a picture of callus obtained from culturing perennial Elymus nutans for 45 days, with the scale bar being 1 cm;

[0023] Figure 2 It is adventitious buds obtained from differentiating and culturing callus of Elymus nutans for 28 days, with the scale bar being 1 cm;

[0024] Figure 3 It is adventitious buds of resistant callus screened on the selection medium, with the scale bar being 1 cm;

[0025] Figure 4 It is a picture of the growth state of transgenic Elymus nutans plants, with the scale bar being 5 cm;

[0026] Figure 5 It is for identifying positive Elymus nutans plants by PCR amplification of the selection resistance gene hygromycin (Hyg) in transgenic plants; among them, M represents Marker DL 5000; 1 represents positive; 2 - 5 represent transgenic plants; 6 represents negative plants; positive represents the positive control, Agrobacterium after transferring the target gene; negative plants are those that did not successfully transfer the gene after infection, but there was an infection process, only the result was that the target gene was not transferred;

[0027] Figure 6Growth status of Elymus nutans in different screening media, where A represents the growth status diagram on the 0th day of culture, B represents the status diagram on the 14th day of growth, and the scale bar is 1 cm;

[0028] Figure 7 It is a culture status diagram of the unwashed callus in Comparative Example 1 on the differentiation and regeneration medium, and the scale bar is 1 cm;

[0029] Figure 8 It is a culture status diagram of the callus in Comparative Example 2 on the differentiation and regeneration medium, and the scale bar is 1 cm;

[0030] Figure 9 It is Figure 8 Microscopic picture of the callus, and the scale bar is 100 μm;

[0031] Figure 10 It is a culture status diagram of the callus in Comparative Example 3 on the differentiation and regeneration medium, and the scale bar is 1 cm;

[0032] Figure 11 It is Figure 10 Microscopic picture of the callus in, and the scale bar is 100 μm;

[0033] Figure 12 It is the screening situation diagram of the regenerated seedlings of the callus with an OD 600 value of 0.4 in Comparative Example 4 on the screening medium, and the scale bar is 1 cm;

[0034] Figure 13 It is the screening situation diagram of the regenerated seedlings of the callus with an OD 600 value of 0.5 in Comparative Example 5 on the screening medium, and the scale bar is 1 cm. Specific implementation manner

[0035] The present invention provides a genetic transformation method for Elymus nutans, comprising the following steps:

[0036] Induce callus culture from mature embryos of Elymus nutans to obtain callus;

[0037] Infect and co-culture the callus with an Agrobacterium infection solution to obtain infected callus; the OD 600 of the Agrobacterium infection solution is 0.6 - 0.8, and the concentration of acetosyringone in the infection solution is 0.2 - 0.25 mM;

[0038] Perform bactericidal treatment and culture on the infected callus to obtain Elymus nutans plants.

[0039] The present invention induces callus culture from mature embryos of Elymus nutans to obtain callus.

[0040] As an alternative embodiment, the method for preparing mature embryos of Elymus nutans in the present invention may include: dehulling the Elymus nutans seeds to obtain mature embryos. The Elymus nutans seeds may be mature seeds of Elymus nutans with plump grains. Before inoculating the mature embryos of Elymus nutans on an induction medium for callus induction culture, it further includes disinfecting the mature embryos of Elymus nutans and rinsing them with sterile water. The solution used for the disinfection treatment may include a sodium hypochlorite solution with a mass concentration of 40%, and the time for the disinfection treatment may be 30 min. The number of times of rinsing with sterile water may be 5 - 7 times, and the function of rinsing with sterile water is to remove the sodium hypochlorite solution and avoid the adverse effects of the sodium hypochlorite solution on the mature embryos. As an alternative embodiment, the present invention may inoculate the disinfected mature embryos on an induction medium for callus induction culture to obtain callus. As an alternative embodiment, the composition of the induction medium in the present invention includes: 4 - 4.43 g / L MS, 4 - 6 mg / L 2,4-D, 40 - 60 mg / L CH, 1 - 3 mg / L ABA, 0.04 - 0.07 mg / L KT, 28 - 30 g / L sucrose, and 7 - 9 g / L agar. The concentration of MS in the induction medium provided by the present invention may be 4 - 4.43 g / L, or may also be 4 - 4.2 g / L. The concentration of 2,4-D in the induction medium provided by the present invention may be 4 - 6 mg / L; in the specific embodiments of the present invention, the concentration of 2,4-D in the induction medium may be 4.0, 4.5, 5.0, 5.5, or 6.0 mg / L. 2,4-D is 2,4-dichlorophenoxy acetic acid, which can induce explants to form callus and promote cell division and growth.

[0041] The concentration of CH in the induction medium provided by the present invention may be 40 - 60 mg / L; in the specific embodiments of the present invention, the concentration of CH in the induction medium may be 40, 45, 50, 55, or 60 mg / L. CH is Hydrolyzed casein, and CH has a good effect on the differentiation of embryoids and adventitious buds.

[0042] The concentration of ABA in the induction medium provided by the present invention may be 1 - 3 mg / L, and in the specific embodiments of the present invention, the concentration of ABA in the induction medium may be 1, 1.5, 2, or 3.0 mg / L. ABA is Abscisic Acid, and ABA can promote the development of seed embryos.

[0043] The concentration of KT in the induction medium provided by the present invention can be 0.04 - 0.07 mg / L. In the specific embodiments of the present invention, the concentration of KT in the induction medium can be 0.04, 0.05, 0.06 or 0.07 mg / L.

[0044] KT is Kinetin, a cytokinin; KT can promote the formation of callus. The concentration of sucrose in the induction medium provided by the present invention is 28 - 30 g / L, or can also be 29 - 30 g / L. The concentration of agar in the induction medium provided by the present invention is 7 - 9 g / L, or can also be 8 - 9 g / L.

[0045] In the present invention, the pH value of the induction medium can be 5.7 - 5.9, or can also be 5.80.

[0046] As an alternative embodiment, the temperature for callus induction culture can be 23 - 27 °C, or can also be 25 °C; the time for callus induction culture can be 30 - 45 d, or can also be 40 - 45 d; the induction culture is dark culture.

[0047] As an alternative embodiment, after obtaining the callus, the present invention can cut the callus. The function of the cutting is to remove the seed embryo and buds, and only retain the compact callus; the callus can be embryogenic callus; the size of the callus after cutting in the present invention can be 50 mm 3 . The surface of the compact callus is smooth and shiny, with a compact structure, mostly light yellow or white, and is easy to regenerate buds.

[0048] As an alternative embodiment, after obtaining the cut callus, the present invention can infect and co-culture the cut callus with an Agrobacterium infection solution to obtain the infected callus. The OD of the Agrobacterium infection solution 600is 0.6 - 0.8. The infection solution contains acetosyringone, and the concentration of acetosyringone in the infection solution is 0.2 - 0.25 mM. The acetosyringone can promote the growth of Agrobacterium and enhance its activity, thereby improving the transformation efficiency. The infection time of Agrobacterium in the present invention can be 8 - 12 min, or 9 - 11 min. After infection, the present invention preferably performs a drying treatment on the callus, and after the drying treatment, it is inoculated into a medium for co-culture. The drying treatment in the present invention can be completed using a dry sterilized filter paper. The co-culture time in the present invention can be 2 - 3 d; the co-culture can be carried out in the dark; the temperature for co-culture can be 25 ± 2 °C. The composition of the medium used for co-culture in the present invention is to add 100 - 200 mg / L TMT to the induction medium. The ticarcillin (TMT) in the present invention can effectively inhibit the growth of Agrobacterium. The composition of the induction medium in the present invention has been described above and will not be elaborated here.

[0049] After obtaining the infected callus, the present invention can perform a sterilization treatment and culture on the infected callus to obtain a callus that produces adventitious buds.

[0050] As an alternative embodiment, the method of the sterilization treatment in the present invention can be to place the infected callus in a cefotaxime solution for sterilization treatment. The cefotaxime in the present invention can be cefotaxime sodium, and the preparation method of the cefotaxime solution in the present invention can include: mixing the cefotaxime sodium with water. The concentration of the cefotaxime solution in the present invention can be 0.4 - 0.6 g / L, or 0.5 g / L. The time for each sterilization treatment in the present invention can be 8 - 10 min, or 8.5 - 9 min; the number of sterilization treatments can be 3 - 5 times, or 4 - 5 times. The cefotaxime solution can be replaced for each sterilization treatment in the present invention. After soaking the infected callus for 5 - 10 min, the infected callus is transferred to a new cefotaxime solution for the second bacteria washing until the bacteria washing is completed 3 - 5 times. The sterilization treatment in the present invention effectively inhibits the browning phenomenon of the callus and enables the callus to resume normal growth.

[0051] As an alternative embodiment, the present invention can transfer the callus obtained after the sterilization treatment to a sterilized filter paper to absorb the cefotaxime solution and then perform culture to obtain Elymus nutans plants.

[0052] As an alternative embodiment, the culture in the present invention can include performing differentiation culture, screening, and rooting culture in sequence to obtain Elymus nutans plants.

[0053] As an alternative embodiment, the composition of the differentiation and regeneration medium for the differentiation culture according to the present invention includes: 2 - 2.2 g / L of MS, 0.01 - 0.03 mg / L of KT, 0.3 - 0.5 mg / L of NAA, 100 - 200 mg / L of TMT, 12.5 - 15 g / L of sucrose, and 7 - 9 g / L of agar. The concentration of MS in the differentiation and regeneration medium provided by the present invention is 2 - 2.2 g / L of MS, and it can also be 2 - 2.1 g / L of MS. The concentration of KT in the differentiation and regeneration medium provided by the present invention is 0.01 - 0.03 mg / L; in specific embodiments of the present invention, the concentration of KT in the differentiation and regeneration medium is 0.01, 0.015, 0.02, or 0.03 mg / L. KT, namely Kinetin, is called kinetin in Chinese. KT can promote the formation of adventitious buds and has a certain ratio with NAA to balance the formation of plant adventitious buds.

[0054] The concentration of NAA in the differentiation and regeneration medium provided by the present invention is 0.3 - 0.5 mg / L; in specific embodiments of the present invention, the concentration of NAA in the differentiation and regeneration medium is 0.3, 0.35, 0.4, 0.45, or 0.5 mg / L. NAA promotes the formation of adventitious buds and jointly promotes the formation of adventitious buds with KT at an appropriate concentration ratio.

[0055] The concentration of TMT in the differentiation and regeneration medium provided by the present invention can be 100 - 200 mg / L. In specific embodiments of the present invention, the concentration of TMT in the differentiation and regeneration medium can be 100, 110, 130, 150, 170, or 200 mg / L. TMT is Timentin (meilunbio®), that is, an antibacterial agent, which can inhibit the growth of bacteria. The concentration of sucrose in the differentiation and regeneration medium provided by the present invention is 12.5 - 15 g / L, and it can also be 13 - 15 g / L. The concentration of agar in the differentiation and regeneration medium provided by the present invention is 7 - 9 g / L, and it can also be 8 - 9 g / L.

[0056] The differentiation culture described in the present invention can be light-dark alternating culture. The light time can be 16 h / d, and the dark time can be 8 h / d. The light intensity of the present invention can be 2,500 lux, and the temperature of the differentiation culture can be 25 ± 2°C. During the process of the differentiation culture of the present invention, the differentiation and regeneration medium is replaced every 14 days to ensure the nutrient supply of the callus. The composition of the differentiation and regeneration medium replaced each time in the present invention is the same. The differentiation culture in the present invention is carried out until the length of the adventitious buds reaches 5 - 8 cm. After the differentiation culture is completed, the adventitious buds in the present invention are transferred to a selection medium for selection culture. After the selection culture is completed, the adventitious buds with good growth are the adventitious buds with resistance. The types of antibiotics in the antibiotic selection medium described in the present invention can include hygromycin, and adventitious buds resistant to hygromycin can be screened. As an alternative embodiment, the composition of the selection medium in the present invention includes: 4 - 4.43 g / L MS, 30 mg / L hygromycin, 28 - 30 g / L sucrose, and 7 - 9 g / L agar. The concentration of MS in the selection medium provided by the present invention is 4 - 4.43 g / L, or can also be 4 - 4.15 g / L. The setting of the hygromycin concentration is conducive to screening transgenic Elymus nutans plants, that is, positive plants. The concentration of sucrose in the selection medium provided by the present invention is 28 - 30 g / L, or can also be 28 - 29 g / L. The concentration of agar in the selection medium provided by the present invention is 7 - 9 g / L, or can also be 8 - 9 g / L.

[0057] The types of antibiotics in the selection medium described in the present invention depend on the resistance of the Agrobacterium tumefaciens infection vector in the plant. The Agrobacterium strain applied in the present invention can be LBA4404, and the vector applied can be pCAMBIA1300. The pCAMBIA1300 vector itself has hygromycin resistance. Therefore, after the LBA4404 infection is successful, the vector invades the plant body, making the transgenic plant have hygromycin resistance and growing well on the culture medium plate with hygromycin resistance, and adventitious buds with good growth are screened.

[0058] As an alternative embodiment, the temperature of the differentiation culture in the present invention is 25 ± 2°C, and the time of the selection culture is 18 - 25 d, or can also be 21 - 23 d. In the specific embodiments of the present invention, the time of the selection culture is 18, 20, 21, 23, or 25 d.

[0059] As an alternative embodiment, the well-growing adventitious buds screened in the present invention are subjected to rooting culture to obtain transgenic Elymus nutans plants. The temperature for the rooting culture in the present invention is 25±2°C; the time for the rooting culture is 12 - 14 days. The composition of the rooting medium in the present invention: 4 - 4.43 g / L MS, 0.3 - 0.5 g / L NAA, 100 - 200 mg / L TMT, 28 - 30 g / L sucrose, and 7 - 9 g / L agar. The concentration of MS in the rooting medium provided by the present invention is 4 - 4.43 g / L, and it can also be 4 - 4.3 g / L. The concentration of NAA in the rooting medium provided by the present invention is 0.3 - 0.5 g / L; in the specific embodiments of the present invention, the concentration of NAA in the differentiation and regeneration medium is 0.3, 0.35, 0.4, 0.45, or 0.5 g / L. NAA is 1-naphthaleneacetic acid, that is, 1-naphthylacetic acid; NAA can promote plant growth and the formation of adventitious roots. The concentration of sucrose in the rooting medium provided by the present invention is 28 - 30 g / L, and it can also be 28 - 29 g / L. The concentration of agar in the rooting medium provided by the present invention is 7 - 9 g / L, and it can also be 29 - 30 g / L.

[0060] As an alternative embodiment, the rooting culture in the present invention yields rooted seedlings, and the rooted seedlings are acclimatized and transplanted to obtain transgenic Elymus nutans plants. The time for acclimatization can be 3 days. The substrate used for transplantation includes peat soil and vermiculite, and the volume ratio of peat soil to vermiculite can be (1 - 1.5):(0.5 - 1), or it can also be 1:1.

[0061] The present invention provides a method for using Agrobacterium tumefaciens ( Agrbacteriumtumefaciens ), mediated Elymus nutansA genetic transformation method, which uses an induction medium to induce callus from mature embryos of Elymus nutans, and obtains dense embryogenic callus through culture; co-cultures the subcultured callus with Agrobacterium, and uses an infection solution to enable Agrobacterium containing a target gene to infect the callus; performs a sterilization treatment on the co-cultured callus; uses a differentiation and regeneration medium to culture the obtained sterilized callus until adventitious buds are differentiated; uses a screening medium to screen the callus that produces adventitious buds to obtain resistant adventitious buds; performs rooting culture on the screened resistant adventitious buds of Elymus nutans to obtain transgenic Elymus nutans seedlings. The present invention can effectively screen transgenic plants, and the positive rate of screening with the screening medium concentration is as high as 100%, and the operation is simple and easy. The Agrobacterium-mediated genetic transformation system established by the present invention provides certain technical guidance for studying the gene functions of Elymus nutans; as a forage grass in the Triticeae tribe, the genetic transformation system of Elymus nutans also provides a reliable heterologous expression system for wheat gene function verification, and at the same time provides a basis for gene improvement of gramineous crops.

[0062] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0063] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments, rather than limiting the protection scope of the present invention. The test methods without specific conditions indicated in the following embodiments are usually carried out under conventional conditions, or according to the conditions recommended by each reagent manufacturer.

[0064] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and any one of the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of the present technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of the present technology and the description of the present invention, any methods, equipment, and materials similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0065] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional technologies in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, gene mutation technology, and related fields in the present technology field.

[0066] In the following examples and comparative examples, Elymus nutans seeds with plump grains were used. The same batch of seeds was deglumed to obtain the mature embryos of the same batch of Elymus nutans.

[0067] Example 1

[0068] An Agrobacterium-mediated genetic transformation method based on the mature embryos of Elymus nutans is as follows:

[0069] 1. Disinfection of mature embryos of Elymus nutans

[0070] The mature embryos of Elymus nutans were obtained by degluming Elymus nutans seeds with plump grains. The degluming treatment was as follows: The seeds were soaked in water for 8 h for manual peeling to obtain the mature embryos of Elymus nutans, and the seeds could not be polished; after polishing, the seed coat was removed and the embryo would be damaged.

[0071] Using the mature embryos of Elymus nutans as explants, they were disinfected by soaking in a sodium hypochlorite solution with a mass concentration of 40% prepared with ultrapure water for 30 min. The available chlorine concentration of the sodium hypochlorite solution with a mass concentration of 40% was 10%. After disinfection, they were placed under sterile conditions in a laminar flow hood and rinsed 10 times with sterile water. Subsequently, the surface of the mature embryos was dried with sterilized filter paper. The disinfection step is relatively simple, without toxic chemicals, and causes less harm to the human environment.

[0072] 2. Callus induction

[0073] The disinfected mature embryos of Elymus nutans were placed in the callus induction medium for constant temperature culture. The temperature of the constant temperature culture was 25 ± 2 °C. The constant temperature culture was dark culture, and the time of the constant temperature culture was 45 d. After culture, tender yellow, dense, embryogenic callus of Elymus nutans was obtained. During this period, timely bud cutting treatment was carried out. The tender yellow, dense, embryogenic callus obtained from the culture of perennial Elymus nutans for 45 d is shown in Figure 1 .

[0074] The composition of the callus induction medium was: 4 g / L MS, 4 mg / L 2,4-D, 50 mg / L CH, 2 mg / L ABA, 0.04 mg / L KT, 30 g / L sucrose, and 8 g / L agar, with a pH of 5.8.

[0075] 3. Agrobacterium culture

[0076] The Agrobacterium carrying the target gene and the selectable resistance gene was plated and cultured in an incubator at 28 °C for 2 d. The Agrobacterium was suspended in MS liquid medium to form an infection solution. The concentration of the infection solution was OD 600 of 0.8. The infection solution contained 0.2 mM acetosyringone (that is, 50 μL of the acetosyringone stock solution was added to 50 mL of the infection solution, and the concentration of the acetosyringone stock solution was 200 mM) to activate the Agrobacterium.

[0077] 4. Agrobacterium infection and co - culture with callus

[0078] Agrobacterium infection is carried out by soaking the compact embryogenic callus with the infection solution for 10 min.

[0079] After that, the callus is taken out from the infection solution, and the excess infection solution is blotted with sterile filter paper to obtain the infected callus. Then the infected callus is placed in the co - culture medium for co - culture. The co - culture is carried out in the dark for 2 d. The composition of the medium used for co - culture is adding 100 mg / L TMT to the induction medium, that is, the composition of the medium used for co - culture is: 4 g / L MS, 4 mg / L 2,4 - D, 50 mg / L CH, 2 mg / L ABA, 0.04 mg / L KT, 100 mg / L TMT, 30 g / L sucrose and 8 g / L agar, and the pH is 5.8. The Agrobacterium strain is LBA4404 and the vector is pCAMBIA1300.

[0080] 5. Callus recovery and bacterium removal treatment

[0081] The callus obtained from the co - culture in step 4 is washed with a 0.5 g / L cephalosporin solution for 10 min each time, and washed 4 times in total. Then the washed callus is transferred to sterile filter paper to absorb the cephalosporin solution, and the washed and dried callus is obtained.

[0082] 6. Callus differentiation and regeneration

[0083] The washed and dried callus is placed in the differentiation and regeneration medium for induced bud formation. The induced bud formation is carried out under light culture, the light intensity is 2000 - 2500 lux, the light time is 16 h / d, the dark time is 8 h / d, and the culture temperature is 25 ± 2℃. The differentiation and regeneration medium is changed every 14 d. The adventitious buds obtained from the differentiation culture of Elymus nutans callus for 28 d are shown in Figure 2 .

[0084] The composition of the differentiation and regeneration medium is: 2 g / L MS, 0.02 mg / L KT, 0.5 mg / L NAA, 200 mg / L TMT, 15 g / L sucrose and 8 g / L agar.

[0085] 7. Screening of positive plants

[0086] When the adventitious buds generated from the callus in step 6 reach a length of 5 - 8 cm, the adventitious buds are transferred to the screening medium for screening culture. The composition of the screening medium is: 4 g / L MS, 30 mg / L Hyg, 30 g / L sucrose and 8 g / L agar, the pH is 5.8, and Hyg is hygromycin. The adventitious buds of the resistant callus obtained on the screening medium are shown inFigure 3 .

[0087] 8. Rooting, hardening and transplanting of regenerated seedlings

[0088] Transfer the well - growing green buds on the screening medium to the rooting medium for rooting culture. The temperature for rooting culture is 25±2°C, and the time for rooting culture is 14 days. After strong roots grow, open the test - tube seedlings for hardening for 3 days, then take out the test - tube seedlings, wash the residual medium on the roots, and transplant them into flower pots in the greenhouse mixed with peat soil and vermiculite in a volume ratio of 1:1 to obtain complete transgenic regenerated Elymus nutans plants, as shown in Figure 4 . The formula of the rooting medium is: 4 g / L MS, 0.35 g / L NAA, 200 mg / L TMT, 30 g / L sucrose and 8 g / L agar, with a pH of 5.8.

[0089] In this example, the callus induction rate of Elymus nutans mature embryos is 65%, and the regeneration and differentiation rate is 46%.

[0090] Induction rate (%)=(Number of explants forming callus / Number of infected explants)×100%;

[0091] Differentiation rate (%)=(Number of callus with buds / Number of infected callus)×100%.

[0092] Example 2

[0093] Screening efficiency of different - concentration hygromycin screening media for regenerated adventitious buds

[0094] Culture adventitious buds of Elymus nutans using the method of steps 1 - 6 in Example 1. After adventitious buds grow from the Elymus nutans callus, transfer the adventitious buds to the screening media of Treatment 1 - Treatment 5 for culture. There are 5 regenerated plants in each petri dish, and 3 replicate experiments are set for each screening concentration. Record the growth of the regenerated plants. Among them, the screening medium for Treatment 1 is: 4 g / L MS, 10 mg / L Hyg, 30 g / L sucrose and 8 g / L agar, with a pH of 5.8; the screening medium for Treatment 2 is: 4 g / L MS, 20 mg / L Hyg, 30 g / L sucrose and 8 g / L agar, with a pH of 5.8; the screening medium for Treatment 3 is: 4 g / L MS, 30 mg / L Hyg, 30 g / L sucrose and 8 g / L agar, with a pH of 5.8; the screening medium for Treatment 4 is: 4 g / L MS, 40 mg / L Hyg, 30 g / L sucrose and 8 g / L agar, with a pH of 5.8; the screening medium for Treatment 5 is: 4 g / L MS, 50 mg / L Hyg, 30 g / L sucrose and 8 g / L agar, with a pH of 5.8.

[0095] After 14 days of culture, the leaves of the plants surviving on the hygromycin screening medium were used to extract genomic DNA. Subsequently, using the plant DNA as a template, PCR was performed to determine whether the hygromycin resistance gene was successfully transferred. The identification results of Elymus nutans positive plants are shown in Figure 5 , where M represents Marker DL 5000; 1 represents the positive control, namely the Agrobacterium tumefaciens after gene transfer; 2 - 5 represent transgenic plants; 6 represents the negative plant.

[0096] The hygromycin positive rates were statistically analyzed respectively.

[0097] Hygromycin positive rate (%) = (number of hygromycin positive plants / number of surviving plants) × 100;

[0098] The results showed that on the 10 mg / L hygromycin screening medium, all 15 regenerated plants survived; on the 20 mg / L hygromycin screening medium, 11 out of 15 regenerated plants survived, but there were hygromycin negative plants among the surviving plants, indicating that this concentration could not play a good screening role; on the 30 mg / L hygromycin screening medium, 3 out of 15 regenerated plants survived, and PCR detection confirmed that all the surviving plants were hygromycin positive plants, indicating that this concentration could play a good screening role; on the 40 mg / L hygromycin screening medium, 1 out of 15 regenerated plants survived, and PCR testing confirmed that the surviving plant was a hygromycin positive plant, indicating that this concentration could also play a screening role, but the survival rate was too low; on the 50 mg / L hygromycin screening medium, all 15 regenerated plants died (Table 1). The growth status of adventitious buds of Elymus nutans in different screening media is shown in Figure 6 , where A represents the growth status diagram at 0 days of culture, and B represents the status diagram at the 14th day of growth; Figure 6 In the hygromycin screening medium plates from left to right, the concentrations of hygromycin are 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L in sequence.

[0099] Table 1 Statistical results of screening with different concentrations of hygromycin

[0100]

[0101] The size of the PCR identification band described above is about 1000 bp, and the primer sequences are as follows

[0102] Table 2 List of PCR amplification primers for Hygromycin gene

[0103]

[0104] In this example, the optimal hygromycin screening concentration was 30 - 40 mg / L, and the transgenic positive screening rate was 100%.

[0105] Comparative Example 1

[0106] An Agrobacterium-mediated genetic transformation method based on the mature embryos of Elymus nutans, the steps are as follows:

[0107] 1. Disinfection of mature embryos of Elymus nutans. The same as Example 1.

[0108] 2. Callus induction. The same as Example 1.

[0109] 3. Agrobacterium culture. The same as Example 1.

[0110] 4. Agrobacterium infection and co-culture with callus. The same as Example 1.

[0111] 5. Callus differentiation and regeneration

[0112] The callus obtained in step 4 was placed in a differentiation and regeneration medium for shoot induction. Shoot induction was carried out under light culture, with a light intensity of 2000 - 2500 lux, a light time of 16 h / d, a dark time of 8 h / d, and a culture temperature of 25 ± 2°C. The callus obtained after 7 days of differentiation culture of Elymus nutans callus is shown in Figure 7 .

[0113] The composition of the differentiation and regeneration medium is: 2 g / L MS, 0.02 mg / L KT, 0.5 mg / L NAA, 200 mg / L TMT, 15 g / L sucrose, and 8 g / L agar.

[0114] According to Figure 7 it can be seen that after the co-culture ended, no bactericidal treatment was carried out, and the callus obtained from the co-culture was directly inoculated on the differentiation and regeneration medium. There were bacteria on the surface of the callus and in the medium, and the browning degree of the callus was high. This state of the callus could not generate regenerated seedlings.

[0115] Comparative Example 2

[0116] Only steps 1 - 6 of Example 1 were carried out, the same as Example 1, and the only difference was that: the callus obtained from the co-culture was washed with a 0.5 g / L cephalosporin solution for 4 minutes each time, and washed 4 times in total. Then the washed callus was transferred to a sterilized filter paper to absorb the cephalosporin solution, and the washed and dried callus was obtained.

[0117] The washed and dried callus was placed in a differentiation and regeneration medium for shoot induction. Pictures of the callus after 13 days of culture are shown in Figure 8 and Figure 9 . It can be seen that there were no traces of bacteria on the differentiation and regeneration medium, but there was still bacterial coverage on the surface of the callus, and the callus could not regenerate.

[0118] Comparative Example 3

[0119] Only perform steps 1 to 6 of Example 1. It is the same as Example 1, and the only difference is that the callus obtained from co-culture is washed with a 0.5 g / L cefotaxime solution for 7 minutes each time, and washed 4 times in total. Then, the washed callus is transferred to sterilized filter paper to absorb the cefotaxime solution, and the callus after being washed and dried is obtained. The callus after being washed and dried is placed in a differentiation and regeneration medium for bud induction. The picture of the callus cultured for 14 days is shown in Figure 10 and Figure 11 . It can be seen that when cultured on the differentiation and regeneration medium, the growth state of the callus is good in the first week, and there is no bacterial growth. However, after one week, bacteria gradually grow around the callus, causing the original bud points of the callus to turn yellow and then white and die, and then the entire callus is completely covered by bacteria and cannot continue to grow.

[0120] Comparative Example 4

[0121] Only perform steps 1 to 7 of Example 1. It is the same as Example 1, and the only difference is that the concentration of the infection solution is OD 600 is 0.4, and the infection solution contains 0.2 mM of acetosyringone.

[0122] The adventitious buds obtained by culturing on the selection medium are shown in Figure 12 . It can be seen that although there is no obvious difference in the growth of the callus on the differentiation and regeneration medium, when the regenerated seedlings produced by the callus are cultured on the selection medium, it is found that all the regenerated seedlings in the appropriate selection medium die. This shows that although bacteria do not grow on the callus under this condition, the regenerated seedlings generated when the concentration of the infection solution is OD 600 is 0.4 do not survive on the selection medium, indicating that the infection success rate of positive seedlings is greatly reduced under this experimental condition.

[0123] Comparative Example 5

[0124] Only perform steps 1 to 7 of Example 1. It is the same as Example 1, and the only difference is that the concentration of the infection solution is OD 600 is 0.5, the infection solution contains 0.2 mM of acetosyringone, and the callus obtained from co-culture is washed with a 0.5 g / L cefotaxime solution for 10 minutes each time, and washed 4 times in total.

[0125] The adventitious buds obtained by culturing on the selection medium are shown in Figure 13 . It can be seen that although the state of the regenerated seedlings on the selection medium is not good, there are still surviving regenerated seedlings compared with Comparative Example 4, but the survival rate of the regenerated seedlings is low, indicating that although the infection efficiency is higher than that of the infection solution with OD 600 being 0.4, it is lower than the infection efficiency when the OD of the infection solution is 600 0.8.

[0126] In summary, the induction rate of the compact callus of the present invention reaches 65%. During the subsequent differentiation culture process, 46% of the callus differentiates into adventitious buds and regenerates plants, and the positive rate of transgenic plant screening is 100%. The Agrobacterium genetic transformation system based on the mature embryos of Elymus nutans established in the present invention provides certain technical guidance for exploring the gene functions of Elymus nutans, and also lays a foundation for the directional molecular design and improvement of Elymus nutans varieties.

[0127] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A genetic transformation method of Elymus nutans, characterized in that: The following steps are involved: sterilizing mature embryos of Elymus nutans and inoculating them into an induction medium for callus induction culture to obtain callus tissue; The induction medium comprises: 4-4.43 g / L MS, 4-6 mg / L 2,4-D, 40-60 mg / L CH, 1-3 mg / LABA, 0.04-0.07 mg / L KT, 28-30 g / L sucrose and 7-9 g / L agar; the disinfection treatment is disinfection and immersion in a 40% sodium hypochlorite solution for 30 min; The callus tissue is infected and co-cultured with the Agrobacterium infection solution to obtain infected callus tissue; the OD600 of the Agrobacterium infection solution is 0.6-0.8; the infection solution contains acetosyringone, and the concentration of acetosyringone is 0.2-0.25 mM; The infected callus tissue is sterilized, differentiated, screened and rooted to obtain a plant of Elymus nutans; the reagent used in the sterilization treatment is a 0.4-0.6 g / L cephalosporin antibiotic solution; the time for each sterilization treatment is 8-10 minutes, and the number of sterilization treatments is 3-5 times; The composition of the differentiation and regeneration medium used in differentiation culture is: 2~2.2g / L MS, 0.01~0.03mg / L KT, 0.3~0.5mg / L NAA, 100~200mg / L TMT, 12.5~15g / L sucrose and 7~9g / L agar; The type of antibiotic in the culture medium used for screening is hygromycin; the composition of the culture medium used for screening is: 4-4.43 g / L MS, 30 mg / L hygromycin, 28-30 g / L sucrose and 7-9 g / L agar; The composition of the culture medium used for the rooting culture is: 4-4.43 g / L MS, 0.3-0.5 g / L NAA, 100-200 mg / L TMT, 28-30 g / L sucrose and 7-9 g / L agar.

2. The genetic transformation method according to claim 1, characterized in that The infection time of Agrobacterium is 8~12min.

3. The genetic transformation method according to claim 1, characterized in that The co-cultivation time is 2 to 3 days; the co-cultivation is dark culture; the co-cultivation temperature is 25±2°C.

4. The genetic transformation method according to claim 1, characterized in that During the differentiation culture, the differentiation and regeneration medium is replaced every 14 days; the differentiation culture is carried out until the length of the adventitious buds is 5-8 cm.

5. The genetic transformation method according to claim 1, characterized in that The callus induction culture time is 30-45 days; the induction culture is dark culture; the induction culture temperature is 25±2°C.

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