Genetic transformation method of perennial elymus nutans

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a genetic transformation method of perennial elymus nutans. According to the genetic transformation method of elymus nutans, mature embryos serve as explants for inducing callus formation, the method has the advantages that material obtaining is convenient, operation is simple and easy, the supply problem of explant material obtaining is solved, and meanwhile the physiological state of the mature embryos is consistent, so that the repeatability of experiments is good; in addition, the infection liquid contains the acetosyringone, and the acetosyringone (AS, Acetosyringone) can promote the growth of agrobacterium tumefaciens and enhance the activity of the agrobacterium tumefaciens, so that the conversion efficiency is improved; the browning phenomenon of the calluses is effectively inhibited through sterilization treatment, the normal growth of the calluses can be recovered, the transgenic elymus nutans plants are obtained through the technical scheme, and the research of researchers on the gene functions of the elymus nutans can be greatly promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular relates to a genetic transformation method of perennial Elymus nutans. Background Art

[0002] Elymus nutans ( Elymus nutans ) belongs to the genus Elymus, Triticeae, Poaceae, subfamily Poaceae, and is a perennial sparse clump herb. Elymus nutans is extremely widely distributed in my country and is adapted to most of my country's grassland ecosystems. Elymus nutans has strong drought and cold resistance, good feeding value, and plays an irreplaceable role in soil and water conservation, grassland construction, and ecological restoration. At the same time, as a plant of the Triticeae family, Elymus nutans retains many of the original traits and genetic characteristics of the plants in this family. Therefore, the establishment of the genetic transformation system of Elymus nutans lays the foundation for the functional genomics research of Poaceae forages.

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

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

[0005] In order to solve the above technical problems, the present invention proposes the following technical solutions: The present invention provides a genetic transformation method of Elymus nutans, comprising the following steps: Perform callus induction culture on mature embryos of Elymus nutans to obtain callus tissue; The callus tissue is infected and co-cultured with the Agrobacterium infection solution to obtain infected callus tissue; the OD of the Agrobacterium infection solution is 600 The infection solution contains acetosyringone, and the concentration of acetosyringone is 0.2-0.25 mM; The infected callus tissue is sterilized and cultured to obtain Elymus nutans plants.

[0006] Preferably, the culture comprises performing differentiation culture, screening and rooting culture in sequence to obtain Elymus nutans plants.

[0007] Preferably, the reagent used in the sterilization treatment includes a 0.4-0.6 g / L cephalosporin antibiotic solution.

[0008] Preferably, the duration of each sterilization treatment is 8 to 10 minutes, and the number of sterilization treatments is 3 to 5 times.

[0009] Preferably, the Agrobacterium infection time is 8 to 12 minutes.

[0010] Preferably, the co-cultivation time is 2 to 3 days; the co-cultivation is dark culture; and the co-cultivation temperature is 25±2°C.

[0011] Preferably, the culture medium used for callus induction culture includes an induction medium, and the composition of the induction medium includes: 4~4.43g / L MS, 4~6mg / L 2,4-D, 40~60mg / L CH, 1~3mg / L ABA, 0.04~0.07mg / L KT, 28~30g / L sucrose and 7~9g / L agar.

[0012] Preferably, the culture medium used in the differentiation culture comprises a differentiation and regeneration culture medium, and the composition of the differentiation and regeneration culture medium comprises: 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 culture medium is replaced every 14 days during the differentiation culture; the differentiation culture is carried out until the length of the adventitious buds is 5-8 cm; The culture medium used for the rooting culture comprises a rooting culture medium, and the composition of the rooting culture medium comprises: 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.

[0013] Preferably, the callus induction culture time is 30-45 days; the induction culture is dark culture; the induction culture temperature is 25±2°C.

[0014] Preferably, the culture medium for screening application includes a screening medium, and the type of antibiotics in the screening medium includes hygromycin; the composition of the screening medium includes: 4~4.43g / L MS, 30mg / L hygromycin, 28~30g / L sucrose and 7~9g / L agar.

[0015] The beneficial effects of the invention are as follows: the invention provides a genetic transformation method for Elymus nutans. In the method provided by the invention, mature seeds of Elymus nutans with full grains are de-glue to obtain mature embryos, and the mature embryos are used as explants for inducing callus formation. The method has the characteristics of convenient material collection and simple and easy operation. The mature embryos are easy to obtain, which avoids the problem of supply of explant materials. At the same time, the physiological state of the mature embryos is consistent, so the repeatability of the experiment is good. In addition, the infection solution contains acetosyringone, and acetosyringone (AS) can promote the growth of Agrobacterium and enhance its Agrobacterium activity, thereby improving the transformation efficiency. The sterilization treatment effectively inhibits the browning phenomenon of callus tissue, and can restore the normal growth of callus tissue. Transgenic Elymus nutans plants are obtained by using the technical solution of the invention, which can greatly promote researchers' research on the gene function of Elymus nutans.

[0016] The invention provides a genetic transformation method based on mature embryos of Elymus nutans and mediated by Agrobacterium tumefaciens, and an Agrobacterium genetic transformation system based on mature embryos of Elymus nutans is established, which provides certain technical guidance for exploring the gene functions of Elymus nutans, lays a foundation for the transfer of target genes of perennial Elymus nutans, the creation and cultivation of new materials, and the study of gene functions, and also lays a foundation for the improvement of Elymus nutans varieties by directed molecular design. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a callus picture of perennial Elymus nutans cultured for 45 days, with a scale of 1 cm; Figure 2 These are the adventitious buds obtained from the callus differentiation culture of Elymus nutans for 28 days. The scale is 1 cm. Figure 3 This is the adventitious buds of callus with resistance screened on the screening medium. The scale is 1 cm; Figure 4 This is a growth status diagram of the transgenic Elymus nutans plant, with a scale of 5 cm; Figure 5 To select the hygromycin resistance gene (Hyg) in PCR amplification of transgenic plants, identify positive plants of Elymus nutans; where M represents Marker DL 5000; 1 represents positive; 2-5 represent transgenic plants; 6 represents negative plants; positive represents positive control, which is Agrobacterium after the target gene is transferred; negative plants are those that have not been successfully transgenic after infection, but the infection process exists, but the target gene is not transferred as a result; Figure 6 The growth status of Elymus nutans in different screening media, where A represents the growth status diagram on the 0th day of culture, and B represents the status diagram on the 14th day of growth, and the scale is 1 cm; Figure 7This is a diagram showing the culture state of the unwashed callus in Comparative Example 1 on the differentiation and regeneration medium, with a scale of 1 cm; Figure 8 This is a diagram showing the culture state of callus tissue on differentiation and regeneration medium in Comparative Example 2, with a scale of 1 cm; Fig. 9 for Figure 8 Microscopic picture of callus tissue, scale bar is 100μm; Fig.10 This is a diagram showing the culture state of callus tissue on differentiation and regeneration medium in Comparative Example 3, with a scale of 1 cm; Fig.11 for Fig.10 Microscopic picture of callus tissue, scale bar is 100 μm; Fig.12 The infection solution OD of Comparative Example 4 600 The figure shows the screening situation of callus regeneration seedlings with a value of 0.4 on the screening medium, with a scale of 1 cm; Fig.13 The infection solution OD of Comparative Example 5 600 The screening situation of callus regeneration seedlings with a value of 0.5 on the screening medium, the scale is 1 cm. DETAILED DESCRIPTION

[0018] The present invention provides a genetic transformation method of Elymus nutans, comprising the following steps: Perform callus induction culture on mature embryos of Elymus nutans to obtain callus tissue; The callus tissue is infected and co-cultured with the Agrobacterium infection solution to obtain infected callus tissue; the OD of the Agrobacterium infection solution is 600 is 0.6-0.8, and the concentration of acetosyringone in the infection solution is 0.2-0.25 mM; The infected callus tissue is sterilized and cultured to obtain Elymus nutans plants.

[0019] The invention carries out callus induction culture on mature embryos of Elymus nutans to obtain callus.

[0020] As an optional embodiment, the preparation method of the mature embryo of Elymus nutans of the present invention may include: de-ingraining the seeds of the Elymus nutans to obtain mature embryos. The seeds of the Elymus nutans may be mature seeds of the Elymus nutans with full grains. Before the mature embryo of the Elymus nutans of the present invention is inoculated in an induction medium for callus induction culture, it also includes disinfecting the mature embryo of the Elymus nutans and rinsing it with sterile water. The solution used for the elimination treatment may include a sodium hypochlorite solution with a mass concentration of 40%, and the time of the disinfection treatment may be 30 min. The number of times of the sterile water rinsing may be 5 to 7 times, and the role of the sterile water washing is to remove the sodium hypochlorite solution and avoid the adverse effects of the sodium hypochlorite solution on the mature embryo. As an optional embodiment, the present invention can inoculate the sterilized mature embryo into an induction medium for callus induction culture to obtain callus. As an optional embodiment, the composition of the induction medium of the present invention includes: 4~4.43g / L MS, 4~6mg / L 2,4-D, 40~60mg / L CH, 1~3mg / LABA, 0.04~0.07mg / L KT, 28~30g / L sucrose and 7~9g / L agar. The concentration of MS in the induction medium provided by the present invention can be 4~4.43g / L, or 4~4.2g / L. The concentration of 2,4-D in the induction medium provided by the present invention can be 4~6mg / L; in a specific embodiment of the present invention, the concentration of 2,4-D in the induction medium can be 4.0, 4.5, 5.0, 5.5 or 6.0mg / L. 2,4-D is 2,4-dichlorophenoxy acetic acid, which can induce explants to form callus tissue and promote cell division and growth.

[0021] The concentration of CH in the induction medium provided by the present invention can be 40-60 mg / L; in a specific embodiment of the present invention, the concentration of CH in the induction medium can be 40, 45, 50, 55 or 60 mg / L. CH is Hydrolyzed casein; CH has a good effect on the differentiation of embryoids and adventitious buds.

[0022] The concentration of ABA in the induction medium provided by the present invention can be 1-3 mg / L. In a specific embodiment of the present invention, the concentration of ABA in the induction medium can be 1, 1.5, 2 or 3.0 mg / L. ABA is Abscisic Acid, which can promote the development of seed embryos.

[0023] The concentration of KT in the induction medium provided by the present invention may be 0.04-0.07 mg / L. In a specific embodiment of the present invention, the concentration of KT in the induction medium may be 0.04, 0.05, 0.06 or 0.07 mg / L.

[0024] KT is Kinetin, which can promote the formation of callus tissue. The concentration of sucrose in the induction medium provided by the present invention is 28-30 g / L, or 29-30 g / L. The concentration of agar in the induction medium provided by the present invention is 7-9 g / L, or 8-9 g / L.

[0025] In the present invention, the pH value of the induction medium may be 5.7-5.9, or 5.80.

[0026] As an optional embodiment, the temperature of the callus induction culture can be 23-27°C, or 25°C; the time of the callus induction culture can be 30-45 days, or 40-45 days; and the induction culture is dark culture.

[0027] As an optional embodiment, the callus tissue is obtained. The present invention can cut the callus tissue into pieces, and the cutting is used to remove the embryo and bud, and only retain the dense structure type callus tissue; the callus tissue can be embryonic callus tissue; the size of the cut callus tissue can be 50mm 3 The surface of the dense callus is smooth and shiny, the structure is dense, and the color is mostly light yellow or white, and it is easy to regenerate buds.

[0028] As an optional embodiment, the cut callus tissue is obtained. In the present invention, the cut callus tissue is infected and co-cultured with Agrobacterium infection solution to obtain the infected callus tissue. The OD of the Agrobacterium infection solution is 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, enhance its Agrobacterium activity, and thus improve the transformation efficiency. The time of Agrobacterium infection of the present invention can be 8-12 min, or 9-11 min. After infection, the present invention preferably performs drying treatment on the callus, and inoculates it into a culture medium for co-cultivation after drying treatment. The drying treatment of the present invention can be completed by using dry sterilized filter paper. The time of co-cultivation of the present invention can be 2-3 d; the co-cultivation can be dark culture; the temperature of the co-cultivation can be 25±2°C. The culture medium composition of the co-cultivation of the present invention is to add 100-200 mg / L TMT to the induction culture medium ...ntin (TMT) of the present invention can effectively inhibit the growth of Agrobacterium. The composition of the induction culture medium of the present invention has been discussed above and will not be repeated here.

[0029] The infected callus tissue is obtained. The present invention can sterilize and culture the infected callus tissue to obtain the callus tissue producing adventitious buds.

[0030] As an optional embodiment, the method for sterilization treatment of the present invention can be to place the infected callus in a cephalosporin solution for sterilization treatment. The cephalosporin of the present invention can be cefotaxime sodium, and the preparation method of the cephalosporin solution of the present invention can include: mixing the cefotaxime sodium with water. The concentration of the cephalosporin solution of the present invention can be 0.4~0.6 g / L, or 0.5g / L. The time of each sterilization treatment of the present invention can be 8~10min, or 8.5~9min; the number of sterilization treatments can be 3~5 times, or 4~5 times. The cephalosporin solution can be replaced for each sterilization treatment of the present invention. After soaking the infected callus for 5~10min, the infected callus is replaced with a new cephalosporin solution for the second sterilization, until the sterilization is completed 3~5 times. The sterilization treatment of the present invention effectively inhibits the browning phenomenon of the callus and enables the callus to resume normal growth.

[0031] As an optional embodiment, the present invention can transfer the callus tissue obtained by the sterilization treatment onto a sterilized filter paper to absorb the cephalosporin solution, and then culture it to obtain the Elymus nutans plant.

[0032] As an optional embodiment, the culture of the present invention may include performing differentiation culture, screening and rooting culture in sequence to obtain Elymus nutans plants.

[0033] As an optional embodiment, the composition of the differentiation and regeneration medium of the differentiation culture of the present invention includes: 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 concentration of MS in the differentiation and regeneration medium provided by the present invention is 2~2.2g / L MS, or 2~2.1g / LMS. The concentration of KT in the differentiation and regeneration medium provided by the present invention is 0.01~0.03mg / L; in a specific embodiment of the present invention, the concentration of KT in the differentiation and regeneration medium is 0.01, 0.015, 0.02 or 0.03mg / L. KT is Kinetin, and its Chinese name is kinetin. KT can promote the formation of adventitious buds, and has a certain ratio with NAA to balance the formation of adventitious buds in plants.

[0034] The concentration of NAA in the differentiation and regeneration medium provided by the present invention is 0.3-0.5 mg / L; in a specific embodiment 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 together with KT at an appropriate concentration ratio promotes the formation of adventitious buds.

[0035] The concentration of TMT in the differentiation and regeneration medium provided by the present invention can be 100~200 mg / L. In a specific embodiment 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®), which is an antibacterial agent that can inhibit the growth of bacteria. The concentration of sucrose in the differentiation and regeneration medium provided by the present invention is 12.5~15g / L, or 13~15g / L. The concentration of agar in the differentiation and regeneration medium provided by the present invention is 7~9g / L, or 8~9g / L.

[0036] The differentiation culture of the present invention can be light-dark alternating culture, the illumination time can be 16h / d, the dark time can be 8h / d, the illumination intensity of the present invention can be 2500 lux, and the temperature of the differentiation culture can be 25±2°C. In the differentiation culture process of the present invention, the differentiation and regeneration medium is replaced every 14d to ensure the nutrient supply of the callus tissue, and the composition of the differentiation and regeneration medium replaced each time of the present invention is the same. The differentiation culture of the present invention is carried out until the length of the adventitious bud is 5~8cm. After the differentiation culture is completed, the present invention transfers the adventitious bud to the screening culture medium for screening culture. The adventitious buds with good growth after the screening culture are resistant adventitious buds. The types of antibiotics in the antibiotic screening culture medium of the present invention can include hygromycin, and adventitious buds resistant to hygromycin can be screened; as an optional embodiment, the composition of the screening culture medium of the present invention includes: 4~4.43g / L MS, 30mg / L hygromycin, 28~30g / L sucrose and 7~9g / L agar. The concentration of MS in the screening medium provided by the present invention is 4-4.43 g / L, or 4-4.15 g / L. The setting of the hygromycin concentration is conducive to screening transgenic Elymus nutans plants, i.e. positive plants. The concentration of sucrose in the screening medium provided by the present invention is 28-30 g / L, or 28-29 g / L. The concentration of agar in the screening medium provided by the present invention is 7-9 g / L, or 8-9 g / L.

[0037] The type of antibiotic in the screening culture medium of the present invention depends on the resistance of the Agrobacterium tumefaciens infection vector in the plant body. The Agrobacterium strain used in the present invention may be LBA4404, and the vector used may be pCAMBIA1300. The pCAMBIA1300 vector itself has hygromycin resistance. Therefore, after LBA4404 infection is successful, the vector invades the plant body, making the transgenic plant hygromycin resistant and growing well on the culture medium plate with hygromycin resistance, and the adventitious buds with good growth are obtained by screening.

[0038] As an optional embodiment, the temperature of the differentiation culture of the present invention is 25±2°C, and the time of the screening culture is 18-25 days, or 21-23 days. In a specific embodiment of the present invention, the time of the screening culture is 18, 20, 21, 23 or 25 days.

[0039] As an optional embodiment, the adventitious buds with good growth obtained by screening in the present invention are subjected to rooting culture to obtain transgenic Elymus nutans plants. The temperature of the rooting culture in the present invention is 25±2°C; the time of the rooting culture is 12~14d. The composition of the rooting medium of the present invention: 4~4.43g / L MS, 0.3~0.5g / L NAA, 100~200mg / LTMT, 28~30g / L sucrose and 7~9g / L agar. The concentration of MS in the rooting medium provided by the present invention is 4~4.43g / L, and can also be 4~4.3g / L. The concentration of NAA in the rooting medium provided by the present invention is 0.3~0.5g / L; in a specific embodiment of the present invention, the concentration of NAA in the differentiation and regeneration medium is 0.3, 0.35, 0.4, 0.45 or 0.5g / L. NAA is 1-naphthaleneacetic acid, i.e., 1-naphthaleneacetic 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, or 28-29 g / L. The concentration of agar in the rooting medium provided by the present invention is 7-9 g / L, or 29-30 g / L.

[0040] As an optional embodiment, the rooting culture of the present invention obtains rooted seedlings, and the rooted seedlings are hardened and transplanted to obtain transgenic Elymus nutans plants. The hardening time can be 3 days, and the substrate used for transplanting includes peat soil and vermiculite, and the volume ratio of the peat soil to the vermiculite can be (1-1.5): (0.5-1), or can be 1:1.

[0041] The present invention provides a method for using Agrobacterium tumefaciens ( Agrbacteriumtumefaciens )-mediated by Elymus nutans ( Elymus nutans) Genetic transformation method, using induction medium, callus induction of mature embryos of Elymus nutans, culture to obtain dense embryonic callus; co-culture the callus after subculture with Agrobacterium, use infection solution to infect the callus with Agrobacterium containing target gene; sterilize the callus after co-culture; culture the obtained sterilized callus with differentiation and regeneration medium until adventitious buds are differentiated; screen the callus producing adventitious buds with screening medium to obtain adventitious buds with resistance; root culture the screened adventitious buds of Elymus nutans with resistance to obtain transgenic Elymus nutans seedlings. The present invention can effectively screen transgenic plants, the screening positive rate of the screening medium concentration is as high as 100%, and the operation is simple and easy. The Agrobacterium genetic transformation system established by the present invention provides certain technical guidance for studying the gene function of Elymus nutans; as a wheat family forage grass, the genetic transformation system of Elymus nutans also provides a reliable heterologous expression system for wheat gene function verification, and also provides a basis for gene improvement of Gramineae crops.

[0042] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] 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 specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the protection scope of the present invention. The test methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the reagent manufacturers.

[0044] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0045] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, gene mutation technology, and conventional techniques in related fields in the technical field.

[0046] In the following examples and comparative examples, seeds of Elymus nutans with full grains were used. The same batch of seeds were deglazed to obtain the same batch of mature embryos of Elymus nutans.

[0047] Example 1 A genetic transformation method based on mature embryos of Elymus nutans and mediated by Agrobacterium, the steps are as follows: 1. Disinfection of mature embryos of Elymus nutans The mature embryos of Elymus nutans are obtained by de-gluming the seeds of Elymus nutans with full grains. The de-gluming process means that the seeds are soaked in water for 8 hours and artificially peeled to obtain the mature embryos of Elymus nutans. The seeds cannot be polished; if the seed coat is removed after polishing, the embryo will be damaged.

[0048] The mature embryos of Elymus nutans were used as explants and disinfected by soaking in a 40% sodium hypochlorite solution prepared with ultrapure water for 30 min. The effective chlorine concentration of the 40% sodium hypochlorite solution was 10%. After disinfection, they were placed under sterile conditions on a clean bench and rinsed 10 times with sterile water. The surface of the mature embryos was then wiped dry with sterilized filter paper. The disinfection steps are relatively simple, without toxic chemicals, and less harm to the human body and the environment.

[0049] 2. Callus Induction The sterilized mature embryos of Elymus nutans were placed in a callus induction medium and cultured at a constant temperature of 25±2°C in a dark culture for 45 days. After culture, the dense and tender yellow embryonic callus of Elymus nutans was obtained. During this period, the buds were cut in time. Figure 1 .

[0050] The callus induction medium consisted of 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.

[0051] 3. Agrobacterium Cultivation Agrobacterium carrying the target gene and the selected resistance gene was plated and cultured in a 28°C incubator for 2 days, and the Agrobacterium was suspended in MS liquid medium to form an infection solution. The concentration of the infection solution was OD 600 The concentration of acetosyringone is 0.8, and the infection solution contains 0.2 mM acetosyringone (i.e., 50 μL of acetosyringone mother solution is added to 50 mL of infection solution, and the concentration of acetosyringone mother solution is 200 mM) to activate Agrobacterium.

[0052] 4. Agrobacterium infection and co-cultivation with callus Agrobacterium infection is carried out by soaking dense embryonic callus tissue in infection solution, and the infection time is 10 minutes.

[0053] Then, the callus tissue was fished out from the infection solution, and the excess infection solution was absorbed by sterile filter paper to obtain the infected callus tissue, and then the infected callus tissue was placed in the co-culture medium for co-culture, the co-culture was dark culture, the co-culture time was 2 days, and the composition of the co-culture medium was 100 mg / L TMT added to the induction medium, that is, the composition of the co-culture medium was: 4g / L MS, 4mg / L 2,4-D, 50mg / L CH, 2mg / L ABA, 0.04mg / L KT, 100mg / L TMT, 30g / L sucrose and 8g / L agar, pH 5.8. The Agrobacterium strain was LBA4404, and the vector was pCAMBIA1300.

[0054] 5. Callus recovery and sterilization The callus tissue obtained by co-cultivation in step 4 was washed with 0.5 g / L cephalosporin solution, each washing lasted for 10 minutes, and the washing was performed 4 times in total. The washed callus tissue was then transferred to sterile filter paper to absorb the cephalosporin solution, thereby obtaining washed and dried callus tissue.

[0055] 6. Callus differentiation and regeneration The washed and dried callus was placed in differentiation and regeneration medium to induce buds. The buds were induced by 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℃. The differentiation and regeneration medium was replaced every 14 days. The adventitious buds obtained from the differentiation culture of Elymus nutans callus for 28 days were Figure 2 .

[0056] The composition of the differentiation and regeneration medium is: 2g / L MS, 0.02mg / L KT, 0.5mg / L NAA, 200mg / L TMT, 15g / L sucrose and 8g / L agar.

[0057] 7. Positive plant screening When the adventitious buds generated by the callus in step 6 are 5-8 cm in length, the adventitious buds are transferred to the screening medium for screening culture. The screening medium consists of: 4 g / L MS, 30 mg / L Hyg, 30 g / L sucrose and 8 g / L agar, with a pH of 5.8, and Hyg is hygromycin. The resistant adventitious buds of the callus obtained by screening on the screening medium are shown in FIG. Figure 3 .

[0058] 8. Rooting, hardening and transplanting of regenerated seedlings The green shoots with good growth on the screening medium were transferred to the rooting medium for rooting culture. The rooting culture temperature was 25±2℃ and the rooting culture time was 14 days. After strong roots grew out, the test tube seedlings were opened and hardened for 3 days. The test tube seedlings were taken out and the residual culture medium on the roots was washed off. They were transplanted into a flower pot in the greenhouse with peat soil and vermiculite in a volume ratio of 1:1 to obtain complete transgenic regenerated plants of Elymus nutans. Figure 4 The rooting medium formula was: 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.

[0059] In this example, the callus induction rate of mature embryos of Elymus nutans was 65%, and the regeneration differentiation rate was 46%.

[0060] Induction rate (%) = (number of healed explants / number of infected explants) × 100%; Differentiation rate (%) = (number of sprouting callus / number of infected callus) × 100%.

[0061] Example 2 Screening efficiency of regenerated adventitious buds in different concentrations of hygromycin screening medium The adventitious buds of Elymus nutans were cultured using the method of steps 1 to 6 in Example 1. After the adventitious buds grew out of the callus tissue of Elymus nutans, the adventitious buds were transferred to the screening medium of treatment 1 to treatment 5 for culture, with 5 regenerated plants in each plate, and 3 repeated experiments were set for each screening concentration. The growth of the regenerated plants was recorded. Among them, the screening medium of treatment 1 was: 4g / L MS, 10mg / L Hyg, 30 g / L sucrose and 8g / L agar, with a pH of 5.8; the screening medium of treatment 2 was: 4g / LMS, 20mg / L Hyg, 30 g / L sucrose and 8g / L agar, with a pH of 5.8; the screening medium of treatment 3 was: 4g / L MS, 30mg / LHyg, 30 g / L sucrose and 8g / L agar, with a pH of 5.8; the screening medium of treatment 4 was: 4g / L MS, 40mg / L Hyg, 30 g / L sucrose and 8g / L agar, with a pH of 5.8; the screening medium of treatment 5 was: 4g / L MS, 50mg / L Hyg, 30 g / L sucrose and 8g / L agar, with a pH of 5.8.

[0062] After 14 days of culture, the leaf DNA of the plants that survived on the hygromycin selection medium was extracted, and then PCR was performed using the plant DNA as a template to determine whether the hygromycin resistance gene was successfully transferred. The results of the positive plant identification of Elymus nutans are shown in Figure 5 , where M represents Marker DL 5000; 1 represents the positive control, i.e., Agrobacterium after gene transfer; 2~5 represent transgenic plants; and 6 represents negative plants.

[0063] The positive rates of hygromycin were counted respectively.

[0064] Hygromycin positive rate (%) = (number of hygromycin positive plants / number of surviving plants) × 100; 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 of the 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 of the 15 regenerated plants survived, and the surviving plants were confirmed to be hygromycin-positive plants by PCR testing, indicating that this concentration can play a good screening role; on the 40 mg / L hygromycin screening medium, 1 of the 15 regenerated plants survived, and the surviving plants were confirmed to be hygromycin-positive plants by PCR testing, indicating that this concentration can also play a screening role, but the survival rate is too low; on the 50 mg / L hygromycin screening medium, all 15 regenerated plants died (Table 1). The growth status of the adventitious buds of Elymus nutans in different screening media is shown in Figure 6 , where A represents the growth state diagram of the 0th day of culture, and B represents the state diagram of the 14th day of growth; Figure 6 The concentrations of hygromycin in the hygromycin selection culture medium plates from left to right are 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, respectively.

[0065] Table 1 Statistics of screening results of different concentrations of hygromycin

[0066] The PCR identification band size is about 1000 bp, and the primer sequences are as follows Table 2 List of primers for PCR amplification of Hygromycin gene

[0067] In this example, the optimal hygromycin screening concentration screened was 30-40 mg / L, and the transgenic positive screening rate was 100%.

[0068] Comparative Example 1 A genetic transformation method based on mature embryos of Elymus nutans and mediated by Agrobacterium, the steps are as follows: 1. Disinfection of mature embryos of Elymus nutans. Same as Example 1.

[0069] 2. Callus induction: Same as Example 1.

[0070] 3. Agrobacterium culture. Same as Example 1.

[0071] 4. Infection of Agrobacterium and co-cultivation with callus tissue. Same as Example 1.

[0072] 5. Callus differentiation and regeneration The callus obtained in step 4 was placed in a differentiation and regeneration medium to induce buds. The buds were induced by 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. Figure 7 .

[0073] 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.

[0074] according to Figure 7 It can be seen that after the co-cultivation, no sterilization treatment was carried out, and the callus tissue obtained by co-cultivation was directly inoculated on the differentiation and regeneration medium. Bacteria existed on the surface of the callus tissue and on the medium, and the callus tissue had a high degree of browning. This callus tissue state could not generate regenerated seedlings.

[0075] Comparative Example 2 Only steps 1 to 6 of Example 1 were performed, which were the same as Example 1, except that the callus obtained by co-cultivation was washed with 0.5 g / L cephalosporin solution, each time for 4 minutes, and a total of 4 times. The washed callus was then transferred to a sterilized filter paper to absorb the cephalosporin solution to obtain washed and dried callus.

[0076] The washed and dried callus tissue was placed in differentiation and regeneration medium to induce buds. The picture of callus tissue after 13 days of culture is shown in Figure 8 and Fig. 9 It can be seen that there are no traces of bacteria on the differentiation and regeneration medium, but there are still bacteria covering the surface of the callus tissue, and the callus tissue cannot regenerate.

[0077] Comparative Example 3 Only 1 to 6 of Example 1 were performed, which was the same as Example 1, with the only difference being that the callus obtained by co-cultivation was washed with 0.5 g / L cephalosporin solution, each time for 7 minutes, and a total of 4 times. The washed callus was then transferred to sterile filter paper to absorb the cephalosporin solution to obtain washed and dried callus. The washed and dried callus was placed in a differentiation and regeneration medium to induce budding. The picture of the callus after 14 days of culture is shown in Fig.10 and Fig.11It can be seen that in the differentiation and regeneration medium, the callus tissue grew well in the first week without bacterial growth, but one week later, bacteria gradually grew around the callus tissue, causing the original buds of the callus tissue to turn yellow and then white and die, and then the entire callus tissue was completely covered with bacteria and could not continue to grow.

[0078] Comparative Example 4 Only 1 to 7 of Example 1 are carried out, which is the same as Example 1, except that the concentration of the infection solution is OD 600 is 0.4, and the infection solution contains 0.2 mM acetosyringone.

[0079] The adventitious buds cultured on the screening medium were Fig.12 It can be seen that although there is no obvious difference when the callus tissue grows on the differentiation and regeneration medium, when the regenerated seedlings produced by the callus tissue are cultured on the screening medium, it is found that all the regenerated seedlings in the appropriate screening medium die. This shows that although the callus tissue does not breed bacteria under this condition, the infection solution concentration is OD 600 The regenerated seedlings generated by the concentration of 0.4 did not survive on the screening medium, indicating that the infection success rate of the positive seedlings was greatly reduced under this experimental condition.

[0080] Comparative Example 5 Only 1 to 7 of Example 1 are carried out, which is the same as Example 1, except that the concentration of the infection solution is OD 600 The concentration of acetosyringone was 0.5, and the infection solution contained 0.2 mM acetosyringone. The callus obtained by co-cultivation was washed with 0.5 g / L cephalosporin solution, each washing lasted 10 minutes, and the washing was performed 4 times in total.

[0081] The adventitious buds cultured on the screening medium were Fig.13 It can be seen that although the state of the regenerated seedlings on the screening medium is not good, there are still regenerated seedlings that survive compared to Comparative Example 4, but the survival rate of the regenerated seedlings is low, indicating that the infection efficiency is higher than OD 600 The infection solution is 0.4, but lower than the infection solution OD 600 The infection efficiency is 0.8.

[0082] In summary, the induction rate of the compact callus of the present invention reaches 65%, and in the subsequent differentiation culture process, 46% of the callus differentiates into adventitious buds and produces regenerated plants, and the positive rate of transgenic plant screening is 100%. The Agrobacterium genetic transformation system based on mature embryos of Elymus nutans established by the present invention provides certain technical guidance for exploring the gene function of Elymus nutans, and also lays a foundation for the directional molecular design to improve the varieties of Elymus nutans.

[0083] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, 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: Perform callus induction culture on mature embryos of Elymus nutans to obtain callus tissue; The callus tissue is infected and co-cultured with the Agrobacterium infection solution to obtain infected callus tissue; the OD of the Agrobacterium infection solution is 600 The infection solution contains acetosyringone, and the concentration of acetosyringone is 0.2-0.25 mM; The infected callus tissue is sterilized and cultured to obtain Elymus nutans plants.

2. The genetic transformation method according to claim 1, characterized in that The culture comprises performing differentiation culture, screening and rooting culture in sequence to obtain Elymus nutans plants.

3. The genetic transformation method according to claim 1, characterized in that The reagents used in the sterilization treatment include 0.4-0.6 g / L cephalosporin antibiotic solution.

4. The genetic transformation method according to claim 1 or 3, characterized in that Each sterilization treatment lasts 8 to 10 minutes, and the number of sterilization treatments is 3 to 5 times.

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

6. 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.

7. The genetic transformation method according to claim 1, characterized in that The culture medium used for callus induction culture comprises an induction culture medium, and the composition of the induction culture medium comprises: 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.

8. The genetic transformation method according to claim 2, characterized in that The culture medium used in the differentiation culture includes a differentiation and regeneration culture medium, and the composition of the differentiation and regeneration culture 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 culture medium is replaced every 14 days during the differentiation culture; the differentiation culture is carried out until the length of the adventitious buds is 5-8 cm; The culture medium used for the rooting culture comprises a rooting culture medium, and the composition of the rooting culture medium comprises: 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.

9. 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.

10. The genetic transformation method according to claim 2, characterized in that: The culture medium used for screening includes a screening culture medium, and the types of antibiotics in the screening culture medium include hygromycin; The screening culture medium comprises: 4-4.43 g / L MS, 30 mg / L hygromycin, 28-30 g / L sucrose and 7-9 g / L agar.

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