Genetic transformation method based on oat immature embryos

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a genetic transformation method based on oat immature embryos, which comprises the following steps: preparing an infection solution containing a target gene; taking immature caryopses of the oats 21-23 days after pollination; infecting the immature embryos with infection liquid, and co-culturing the immature embryos after infection is completed; after co-culture is completed, immature embryos are sequentially transferred into an L3-T culture medium and an L3-B5 culture medium to induce formation of calluses, and after the calluses are formed, the calluses are sequentially induced to be differentiated, rooted and hardened. According to the method, efficient genetic transformation of exogenous genes is realized by selecting proper oat immature embryos, proper culture conditions and proper transformation conditions. By means of the genetic transformation method, the genetic transformation efficiency of the oats is improved to 25% from 2%-3%. And meanwhile, the immature embryo is used as a transformation acceptor material, and the complete plant can be efficiently and stably regenerated by the method disclosed by the invention.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and particularly relates to a genetic transformation method based on oat immature embryos. Background Art

[0002] Oat is an important crop that can be used as both food and forage, and plays an important role in ensuring a stable supply of forage and providing miscellaneous grain foods with rich nutritional value. However, due to the lack of an efficient and stable genetic transformation system in oats at present, the basic biological research of oats lags far behind that of other important gramineous crops such as maize, wheat, and rice, seriously hindering the research of oat functional genes and the breeding process of new oat varieties with high yield, high quality, and stress tolerance. Therefore, establishing an efficient and stable oat genetic transformation system is the basic work for studying oat gene functions, carrying out molecular design breeding using excellent gene resources, and improving oat yield and environmental adaptability.

[0003] Although there are currently a few research reports on oat genetic transformation, according to the existing technical status, oat genetic transformation still faces many technical problems, mainly the low efficiency of oat genetic transformation: the low efficiency of oat genetic transformation is one of the key factors restricting its genetic improvement, which may be related to various factors such as oat genotype, explant selection, and culture conditions. According to current research, the genetic transformation efficiency of oats is only about 2% - 3%.

[0004] Therefore, there is an urgent need to construct an efficient and stable oat genetic transformation system at present. This is the basic work for studying oat functional genes and carrying out molecular design breeding using excellent gene resources, and is of great significance for further carrying out basic biological research on oats and cultivating new oat varieties with high yield, high quality, and stress tolerance. Summary of the Invention

[0005] The purpose of the present invention is to provide a genetic transformation method based on oat immature embryos, which solves the problem of low efficiency of oat genetic transformation in the prior art.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The present invention provides a genetic transformation method based on oat immature embryos, comprising the following steps:

[0008] Prepare an infection solution containing the target gene; take immature caryopses of oat ears 21 to 23 days after pollination, and collect the immature embryos of the immature caryopses under sterile conditions; use the infection solution to infect the immature embryos, and perform co-culture on the immature embryos after the infection is completed; after the co-culture is completed, transfer the immature embryos to L3-T medium and L3-B5 medium in sequence to induce the formation of callus. Each liter of L3-T medium contains 4.55 g to 4.65 g of L3 medium basal salts containing vitamins, 29.95 g to 30.05 g of maltose, 3.95 g to 4.05 g of phytagel, 1.90 mL to 2.10 mL of 2,4-D at 1 mg / mL, 0.90 mL to 1.10 mL of dicamba at 1 mg / mL, and 1.00 mL to 1.20 mL of ticarcillin at 200 mg / mL, and the rest is made up with water; each liter of L3-B5 medium contains 4.55 g to 4.65 g of L3 medium basal salts containing vitamins, 29.95 g to 30.05 g of maltose, 3.95 g to 4.05 g of phytagel, 1.90 mL to 2.10 mL of 2,4-D at 1 mg / mL, 0.90 mL to 1.10 mL of dicamba at 1 mg / mL, 1.00 mL to 1.20 mL of ticarcillin at 200 mg / mL, 200 μL to 210 μL of glufosinate at 25 mg / mL, and the rest is made up with water; after the callus is formed, induce the differentiation of the callus and root hardening and seedling raising in sequence.

[0009] Preferably, each liter of L3-T medium contains 4.6 g of L3 medium basal salts containing vitamins, 30 g of maltose, 4 g of phytagel, 2 mL of 2,4-D at 1 mg / mL, 1 mL of dicamba at 1 mg / mL, and 1 mL of ticarcillin at 200 mg / mL, and the rest is made up with water; each liter of L3-B5 medium contains 4.6 g of L3 medium basal salts containing vitamins, 30 g of maltose, 4 g of phytagel, 2 mL of 2,4-D at 1 mg / mL, 1 mL of dicamba at 1 mg / mL, 1 mL of ticarcillin at 200 mg / mL, 200 μL of glufosinate at 25 mg / mL, and the rest is made up with water.

[0010] Preferably, the process of infecting the immature embryos is: soak the pretreated immature embryos in the infection solution and let them stand at room temperature for 15 min to 20 min; the pretreatment conditions are 4°C, 12,000 rpm, and centrifuge for 10 min.

[0011] Preferably, the process of inducing the formation of the callus is: transfer the co-cultured immature embryos to L3-T medium and culture them in the dark at 25°C ± 2°C for 5 days to 7 days, and then transfer them to L3-B5 medium and culture them in the dark at 25°C ± 2°C for 5 weeks to 6 weeks.

[0012] Preferably, the culture medium formulation for the co-culture is as follows: per liter of the culture medium, it contains 4.3 g of Linsmaier & Skoog basal salts, 10 g of glucose, 0.5 g of 2-(N-morpholino)ethanesulfonic acid, 100 μL of 12.5 g / L CuSO 4 ·5H 2 O 100 μL, 4 g of phytagel, 100 μL of 1000×MS vitamin solution, 1 mL of 100 mM acetosyringone, and 100 μL of 8.5 g / L AgNO 3 100 μL, and the rest is made up with water.

[0013] Preferably, the culture medium formulation for inducing the differentiation of the callus is as follows: per liter of the culture medium, it contains 4.6 g of L3 medium basal salts with vitamins, 20 g of sucrose, 0.5 g of 2-(N-morpholino)ethanesulfonic acid, 200 μL of 12.5 g / L CuSO 4 ·5H 2 O 200 μL, 4 g of phytagel, 5 mL of 1 mg / mL zeatin, 1 mL of 200 mg / mL ticarcillin, and 200 μL of 25 mg / mL glufosinate, and the rest is made up with water.

[0014] Preferably, the culture medium formulation for the rooting and acclimatization of seedlings is as follows: per liter of the culture medium, it contains 4.6 g of L3 medium basal salts with vitamins, 15 g of sucrose, 0.5 g of 2-(N-morpholino)ethanesulfonic acid, 4 g of phytagel, 20 μL of 10 mg / mL IBA, 1 mL of 200 mg / mL ticarcillin, 200 μL of 25 mg / mL glufosinate, and the rest is made up with water.

[0015] Preferably, the process for preparing the infection solution is as follows: Take the positive monoclonal Agrobacterium colony and culture it in YEP liquid medium, collect the bacterial cells, resuspend them in LS-liq medium, and add acetosyringone with a final concentration of 100 μM; the positive monoclonal is the plasmid containing the target gene.

[0016] Preferably, the formulation of the LS-liq medium is as follows: per liter of the LS-liq medium, it contains 4.3 g of Linsmaier & Skoog basal salts, 100 μL of 1000×MS vitamin solution, 10 g of glucose, 0.5 g of 2-(N-morpholino)ethanesulfonic acid, and the rest is made up with water.

[0017] Preferably, the conditions for inducing the differentiation of the callus are: 25 °C, 70 μmol / m 2 / s, culture for 3 weeks; the conditions for the rooting and acclimatization of seedlings are 25 °C, 70 μmol / m 2 / s, culture for 3 weeks.

[0018] Preferably, the target gene includes the GUS reporter gene or the gene for genetic transformation.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention provides a genetic transformation method based on oat immature embryos, comprising the following steps:

[0021] Prepare an infection solution containing a target gene; take immature caryopses of oat spikes 21 to 23 days after oat pollination, and collect the immature embryos of the immature caryopses under sterile conditions; infect the immature embryos with the infection solution, and perform co-culture on the immature embryos after the infection is completed; after the co-culture is completed, transfer the immature embryos to L3-T medium and L3-B5 medium in sequence to induce callus formation. Each liter of L3-T medium contains 4.55 g to 4.65 g of L3 medium basal salts containing vitamins, 29.95 g to 30.05 g of maltose, 3.95 g to 4.05 g of phytagel, 1.90 mL to 2.10 mL of 2,4-D at 1 mg / mL, 0.90 mL to 1.10 mL of dicamba at 1 mg / mL, and 1.00 mL to 1.20 mL of ticarcillin at 200 mg / mL, and the rest is made up with water; each liter of L3-B5 medium contains 4.55 g to 4.65 g of L3 medium basal salts containing vitamins, 29.95 g to 30.05 g of maltose, 3.95 g to 4.05 g of phytagel, 1.90 mL to 2.10 mL of 2,4-D at 1 mg / mL, 0.90 mL to 1.10 mL of dicamba at 1 mg / mL, 1.00 mL to 1.20 mL of ticarcillin at 200 mg / mL, 200 μL to 210 μL of glufosinate at 25 mg / mL, and the rest is made up with water; after callus formation, induce callus differentiation and rooting and acclimatization in sequence.

[0022] The present invention realizes the efficient genetic transformation of foreign genes by selecting suitable oat immature embryos, appropriate culture conditions and transformation conditions, including culture medium, infection method and culture conditions. Through the genetic transformation method described in the present invention, the genetic transformation efficiency of oats is increased from the original 2% - 3% to 25%.

[0023] In the prior art of oat genetic transformation, it is difficult to select transformation receptors, and the selection of transformation receptors is one of the key factors affecting transformation efficiency. However, the sources of oat transformation receptors are limited, and different receptors also have differences in adaptability to transformation conditions. In previous studies, researchers carried out oat genetic transformation using explants such as oat mature embryos, young leaves, hypocotyls, root tips, anthers, ovaries, glumes, etc., but the success rate was extremely low. The present invention uses immature embryos as transformation receptor materials. The immature embryos are easily obtained and large in quantity as transformation receptor materials, which is conducive to large-scale genetic transformation experiments; their transformation receptor regeneration ability is strong, and complete plants can be regenerated efficiently and stably under the culture conditions and transformation conditions provided by the present invention.

[0024] The type of transformation receptor is a major factor affecting plant genetic transformation. Selecting a suitable transformation receptor is an important prerequisite for the success of genetic transformation. There are many options for the transformation receptor in the oat genetic transformation process, including immature embryos, young leaves, hypocotyls, root tips, anthers, ovaries, glumes, etc., but there are few successful cases at present. Initially, some scholars at home and abroad mostly used embryogenic callus as the transformation receptor, but the transformation results were not very satisfactory. The fertility of the regenerated plants was not high, and albino seedlings appeared. Moreover, using embryogenic callus as the receptor had a long cycle. Therefore, in recent years, some researchers have used apical meristems, immature embryos, and leaf base segments as transformation receptors. The problem of low genetic transformation efficiency faced by using immature embryos as transformation receptors may be due to unsolved problems in multiple key links. For example, it is difficult to accurately determine the sampling period. If the sampling is too early, the immature embryo cells have a low degree of differentiation and unstable cell activity, which may lead to difficulties in normal cell division and development after transformation. If the sampling is too late, the immature embryo cells may have already started to highly differentiate, the cell wall thickens, which will hinder the introduction of foreign genes, and the cell totipotency decreases, and the regeneration ability weakens. The leaf base is the base of the plant leaf, which is easy to separate and obtain from the plant and does not require complex tissue culture techniques. Compared with immature embryos, the leaf base has a weaker regeneration ability and may not be able to efficiently regenerate complete plants. Immature embryos usually have a strong regeneration ability and can efficiently and stably regenerate complete plants under tissue culture conditions. Moreover, the immature embryo cells have a low degree of differentiation and high totipotency, so they are more likely to accept the integration and expression of foreign genes. Transgenic plants obtained by immature embryo transformation usually have good genetic stability and can stably transmit foreign genes to the offspring.

[0025] Plant genetic transformation technology is a basic biological technology for analyzing gene functions and improving plant traits. By genetic transformation, transgenic plants can be constructed to achieve the directional improvement of some excellent agronomic traits. However, at present, an efficient and stable genetic transformation system has not been established in oats. Therefore, the present invention aims to establish an efficient and stable oat genetic transformation system that can obtain oat transgenic plants, which is of great significance for the development of oat basic biological research. Brief Description of the Drawings

[0026] Figure 1 It is a flow chart of the genetic transformation of oat immature embryos. Among them, a: oat immature embryos placed in the infection solution; b: the infected immature embryos are dried on sterile filter paper; c: the infected immature embryos are co-cultured on the LS-AS medium; d: callus induced on the L3-B5 medium; e: the callus differentiates into regenerated seedlings on the L3L-B5 medium; f: the regenerated seedlings are rooted on the L3R-B5 medium; g: the seedlings are transplanted; h: the transgenic positive seedlings are detected by the transgenic PAT / bar colloidal gold test strip. From left to right, the first 20 are 20 transgenic seedlings, and the last 2 are wild types. Detailed Implementation Modes

[0027] The present invention will be further described below through specific embodiments, but the scope of the present invention is not limited thereby. Modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0028] The inventive concept of the present invention is as follows:

[0029] In the prior art, there are many technical problems in oat genetic transformation. The main problem is the low efficiency of oat genetic transformation: the low efficiency of oat genetic transformation is one of the key factors restricting its genetic improvement, which may be related to various factors such as the genotype of oats, the selection of explants, and the culture conditions. According to current research, the genetic transformation efficiency of oats is only about 2% - 3%. Therefore, there is an urgent need to construct an efficient and stable oat genetic transformation system.

[0030] Based on this, the present invention provides a genetic transformation method based on oat immature embryos, comprising the following steps:

[0031] Prepare an infection solution containing a target gene; take immature caryopses of oat ears 21 - 23 days after pollination of oats, and collect immature embryos of the immature caryopses under sterile conditions; use the infection solution to infect the immature embryos, and after the infection is completed, co - culture the immature embryos; after the co - culture is completed, transfer the immature embryos to L3 - T medium and L3 - B5 medium in sequence to induce the formation of callus. Each liter of L3 - T medium contains 4.55 g - 4.65 g of L3 medium basal salts containing vitamins, 29.95 g - 30.05 g of maltose, 3.95 g - 4.05 g of phytagel, 1.90 mL - 2.10 mL of 2,4 - D at 1 mg / mL, 0.90 mL - 1.10 mL of dicamba at 1 mg / mL, and 1.00 mL - 1.20 mL of ticarcillin at 200 mg / mL, and the rest is made up with water; each liter of L3 - B5 medium contains 4.55 g - 4.65 g of L3 medium basal salts containing vitamins, 29.95 g - 30.05 g of maltose, 3.95 g - 4.05 g of phytagel, 1.90 mL - 2.10 mL of 2,4 - D at 1 mg / mL, 0.90 mL - 1.10 mL of dicamba at 1 mg / mL, 1.00 mL - 1.20 mL of ticarcillin at 200 mg / mL, 200 μL - 210 μL of glufosinate at 25 mg / mL, and the rest is made up with water; after the callus is formed, induce the differentiation of the callus and rooting and acclimatization of the seedlings in sequence.

[0032] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with specific embodiments. In the description of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0033] Preparation method of the culture medium used in the present invention:

[0034] (1) For every liter of YEP medium: 10 g of YEAST EXTRACT, 10 g of TRYPTONE, 5 g of NaCl, and make up the volume to 1 L with water.

[0035] (2) For every liter of LS-liq medium: 4.3 g of Linsmaier & Skoog Base Salts, 100 μL of 1000×MS vitamin solution, 10 g of Glucose, 0.5 g of 2-(N-morpholino)ethanesulfonic acid, make up the volume to 1 L with water, adjust the pH to 5.8 with 1 mol / L KOH, and filter-sterilize by suction.

[0036] (3) For every liter of LS-AS medium: 4.3 g of Linsmaier & Skoog basal salts, 10 g of Glucose, 0.5 g of 2-(N-morpholino)ethanesulfonic acid, 100 μL of 12.5 g / L CuSO 4 ·5H 2 O, 4 g of Phytagel, adjust the pH to 5.8 with 1 mol / L KOH, autoclave at 121 °C for 15 min, after cooling to room temperature, add 100 μL of 1000×MS vitamin solution, 1 mL of 100 mM acetosyringone, 100 μL of 8.5 g / L AgNO 3 100 μL.

[0037] (4) For every liter of L3-T medium: 4.6 g of L3 Base Salts with vitamins, 30 g of Maltose, 4 g of Phytagel, adjust the pH to 5.8 with 1 mol / L KOH, autoclave at 121 °C for 15 min, after cooling to room temperature, add 2 mL of 1 mg / mL 2,4-D, 1 mL of 1 mg / mL Dicamba, 1 mL of 200 mg / mL Timentin.

[0038] (5) For every liter of L3-B5 medium: 4.6 g of L3 Base Salts with vitamins, 30 g of Maltose, 4 g of Phytagel, adjust the pH to 5.8 with 1 mol of KOH, autoclave at 121 °C for 15 min, after cooling to room temperature, add 2 mL of 1 mg / mL 2,4-D, 1 mL of 1 mg / mL Dicamba, 1 mL of 200 mg / mL Timentin, 200 μL of 25 mg / mL Basta.

[0039] (6) L3 Base Salts with vitamins 4.6 g, Sucrose 20 g, 2-(N-morpholino)ethanesulfonic acid 0.5 g, 12.5 g / L CuSO per liter 4 ·5H 2 O 200μL, Phytagel 4g, adjust the pH to 5.8 with 1mol / L KOH, sterilize at 121℃ for 15min, add 1mg / mL Zeatin 5mL, 200mg / mL Timentin 1mL, and 25mg / mL Basta 200μL after cooling to room temperature.

[0040] (7) Per liter of L3R-B5 medium: L3 Base Salts with vitamins 4.6 g, Sucrose 15 g, 2-(N-morpholine)ethanesulfonic acid 0.5 g, Phytagel 4 g, adjust the pH to 5.8 with 1 mol / L KOH, sterilize at 121°C for 15 min, and after cooling to room temperature, add 20 μL of 10 mg / mL IBA, 1 mL of 200 mg / mL Timentin, and 200 μL of 25 mg / mL Basta.

[0041] 1000×MS vitamin solution was purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd. with the product number MV2520S.

[0042] L3 Base Salts withvitamins was purchased from CHINOOK with the product number CN260600-25L.

[0043] The abbreviations / Chinese-English comparison table of the present invention is shown in Table 1.

[0044] Table 1 Abbreviations / English-Chinese correspondence

[0045]

[0046]

[0047] Example 1

[0048] A genetic transformation method based on oat immature embryos is described as follows:

[0049] The present invention uses oat embryos with strong regeneration ability as transformation receptors, and successfully obtains transgenic positive seedlings by improving the culture medium formula, infection method and culture conditions. Except for the planting of oats and the separation of immature caryopsis, other operations are completed in a clean bench, and the specific steps are as follows:

[0050] (1) Prepare infection solution containing target gene:

[0051] The Ubi::GUS plasmid carrying the GUS reporter gene was transferred into Agrobacterium tumefaciens GV3101 by the liquid nitrogen freeze-thaw method, and PCR detection was carried out to screen for positive Agrobacterium colonies containing the Ubi::GUS plasmid.

[0052] Positive monoclonal Agrobacterium colonies were picked into 10 mL of YEP liquid medium and cultured overnight at 28 °C and 220 rpm in a shaker for 16 h. The bacteria were collected by centrifugation at 5000 rpm for 10 min at room temperature, and the supernatant was discarded.

[0053] Add 1 mL of LS-liq medium to resuspend, adjust OD 600 to about 1.0, and add acetosyringone with a final concentration of 100 μM for use.

[0054] (2) Take the immature caryopses of oat spikes 21 days after pollination, and collect the immature embryos of the immature caryopses under sterile conditions:

[0055] Oat cultivation: Oat plants were potted in an artificial climate chamber. The light conditions in the climate chamber were 16 h light / 8 h dark, the light intensity was set to 100%, 1000 μmol / m 2 / s, the humidity was set to 60%, the temperatures during the light period and the dark period were set to 25 °C and 18 °C respectively, and the plants were watered once every 5 days during the growth period.

[0056] Isolation of immature caryopses: Take oat spikes 21 days after heading and pollination, select the lowest seed in each oat floret, remove the glumes, lemma, and palea wrapped outside the seed, and only keep the caryopsis.

[0057] Disinfection of immature caryopses: In a sterile laminar flow hood, place the isolated caryopses in 75% (v / v) alcohol for 1 min, wash 3 times with sterile water, then place them in 1% (v / v) sodium hypochlorite solution for 10 min, shake well during this period, and wash 5 times with sterile water for use.

[0058] Sampling of immature embryos in immature caryopses: In a sterile laminar flow hood, use a sterile scalpel to dissect the immature embryos in the disinfected immature caryopses under a stereomicroscope, and place the immature embryos into a 2 mL centrifuge tube containing LS-liq medium.

[0059] (3) Infect the immature embryos with the infection solution:

[0060] Invert the 2 mL centrifuge tube containing the immature embryos several times and then pour out the LS-liq medium. Add 1 mL of LS-liq medium, centrifuge at 4 °C and 12000 rpm for 10 min, and pour out the LS-liq medium in the centrifuge tube. Add 1 mL of the infection solution prepared in (1) above, immerse the immature embryos in the infection solution, and let stand at room temperature for 15 min.

[0061] (4) After the infection is completed, co-culture the immature embryos:

[0062] Pour out the infection solution, and use sterile forceps to transfer the immature embryos to sterile filter paper to dry for 2 min.

[0063] Use sterile forceps to transfer the dried immature embryos to LS-AS medium, with the cut surface facing down and closely attached to the medium, and co-culture in a 25 °C dark incubator for 2 d.

[0064] (5) After the co-culture is completed, transfer the immature embryos to L3-T medium and L3-B5 medium to induce callus formation:

[0065] Use sterile forceps to transfer the immature embryos after 2 d of co-culture to L3-T medium, with the cut surface facing down and closely attached to the medium, and culture in a 25 °C dark incubator for 5 days.

[0066] Use sterile forceps to transfer the immature embryos to L3-B5 medium, with the cut surface facing down and closely attached to the medium, and culture in a 25 °C dark incubator for 5 weeks. During this period, if there are elongated buds, use sterile scissors to remove the elongated buds. Replace the fresh L3-B5 solid medium every 2 weeks.

[0067] (6) After the callus is formed, induce the differentiation of the callus:

[0068] Transfer the callus induced in the above (5) to L3L-B5 medium with sterile forceps. Culture in a 25 °C incubator under full light, with a light intensity of 70 μmol / m 2 / s, and culture for 3 weeks until the differentiated seedlings grow to about 3 cm.

[0069] (7) Rooting and hardening off:

[0070] Transfer the regenerated seedlings to L3R-B5 solid medium, culture in a 25 °C incubator under full light, with a light intensity of 70 μmol / m 2 / s, and culture for 3 weeks. After rooting, transplant.

[0071] Transplant the rooted plant seedlings into flower pots filled with nutrient soil, where the weight ratio of nutrient soil to vermiculite is 1:1. Place them in an artificial climate chamber for growth. The conditions of the climate chamber are set the same as those in the above (2). Cover the flower pots with plastic wrap to keep them moist for about one week and then remove it. After the seedlings grow healthily for 3 weeks, wait for detection. The whole process is shown in Figure 1 a - g of

[0072] Detect the oat seedlings prepared by the method described in Example 1. The transgenic PAT / bar colloidal gold test strip method is used. The test kit is purchased from Wuhan Boyuan Yanjuan. The specific process is as follows:

[0073] Cut a young and tender leaf about 2 cm long and place it in a 1.5 mL centrifuge tube, making a mark.

[0074] Use a disposable grinding rod to rotate and crush the leaf for 30 s.

[0075] Add 0.2 mL of extraction buffer to the centrifuge tube, and use the grinding rod to repeat the crushing to fully contact and mix the sample with the extraction buffer to extract the antigen protein in the sample.

[0076] Insert the transgenic PAT / bar colloidal gold test strip directly into the sample and start timing. After 10 min, place the test strip horizontally in front of the observer to analyze the result. Negative: The T line does not show color; Positive: The T line shows visible color to the naked eye; Invalid: The C line does not appear, which may be due to improper operation or the test strip being invalid. In this case, the instruction manual should be read again and the test should be repeated with a new test strip. The test results are shown in Figure 1 h.

[0077] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0078] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A genetic transformation method based on oat immature embryos, characterized in that: The following steps are involved: preparing an infection solution containing the target gene; Take the immature caryopsis of oat ears 21 to 23 days after oat pollination, and collect the young embryos of the immature caryopsis under sterile conditions; Infect young embryos with infection solution, and co-culture the young embryos after infection; After the co-culture is completed, the immature embryos are transferred to L3-T medium and L3-B5 medium in sequence to induce callus formation. Each liter of L3-T medium contains 4.55 g to 4.65 g of L3 medium base salt containing vitamins, 29.95 g to 30.05 g of maltose, 3.95 g to 4.05 g of plant gel, and 1 mg / mL of 2,4-D. 1.90mL~2.10mL, 0.90mL~1.10mL of 1mg / mL dicamba and 1.00mL~1.20mL of 200mg / mL timentin, and the rest is supplemented with water; each liter of L3-B5 culture medium contains 4.55g~4.65g of L3 culture medium base salt containing vitamins, 29.95g~30.05g of maltose, 3.95g~4.05g of plant gel, 1.90mL~2.10mL of 1mg / mL 2,4-D, 0.90mL~1.10mL of 1mg / mL dicamba, 1.00mL~1.20mL of 200mg / mL timentin, 200μL~210μL of 25mg / mL glufosinate, and the rest is supplemented with water; After callus formation, callus differentiation and rooting and seedling hardening are induced in sequence.

2. The genetic transformation method according to claim 1, characterized in that Each liter of L3-T culture medium contains 4.6 g of L3 culture medium base salt containing vitamins, 30 g of maltose, 4 g of plant gel, 2 mL of 1 mg / mL 2,4-D, 1 mL of 1 mg / mL dicamba, and 1 mL of 200 mg / mL timentin, and the rest is made up with water; Each liter of the L3-B5 culture medium contains 4.6 g of L3 culture medium basal salt containing vitamins, 30 g of maltose, 4 g of plant gel, 2 mL of 1 mg / mL 2,4-D, 1 mL of 1 mg / mL dicamba, 1 mL of 200 mg / mL timentin, 200 μL of 25 mg / mL glufosinate ammonium, and the rest is made up with water.

3. The genetic transformation method according to claim 1, characterized in that The process of infecting the young embryos is as follows: immersing the pretreated young embryos in the invading dye solution and standing at room temperature for 15 to 20 minutes; the pretreatment conditions are 4° C., 12,000 rpm, and centrifugation for 10 minutes.

4. The genetic transformation method according to claim 1, characterized in that The process of inducing callus formation is as follows: transferring the co-cultured immature embryos to L3-T medium, culturing at 25°C±2°C in the dark for 5 to 7 days, and then transferring to L3-B5 medium, culturing at 25°C±2°C in the dark for 5 to 6 weeks.

5. The genetic transformation method according to claim 1, characterized in that The culture medium formula for the co-cultivation is as follows: each liter of culture medium contains 4.3 g of Linsmaier & Skoog basal salts, 10 g of glucose, 0.5 g of 2-(N-morpholine)ethanesulfonic acid, 100 μL of 12.5 g / L CuSO4·5H2O, 4 g of plant gel, 100 μL of 1000×MS vitamin solution, 1 mL of 100 mM acetosyringone and 100 μL of 8.5 g / L AgNO3, and the rest is made up with water.

6. The genetic transformation method according to claim 1, characterized in that The culture medium formula for inducing callus differentiation is as follows: each liter of culture medium contains 4.6 g of L3 medium base salt containing vitamins, 20 g of sucrose, 0.5 g of 2-(N-morpholine)ethanesulfonic acid, 200 μL of 12.5 g / L CuSO4·5H2O, 4 g of plant gel, 5 mL of 1 mg / mL zeatin, 1 mL of 200 mg / mL timentin and 200 μL of 25 mg / mL glufosinate ammonium, and the rest is made up with water.

7. The genetic transformation method according to claim 1, characterized in that The culture medium formula for the rooting and seedling hardening is: each liter of culture medium contains 4.6g of L3 culture medium base salt containing vitamins, 15g of sucrose, 0.5g of 2-(N-morpholine)ethanesulfonic acid, 4g of plant gel, 20μL of 10mg / mL IBA, 1mL of 200mg / mL Timentin, 200μL of 25mg / mL glufosinate ammonium, and the rest is made up with water.

8. The genetic transformation method according to claim 1, characterized in that The process of preparing the infection solution is: taking a positive monoclonal Agrobacterium colony to culture in a YEP liquid culture medium, collecting the bacteria, adding LS-liq culture medium to resuspend, and adding acetosyringone with a final concentration of 100 μM; the positive monoclonal is a plasmid containing the target gene.

9. The genetic transformation method according to claim 8, characterized in that The formula of the LS-liq culture medium is as follows: each liter of the LS-liq culture medium contains 4.3 g of Linsmaier & Skoog basal salt, 100 μL of 1000×MS vitamin solution, 10 g of glucose, and 0.5 g of 2-(N-morpholine)ethanesulfonic acid, and the rest is made up with water.

10. The genetic transformation method according to claim 1, characterized in that The conditions for inducing callus differentiation are: 25°C, 70 μmol / m 2 / s, cultured for 3 weeks; The conditions for rooting and hardening the seedlings were 25°C, 70 μmol / m 2 / s, cultured for 3 weeks.

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