Differential medium for screening corn transformants and method for screening corn positive seedlings

Abstract

The invention provides a differential medium for screening corn transformants and a method for screening corn positive seedlings, and belongs to the technical field of plant genetic engineering. The invention provides a differential medium for screening corn transformants. The differential medium contains glufosinate-ammonium or glufosinate-ammonium pesticide. The differential medium provided by the invention is suitable for the corn transformant carrying a target exogenous gene and a glufosinate-ammonium resistance gene. The method comprises the following steps: inoculating corn buds obtained by recombinant agrobacterium tumefaciens-mediated transformation to a differential medium containing glufosinate-ammonium or glufosinate-ammonium pesticide with specific concentration, and culturing; in the culture process, corn buds with glufosinate-ammonium resistance genes (co-transformed with target exogenous genes) successfully introduced can normally grow on the culture medium to form positive seedlings with complete plant morphology. Through the differential culture medium, positive seedlings can be screened out from a large number of transformed seedlings in a short time, and it is ensured that high-proportion positive plants are obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to a differentiation medium for screening of corn transformants and a method for screening of corn positive seedlings. BACKGROUND

[0002] In the process of corn genetic engineering research and new variety breeding, Agrobacterium-mediated transformation is a commonly used method for introducing foreign genes into the corn genome. However, how to quickly and accurately screen the positive seedlings successfully introduced with foreign genes after transformation has been a difficult problem for researchers and breeders. SUMMARY

[0003] The present application aims to provide a differentiation medium for screening of corn transformants and a method for screening of corn positive seedlings, which can quickly and efficiently screen the corn positive seedlings.

[0004] The present application provides a differentiation medium for screening of corn transformants, which contains glufosinate or a glufosinate pesticide; the concentration of the glufosinate pesticide in the differentiation medium is 0.4-0.6 ml / L.

[0005] Preferably, the concentration of the glufosinate pesticide in the differentiation medium is 0.5 ml / L; the mass concentration of glufosinate in the glufosinate pesticide is 18%.

[0006] Preferably, the differentiation medium comprises the following components at the following concentrations: potassium nitrate 1700-2000 mg / L, ammonium nitrate 1550-1700 mg / L, potassium dihydrogen phosphate 150-180 mg / L, magnesium sulfate 350-380 mg / L, calcium chloride 430-460 mg / L, ethylenediaminetetraacetic acid disodium 36.9-37.5 mg / L, ferrous sulfate 25-29 mg / L, manganese sulfate 21.9-22.5 mg / L, zinc sulfate 8.5-8.8 mg / L, boric acid 6.0-6.5 mg / L, potassium iodide 0.79-0.85 mg / L, vitamin B1 0.08-0.11 mg / L, vitamin B6 0.45-0.55 mg / L, glycine 1.6-2.2 mg / L, sodium molybdate 0.22-0.3 mg / L, copper sulfate 0.02-0.03 mg / L, inositol 91-102 mg / L, cobalt chloride 0.022-0.03 mg / L, nicotinic acid 0.45-0.55 mg / L, copper sulfate 1.5-3 mg / L, MES 0.3-0.8 g / L, zeatin 1-3 mg / L, sucrose 15-30 g / L, agar 4.5-6 g / L, temin 180-220 mg / L, and glufosinate pesticide 0.4-0.6 ml / L; the pH value of the differentiation medium is 5.8-6.2.

[0007] The application also provides the application of the differentiation medium in the screening of corn transformants, and the corn transformation vector used for constructing the corn transformants carries the target foreign gene and the glufosinate-ammonium resistance gene.

[0008] Preferably, the corn variety includes one or more of B104, B73 and LH244.

[0009] The application also provides a method for screening corn positive seedlings, including the following steps:

[0010] The corn seedlings obtained through the transformation mediated by the recombinant agrobacterium are inoculated into the differentiation medium to perform differentiation culture, and the normally grown and differentiated seedlings are the corn positive seedlings.

[0011] The recombinant agrobacterium includes a corn transformation vector.

[0012] The corn transformation vector carries the target foreign gene and the glufosinate-ammonium resistance gene.

[0013] Preferably, the differentiation culture time is 15-25 days.

[0014] Preferably, the construction method of the corn seedlings obtained through the transformation mediated by the recombinant agrobacterium includes the following steps:

[0015] After the corn young embryos are infected by the recombinant agrobacterium, the corn young embryos are placed on the co-culture medium to perform co-culture.

[0016] The corn young embryos after the co-culture are transferred to the screening medium to perform the first screening culture, and the yellow and vigorous growing callus is selected and transferred to the screening medium to perform the second screening culture.

[0017] The callus after the second screening culture is transferred to the differentiation medium without glufosinate-ammonium to perform differentiation culture, and the corn seedlings are obtained.

[0018] Preferably, the co-culture medium comprises the following components at the following concentrations: potassium nitrate 1700–2000 mg / L, ammonium nitrate 1550–1700 mg / L, potassium dihydrogen phosphate 150–180 mg / L, magnesium sulfate 350–380 mg / L, calcium chloride 430–460 mg / L, disodium EDTA 36.9–37.5 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 21.9–22.5 mg / L, zinc sulfate 8.5–8.8 mg / L, boric acid 6.0–6.5 mg / L, potassium iodide 0.79–0.85 mg / L, vitamin B1 (thiamine hydrochloride) 0.08–0.11 mg / L, and vitamin B6 (pyrimidine hydrochloride) The co-culture medium contains: sodium molybdate (0.45–0.55 mg / L), glycine (1.6–2.2 mg / L), sodium molybdate (0.22–0.3 mg / L), copper sulfate (0.02–0.03 mg / L), inositol (91–102 mg / L), cobalt chloride (0.022–0.03 mg / L), nicotinic acid (0.45–0.55 mg / L), 2,4-dichlorophenoxyacetic acid (1.2–1.8 mg / L), proline (0.5–0.9 g / L), sucrose (32–37 g / L), plant gel (3–4 g / L), acetylsyl syringone (100–150 mg / L), silver nitrate (5–7 mg / L), and cysteine ​​hydrochloride (300–400 mg / L); the pH of the co-culture medium is 5.6–5.9.

[0019] Preferably, the screening medium comprises the following components at the following concentrations: potassium nitrate 2200–3000 mg / L, ammonium nitrate 450–480 mg / L, potassium dihydrogen phosphate 380–410 mg / L, magnesium sulfate 170–190 mg / L, calcium chloride 150–170 mg / L, disodium EDTA 30–40 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 3.9–5.0 mg / L, zinc sulfate 1.1–1.6 mg / L, boric acid 1.2–1.6 mg / L, and potassium iodide 0.6–0.9 mg / L. The screening medium contains: vitamin B1 (thiamine hydrochloride) 0.8–1.1 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.4–0.8 mg / L, glycine 1.8–2.1 mg / L, proline 0.5–0.9 g / L, hydrolyzed casein 0.4–0.7 g / L, 2,4-dichlorophenoxyacetic acid 1.2–1.8 mg / L, sucrose 32–37 g / L, plant gel 1.5–3 g / L, termethin 180–220 mg / L, and diammonium phosphate 1–2 mg / L; the pH of the screening medium is 5.6–5.9.

[0020] This invention provides a differentiation medium for screening maize transformants, wherein the differentiation medium contains glufosinate or glufosinate pesticide; the concentration of glufosinate pesticide in the differentiation medium is 0.4–0.6 ml / L. The differentiation medium of this invention is suitable for maize transformants carrying a target exogenous gene and a glufosinate resistance gene. In this invention, maize shoots obtained through Agrobacterium-mediated transformation are inoculated onto a differentiation medium containing a specific concentration of glufosinate or glufosinate pesticide for cultivation. During cultivation, untransformed maize shoots, lacking resistance to the pesticide, experience significant growth inhibition and gradually die; while maize shoots successfully inoculated with the glufosinate resistance gene (co-transformed with the target exogenous gene) can grow normally on this medium, forming positive seedlings with complete plant morphology. The differentiation medium of this invention allows for the selection of positive seedlings from a large number of transformed seedlings in a relatively short time. In conventional maize genetic transformation experiments, seedling differentiation and positive seedling selection are performed separately. First, seedling differentiation is performed, and then the resulting seedlings are screened using a herbicide. Clearly, conventional methods are cumbersome and wasteful of manpower and resources. Therefore, the differentiation medium of this invention can be used for rapid and efficient screening of positive maize seedlings and ensures a high proportion of positive plants, which is of great significance for advancing maize genetic engineering research and breeding. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a comparison diagram of callus differentiation states. Detailed Implementation

[0023] The present invention provides a differentiation culture medium for screening maize transformants, wherein the differentiation culture medium contains glufosinate or glufosinate pesticide; the concentration of glufosinate pesticide in the differentiation culture medium is 0.4-0.6 ml / L.

[0024] In one embodiment, the concentration of glufosinate pesticide in the differentiation medium is 0.5 ml / L. When glufosinate at this concentration is used as a screening medium, no significant inhibition of callus is found and no negative seedlings are detected. Therefore, it can be used as the optimal concentration for screening the differentiation medium.

[0025] As one implementation method, the brand of the glufosinate pesticide is [brand name missing]. The active ingredient and its content are: glufosinate 18% (w / w, g / g).

[0026] Compared with traditional screening methods, this invention utilizes a differentiation medium supplemented with glufosinate-ammonia for screening. This method can rapidly eliminate a large number of unconverted embryos in the early stages, shortening the screening cycle and improving work efficiency. Furthermore, by precisely controlling the type and concentration of pesticide in the differentiation medium, this invention effectively ensures that over 99% of the screened seedlings are positive plants, greatly reducing subsequent ineffective culture of false-positive plants and saving significant manpower, material resources, and time costs. This provides a reliable source of positive materials for maize genetic engineering research and breeding.

[0027] In one embodiment, the differentiation culture medium comprises the following components at the following concentrations: potassium nitrate 1700–2000 mg / L, ammonium nitrate 1550–1700 mg / L, potassium dihydrogen phosphate 150–180 mg / L, magnesium sulfate 350–380 mg / L, calcium chloride 430–460 mg / L, disodium ethylenediaminetetraacetate 36.9–37.5 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 21.9–22.5 mg / L, zinc sulfate 8.5–8.8 mg / L, and boric acid. 6.0–6.5 mg / L, potassium iodide 0.79–0.85 mg / L, vitamin B1 0.08–0.11 mg / L, vitamin B6 0.45–0.55 mg / L, glycine 1.6–2.2 mg / L, sodium molybdate 0.22–0.3 mg / L, copper sulfate 0.02–0.03 mg / L, inositol 91–102 mg / L, cobalt chloride 0.022–0.03 mg / L, niacin 0.45–0.55 mg / L, copper sulfate 1.5–3 mg / L, MES 0.3–0.8 g / L, zeatin 1–3 mg / L, sucrose 15–30 g / L, agar 4.5–6 g / L, termethin 180–220 mg / L, and glufosinate pesticide 0.4–0.6 ml / L; further, the differentiation medium comprises the following components at the following concentrations: potassium nitrate 2000 mg / L, ammonium nitrate 1600 mg / L, potassium dihydrogen phosphate 180 mg / L, magnesium sulfate 360 ​​mg / L, calcium chloride 450 mg / L, disodium ethylenediaminetetraacetate 37.0 mg / L, sulfur The culture medium contained the following components: ferrous sulfate 28 mg / L, manganese sulfate 22.0 mg / L, zinc sulfate 8.5 mg / L, boric acid 6.5 mg / L, potassium iodide 0.8 mg / L, vitamin B1 (thiamine hydrochloride) 0.9 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.5 mg / L, glycine 1.8 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 2.0 mg / L, inositol 100 mg / L, cobalt chloride 0.03 mg / L, nicotinic acid 0.5 mg / L, copper sulfate 0.03 mg / L, MES 0.4 g / L, zeatin 1.5 mg / L, sucrose 22 g / L, agar 6 g / L, termethin 200 mg / L, and glufosinate pesticide 0.5 ml / L; the pH of the differentiation medium was 5.8–6.2.

[0028] This invention also provides the application of the differentiation medium described above in the screening of maize transformants, wherein the maize transformation vector used to construct the maize transformants contains a glufosinate resistance gene and a target gene.

[0029] In one embodiment, the maize variety includes one or more of B104, B73, and LH244. The differentiation medium formulation of the present invention is highly compatible with B104, B73, and LH244.

[0030] This invention also provides a method for screening positive maize seedlings, comprising the following steps:

[0031] The maize seedlings obtained by recombinant Agrobacterium-mediated transformation were inoculated into the differentiation medium described in the above scheme for differentiation culture. Seedlings that grow and differentiate normally are maize positive seedlings. The recombinant Agrobacterium contains a maize transformation vector. The maize transformation vector contains a glufosinate resistance gene and a target gene.

[0032] This invention introduces a vector carrying a target exogenous gene and a glufosinate resistance gene into maize immature embryos via Agrobacterium-mediated transformation. During the differentiation culture stage of the transformed maize immature embryos, they are cultured in a differentiation medium supplemented with glufosinate pesticide. Untransformed maize cells are inhibited in growth due to their sensitivity to pesticides, while successfully transformed maize cells grow and differentiate normally to form positive seedlings due to their pesticide resistance. The proportion of positive plants among the screened positive seedlings is over 98%.

[0033] The method of this invention can significantly shorten the screening cycle and ensure that more than 99% of the seedlings obtained from screening are maize positive plants, which greatly improves the efficiency and accuracy of maize transformant screening, while saving costs and avoiding wasting time, manpower and resources on ineffective plants.

[0034] In one implementation, the differentiation culture time is 15-25 days, more specifically 18-20 days.

[0035] In one embodiment, the maize transformation vector includes a gene editing vector or an overexpression vector; the gene editing vector includes the posCas9 vector; the overexpression vector includes the pCAMBIA3301 vector, purchased from Bioman (Beijing) Technology Co., Ltd.

[0036] As one implementation method, the method for constructing maize sprouts obtained after Agrobacterium-mediated transformation includes the following steps:

[0037] After infecting maize embryos with recombinant Agrobacterium, they were placed on a co-culture medium for co-culture. The co-cultured maize embryos were then transferred to a screening medium for the first screening culture. Callus tissues that were bright yellow and growing vigorously were selected and transferred to the screening medium for the second screening culture. The callus tissues after the second screening culture were then transferred to a differentiation medium without glufosinate for differentiation culture to obtain maize shoots.

[0038] This invention first involves infecting corn embryos with recombinant Agrobacterium tumefaciens, then placing them on a co-culture medium for co-culture.

[0039] This invention utilizes a conventional Agrobacterium-mediated transformation method to co-culture recombinant Agrobacterium carrying the target exogenous gene and glufosinate resistance gene with maize immature embryos, thereby integrating the exogenous gene and glufosinate resistance gene into the maize genome.

[0040] The present invention does not impose any special restrictions on the method for obtaining the corn embryo; conventional methods in the field can be used.

[0041] The present invention does not impose any special restrictions on the construction method of the recombinant Agrobacterium, and conventional methods in the field can be used.

[0042] As one implementation method, the method of infecting corn embryos with recombinant Agrobacterium includes: mixing a suspension of recombinant Agrobacterium with corn embryos and introducing it into an infection culture medium for infection; the infection time is 10 min.

[0043] In one embodiment, the infection culture medium comprises the following components at the following concentrations: potassium nitrate 2200–3000 mg / L, ammonium nitrate 450–480 mg / L, potassium dihydrogen phosphate 380–410 mg / L, magnesium sulfate 170–190 mg / L, calcium chloride 150–170 mg / L, disodium ethylenediaminetetraacetate 30–40 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 3.9–5 mg / L, zinc sulfate 1.1–1.6 mg / L, and boric acid 1.2–1 mg / L. 0.6 mg / L, potassium iodide 0.6–0.9 mg / L, vitamin B1 (thiamine hydrochloride) 0.8–1.1 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.4–0.8 mg / L, glycine 1.8–2.1 mg / L, 2,4-dichlorophenoxyacetic acid 1.2–1.8 mg / L, proline 0.5–0.9 g / L, sucrose 65–75 g / L, glucose 33–38 g / L, acetylsylgenone 100–150 mg / L, pH 5.1–5.5.

[0044] As one embodiment, the method for preparing the recombinant Agrobacterium suspension includes: inoculating recombinant Agrobacterium on LB medium, culturing, collecting and resuspending the bacterial cells to obtain a recombinant Agrobacterium suspension; the culture temperature is 28°C; the culture is a dark culture; the culture time is 24 hours; the reagent used for resuspending the bacterial cells is an infiltration medium; the OD of the recombinant Agrobacterium suspension is... 600 The pH value is 0.2–0.3; the LB medium comprises the following components at the following concentrations: peptone 10 g / L, NaCl 15 g / L, yeast extract 10 g / L, agar 15 g / L, rifampin 50 mg / L, spectinomycin 50 mg / L and kanamycin 50 mg / L; the pH value of the LB medium is 5.6–5.9.

[0045] In one embodiment, placing the corn embryo scutellariae onto the co-culture medium includes placing the corn embryo scutellariae face up onto the co-culture medium.

[0046] In one embodiment, the co-culture medium comprises the following components at the following concentrations: potassium nitrate 1700–2000 mg / L, ammonium nitrate 1550–1700 mg / L, potassium dihydrogen phosphate 150–180 mg / L, magnesium sulfate 350–380 mg / L, calcium chloride 430–460 mg / L, disodium ethylenediaminetetraacetate 36.9–37.5 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 21.9–22.5 mg / L, zinc sulfate 8.5–8.8 mg / L, and boron. The concentrations of the following components are listed: acid 6.0–6.5 mg / L, potassium iodide 0.79–0.85 mg / L, vitamin B1 (thiamine hydrochloride) 0.08–0.11 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.45–0.55 mg / L, glycine 1.6–2.2 mg / L, sodium molybdate 0.22–0.3 mg / L, copper sulfate 0.02–0.03 mg / L, inositol 91–102 mg / L, cobalt chloride 0.022–0.03 mg / L, and nicotinic acid 0.45–0.55 mg / L. The co-culture medium comprises the following components: 2,4-dichlorophenoxyacetic acid 1.2–1.8 mg / L, proline 0.5–0.9 g / L, sucrose 32–37 g / L, plant gel 3–4 g / L, acetylsylgenin 100–150 mg / L, silver nitrate 5–7 mg / L, and cysteine ​​hydrochloride 300–400 mg / L; furthermore, the co-culture medium comprises the following components at the following concentrations: potassium nitrate 1900 mg / L, ammonium nitrate 1620 mg / L, potassium dihydrogen phosphate 180 mg / L, magnesium sulfate 350 mg / L, calcium chloride 450 mg / L, disodium EDTA 37 mg / L, ferrous sulfate 28 mg / L, manganese sulfate 22 mg / L, and zinc sulfate 8.5 mg / L. / L, boric acid 6.5mg / L, potassium iodide 0.8mg / L, vitamin B1 (thiamine hydrochloride) 0.1mg / L, vitamin B6 (pyridoxine hydrochloride) 0.5mg / L, glycine 2.0mg / L, sodium molybdate 0.25mg / L, copper sulfate 0.02mg / L, inositol 100mg / L, cobalt chloride 0.03mg / L, nicotinic acid 0.5mg / L, 2,4-dichlorophenoxyacetic acid 1.5mg / L, proline 0.8g / L, sucrose 37g / L, plant gel 3g / L, acetylsuccione 100mg / L, silver nitrate 5mg / L and cysteine ​​hydrochloride 300mg; the pH of the co-culture medium is 5.6-5.9.

[0047] In one implementation, the co-culture temperature is 22°C; the co-culture is a dark culture; and the co-culture time is 2 to 3 days.

[0048] After co-culture, the present invention transfers the co-cultured maize embryos to a screening medium for the first screening culture, and selects callus tissues that are bright yellow and growing vigorously to transfer to the screening medium for the second screening culture.

[0049] In one embodiment, the screening culture medium comprises the following components at the following concentrations: potassium nitrate 2200–3000 mg / L, ammonium nitrate 450–480 mg / L, potassium dihydrogen phosphate 380–410 mg / L, magnesium sulfate 170–190 mg / L, calcium chloride 150–170 mg / L, disodium ethylenediaminetetraacetate 30–40 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 3.9–5.0 mg / L, and zinc sulfate 1.1–1.6 mg / L. L, boric acid 1.2–1.6 mg / L, potassium iodide 0.6–0.9 mg / L, vitamin B1 (thiamine hydrochloride) 0.8–1.1 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.4–0.8 mg / L, glycine 1.8–2.1 mg / L, proline 0.5–0.9 g / L, hydrolyzed casein 0.4–0.7 g / L, 2,4-dichlorophenoxyacetic acid 1.2–1.8 mg / L, sucrose 32–37 g / L, plant gel 1.5– 3 g / L, termethin 180–220 mg / L and diammonium phosphate 1–2 mg / L; further, the screening medium comprises the following components at the following concentrations: potassium nitrate 2500 mg / L, ammonium nitrate 450 mg / L, potassium dihydrogen phosphate 380 mg / L, magnesium sulfate 180 mg / L, calcium chloride 150 mg / L, disodium ethylenediaminetetraacetate 35 mg / L, ferrous sulfate 28 mg / L, manganese sulfate 4.5 mg / L, zinc sulfate 1.5 mg / L, boric acid 1.6 g / L. The screening medium contained the following components: mg / L potassium iodide, 0.8 mg / L thiamine hydrochloride, 0.9 mg / L vitamin B1 (thiamine hydrochloride), 0.8 mg / L vitamin B6 (pyridoxine hydrochloride), 1.8 mg / L glycine, 0.5 g / L proline, 0.6 g / L hydrolyzed casein, 1.5 mg / L 2,4-dichlorophenoxyacetic acid, 37 g / L sucrose, 3 g / L plant gel, 200 mg / L termethin, and 1 mg / L diammonium phosphate; the pH of the screening medium was 5.6–5.9.

[0050] In one implementation, the temperatures of the first screening culture and the second screening culture are 28°C, respectively; the first screening culture and the second screening culture are dark cultures; the time of the first screening culture is 10 to 12 days; and the time of the second screening culture is 12 to 16 days, further up to 15 days.

[0051] After the second screening culture, the callus tissue after the second screening culture was transferred to a differentiation medium without glufosinate for differentiation culture to obtain maize seedlings.

[0052] In one embodiment, the callus tissue is a callus tissue with differentiation capacity; before the callus tissue after the second screening culture, the callus tissue with differentiation capacity is cut into pieces with a diameter of 2-3 mm; the temperature of the differentiation culture is 25°C; the differentiation culture is carried out under light conditions; the differentiation culture time is 20 days, based on the differentiation of buds.

[0053] This invention involves inoculating maize seedlings obtained through recombinant Agrobacterium-mediated transformation into the differentiation medium described in the above-mentioned scheme for differentiation culture. Seedlings that fail to transform gradually turn yellow and die under the influence of glufosinate; seedlings that differentiate normally are maize positive seedlings, capable of developing into complete plants and growing vigorously. After the plants grow to a certain height, molecular biological methods are used to verify whether the exogenous gene has been successfully integrated and expressed; the molecular biological methods are PCR amplification or Southern hybridization. Extensive experimental verification shows that the proportion of positive plants among those screened using this method is over 99%.

[0054] Currently, most maize transformation vectors use glufosinate resistance genes. Therefore, this invention adds a specific concentration of glufosinate to the differentiation medium during the differentiation culture of the transformed embryos. Maize shoots obtained through Agrobacterium-mediated transformation are inoculated onto a differentiation medium containing glufosinate or glufosinate pesticide for cultivation. During the culture process, untransformed maize shoots, lacking resistance to glufosinate, experience significantly inhibited growth and gradually die; while maize shoots successfully infused with the glufosinate resistance gene (co-transformed with the target exogenous gene) can grow normally on this medium, forming positive seedlings with complete plant morphology. In this way, positive seedlings can be screened from a large number of transformed seedlings in a relatively short time.

[0055] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a differentiation culture medium for screening maize transformants and a method for screening positive maize seedlings provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0056] Information on the glufosinate pesticide used in the following examples and comparative examples: Country of origin: Germany; Brand: The active ingredient and its content are: glufosinate 18%; Registration certificate holder: BASF Europe; Packaging unit: Zhuhai Special Economic Zone Ruinong Plant Protection Technology Co., Ltd.; Pesticide registration certificate number: PD20096847; Production license number: Pesticide Production Permit (Guangdong) 0032; Production quality standard number: Q / BASF H111-2022.

[0057] Example 1

[0058] (1) Obtaining the embryo

[0059] Take corn ears that have been artificially pollinated for 12-15 days. Remove the husks, disinfect the surface with 75% alcohol for 1 minute, and use a scalpel to remove about 2 / 3 of the corn kernels to obtain the embryo, which should be about 1.5 mm in size.

[0060] (2) Prepare Agrobacterium tumefaciens bacterial solution

[0061] The recombinant Agrobacterium strain was evenly spread on LB medium supplemented with appropriate antibiotics (the antibiotics used should be based on the prokaryotic resistance of the vector; for example, for the posCas9 vector, rifampin and kanamycin are required. Kanamycin is the vector's own prokaryotic resistance, while rifampin is Agrobacterium's own resistance). The culture was incubated in the dark at 28°C for 24 hours. The recombinant Agrobacterium was collected using an inoculation loop (see [Maize smart-canopy architecture enhances yield at highdensities. Nature]), suspended in inoculation medium, and the OD was adjusted. 600 Up to 0.2-0.3.

[0062] LB medium: peptone 10 g / L, NaCl 15 g / L, yeast extract 10 g / L, agar 15 g / L, rifampin 50 mg / L, spectinomycin 50 mg / L and kanamycin 50 mg / L, pH 5.6–5.9.

[0063] (3) Recombinant Agrobacterium infection

[0064] The recombinant Agrobacterium suspension was mixed with the collected corn embryos and introduced into the infection medium for about 10 minutes for infection.

[0065] (4) Co-culture of immature embryos

[0066] After the inoculation was completed, the corn embryo scutellum was placed face up on the co-culture medium and incubated in the dark at 22°C for 3 days.

[0067] Co-culture medium: potassium nitrate 1900 mg / L, ammonium nitrate 1620 mg / L, potassium dihydrogen phosphate 180 mg / L, magnesium sulfate 350 mg / L, calcium chloride 450 mg / L, disodium EDTA 37 mg / L, ferrous sulfate 28 mg / L, manganese sulfate 22 mg / L, zinc sulfate 8.5 mg / L, boric acid 6.5 mg / L, potassium iodide 0.8 mg / L, vitamin B1 (thiamine hydrochloride) 0.1 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.5 mg / L, glycine 2.0 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.02 mg / L, inositol 100 mg / L, cobalt chloride 0.03 mg / L, nicotinic acid 0.5 mg / L. 2,4-Dichlorophenoxyacetic acid 1.5 mg / L, proline 0.8 g / L, sucrose 37 g / L, plant gel 3 g / L, acetylsuccinone 100 mg / L, silver nitrate 5 mg / L and cysteine ​​hydrochloride 300 mg / L, pH 5.8.

[0068] (5) Screening and Cultivation

[0069] After 3 days of co-culture, the embryos were transferred to selection medium and cultured for the first selection at 28°C in the dark for 10 days. After the first selection culture, bright yellow and vigorously growing callus tissues were selected and transferred to selection medium for the second selection culture for 15 days.

[0070] Screening medium: Potassium nitrate 2500 mg / L, ammonium nitrate 450 mg / L, potassium dihydrogen phosphate 380 mg / L, magnesium sulfate 180 mg / L, calcium chloride 150 mg / L, disodium EDTA 35 mg / L, ferrous sulfate 28 mg / L, manganese sulfate 4.5 mg / L, zinc sulfate 1.5 mg / L, boric acid 1.6 mg / L, potassium iodide 0.8 mg / L, vitamin B1 (thiamine hydrochloride) 0.9 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.8 mg / L, glycine 1.8 mg / L, proline 0.5 g / L, hydrolyzed casein 0.6 g / L, 2,4-dichlorophenoxyacetic acid 1.5 mg / L, sucrose 37 g / L, plant gel 3 g / L, termethin 200 mg / L, and diammonium phosphate 1 mg / L. pH 5.8.

[0071] (6) Differentiation culture

[0072] After the second screening culture was completed, the callus tissue with differentiation capacity was cut into pieces with a diameter of 2-3 mm and transferred to a pesticide-free differentiation medium. It was then cultured for another 20 days under 25°C light conditions. After 20 days, the differentiated shoots were transferred to a differentiation medium containing 0.5 ml / L glufosinate pesticide.

[0073] Differentiation medium: Potassium nitrate 2000 mg / L, ammonium nitrate 1600 mg / L, potassium dihydrogen phosphate 180 mg / L, magnesium sulfate 360 ​​mg / L, calcium chloride 450 mg / L, disodium EDTA 37.0 mg / L, ferrous sulfate 28 mg / L, manganese sulfate 22.0 mg / L, zinc sulfate 8.5 mg / L, boric acid 6.5 mg / L, potassium iodide 0.8 mg / L, vitamin B1 (thiamine hydrochloride) 0.9 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.5 mg / L, glycine 1.8 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 2.0 mg / L, inositol 100 mg / L, cobalt chloride 0.03 mg / L, nicotinic acid 0.5 mg / L, copper sulfate 0.03 mg / L, MES 0.4 g / L, zeatin 1.5 mg / L, sucrose 22 g / L, agar 6 g / L and termethin 200 mg / L; pH 6.0. A culture medium containing glufosinate pesticide was prepared by adding 0.5 ml / L of (Promethazine) glufosinate to the differentiation medium.

[0074] After being cultured on a differentiation medium containing glufosinate pesticide for about one week, the unconverted seedlings gradually turned yellow and died under the influence of glufosinate, while the successfully converted seedlings developed into complete plants and grew vigorously. Once the plants reached a certain height, molecular biology methods were used to verify whether the exogenous gene was successfully integrated and expressed. Extensive experimental verification showed that the proportion of positive plants obtained through this method was over 99%.

[0075] Comparative Example 1

[0076] By adding different concentrations of glufosinate to the differentiation medium in Example 1 for screening, it was found that using 0.5 ml / L glufosinate as the screening medium did not result in significantly inhibited callus formation, and no negative seedlings were detected. This concentration can be considered as the optimal concentration for screening the differentiation medium, as shown in Table 1. Figure 1 (All values ​​are averages of 3 replicates):

[0077] Table 1 shows the screening results of adding different concentrations of glufosinate to the differentiation culture medium in Example 1.

[0078]

[0079] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A differentiation culture medium for screening maize transformants, characterized in that, The differentiation medium contains glufosinate or glufosinate pesticide; the concentration of glufosinate pesticide in the differentiation medium is 0.4–0.6 ml / L.

2. The differentiation culture medium according to claim 1, characterized in that, The concentration of glufosinate pesticide in the differentiation culture medium was 0.5 ml / L; the mass concentration of glufosinate pesticide in the glufosinate pesticide was 18%.

3. The differentiation culture medium according to claim 1, characterized in that, The differentiation medium comprises the following components at the following concentrations: potassium nitrate 1700–2000 mg / L, ammonium nitrate 1550–1700 mg / L, potassium dihydrogen phosphate 150–180 mg / L, magnesium sulfate 350–380 mg / L, calcium chloride 430–460 mg / L, disodium ethylenediaminetetraacetate 36.9–37.5 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 21.9–22.5 mg / L, zinc sulfate 8.5–8.8 mg / L, and boric acid 6.0–6 mg / L. The culture medium contained the following components: 0.5 mg / L potassium iodide, 0.79–0.85 mg / L vitamin B1, 0.08–0.11 mg / L vitamin B6, 0.45–0.55 mg / L glycine, 1.6–2.2 mg / L sodium molybdate, 0.22–0.3 mg / L copper sulfate, 0.02–0.03 mg / L inositol, 91–102 mg / L cobalt chloride, 0.022–0.03 mg / L nicotinic acid, 0.45–0.55 mg / L copper sulfate, 0.5–3 mg / L MES, 1–3 mg / L zeatin, 15–30 g / L sucrose, 4.5–6 g / L agar, 180–220 mg / L termethin, and 0.4–0.6 ml / L glufosinate pesticide; the pH of the differentiation medium was 5.8–6.

2.

4. The application of the differentiation medium according to any one of claims 1 to 3 in the screening of maize transformants, characterized in that, The maize transformation vector used to construct the maize transformant carries the target exogenous gene and the glufosinate resistance gene.

5. The application according to claim 4, characterized in that, The corn varieties include one or more of B104, B73, and LH244.

6. A method for screening positive maize seedlings, characterized in that, Includes the following steps: The corn seedlings obtained by recombinant Agrobacterium-mediated transformation are inoculated into the differentiation medium described in any one of claims 1 to 3 for differentiation culture, and the seedlings that grow and differentiate normally are corn positive seedlings. The recombinant Agrobacterium contains a maize transformation vector; The maize transformation vector carries the target exogenous gene and the glufosinate resistance gene.

7. The method according to claim 6, characterized in that, The differentiation culture time is 15–25 days.

8. The method according to claim 6, characterized in that, The method for constructing maize sprouts obtained after Agrobacterium-mediated transformation includes the following steps: After infecting maize embryos with recombinant Agrobacterium, they were placed on a co-culture medium for co-culture. The co-cultured maize embryos were transferred to a screening medium for the first screening culture, and the bright yellow and vigorously growing callus tissues were selected and transferred to the screening medium for the second screening culture. The callus tissue after the second screening culture was transferred to a differentiation medium without glufosinate for differentiation culture to obtain maize shoots.

9. The method according to claim 8, characterized in that, The co-culture medium comprises the following components at the following concentrations: potassium nitrate 1700–2000 mg / L, ammonium nitrate 1550–1700 mg / L, potassium dihydrogen phosphate 150–180 mg / L, magnesium sulfate 350–380 mg / L, calcium chloride 430–460 mg / L, disodium EDTA 36.9–37.5 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 21.9–22.5 mg / L, zinc sulfate 8.5–8.8 mg / L, boric acid 6.0–6.5 mg / L, potassium iodide 0.79–0.85 mg / L, vitamin B1 (thiamine hydrochloride) 0.08–0.11 mg / L, and vitamin B6 (pyridoxine hydrochloride). The co-culture medium contains 0.45–0.55 mg / L of sodium molybdate, 1.6–2.2 mg / L of glycine, 0.22–0.3 mg / L of sodium molybdate, 0.02–0.03 mg / L of copper sulfate, 91–102 mg / L of inositol, 0.022–0.03 mg / L of cobalt chloride, 0.45–0.55 mg / L of nicotinic acid, 1.2–1.8 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5–0.9 g / L of proline, 32–37 g / L of sucrose, 3–4 g / L of plant gel, 100–150 mg / L of acetylsuccione, 5–7 mg / L of silver nitrate, and 300–400 mg / L of cysteine ​​hydrochloride; the pH of the co-culture medium is 5.6–5.

9.

10. The method according to claim 8, characterized in that, The screening medium comprises the following components at the following concentrations: potassium nitrate 2200–3000 mg / L, ammonium nitrate 450–480 mg / L, potassium dihydrogen phosphate 380–410 mg / L, magnesium sulfate 170–190 mg / L, calcium chloride 150–170 mg / L, disodium ethylenediaminetetraacetate 30–40 mg / L, ferrous sulfate 25–29 mg / L, manganese sulfate 3.9–5.0 mg / L, zinc sulfate 1.1–1.6 mg / L, boric acid 1.2–1.6 mg / L, and potassium iodide 0.6–0.9 mg / L. The screening medium contained the following components: vitamin B1 (thiamine hydrochloride) 0.8–1.1 mg / L, vitamin B6 (pyridoxine hydrochloride) 0.4–0.8 mg / L, glycine 1.8–2.1 mg / L, proline 0.5–0.9 g / L, hydrolyzed casein 0.4–0.7 g / L, 2,4-dichlorophenoxyacetic acid 1.2–1.8 mg / L, sucrose 32–37 g / L, plant gel 1.5–3 g / L, termethin 180–220 mg / L, and diammonium phosphate 1–2 mg / L; the pH of the screening medium was 5.6–5.9.

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