An agrobacterium-mediated maize commercial inbred line embryogenic callus genetic transformation and seedling regeneration system and application

Abstract

The present application belongs to the field of plant biotechnology and bioengineering breeding, and particularly relates to a kind of agrobacterium-mediated maize commercial backbone inbred line embryogenic callus genetic transformation and seedling regeneration system and application.The present application constructs maize callus genetic transformation system with commercial maize breeding backbone inbred line as background material and not dependent on seasonal restriction, effectively overcomes the problems of low maize callus induction frequency, difficulty in long-term subculture of embryogenic callus, low transformation and seedling regeneration frequency of callus, can efficiently and quickly produce transgenic plants in batches, and has important value for genetic improvement and gene function research of maize.

Description

Technical Field

[0001] This invention belongs to the fields of plant biotechnology and bioengineering breeding, specifically relating to an Agrobacterium-mediated genetic transformation and seedling regeneration system for embryogenic callus of commercial maize backbone inbred lines and its application. Background Technology

[0002] Genetic transformation is a key tool in the field of plant genetic engineering. Over the years, maize genetic transformation technology has evolved into various approaches, including protoplast methods, electroporation, and gene gun bombardment. Gene gun bombardment, due to its simplicity, was once widely adopted and successfully produced some fertile transgenic plants. However, this method suffers from problems such as high-copy insertion of exogenous DNA and sequence rearrangements, leading to a high proportion of malformed or sterile transgenic plants, ultimately limiting its widespread application.

[0003] With the deepening research into the mechanism of T-DNA transfer, Agrobacterium-mediated transformation technology is increasingly being applied to the genetic transformation of cereal crops. However, almost all reported effective maize transformation systems currently use immature embryos as recipients. The core determinants of successful immature embryo transformation include the embryo's response in tissue culture, the type of cells differentiated and grown, and subsequent proliferation and regeneration characteristics. Establishing an efficient genetic transformation system using embryonic callus tissue as a recipient can not only overcome the bottleneck of seasonality and developmental time limitations of immature embryo recipients, but also save the large amount of manpower and greenhouse space required for immature embryo transformation, thereby improving the efficiency of maize genetic engineering breeding.

[0004] The efficiency of Agrobacterium-mediated genetic transformation of maize callus mainly depends on three key factors: (1) the ability to induce and maintain high-quality embryogenic callus; (2) the sensitivity of recipient genotype materials to Agrobacterium infection; and (3) the regeneration capacity of callus after infection. Among these, genotype dependence remains the main bottleneck in maize genetic transformation. Most maize genotypes, especially the superior genotype materials currently used in breeding, generally suffer from limiting factors such as low callus induction frequency, poor tissue culture characteristics, low Agrobacterium infection efficiency, and difficulty in regeneration. This makes it extremely difficult to obtain a large number of transgenic plants using callus from commercial maize backbone inbred lines as material. There is a severe lack of technical systems that can be directly used for the transformation of commercial maize backbone inbred lines. Moreover, the existing systems have a single recipient type and rely heavily on immature embryos, which has obvious drawbacks and has become an important factor restricting the development of maize genetic engineering breeding.

[0005] Therefore, to address the key constraints on Agrobacterium-mediated genetic transformation of maize callus, it is crucial to establish a high-quality embryogenic callus induction and sustainable subculture system, improve the infection efficiency of Agrobacterium on callus, establish an efficient and rapid seedling regeneration system, integrate a highly efficient Agrobacterium-mediated genetic transformation technology system for embryogenic callus of commercial maize backbone inbred lines, and expand the maize genotypes and recipient types that can be effectively transformed. This is of great significance for improving the efficiency of maize genetic breeding. Summary of the Invention

[0006] To address the shortcomings of the existing technology, the purpose of this invention is to provide an Agrobacterium-mediated genetic transformation and seedling regeneration system for embryogenic callus of commercial maize backbone inbred lines, and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for Agrobacterium-mediated genetic transformation of maize callus, comprising the following steps: (1) Obtaining maize embryos; (2) Place the corn embryo on the callus induction medium to induce embryogenic callus, and then propagate it on the callus subculture medium to obtain embryogenic callus. (3) Activate the Agrobacterium containing the infection vector and suspend the activated Agrobacterium in the Agrobacterium infection culture medium to obtain the Agrobacterium infection culture medium; (4) Take the above Agrobacterium infection solution and use Agrobacterium-mediated method to infect embryonic callus to obtain infected callus. (5) The infected callus was placed on a co-culture medium and co-cultured to obtain the co-cultured callus; (6) Transfer the co-cultured callus to a recovery culture medium for antibacterial and recovery culture; (7) The callus tissue after antibacterial and recovery culture was transferred to the screening medium for screening to obtain resistant callus tissue; (8) Transfer the resistant callus to a differentiation medium to induce differentiation and obtain differentiated callus; (9) Transfer the differentiated callus tissue blocks to the seedling culture medium to differentiate seedlings; (10) Selective agents were used to screen and molecular identification of seedlings to obtain positive seedlings.

[0008] In the above method, in (1), the method for obtaining maize embryos includes: selecting maize inbred lines or maize hybrids that have been pollinated for 10-12 days (e.g., 10, 11, or 12 days) and sterilizing the surface of the ears, then picking out the embryos and placing them in the embryo storage culture medium.

[0009] Furthermore, the long axis length of the corn embryo is 1 to 1.4 mm (e.g., 1.1-1.3 mm, 1.1 mm, 1.2 mm, 1.3 mm, or 1.4 mm).

[0010] Furthermore, the specific composition of the temporary embryo culture medium is as follows: N6 salt 3.8–4.2 g / L (e.g., 3.9–4.1 g / L, 4.0–4.1 g / L, 3.9 g / L, 3.95 g / L, 4.0 g / L, 4.01 g / L, 4.03 g / L, 4.07 g / L, or 4.1 g / L), sodium citrate 10–20 mg / L (e.g., 12–18 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L), glutamate 90–110 mg / L (e.g., 95–105 mg / L, 90 mg / L, 95 mg / L, 96 mg / L, 97 mg / L, or 98 mg / L). mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), L-proline 0.4-0.8 g / L (e.g. 0.5-0.7 g / L or 0.4 g / L or 0.5 g / L or 0.6 g / L or 0.7 g / L or 0.8 g / L), hydrolyzed casein 90-110 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), MES 0.4-0.6 g / L (e.g., 0.4 g / L, 0.5 g / L, or 0.6 g / L), sucrose 30-50 g / L (e.g., 35-45 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, or 50 g / L), AgNO3 3-6 mg / L (e.g., 3.5-5.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, or 5 mg / L).5 mg / L or 6 mg / L), cephalosporins 280-320 mg / L (e.g., 290-310 mg / L or 295-305 mg / L or 280 mg / L or 290 mg / L or 295 mg / L or 296 mg / L or 297 mg / L or 298 mg / L or 299 mg / L or 300 mg / L or 301 mg / L or 302 mg / L or 303 mg / L or 304 mg / L or 305 mg / L or 310 mg / L or 320 mg / L), 2,4-D 2-4 mg / L (e.g., 2.5-3.5 mg / L or 2 mg / L or 2.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L), NAA 0.4-0.6 mg / L (e.g., 0.4 mg / L or 0.45 mg / L or 0.5 mg / L). (mg / L or 0.55mg / L or 0.6 mg / L), pH 6.0.

[0011] In the above method, step (2), which involves placing the corn embryos on a callus induction medium to induce embryogenic callus, includes: placing the collected corn embryos on sterile filter paper, absorbing excess liquid from the surface, and placing them with the scutellum facing upwards on callus induction medium A, and incubating them in the dark at 28°C for 1 week. After incubation, the embryos are dehisced and transferred to callus induction medium B, and subcultured every 7 days. During the incubation process, the embryos are dehisced promptly, and incubated in the dark at 28°C for 2-4 weeks.

[0012] Furthermore, the callus induction medium includes callus induction medium A and callus induction medium B; The formula for the callus induction culture medium A is as follows: N6 salt 3.8-4.2 g / L (e.g., 3.9-4.1 g / L, 4.0-4.1 g / L, 3.9 g / L, 3.95 g / L, 4.0 g / L, 4.01 g / L, 4.03 g / L, 4.07 g / L, or 4.1 g / L), sodium citrate 10-20 mg / L (e.g., 12-18 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L), glutamate 90-110 mg / L (e.g., 95-105 mg / L, 90 mg / L, 95 mg / L, 96 mg / L, 97 mg / L, 98 mg / L, or 99 mg / L). mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), L-proline 0.4-0.8 g / L (e.g. 0.5-0.7 g / L or 0.4 g / L or 0.5 g / L or 0.6 g / L or 0.7 g / L or 0.8 g / L), hydrolyzed casein 90-110 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), MES 0.4-0.6 g / L (e.g. 0.4 ... g / L or 0.5 g / L or 0.6 g / L), sucrose 30-50 g / L (e.g. 35-45 g / L or 30 g / L or 31 g / L or 32 g / L or 33 g / L or 34 g / L or 35 g / L or 36 g / L or 37 g / L or 38 g / L or 39 g / L or 40 g / L or 41 g / L or 42 g / L or 43 g / L or 44 g / L or 45 g / L or 46 g / L or 47 g / L or 48 g / L or 49 g / L or 50 g / L), AgNO3 3-6 mg / L (e.g. 3.5-5.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L or 4.5 mg / L or 5 mg / L or 5 mg / L).5 mg / L or 6 mg / L), cephalosporins 280-320 mg / L (e.g., 290-310 mg / L or 295-305 mg / L or 280 mg / L or 290 mg / L or 295 mg / L or 296 mg / L or 297 mg / L or 298 mg / L or 299 mg / L or 300 mg / L or 301 mg / L or 302 mg / L or 303 mg / L or 304 mg / L or 305 mg / L or 310 mg / L or 320 mg / L), 2,4-D 2-4 mg / L (e.g., 2.5-3.5 mg / L or 2 mg / L or 2.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L), NAA 0.4-0.6 mg / L (e.g., 0.4 mg / L or 0.45 mg / L or 0.5 mg / L). mg / L or 0.55 mg / L or 0.6 mg / L), agar 5.5-6.5 g / L (e.g. 5.5 g / L or 6 g / L or 6.5 g / L), pH 6.0; The formula for the callus induction medium B is as follows: N6 salt 3.8-4.2 g / L (e.g., 3.9-4.1 g / L, 4.0-4.1 g / L, 3.9 g / L, 3.95 g / L, 4.0 g / L, 4.01 g / L, 4.03 g / L, 4.07 g / L, or 4.1 g / L), sodium citrate 10-20 mg / L (e.g., 12-18 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L), glutamate 90-110 mg / L (e.g., 95-105 mg / L, 90 mg / L, 95 mg / L, 96 mg / L, 97 mg / L, 98 mg / L, or 99 mg / L). mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), L-proline 0.4-0.8 g / L (e.g. 0.5-0.7 g / L or 0.4 g / L or 0.5 g / L or 0.6 g / L or 0.7 g / L or 0.8 g / L), hydrolyzed casein 90-110 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), MES 0.4-0.6 g / L (e.g. 0.4 ... g / L or 0.5 g / L or 0.6 g / L), sucrose 30-50 g / L (e.g. 35-45 g / L or 30 g / L or 31 g / L or 32 g / L or 33 g / L or 34 g / L or 35 g / L or 36 g / L or 37 g / L or 38 g / L or 39 g / L or 40 g / L or 41 g / L or 42 g / L or 43 g / L or 44 g / L or 45 g / L or 46 g / L or 47 g / L or 48 g / L or 49 g / L or 50 g / L), AgNO3 3-6 mg / L (e.g. 3.5-5.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L or 4.5 mg / L or 5 mg / L or 5 mg / L).5 mg / L or 6 mg / L), cephalosporins 280-320 mg / L (e.g., 290-310 mg / L or 295-305 mg / L or 280 mg / L or 290 mg / L or 295 mg / L or 296 mg / L or 297 mg / L or 298 mg / L or 299 mg / L or 300 mg / L or 301 mg / L or 302 mg / L or 303 mg / L or 304 mg / L or 305 mg / L or 310 mg / L or 320 mg / L), 2,4-D 1-3 mg / L (e.g., 1.5-2.5 mg / L or 1 mg / L or 1.5 mg / L or 2 mg / L or 2.5 mg / L or 3 mg / L), NAA 0.4-0.6 mg / L (e.g., 0.4 mg / L or 0.45 mg / L or 0.5 mg / L). (mg / L or 0.55 mg / L or 0.6 mg / L), agar 5.5-6.5 g / L (e.g. 5.5 g / L or 6 g / L or 6.5 g / L), pH 6.0.

[0013] In the above method, in step (2), the conditions for propagation culture on callus subculture medium after induction are: subculture once every 7 days, and culture in the dark at 28°C.

[0014] The formula for the callus subculture medium is as follows: N6 salt 3.8-4.2 g / L (e.g., 3.9-4.1 g / L, 4.0-4.1 g / L, 3.9 g / L, 3.95 g / L, 4.0 g / L, 4.01 g / L, 4.03 g / L, 4.07 g / L, or 4.1 g / L), sodium citrate 10-20 mg / L (e.g., 12-18 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L), glutamate 90-110 mg / L (e.g., 95-105 mg / L, 90 mg / L, 95 mg / L, 96 mg / L, 97 mg / L, 98 mg / L, or 99 mg / L). mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), L-proline 0.4-0.8 g / L (e.g. 0.5-0.7 g / L or 0.4 g / L or 0.5 g / L or 0.6 g / L or 0.7 g / L or 0.8 g / L), hydrolyzed casein 90-110 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), MES 0.4-0.6 g / L (e.g. 0.4 ... g / L or 0.5 g / L or 0.6 g / L), sucrose 30-50 g / L (e.g. 35-45 g / L or 30 g / L or 31 g / L or 32 g / L or 33 g / L or 34 g / L or 35 g / L or 36 g / L or 37 g / L or 38 g / L or 39 g / L or 40 g / L or 41 g / L or 42 g / L or 43 g / L or 44 g / L or 45 g / L or 46 g / L or 47 g / L or 48 g / L or 49 g / L or 50 g / L), AgNO3 3-6 mg / L (e.g. 3.5-5.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L or 4.5 mg / L or 5 mg / L or 5 mg / L).5 mg / L or 6 mg / L), cephalosporins 280-320 mg / L (e.g., 290-310 mg / L or 295-305 mg / L or 280 mg / L or 290 mg / L or 295 mg / L or 296 mg / L or 297 mg / L or 298 mg / L or 299 mg / L or 300 mg / L or 301 mg / L or 302 mg / L or 303 mg / L or 304 mg / L or 305 mg / L or 310 mg / L or 320 mg / L), 2,4-D 1-2 mg / L (e.g., 1 mg / L or 1.5 mg / L or 2 mg / L), NAA 0.4-0.6 mg / L (e.g., 0.4 mg / L or 0.45 mg / L or 0.5 mg / L or 0.55 mg / L or 0.6 mg / L), agar 5.5-6.5 g / L (e.g., 5.5 g / L, 6 g / L, or 6.5 g / L), pH 6.0.

[0015] In the above method, step (3) involves activating the Agrobacterium containing the infection vector by: culturing the Agrobacterium in YEP culture medium with added antibiotics at 28°C with shaking, allowing the Agrobacterium to OD250. 600 = 0.6~0.8.

[0016] The formulation of the Agrobacterium infection culture medium is as follows: N6 salt 3.8~4.2 g / L (e.g. 3.9~4.1 g / L or 4.0~4.1 g / L or 3.9 g / L or 3.95 g / L or 4.0 g / L or 4.01 g / L or 4.03 g / L or 4.07 g / L or 4.1 g / L), sodium citrate 10-20 mg / L (e.g. 12-18 mg / L or 10 mg / L or 11 mg / L or 12 mg / L or 13 mg / L or 14 mg / L or 15 mg / L or 16 mg / L or 17 mg / L or 18 mg / L or 19 mg / L or 20 mg / L), glutamate 100 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L). mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), L-proline 0.4-0.8 g / L (e.g. 0.5-0.7 g / L or 0.4 g / L or 0.5 g / L or 0.6 g / L or 0.7 g / L or 0.8 g / L), hydrolyzed casein 100 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), MES 0.4-0.6 g / L (e.g. 0.4 ... 40-60 g / L (e.g., 45-55 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, or 60 g / L), AgNO3 3-6 mg / L (e.g., 3.5-5.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, 5 mg / L, 5.5 mg / L, or 6 mg / L), 2,4-D 1-2 mg / L (e.g., 1 mg / L, 0.5 mg / L, 0.6 ...5 mg / L or 1.5 mg / L or 2 mg / L), NAA 0.4-0.6 mg / L (e.g. 0.4 mg / L or 0.45 mg / L or 0.5 mg / L or 0.55 mg / L or 0.6 mg / L), AS 15-25 mg / L (e.g., 18-22 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, or 25 mg / L), pH 6.0.

[0017] Furthermore, the infection vector is a vector containing the target gene, selected from any one of plant overexpression vectors, RNAi vectors, or gene editing vectors.

[0018] In the above method, in step (4), the Agrobacterium-mediated method is selected from any one of the following: Agrobacterium immersion method, dip method, ultrasonic treatment Agrobacterium immersion method, or Agrobacterium vacuum negative pressure immersion method.

[0019] Furthermore, the Agrobacterium-mediated method is the Agrobacterium vacuum negative pressure impregnation method.

[0020] Furthermore, the infection conditions were immersion in a negative pressure of 0.05-0.06 MPa for 20 min.

[0021] In the above method, in step (5), the conditions for co-culturing the infected callus on the co-culture medium are: culturing at 22°C in the dark for 3 days.

[0022] The co-culture medium is formulated as follows: N6 salt 3.8–4.2 g / L (e.g., 3.9–4.1 g / L, 4.0–4.1 g / L, 3.9 g / L, 3.95 g / L, 4.0 g / L, 4.01 g / L, 4.03 g / L, 4.07 g / L, or 4.1 g / L), sodium citrate 10–20 mg / L (e.g., 12–18 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L), glutamate 90–110 mg / L (e.g., 95–105 mg / L, 90 mg / L, 95 mg / L, 96 mg / L, 97 mg / L, 98 mg / L, or 99 mg / L). mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), L-proline 0.4-0.8 g / L (e.g. 0.5-0.7 g / L or 0.4 g / L or 0.5 g / L or 0.6 g / L or 0.7 g / L or 0.8 g / L), hydrolyzed casein 90-110 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), MES 0.4-0.6 g / L (e.g. 0.4 ... g / L or 0.5 g / L or 0.6 g / L), sucrose 40-60 g / L (e.g. 45-55 g / L or 40 g / L or 41 g / L or 42 g / L or 43 g / L or 44 g / L or 45 g / L or 46 g / L or 47 g / L or 48 g / L or 49 g / L or 50 g / L or 51 g / L or 52 g / L or 53 g / L or 54 g / L or 55 g / L or 56 g / L or 57 g / L or 58 g / L or 59 g / L or 60 g / L), AgNO3 3-6 mg / L (e.g. 3.5-5.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L or 4.5 mg / L or 5 mg / L or 5.5 mg / L or 6 mg / L), 2,4-D 1-2 mg / L (e.g. 1 mg / L or 1.5 mg / L or 2 mg / L), NAA 0.4-0.6 mg / L (e.g. 0.4 mg / L or 0.45 mg / L or 0.5 mg / L or 0.55 mg / L or 0.6 mg / L), AS 15-25 mg / L (e.g., 18-22 mg / L or 15 mg / L or 16 mg / L or 17 mg / L or 18 mg / L or 19 mg / L or 20 mg / L or 21 mg / L or 22 mg / L or 23 mg / L or 24 mg / L or 25 mg / L), agar 5.5-6.5 g / L (e.g., 5.5 g / L or 6 g / L or 6.5 g / L), pH 6.0; In the above method, in step (6), the conditions for transferring the co-cultured callus to the recovery culture medium for antibacterial and recovery culture are: cultured at 28°C in the dark for 7 days.

[0023] The formulation of the recovery culture medium is as follows: N6 salt 3.8-4.2 g / L (e.g., 3.9-4.1 g / L, 4.0-4.1 g / L, 3.9 g / L, 3.95 g / L, 4.0 g / L, 4.01 g / L, 4.03 g / L, 4.07 g / L, or 4.1 g / L), sodium citrate 10-20 mg / L (e.g., 12-18 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L), glutamate 90-110 mg / L (e.g., 95-105 mg / L, 90 mg / L, 95 mg / L, 96 mg / L, 97 mg / L, 98 mg / L, or 99 mg / L). mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), L-proline 0.4-0.8 g / L (e.g. 0.5-0.7 g / L or 0.4 g / L or 0.5 g / L or 0.6 g / L or 0.7 g / L or 0.8 g / L), hydrolyzed casein 90-110 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), MES 0.4-0.6 g / L (e.g. 0.4 ... 0.5 g / L or 0.6 g / L), sucrose 30-50 g / L (e.g. 35-45 g / L or 30 g / L or 31 g / L or 32 g / L or 33 g / L or 34 g / L or 35 g / L or 36 g / L or 37 g / L or 38 g / L or 39 g / L or 40 g / L or 41 g / L or 42 g / L or 43 g / L or 44 g / L or 45 g / L or 46 g / L or 47 g / L or 48 g / L or 49 g / L or 50 g / L), AgNO3 3-6 mg / L (e.g. 3.5-5.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L or 4.5 mg / L or 5 mg / L or 5.5 mg / L or 6 mg / L), 2,4-D 1-2 mg / L (e.g. 1 mg / L or 1.5 mg / L or 2 mg / L), NAA 0.4-0.6 mg / L (e.g. 0.4 mg / L or 0.45 mg / L or 0.5 mg / L or 0.55 mg / L or 0.6 mg / L), AS 15-25 mg / L (e.g., 18-22 mg / L or 15 mg / L or 16 mg / L or 17 mg / L or 18 mg / L or 19 mg / L or 20 mg / L or 21 mg / L or 22 mg / L or 23 mg / L or 24 mg / L or 25 mg / L), cephalosporin 400-500 mg / L (e.g., 450-500 mg / L or 400 mg / L or 410 mg / L or 420 mg / L or 430 mg / L or 440 mg / L or 450 mg / L or 460 mg / L or 470 mg / L or 480 mg / L or 490 mg / L or 500 mg / L), agar 5.5-6.5 g / L (e.g., 5.5 g / L or 6 g / L or 6.5 g / L), pH 6.0.

[0024] In the above method, in step (7), the screening conditions are: dark culture at 28℃, transformation material with herbicide resistance gene (Bar) as screening marker, glufosinate as screening agent, 3 generations, 7 days / generation; transformation material with hygromycin resistance gene (HygR) as screening marker, hygromycin as screening agent, 16 days, 8 days / generation.

[0025] The screening medium is formulated as follows: N6 salt 3.8–4.2 g / L (e.g., 3.9–4.1 g / L, 4.0–4.1 g / L, 3.9 g / L, 3.95 g / L, 4.0 g / L, 4.01 g / L, 4.03 g / L, 4.07 g / L, or 4.1 g / L), sodium citrate 10–20 mg / L (e.g., 12–18 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, or 20 mg / L), glutamate 90–110 mg / L (e.g., 95–105 mg / L, 90 mg / L, 95 mg / L, 96 mg / L, 97 mg / L, 98 mg / L, or 99 mg / L). mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), L-proline 0.4-0.8 g / L (e.g. 0.5-0.7 g / L or 0.4 g / L or 0.5 g / L or 0.6 g / L or 0.7 g / L or 0.8 g / L), hydrolyzed casein 90-110 mg / L (e.g. 95-105 mg / L or 90 mg / L or 95 mg / L or 96 mg / L or 97 mg / L or 98 mg / L or 99 mg / L or 100 mg / L or 101 mg / L or 102 mg / L or 103 mg / L or 104 mg / L or 105 mg / L or 110 mg / L), MES 0.4-0.6 g / L (e.g. 0.4 ... 0.5 g / L or 0.6 g / L), sucrose 30-50 g / L (e.g. 35-45 g / L or 30 g / L or 31 g / L or 32 g / L or 33 g / L or 34 g / L or 35 g / L or 36 g / L or 37 g / L or 38 g / L or 39 g / L or 40 g / L or 41 g / L or 42 g / L or 43 g / L or 44 g / L or 45 g / L or 46 g / L or 47 g / L or 48 g / L or 49 g / L or 50 g / L), AgNO3 3-6 mg / L (e.g. 3.5-5.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L or 4.5 mg / L or 5 mg / L or 5.5 mg / L or 6 mg / L), 2,4-D 1-2 mg / L (e.g. 1 mg / L or 1.5 mg / L or 2 mg / L), NAA 0.4-0.6 mg / L (e.g. 0.4 mg / L or 0.45 mg / L or 0.5 mg / L or 0.55 mg / L or 0.The concentrations are as follows: 6 mg / L for glufosinate and 280-320 mg / L for cephalosporins (e.g., 290-310 mg / L, 295-305 mg / L, 280 mg / L, 290 mg / L, 295 mg / L, 296 mg / L, 297 mg / L, 298 mg / L, 299 mg / L, 300 mg / L, 301 mg / L, 302 mg / L, 303 mg / L, 304 mg / L, 305 mg / L, 310 mg / L, or 320 mg / L); 10 mg / L for screening reagent; 5.5-6.5 g / L for agar (e.g., 5.5 g / L, 6 g / L, or 6.5 g / L); pH 6.0; the screening reagent is glufosinate or hygromycin.

[0026] In the above method, the conditions for inducing differentiation in (8) are: 28℃, light intensity 15000 LUX, photoperiod 16h (daytime) / 8h (nighttime), and culture for 7-10 days.

[0027] The differentiation medium formulation is as follows: MS salt 2-2.5 g / L (e.g., 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, or 2.5 g / L), MES 0.4-0.6 g / L (e.g., 0.4 g / L, 0.5 g / L, or 0.6 g / L), sucrose 30-50 g / L (e.g., 35-45 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, or 50 g / L), AgNO3 3-6 mg / L (e.g., 3.5-5.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L or 4.5 mg / L or 5 mg / L or 5.5 mg / L or 6 mg / L), 6-BA 1-2 mg / L (e.g., 1 mg / L or 1.5 mg / L or 1.8 mg / L or 2 mg / L), cephalosporins 280-320 mg / L (e.g., 290-310 mg / L or 295-305 mg / L or 280 mg / L or 290 mg / L or 295 mg / L or 296 mg / L or 297 mg / L or 298 mg / L or 299 mg / L or 300 mg / L or 301 mg / L or 302 mg / L or 303 mg / L or 304 mg / L or 305 mg / L or 310 mg / L or 320 mg / L), agar 5.5-6.5 g / L (e.g., 5.5 g / L, 6 g / L, or 6.5 g / L), pH 6.0.

[0028] Furthermore, adding a low concentration of Bar (3 mg / L) to the differentiation medium can inhibit the differentiation of uninfected callus cells and the frequency of seedling regeneration, thereby further increasing the proportion of positive seedlings.

[0029] In the above method, the differentiation conditions in (9) are: 28℃, photoperiod 16 h (daytime) / 8 h (nighttime), and light intensity 15000 LUX.

[0030] The formulation of the seedling culture medium is as follows: MS salt 2-2.5 g / L (e.g., 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, or 2.5 g / L), MES 0.4-0.6 g / L (e.g., 0.4 g / L, 0.5 g / L, or 0.6 g / L), sucrose 30-50 g / L (e.g., 35-45 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, or 50 g / L), AgNO3 3-6 mg / L (e.g., 3.5-5.5 mg / L or 3 mg / L or 3.5 mg / L or 4 mg / L or 4.5 mg / L or 5 mg / L or 5.5 mg / L or 6 mg / L), cephalosporin 280-320 mg / L (e.g., 290-310 mg / L or 295-305 mg / L or 280 mg / L or 290 mg / L or 295 mg / L or 296 mg / L or 297 mg / L or 298 mg / L or 299 mg / L or 300 mg / L or 301 mg / L or 302 mg / L or 303 mg / L or 304 mg / L or 305 mg / L or 310 mg / L or 320 mg / L), agar 5.5-6.5 g / L (e.g., 5.5 g / L or 6 g / L or 6.5 g / L), pH 6.0.

[0031] In the above method, in step (10), the molecular identification method includes PCR amplification, Southern hybridization, Northern hybridization and Western blot hybridization; the selector is glyphosate, chlorsulfuron-methyl or hygromycin.

[0032] A second aspect of the present invention provides a kit for genetic transformation of maize inbred lines, comprising the callus induction medium, callus subculture medium, co-culture medium, recovery medium, screening medium, differentiation medium, and seedling medium described in the first aspect.

[0033] A third aspect of the invention provides the use of the kit described in the second aspect in any of the following: (1) Genetic transformation of maize inbred lines; (2) Preparation of products from genetic transformation of maize inbred lines; (3) Tissue culture of maize inbred lines; (4) Preparation of products from tissue culture of maize inbred lines; The maize inbred lines include Qi 319 and Zheng 58.

[0034] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: 1. This invention establishes a highly efficient and high-quality long-term subculture system for maize embryogenic callus. Based on this system, Agrobacterium-mediated genetic transformation is performed using embryogenic callus as the recipient, and transgenic plants can be obtained in batches after regeneration. Compared with genetic transformation methods using immature embryos as recipients, its advantages lie in overcoming the seasonality and developmental stage limitations of immature embryos, as well as providing the intensive labor and greenhouse space required for immature embryos, significantly reducing the cost of maize genetic transformation and improving its efficiency. Compared with genetic transformation methods using shoot tips as recipients, its advantages are avoiding the problems of numerous chimeras and genetic instability in the transformed offspring, as well as the low transformation rate and screening efficiency of maize shoot tips.

[0035] 2. The maize callus genetic transformation system constructed in this invention maintains the high embryogenicity and regeneration capacity of callus, improves transfection efficiency, and can effectively transform commercial maize breeding backbone inbred lines such as Qi 319 and Zheng 58. To a certain extent, it breaks through the genotype limitation of maize genetic transformation. The transformation material does not need to undergo multiple generations of backcrossing and self-pollination homozygosity. The transformed individuals can be directly used for transgenic genetic breeding. This avoids the linkage burden and loss of parental desirable traits that accompany multiple generations of backcrossing and transformation, shortens the breeding cycle, and greatly improves the efficiency of maize transgenic breeding.

[0036] 3. This invention establishes a highly efficient rapid seedling regeneration system independent of exogenous hormones, with a high seedling regeneration frequency, averaging >3 seedlings per callus block. Simultaneously, this system enables rapid rooting and sprouting on the regeneration medium, yielding a large number of transplantable seedlings within 3 weeks without the need to change the medium. The seedlings exhibit good growth and are largely free of deformities. Furthermore, adding a low concentration of Bar (3 mg / L) to the differentiation medium can further increase the proportion of positive seedlings, achieving a transgenic positive rate of over 60% in the regenerated seedlings, providing strong technical support for gene function identification and multi-gene synergistic improvement. Attached Figure Description

[0037] Figure 1 This diagram illustrates the main process of genetic transformation of maize callus.

[0038] Figure 2The diagram shows the process of inducing embryonic callus on Qi 319 after 11 days of pollination. (A) shows the embryonic Qi 319 after 11 days of pollination, with the scutellum facing upward on callus induction medium A; (B) shows the state of the embryonic embryo after 1 week of culture in the dark at 28°C on callus induction medium A, with the embryo swelling, nodular protrusions appearing on the edges, and some embryonic embryos growing radicles; (C) shows the large amount of pale yellow embryonic callus produced after 3 weeks of transfer to callus induction medium B (subcultured every 7 days).

[0039] Figure 3 A diagram illustrating high-quality subculture of embryogenic callus from the maize backbone inbred line Qi 319.

[0040] Figure 4 The removal of the callus washing step and the addition of acetylsuccinone to the recovery culture process after Agrobacterium co-culture improved the ratio of Qi 319 resistant callus. Among them, (A) is a photo of callus washed twice with sterile water on a laminar flow hood after 3 days of Agrobacterium co-culture, transferred to recovery culture medium without acetylsuccinone, and then screened for 3 generations (7 days / generation) on selection medium containing 10 mg / L glufosinate after recovery culture; (B) is a photo of callus washing step removed after 3 days of Agrobacterium co-culture, followed by recovery culture on recovery culture medium containing acetylsuccinone and then screened for 3 generations (7 days / generation) on selection medium containing 10 mg / L glufosinate after recovery culture. In the figure, the untransformed callus is browned and necrotic, while the transformed callus maintains a good growth state.

[0041] Figure 5 This image shows the recovery culture of Qi 319 resistant callus after two generations following herbicide screening.

[0042] Figure 6 This image shows the Qi 319 resistant callus tissue after 10 days of light-induced differentiation to restore its culture.

[0043] Figure 7 This image shows the seedling status of Qi 319 callus blocks after light-induced differentiation on regeneration culture medium.

[0044] Figure 8The following diagrams illustrate the transplanting and identification of Qi 319 regenerated seedlings. In (A), the regenerated seedlings are transplanted into disposable paper cups containing nutrient soil, covered with disposable plastic gloves, and kept moist to improve the survival rate. In (B), the disposable transparent gloves are removed after the seedlings have recovered (about one week). In (C) and (D), 1.25‰ glufosinate is evenly applied to both sides of a certain area of ​​the fully expanded leaves of the seedlings. After screening for about 8-10 days, obvious yellow spots appear on the leaves of non-positive seedlings, while the leaves of resistant seedlings are basically unaffected. In (E), DNA is extracted from the leaves of the seedlings and PCR amplification is performed. In the diagram, M: DNA Marker, molecular weight 100~5000 bp, (+) is the positive plasmid control, CK1 and CK2 are the negative controls without transformation, and lanes 1-19 are the PCR amplification results of the regenerated seedlings.

[0045] Figure 9 The images illustrate the induction of embryogenic callus from Zheng 58, the screening of resistant callus after Agrobacterium infection, and seedling regeneration. (A) shows the scutellum of an 11-day-old Zheng 58 embryo placed face up on callus induction medium A; (B) shows the large amount of pale yellow embryogenic callus produced after transfer to callus induction medium B (subcultured every 7 days); (C) shows the seedlings after Agrobacterium infection and co-culture, 10... (D) Photographs of 3 generations (7 days / generation) screened on screening medium containing mg / L glufosinate; (E) Photographs of Zheng 58 resistant callus after recovery culture transferred to differentiation medium for 10 days to induce differentiation, the callus turned green and a large number of buds were produced on the surrounding nodular protrusions; (F) Photographs of Zheng 58 callus blocks after light-induced differentiation, transferred to canning jars containing regeneration medium, and sterile seedlings produced about 3 weeks later; (F) Photographs of transgenic positive seedlings detected by commercial Bar test strips, the rightmost one is the untransformed wild-type control of Zheng 58, and the other two are the detection results of transgenic positive seedlings. A clear target band can be seen below the standard band. Detailed Implementation

[0046] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0048] Qi 319 in the following examples is an important backbone inbred line in maize breeding, bred by the Maize Research Institute of Shandong Academy of Agricultural Sciences. Its breeding process, characteristics and applications can be found in the core literature: Zhang Fajun et al., Breeding and application of maize inbred line Qi 319 [J]. Maize Science, 2004, 12(Supplement): 1-2. If objective data is needed, the Plant Variety Rights Announcement (Variety Rights No.: CNA20020287.9) can be consulted. Zheng 58 is the female parent of the famous maize hybrid Zhengdan 958. Its breeding background, parental source and characteristics can be found in the core literature: Du Chunxin et al., Breeding and promotion of maize hybrid Zhengdan 958 [J]. Maize Science, 2001, 9(1): 24-26. If objective trait data is needed, the Plant Variety Rights Announcement (Variety Rights No.: CNA20010168.1) can be consulted. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating the relevant experiments of this invention and may not be used for any other purpose.

[0049] The backbone vector carrying the target gene fragment and glufosinate resistance gene used in the following examples is pCAMBIA3300-Ubi-MCS-35S-Bar, the target gene is ZmHMGR1, and the Agrobacterium is Agrobacterium LB4404. These examples use the ZmHMGR1 gene as an example of a foreign gene, but the present invention is not limited to specific foreign genes. The Agrobacterium gene introduction method, vector construction method, and plasmids used in this embodiment are well known to those skilled in the art. Related materials are publicly available from the applicant and are used only for repeating the relevant experiments of this invention; they should not be used for other purposes.

[0050] The N6 salt involved in the following examples (PhytoTechnology Laboratories) TM , CHU's N6 BasalSalt Mixture), MS salt (PhytoTechnology Laboratories TM Reagents required for experiments such as M&S Basal Salt Mixture are available to the public from well-known sales channels or can be prepared according to their well-known formulas.

[0051] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0052] Example 1: Rapid seedling regeneration system of maize backbone inbred lines with callus induction, Agrobacterium-mediated infection, and independence from exogenous hormones. This embodiment provides a method for constructing a rapid seedling regeneration system based on callus induction, Agrobacterium-mediated infection, and exogenous hormone-free process of maize backbone inbred lines. The main flowchart is shown below. Figure 1 As shown, the specific steps are as follows: 1. Establishment of an efficient system for inducing embryogenic callus in commercial maize backbone inbred lines Explant selection: Through numerous experiments, it was found that the long axis length of the young embryo is about 1.2 mm (about 11 days after field pollination in summer) with the best induction efficiency. Therefore, young embryos that have been pollinated in the field for 11 days (the time of embryo harvesting varies slightly depending on the genotype and can be adjusted according to the size of the young embryo) are used as recipients to carry out high-efficiency induction culture of embryogenic callus.

[0053] Selection of substrate medium: N6 medium has a low ammonium nitrogen and high nitrate nitrogen source. Due to the high concentration of NH4+... + It is toxic to many plant cells (especially grasses including maize), inhibiting callus growth and causing browning, while N6 medium contains low NH4. + High NO3 - The ratio of NH4 not only greatly reduces the amount of NH4 + It reduces the toxicity of nitrogen and provides plant cells with a more easily absorbed and utilized form of nitrogen (NO3). - This medium provides a sufficient nitrogen source while minimizing toxicity. Furthermore, the ample supply of glycine, vitamins, and other amino acids, as well as organic components, further meets the needs of rapid cell proliferation. Secondly, N6 medium works exceptionally well with the potent auxin 2,4-D, strongly inducing cell dedifferentiation while maintaining cell viability. Therefore, N6 medium was chosen as the substrate medium.

[0054] Optimization and formulation of high-efficiency induction medium for embryogenic callus: Based on N6 medium, systematic optimization and improvement were carried out, as detailed below: 1) Hydrolyzed casein, L-proline and glutamic acid were added to the N6 medium to provide more comprehensive nutrients, osmotic stress protection substances and growth regulators to regulate the physiological state of plant cells.

[0055] 2) Sodium citrate and 2-morpholinoethanesulfonic acid (MES) were further optimized in the culture medium. Sodium citrate and MES, as mild metal ion chelators and chemically inert and cell membrane-impermeable pH buffer systems, respectively, can create more favorable conditions for callus induction, growth and genetic transformation by optimizing the physicochemical environment.

[0056] 3) Adding an appropriate concentration of sucrose to the culture medium not only provides sufficient carbon and energy sources for tissue culture, driving cell division and growth, but also maintains the osmotic pressure of the medium, preventing excessive water absorption or dehydration of cells, which could affect callus induction and growth. Furthermore, as a sugar signaling molecule, sucrose can also regulate cell growth and development by influencing gene expression, maintaining the proliferative capacity of callus. High sucrose concentrations (e.g., 4%-6%) can, to some extent, inhibit excessive cell division, promoting the formation of compact, dry callus tissue, and even promoting the formation of embryogenic callus. Low sucrose concentrations (e.g., 1%-2%) result in lower osmotic pressure, easily promoting rapid cell division and forming loose, water-rich, and fragile non-embryonic callus tissue. Therefore, selecting an appropriate sucrose concentration is one of the key factors for successful induction of embryogenic callus and long-term high-quality subculture. Different genotypes of material have different sucrose concentration requirements during culture, and the sucrose concentration can be appropriately adjusted within a certain range according to the state of the callus tissue.

[0057] 4) Add appropriate concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA) to the culture medium. By designing the combined use of 2,4-D and NAA, and employing a relatively high concentration of 2,4-D during callus induction, followed by a gradual reduction in the 2,4-D concentration, and a relatively low concentration of 2,4-D during callus maintenance and subculture, while maintaining NAA at a relatively low and stable concentration as a supplement, this approach ensures efficient callus induction while reducing the toxic side effects of high concentrations of 2,4-D, achieving the goal of sustained healthy proliferation and high regenerative capacity of subsequent embryogenic callus.

[0058] 5) Add an appropriate concentration of AgNO3 to the culture medium. Ethylene is an important gaseous hormone in plants. Plant cells accelerate ethylene synthesis under stress (such as explant segmentation and tissue culture stress). Under the closed conditions of aseptic culture, ethylene gas easily accumulates, stimulating the synthesis of phenolic substances and the activity of polyphenol oxidase, exacerbating enzymatic browning, and negatively impacting tissue culture. AgNO3, as an inhibitor of ethylene action, competitively binds to ethylene receptors, blocking ethylene signaling pathways, preventing or slowing tissue browning, and better maintaining the viability of explants or callus tissue. Furthermore, the silver ions released from the dissolved AgNO3, as heavy metal ions, can inhibit the growth of endogenous bacteria and reduce the probability of contamination. Therefore, adding an appropriate concentration of AgNO3 to the culture medium can positively promote callus induction and high-quality continuous culture in ethylene-sensitive or difficult-to-regenerate plant species (including maize).

[0059] 6) Add an appropriate concentration of cephalosporin to the culture medium. Cephalosporin is a β-lactam antibiotic that binds specifically to key enzymes in bacterial cell wall synthesis, preventing bacteria from forming a complete cell wall and causing them to die. Although plant cells also have cell walls, their synthesis mechanism is completely different from that of bacteria. Therefore, cephalosporins are essentially non-toxic to plant cells and can effectively kill or inhibit the growth of Agrobacterium and endophytes without harming plant cells, providing sterility for plant cultures. Therefore, an appropriate concentration of cephalosporins needs to be added during tissue culture processes such as callus induction, subculture, and post-transformation antibacterial treatment to ensure successful genetic transformation experiments.

[0060] Considering the above points 1) to 6), using the immature embryos of commercial maize backbone inbred lines Qi 319 and Zheng 58 11 days after pollination as recipients and N6 medium as the substrate medium, the optimal concentration range of each component in the medium was determined through a combination of single-factor and multi-factor orthogonal experiments. A highly efficient system for inducing embryogenic callus and for long-term, high-quality subculture was established. Based on this, a highly efficient Agrobacterium-mediated genetic transformation technology system for callus from commercial maize backbone inbred lines was developed. Details are as follows: After sterilization of the corn ears 11 days after pollination, immature embryos were selected and placed in a temporary embryo culture medium. The specific composition of the temporary embryo culture medium was as follows: N6 salt 4.07 g / L, sodium citrate 20 mg / L, glutamate 90 mg / L, L-proline 0.5 g / L, hydrolyzed casein 110 mg / L, MES 0.6 g / L, sucrose 30-50 g / L (adjusted according to different genotypes), AgNO3 3 mg / L, cephalosporin 300 mg / L, 2,4-D 3 mg / L, NAA 0.5 mg / L, pH 6.0.

[0061] Discard the temporary culture medium for immature embryos, place the collected immature embryos on sterile filter paper, blot off excess liquid from the surface, and place them with the scutellum facing upwards on callus induction medium A. Incubate in the dark at 28°C for 1 week. A relatively high concentration of 2,4-D can effectively initiate cell dedifferentiation and induce callus formation. The specific composition of callus induction medium A is as follows: N6 salt 4.07 g / L, sodium citrate 20 mg / L, glutamate 90 mg / L, L-proline 0.5 g / L, hydrolyzed casein 110 mg / L, MES 0.6 g / L, sucrose 30-50 g / L (adjust appropriately according to different maize genotypes. For example, Qi 319 40 g / L; Zheng 58 50 g / L), AgNO3 3 mg / L, cephalosporin 300 mg / L, 2,4-D 3 mg / L, NAA 0.5 mg / L, agar 6 g / L, pH 6.0.

[0062] The radicles of the immature embryos were removed, and the embryos were transferred to callus induction medium B. Subculture was performed every 7 days, with timely removal of the radicles during the culture process. The embryos were cultured in the dark at 28°C for 2-4 weeks (the duration depending on the callus production of different maize genotypes) until a large amount of embryogenic callus was produced. The 2,4-D concentration in the medium was reduced to 2.0 mg / L. This moderate reduction in 2,4-D concentration maintained the dedifferentiation state of the cells, promoted the formation of high-quality embryogenic callus with regenerative potential, and avoided the side effect of continuously high 2,4-D concentrations that could inhibit subsequent organogenesis. The specific composition of callus induction medium B is as follows: N6 salt 4.07 g / L, sodium citrate 20 mg / L, glutamic acid 90 mg / L, L-proline 0.5 g / L, hydrolyzed casein 110 mg / L, MES 0.6 g / L, sucrose 30-50 g / L (adjusted appropriately according to different maize genotypes. For example, Qi 319 40 g / L; Zheng 58 50 g / L), AgNO3 3 mg / L, cephalosporin 300 mg / L, 2,4-D 2 mg / L, NAA 0.5 mg / L, agar 6 g / L, pH 6.0.

[0063] 2. Construction of a high-quality embryogenic callus long-term subculture system Relatively high concentrations of 2,4-D are a potent initiation signal for callus formation, but prolonged maintenance of this signal can lead to excessive callus proliferation, resulting in loose, fragile, and highly water-containing callus that transforms into non-embryonic callus and even loses its regenerative capacity. Therefore, after callus induction, it should be promptly transferred to a callus subculture medium containing a lower concentration of 2,4-D. Through callus culture experiments at different 2,4-D concentrations, it was found that embryogenic callus can be subcultured at a concentration of 1.5 mg / L for a long period with high quality. The embryogenicity and regenerative capacity of the callus remained essentially unchanged after more than 18 months of subculture. Therefore, by selecting immature embryos once a year during the appropriate summer season to induce and renew the callus, high-quality callus culture and propagation can be ensured year-round, ready for use in maize genetic transformation. Callus is typically subcultured every 7 days in a dark culture at 28°C. When not transforming, only a small amount of callus needs to be retained for subculture; when transformation is needed, large-scale propagation can be achieved within 2 weeks.

[0064] The specific composition of the callus subculture medium is as follows: N6 salt 4.07 g / L, sodium citrate 20 mg / L, glutamic acid 90 mg / L, L-proline 0.5 g / L, hydrolyzed casein 110 mg / L, MES 0.6 g / L, sucrose 30-50 g / L (adjusted appropriately according to different maize genotypes. For example, Qi 319 40 g / L; Zheng 58 50 g / L), AgNO3 3 mg / L, cephalosporin 300 mg / L, 2,4-D 1.5 mg / L, NAA 0.5 mg / L, agar 6 g / L, pH 6.0.

[0065] 3. Agrobacterium-mediated maize embryonic callus infection system 1) Cultivation and collection of Agrobacterium Agrobacterium bacterial suspension carrying a plant expression vector (containing the target gene and a selector resistance gene) was cultured in YEP medium supplemented with antibiotics at 28°C with shaking at 200 rpm to induce the Agrobacterium to enter the logarithmic growth phase (OD). 600 = 0.6~0.8). Subsequently, the bacteria were collected by centrifugation at 5000 rpm for 10 min, and the bacterial suspension was resuspended in an equal volume of Agrobacterium infection medium containing 20 mg / L acetylsuccinone (AS, which activates the Vir gene virulence system of Agrobacterium). After standing in the dark at 22°C for 1 h, it was used for transformation.

[0066] The specific composition of the Agrobacterium-mediated infection culture medium is as follows: N6 salt 4.07 g / L, sodium citrate 20 mg / L, glutamic acid 90 mg / L, L-proline 0.5 g / L, hydrolyzed casein 110 mg / L, MES 0.6 g / L, sucrose 40-60 g / L (adjusted appropriately according to different maize genotypes. For example, Qi 319 50 g / L; Zheng 58 60 g / L), AgNO3 3 mg / L, 2,4-D 1.5 mg / L, NAA 0.5 mg / L, AS 20 mg / L, pH 6.0.

[0067] 2) Infection of maize callus tissue Embryogenic callus was clipped into small pieces and placed in the above-mentioned Agrobacterium tumefaciens bacterial solution. Incubation was carried out under a negative pressure of 0.05-0.06 MPa for 20 min to improve the Agrobacterium tumefaciens infection efficiency. Afterwards, the callus was blotted dry on sterile filter paper and transferred to a co-culture medium, incubated at 22°C in the dark for 3 days. The specific composition of the co-culture medium was as follows: N6 salt 4.07 g / L, sodium citrate 20 mg / L, glutamate 90 mg / L, L-proline 0.5 g / L, hydrolyzed casein 110 mg / L, MES 0.6 g / L, sucrose 40-60 g / L (adjusted appropriately according to different maize genotypes. For example, Qi 319 50 g / L; Zheng 58 60 g / L), AgNO3 3 mg / L, 2,4-D 1.5 mg / L, NAA 0.5 mg / L, AS 20 mg / L, agar 6 g / L, pH 6.0.

[0068] Compared to the callus subculture medium, the co-culture medium had a correspondingly higher sucrose concentration. A moderate increase in sucrose concentration lowers the water potential of the medium, creating a mild hypertonic environment. This environment effectively inhibits the excessive proliferation of Agrobacterium on the callus surface, preventing the formation of a thick bacterial film that could excessively damage the callus cells. Furthermore, because the callus has just been infected by Agrobacterium, its high water content can easily lead to callus maceration, affecting subsequent embryogenicity and regeneration. Therefore, this mild osmotic stress not only better regulates the physiological state of plant cells but also causes subtle changes in the structure of the cell membrane and cell wall, facilitating Agrobacterium attachment and T-DNA transfer. This places the cells in a more "sensitive state" conducive to the stable integration of exogenous genes, thus improving Agrobacterium infection efficiency. Additionally, the co-culture medium removed cephalosporins and added 20 mg / L of Aspergillus sulfadiazine (AS) to strongly activate the Vir gene system of Agrobacterium, overcoming the natural barrier between monocotyledonous plants and Agrobacterium, allowing for the efficient introduction of exogenous genes into maize cells and enhancing Agrobacterium infection efficiency.

[0069] 3) Recovery culture of maize callus tissue After co-culturing for 3 days, the callus tissue was transferred to recovery medium for inhibition and recovery culture, and cultured in the dark at 28°C for 7 days. Following the Agrobacterium infection and co-culture stages, a significant amount of Agrobacterium adhered to the callus tissue. To simplify the experimental procedures and reduce the risk of callus hydration while still achieving antibacterial effects, after repeated experiments and explorations, the step of washing the callus surface with sterile water to remove excess Agrobacterium was omitted. The concentration of cephalosporin was increased to 400 mg / L, achieving antibacterial effects while minimizing its impact on callus growth. Furthermore, based on the characteristic of Agrobacterium to survive under appropriate selection pressure by increasing its infection efficiency on host cells, 20 mg / L of cephalosporin (AS) was added to the recovery medium to allow Agrobacterium to continue infecting the callus tissue and enhance infection efficiency in the early stages of inhibition.

[0070] The specific composition of the recovery medium is as follows: N6 salt 4.07 g / L, sodium citrate 20 mg / L, glutamic acid 90 mg / L, L-proline 0.5 g / L, hydrolyzed casein 110 mg / L, MES 0.6 g / L, sucrose 30-50 g / L (adjusted appropriately according to different maize genotypes. For example, Qi 319 40 g / L; Zheng 58 50 g / L), AgNO3 3 mg / L, 2,4-D 1.5 mg / L, NAA 0.5 mg / L, AS 20 mg / L, cephalosporin 400 mg / L, agar 6 g / L, pH 6.0.

[0071] 4. Enhance the screening strength of embryogenic callus after Agrobacterium infection and improve the positive rate of regenerated seedlings. After one week of recovery culture, the callus tissue was transferred to selection medium and cultured in the dark at 28°C for screening. Through continuous experimentation and exploration, the selection concentration of the transformed material using the herbicide resistance gene (Bar) as the selection marker was increased from 3, 5, and 5 mg / L glufosinate, each for one generation (7 days / generation), to 10 mg / L for three generations (7 days / generation). The PCR amplification results of the regenerated seedlings showed that the positive rate increased from about 26% to over 60%. For the transformed material using the hygromycin resistance gene (HygR) as the selection marker, the selection intensity was changed from 10 mg / L hygromycin for 10 days to 16 days (8 days / generation). The PCR amplification positive rate of the regenerated seedlings increased from about 40% to about 70%.

[0072] The specific composition of the screening medium is as follows: N6 salt 4.07 g / L, sodium citrate 20 mg / L, glutamic acid 90 mg / L, L-proline 0.5 g / L, hydrolyzed casein 110 mg / L, MES 0.6 g / L, sucrose 30-50 g / L (adjusted appropriately according to different maize genotypes. For example, Qi 319 40 g / L; Zheng 58 50 g / L), AgNO3 3 mg / L, 2,4-D 1.5 mg / L, NAA 0.5 mg / L, cephalosporin 300 mg / L, Hpt / Bar 10 mg / L, agar 6 g / L, pH 6.0.

[0073] 5. Subculture and propagation system of resistant callus Select the above-mentioned resistant callus tissue and transfer it to callus subculture medium. Incubate at 28°C in the dark for 7-14 days (7 days / generation). The specific time depends on the recovery status of the callus tissue.

[0074] 6. Preparation of Differentiation Culture Medium After recovery, the callus tissue was transferred to differentiation medium. The callus tissue blocks were clipped to a diameter of 2-3 mm and cultured at 28°C with a light intensity of 15000 LUX and a photoperiod of 16 h (daytime) / 8 h (nighttime) for 7-10 days (the specific time depends on the differentiation of different maize genotypes) until obvious green sprouting appeared during the culture process.

[0075] Based on the different nutritional and hormone requirements at different culture stages, N6 medium was used as the substrate medium during the induction / proliferation stage, and 1 / 2 MS medium was used as the substrate medium during the differentiation stage. During the induction / proliferation stage, the low ammonium nitrogen formulation of N6 medium avoided ammonia toxicity, while the high nitrate nitrogen promoted cell division and had excellent synergistic effects with 2,4-D. During the differentiation stage, embryogenic callus tissue differentiated into shoots and roots, regenerating complete plants. The higher ammonium nitrogen and more comprehensive nutrient support organs in MS medium, along with the more balanced ionic environment, were conducive to morphogenesis. During the differentiation stage, 2,4-D must be removed to relieve its inhibitory effect on differentiation, and cytokinins (such as 6-BA) must be added simultaneously to promote shoot formation, which is crucial for successful differentiation and regeneration. Finally, the optimized composition of the differentiation medium was determined to be: MS salt 2.2 g / L, MES 0.6 g / L, sucrose 30-50 g / L (adjusted appropriately according to different maize genotypes, for example, Qi 319 40 g / L; Zheng 58 50 g / L), AgNO3 3 mg / L, 6-BA 1.5 mg / L, cephalosporin 300 mg / L, agar 6 g / L, pH 6.0.

[0076] 7. Construction of a rapid seedling system that does not rely on exogenous hormones Callus tissue blocks exhibiting clear budding after induced differentiation were transferred to canning jars containing regeneration medium. Seedlings were incubated at 28°C with a photoperiod of 16 h (daytime) / 8 h (nighttime) and a light intensity of 15000 LUX until they were ready for transplanting. The seedling culture medium consisted of: MS salt 2.2 g / L, MES 0.6 g / L, sucrose 30-50 g / L (adjusted according to different maize genotypes, e.g., Qi 319 40 g / L; Zheng 58 50 g / L), AgNO3 3 mg / L, cephalosporin 300 mg / L, agar 6 g / L, pH 6.0.

[0077] The above genetic transformation system produces seedlings with a very high regeneration frequency, which can generate a large number of regenerated seedlings in a short period of time. To reduce the screening work for positive seedlings in the later stage, a low concentration of Bar (3 mg / L) can be added to the differentiation medium to inhibit the differentiation of uninfected callus cells and the seedling regeneration frequency, thereby further increasing the proportion of positive seedlings.

[0078] 8. Transplanting, resistance screening, and molecular identification of test-tube seedlings Before transplanting the regenerated seedlings, remove the sealing film and add a small amount of sterile water to the jars to soak the culture medium. Harden the seedlings for 1-2 days, then transplant them into paper cups filled with seedling soil. Cultivate until approximately 4 leaves, then apply a 1.25‰ solution of Bar herbicide to both sides of a specific area of ​​the leaves. Screening is performed after about 10 days. Non-positive seedlings show obvious yellow spots or even necrosis, while positive seedlings are largely unaffected. Harvest the leaves of herbicide-resistant seedlings, extract DNA, and perform further PCR testing. Seedlings with positive amplification results are transplanted to a greenhouse or field for self-pollination to harvest transgenic progeny materials.

[0079] Example 2: Genetic transformation and seedling regeneration of embryogenic callus from the commercial maize backbone inbred line Qi 319 This embodiment provides a method for genetic transformation and seedling regeneration of embryogenic callus from the commercial maize backbone inbred line Qi 319. The formulations of the callus induction medium, callus subculture medium, Agrobacterium tumefaciens inoculation medium, co-culture medium, recovery medium, screening medium, differentiation medium, and seedling culture medium used in this embodiment are the same as in Example 1. The specific steps are as follows: 1. Extraction of immature embryos and induction of embryogenic callus from the maize inbred line Qi 319 The maize inbred line Qi 319 was planted in the field. Approximately 11 days after self-pollination, the ears were harvested, the husks removed, and the ears were surface-sterilized by soaking in 70% alcohol for 15 minutes. Using forceps inserted into the corn cob as handles, the upper two-thirds of the kernels were removed with a scalpel. The embryos were then picked up and placed in a temporary embryo culture medium. After collecting a sufficient number of embryos, excess liquid was removed. The embryos were then placed on sterile filter paper in a laminar flow hood, and excess culture medium was blotted off. With the scutellum facing upwards, the embryos were arranged orderly on callus induction medium A. Figure 2 As shown in A, approximately 50-100 embryos are placed in each dish and cultured at 28°C in the dark for one week.

[0080] During the induction of callus tissue, some immature embryos will develop radicles, such as... Figure 2 As shown in section B, the radicle should be removed promptly. After culturing the immature embryo on induction medium A for one week, the radicle is removed and the embryo is transferred to callus induction medium B. Subculture is performed every 7 days, and the tissue blocks are cut into appropriate sizes when changing the medium. A large amount of pale yellow embryogenic callus will be produced in about 3 weeks. Figure 2 As shown in C.

[0081] 2. High-quality subculture and propagation of Qi 319 embryogenic callus The embryogenic callus produced in step 1 was cut into tissue blocks with a diameter of 2-3 mm and transferred to callus subculture medium for subculture and propagation (dark culture at 28℃). After one week, a large number of high-quality, pale yellow embryogenic callus blocks with a diameter of 4-6 mm were produced. Figure 3 As shown, the callus tissue can be cut into 2-3 mm pieces for Agrobacterium-mediated genetic transformation. If transformation is not needed at this stage, only a small amount of embryogenic callus tissue needs to be retained for subculture; it can then be propagated for transformation when needed.

[0082] 3. Activation and culture of Agrobacterium Add 150 μL of Agrobacterium LB4404 bacterial culture containing the target plasmid (carrying the target gene fragment and glufosinate resistance gene) to 25 mL of YEP culture medium containing 50 mg / L rifamycin and 50 mg / L kanamycin, and incubate overnight at 28°C with shaking at 200 rpm. The next day, transfer 2-3 mL of the bacterial culture to a fresh 25 mL YEP culture medium (50 mg / L rifamycin + 50 mg / L kanamycin) and incubate until the bacterial concentration reaches OD500. 600 = 0.6~0.8 (approximately 5-6 hours), centrifuge at 5000 rpm for 10 min to collect bacteria, then suspend the bacterial culture in an equal volume of Agrobacterium infection medium containing 100 μM acetylsyl syringone, and incubate at 22℃ in the dark for 1 h before transformation.

[0083] 4. Agrobacterium infection and recovery culture of Qi 319 embryogenic callus Qi 319 embryogenic callus was clipped into 2-3 mm diameter pieces and placed in Erlenmeyer flasks containing the previously settled Agrobacterium LB4404 bacterial suspension. The flasks were sealed with a sealing film and placed in a negative pressure chamber at 0.05-0.06 MPa for 20 min to improve Agrobacterium infection efficiency. Afterward, the bacterial suspension was discarded, and the callus was blotted dry on sterile filter paper before being transferred to co-culture medium and co-cultured at 22°C in the dark for 3 days. Following co-culture, the callus was clipped into 2-3 mm diameter pieces and divided into two groups. Group A was treated as follows: the callus pieces were washed twice with sterile water on a laminar flow hood and transferred to recovery medium without acetylsylgenin, and recovered at 28°C in the dark for 7 days. Group B was treated as follows: the sterile water washing process was omitted, and the callus pieces were transferred to recovery medium with acetylsylgenin, and recovered at 28°C in the dark for approximately 7 days.

[0084] 5. Herbicide resistance screening and propagation of Qi 319 embryogenic callus After recovery culture in step 4, the Qi 319 embryogenic callus was cut into tissue blocks with a diameter of 2-3 mm and transferred to selection medium containing 10 mg / L glufosinate. Selection was carried out at 28°C in the dark for 3 generations, with each generation lasting 7 days. Figure 4 As shown, during the screening process, untransformed callus gradually browned and necrotized, while transformed callus maintained good growth. It is noteworthy that after co-culturing with Agrobacterium, the callus was washed twice with sterile water and transferred to recovery medium without the addition of acetylsyleugenol for further culture. Figure 4 Compared to A), the callus cleaning step was removed during the recovery culture process and acetylsyleugenone was added. Figure 4 B) The significant increase in the ratio and growth status of resistant callus indicates that the above-mentioned improvement measures have significantly improved the infection efficiency of maize embryogenic callus, which is crucial for improving the efficiency of maize genetic transformation.

[0085] During the selection and culture of callus tissue, browned tissue was removed, and pale yellow embryogenic callus tissue was retained. After three consecutive generations of intensive selection, the proportion of Qi 319-resistant callus tissue increased. Good-condition pale yellow embryogenic callus tissue was selected, cut into tissue blocks with a diameter of 2-3 mm, and cultured in the dark at 28℃ for two generations, with a frequency of 7 days per generation. Figure 4 As shown, after two generations of recovery culture (7 days / generation), the browned and necrotic callus tissue has been basically removed, the resistant callus tissue has recovered to a healthy physiological state, and a large number of nodular protrusions appear around the callus tissue block, which can carry out subsequent differentiation.

[0086] 6. Photoinduced differentiation of resistant callus The Qi 319 embryogenic callus tissue, after resistance selection and propagation in step 5, was cut into callus tissue blocks with a diameter of about 2-3 mm, transferred to differentiation medium, and cultured at 28°C under light with a light intensity of 15000 LUX and a photoperiod of 16 hours of daytime and 8 hours of darkness for 8-10 days. Figure 6 As shown, the Qi 319 resistant callus tissue after recovery culture was transferred to differentiation medium and induced to differentiate for 10 days. The callus tissue turned green as a whole, and a large number of dark green buds were produced on the surrounding nodular protrusions, which can be used for subsequent induction of seedlings.

[0087] 7. Rapid seedling growth without dependence on exogenous hormones The callus tissue blocks after light-induced differentiation in step 6 were transferred to regeneration medium in canned jars, with one-third of the callus tissue submerged in the medium. They were cultured at 28°C under light, with a light intensity of 15000 LUX and a photoperiod of 16 h during the day and 8 h in the dark, to promote seedling growth. Figure 7 As shown, after photoinduced differentiation of Qi 319 callus blocks, they were transferred to canning jars containing regeneration culture medium. Ten days later, the callus blocks differentiated and produced a large number of seedlings. By 20 days, most of the seedlings' leaves had reached the sealing film, and they could be transplanted.

[0088] 8. Transplanting and screening of test-tube seedlings Remove the sealing film from the canning jar, add a small amount of sterile water, soak the culture medium, and harden the seedlings for 1-2 days. Then, remove the seedlings from the canning jar, wash off the culture medium from the roots, and transplant them into paper cups filled with seedling soil. Cover the top of the paper cup with disposable transparent gloves, cutting off some of the fingertips to both retain moisture and ensure air permeability. Figure 8 A). After the seedlings have recovered from transplant shock, remove the disposable transparent gloves. Figure 8 B), once the plants have grown to a certain size, apply a 1.25‰ solution of Bar herbicide evenly to both sides of a certain area of ​​the leaves. After screening for about 8-10 days, non-positive seedlings will show obvious yellow spots or even necrosis, while positive seedlings will be basically unaffected. Figure 8 C and D). DNA was extracted from the leaves of herbicide-resistant seedlings and subjected to PCR detection. Figure 8 E), seedlings with positive amplification results are transplanted to greenhouses or fields for self-pollination and harvesting of transgenic progeny materials.

[0089] Example 3: Genetic transformation and seedling regeneration of embryogenic callus from commercial maize backbone inbred line Zheng 58 This embodiment provides a method for genetic transformation and seedling regeneration of embryogenic callus from the commercial maize backbone inbred line Zheng 58. The formulations of the callus induction medium, callus subculture medium, Agrobacterium tumefaciens inoculation medium, co-culture medium, recovery medium, screening medium, differentiation medium, and seedling culture medium used in this embodiment are the same as in Example 1. The specific steps are as follows: 1. Induction of embryogenic callus: Maize inbred line Zheng 58 was planted in the field. Approximately 11 days after self-pollination, ears were harvested, surface sterilized, and young embryos were selected and placed with the scutellum facing upwards on callus induction medium A. Figure 9 Cultured in the dark at 28°C for 1 week (A). The radicle was removed, and the tissue was transferred to callus induction medium B. Subcultured every 7 days, and after approximately 3 weeks, a large amount of pale yellow embryogenic callus was produced. Figure 9 B). Embryogenic callus was cut into tissue blocks with a diameter of 2-3 mm for Agrobacterium-mediated genetic transformation.

[0090] 2. Preparation of bacterial culture: Agrobacterium LB4404 bacterial culture carrying the target gene fragment and glufosinate resistance gene plasmid was cultured overnight at 28℃ and 200 rpm with shaking. The next day, 2-3 mL of the bacterial culture was transferred to a new 25 mL YEP medium and cultured until the bacterial concentration reached OD500. 600 = 0.6~0.8 (approximately 5-6 hours), centrifuge at 5000 rpm for 10 min to collect bacteria, then suspend the bacterial culture in an equal volume of Agrobacterium infection medium containing 100 μM acetylsyl syringone, and incubate at 22℃ in the dark for 1 h before transformation.

[0091] 3. Agrobacterium-mediated callus transformation and recovery culture: Zheng 58 embryogenic callus was cut into pieces with a diameter of 2-3 mm and placed in the above-mentioned static Agrobacterium LB4404 bacterial solution. The mixture was incubated under a negative pressure of 0.05-0.06 MPa for 20 min. The bacterial solution was then discarded, excess solution was aspirated from the callus surface, and the callus was transferred to a co-culture medium and co-cultured at 22°C in the dark for 3 days. After co-culture, the callus was cut into pieces with a diameter of 2-3 mm and transferred to a recovery medium, where it was recovered and cultured at 28°C in the dark for approximately 7 days.

[0092] 4. Screening of resistant callus: After recovery culture, callus tissue was cut into pieces with a diameter of 2-3 mm and transferred to screening medium containing 10 mg / L glufosinate. Screening was carried out at 28℃ in the dark for 3 generations, with each generation lasting 7 days. During the screening process, untransformed callus tissue gradually browned and died, while transformed callus tissue maintained good growth. Figure 9 C) 5. Differentiation and seedling growth of resistant callus: The screened resistant callus was transferred to a subculture medium and cultured in the dark at 28°C for 7 days. Then, callus pieces with a diameter of approximately 2-3 mm were cut and transferred to a differentiation medium. The medium was then cultured at 28°C with a light intensity of 15000 LUX and a photoperiod of 16 h / 8 h light / dark for 10 days. At this point, the callus turned green and produced numerous nodular protrusions and buds. Figure 9D), and then transferred to canning jars containing regeneration medium, resulting in a large number of sterile seedlings after 3 weeks ( Figure 9 E). Transplant the seedlings into nursery soil. After they have recovered from transplant shock, take a small amount of leaves, grind them, and test the transgenic positive seedlings with a Bar test strip. A positive seedling will produce a new band below the standard band. Figure 9 F).

[0093] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for Agrobacterium-mediated genetic transformation of maize callus, characterized in that, Includes the following steps: (1) Obtaining maize embryos; (2) Place the corn embryo on the callus induction medium to induce embryogenic callus, and then propagate it on the callus subculture medium to obtain embryogenic callus. (3) Activate the Agrobacterium containing the infection vector and suspend the activated Agrobacterium in the Agrobacterium infection culture medium to obtain the Agrobacterium infection culture medium; (4) Take the above Agrobacterium infection solution and use Agrobacterium-mediated method to infect embryonic callus to obtain infected callus. (5) The infected callus was placed on a co-culture medium and co-cultured to obtain co-cultured callus; (6) Transfer the co-cultured callus to a recovery culture medium for antibacterial and recovery culture; (7) The callus tissue after inhibition and recovery culture was transferred to the screening medium for screening to obtain resistant callus tissue; (8) Transfer the resistant callus to a differentiation medium to induce differentiation and obtain differentiated callus; (9) Transfer the differentiated callus tissue blocks to the seedling culture medium to differentiate seedlings; (10) Selective agents were used to screen and molecular identification of seedlings to obtain positive seedlings.

2. The method as described in claim 1, characterized in that, The callus induction culture medium includes callus induction culture medium A and callus induction culture medium B; The formula for the callus induction culture medium A is as follows: N6 salt 3.8-4.2 g / L, sodium citrate 10-20 mg / L, glutamic acid 90-110 mg / L, L-proline 0.4-0.8 g / L, hydrolyzed casein 90-110 mg / L, MES 0.4-0.6 g / L, sucrose 30-50 g / L, AgNO3 3-6 mg / L, cephalosporin 280-320 mg / L, 2,4-D 2-4 mg / L, NAA 0.4-0.6 mg / L, agar 5.5-6.5 g / L, pH 6.0; The formula for the callus induction culture medium B is as follows: N6 salt 3.8-4.2 g / L, sodium citrate 10-20 mg / L, glutamic acid 90-110 mg / L, L-proline 0.4-0.8 g / L, hydrolyzed casein 90-110 mg / L, MES 0.4-0.6 g / L, sucrose 30-50 g / L, AgNO3 3-6 mg / L, cephalosporin 280-320 mg / L, 2,4-D 1-3 mg / L, NAA 0.4-0.6 mg / L, agar 5.5-6.5 g / L, pH 6.0; The formula for the callus subculture medium is as follows: N6 salt 3.8-4.2 g / L, sodium citrate 10-20 mg / L, glutamic acid 90-110 mg / L, L-proline 0.4-0.8 g / L, hydrolyzed casein 90-110 mg / L, MES 0.4-0.6 g / L, sucrose 30-50 g / L, AgNO3 3-6 mg / L, cephalosporin 280-320 mg / L, 2,4-D 1-2 mg / L, NAA 0.4-0.6 mg / L, agar 5.5-6.5 g / L, pH 6.

0. The formulation of the co-culture medium is as follows: N6 salt 3.8-4.2 g / L, sodium citrate 10-20 mg / L, glutamic acid 90-110 mg / L, L-proline 0.4-0.8 g / L, hydrolyzed casein 90-110 mg / L, MES 0.4-0.6 g / L, sucrose 40-60 g / L, AgNO3 3-6 mg / L, 2,4-D 1-2 mg / L, NAA 0.4-0.6 mg / L, AS 15-25 mg / L, agar 5.5-6.5 g / L, pH 6.0; The formulation of the recovery culture medium is as follows: N6 salt 3.8-4.2 g / L, sodium citrate 10-20 mg / L, glutamic acid 90-110 mg / L, L-proline 0.4-0.8 g / L, hydrolyzed casein 90-110 mg / L, MES 0.4-0.6 g / L, sucrose 30-50 g / L, AgNO3 3-6 mg / L, 2,4-D 1-2 mg / L, NAA 0.4-0.6 mg / L, AS 15-25 mg / L, cephalosporin 400-500 mg / L, agar 5.5-6.5 g / L, pH 6.0; The screening medium is formulated as follows: N6 salt 3.8-4.2 g / L, sodium citrate 10-20 mg / L, glutamic acid 90-110 mg / L, L-proline 0.4-0.8 g / L, hydrolyzed casein 90-110 mg / L, MES 0.4-0.6 g / L, sucrose 30-50 g / L, AgNO3 3-6 mg / L, 2,4-D 1-2 mg / L, NAA 0.4-0.6 mg / L, cephalosporin 280-320 mg / L, screening agent 10 mg / L, agar 6 g / L, pH 6.0; the screening agent is glufosinate or hygromycin. The differentiation medium formulation is as follows: MS salt 2-2.5 g / L, MES 0.4-0.6 g / L, sucrose 30-50 g / L, AgNO3 3-6 mg / L, 6-BA 1-2 mg / L, cephalosporin 280-320 mg / L, agar 5.5-6.5 g / L, pH 6.0; The formulation of the seedling culture medium is as follows: MS salt 2-2.5 g / L, MES 0.4-0.6 g / L, sucrose 30-50 g / L, AgNO3 3-6 mg / L, cephalosporin 280-320 mg / L, agar 5.5-6.5 g / L, pH 6.

0.

3. The method as described in claim 1, characterized in that, The corn embryo is taken from the embryo of a corn inbred line or a corn hybrid; preferably, the corn embryo is taken from the embryo of a corn inbred line or a corn hybrid 10-12 days after pollination.

4. The method as described in claim 1, characterized in that, The long axis length of the corn embryo is 1~1.4 mm.

5. The method as described in claim 1, characterized in that, The infection vector is selected from any one of plant overexpression vectors, RNAi vectors, or gene editing vectors.

6. The method as described in claim 1, characterized in that, The Agrobacterium-mediated method is selected from any one of the following: Agrobacterium immersion method, dip method, ultrasonic treatment Agrobacterium immersion method, or Agrobacterium vacuum negative pressure immersion method.

7. The method as described in claim 1, characterized in that, The molecular identification methods include PCR amplification, Southern hybridization, Northern hybridization, and Western blot hybridization.

8. The method as described in claim 1, characterized in that, The selector is glyphosate, chlorsulfuron-methyl, or hygromycin.

9. A kit for genetic transformation of maize inbred lines, characterized in that, It includes the callus induction culture medium of claim 1, the callus subculture culture medium of claim 1, the co-culture culture medium of claim 1, the recovery culture medium of claim 1, the screening culture medium of claim 1, the differentiation culture medium of claim 1, and the seedling culture medium of claim 1.

10. The use of the kit according to claim 9 in any of the following: (1) Genetic transformation of maize inbred lines; (2) Preparation of products from genetic transformation of maize inbred lines; (3) Tissue culture of maize inbred lines; (4) Preparation of products from tissue culture of maize inbred lines; The maize inbred lines include Qi 319 and Zheng 58.

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