A method for obtaining millet embryonic callus and transgenic plants

Abstract

The invention discloses a method for obtaining millet embryonic callus, comprising the following steps: (a1) dehulling and disinfecting mature millet seeds and inoculating them on an induction culture medium for induction culture to obtain primary callus; (a2) selecting primary callus in good condition in (a1) for subculture culture, wherein the culture medium used for subculture culture is the subculture culture medium, and obtaining embryonic callus; (a3) ​​selecting pale yellow and dense embryonic callus in (a2) for differentiation and regeneration culture until green buds are differentiated, so as to evaluate the regeneration ability of different millet seeds. By optimizing the culture medium components, red embryonic callus suitable for genetic transformation is induced, a red genetic transformation system based on Agrobacterium-mediated gene transfer is established, and transgenic plants using hygromycin as a screening marker are obtained. The invention is of great significance for exploring millet stress resistance functional genes and establishing a millet biological breeding system.

Description

Technical Field

[0001] The invention relates to the field of plant tissue culture and plant genetic engineering, and in particular to a method for obtaining embryonic callus tissue of millet for genetic transformation and genetic transformation mediated by Agrobacterium. Background Art

[0002] Broomcorn Millet / Proso Millet, Panicum miliaceum L., belongs to the genus Panicum L. of the Poaceae family, and is an annual herb. Broomcorn Millet originated in northern my country and is one of the oldest crops to date, with a cultivation history of more than 10,000 years. It spread to Europe and all over the world about 3,000 years ago and is cultivated in Asia, Africa, Europe, and the Americas (Chai Yan et al., 2007; Liu Minxuan et al., 2020; Lu et al., 2009). Broomcorn Millet was once the staple food of ancient Chinese ancestors, and it is still one of the main food crops in northern arid and semi-arid areas such as Shanxi, Shaanxi, Gansu, Ningxia, Inner Mongolia, and Hebei (Cheng Bingwen et al., 2019). Millet is drought-resistant, salt-alkali-tolerant, high-temperature-tolerant, barren-tolerant, tolerant to high-concentration carbon dioxide, and has a short growth period but a long range of variation. It is a pioneer crop and staple food crop for stress resistance in arid and semi-arid areas. It is also an internationally recognized strategic reserve crop and environmentally friendly crop for coping with climate warming and drought. It is also a "disaster relief crop" for coping with natural disasters (Feng Baili, 2016; Lan Qian et al., 2023; Shi et al., 2019; Zou et al., 2019; Hunt et al., 2014; Shan et al., 2020; Luo et al., 2022; Pardo et al., 2021). Compared with other gramineous crops such as corn, wheat, sorghum and millet, millet requires the least water, and only 220–300 mm of annual rainfall is needed to produce grains (Pardo et al., 2021; Wu Jing et al., 2023). After years of research on wheat, barley, corn, sorghum, millet and broomcorn, Shantz et al. found that broomcorn has the highest water use efficiency, consuming 1.702g of dry matter per liter of water, followed by broomcorn and sorghum, which are 1.021 and 0.7829g respectively, and barley, corn and wheat are 0.7829, 0.6808 and 0.5106g respectively; when seeds germinate, broomcorn only needs to absorb 25% of the seed weight of water to germinate, which is less than the amount of water absorbed by crops such as wheat (50%), corn (70%) and sorghum (75%). In addition, the mature broomcorn ear accounts for 55% of the total dry matter weight of the plant, indicating that broomcorn has a very high water utilization rate (Wang Yutang, 2011; Diao Xianmin, 2019; Shantz et al., 1927; Ventura et al., 2020). Millet has both nutritional and medicinal value. Its protein content is 8.6% to 15.5%, which is significantly higher than rice, millet, sorghum, corn, barley and highland barley. The grains are also rich in thiamine (vitamin B1), riboflavin (vitamin B2), xanthin and vitamin E, which give millet extremely diverse health functions.The yellow rice after shelling has important medicinal value such as tonifying the middle and replenishing qi, strengthening the spleen and lungs, removing heat and healing sores, and is mainly used to treat symptoms such as spleen and stomach weakness. Millet protein does not contain gluten and has great potential for food application. Therefore, millet is also listed as one of the "Future Smart Foods" (Siddique et al., 2021). According to statistics, the global millet planting area in 2018 was about 6 million hm2, of which Russia had the largest cultivated area, followed by Ukraine and China. The millet planting area in my country was about 530,000 hm2 (Cheng Bingwen et al., 2019; Wang et al., 2016).

[0003] At present, my country's national germplasm bank has collected and preserved about 9,800 millet genetic resources, ranking first in the world. At the same time, my country's research on millet is also at the forefront of the world (Wang Lun et al., 2015; Wang Ruiyun, 2017). In 2019, Zhang Heng and Zhu Jiankang's research group at the Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, combined PacBio, Illumina, HiC and high-density genetic linkage map construction technology to obtain a set of millet Pm0390 (National Crop Germplasm Bank registration number 00000390) genome fine map, with a total length of 855Mb, 18 chromosomes (Contig N50 = 0.36Mb, Scaffold N50 = 45.56Mb), annotated 55,930 protein-coding genes, of which 99.3% of the genes can be located on chromosomes, and the relevant research was published in the journal Nature Communication (Zou et al., 2019). At the same time, the journal also published the genome assembly work of the commercial millet variety Longmi 4 by Lai Jinsheng's research group at China Agricultural University. The study obtained a high-quality genome sequence (Contig N50 = 2.55Mb, Scaffold N50 = 8.24Mb), and annotated 63,671 protein-coding genes, 14% more than millet Pm0390 (Shi et al., 2019). Shi Junpeng's team at Sun Yat-sen University used 51× PacBio HiFi sequencing data and integrated the previous version of the Longmi 4 genome (Longmi_v1) to obtain a higher-quality Longmi 4 genome Longmi_v2. The upgraded millet genome Longmi_v2 is about 846.0Mb in size, with a ContigN50 of about 26.2Mb, filling the 525 gaps left by the Longmi_v1 genome (Wang et al., 2023).

[0004] Since the release of the millet genome, the restriction site-associated DNA sequencing technology (RAD-seq) or specific focused amplified fragment sequencing technology (SLAF-seq) has been used to perform genotyping analysis on SNP markers for association analysis of millet agronomic and seed traits, which has greatly promoted the genetic research of millet (Boukail et al., 2021; Khound et al., 2022). At the same time, researchers have used techniques such as map-based cloning and multi-omics joint analysis to analyze some drought resistance, salinity resistance, and low nitrogen resistance genes in millet, laying a good foundation for analyzing the stress resistance mechanism of millet (Wang Ruiyun et al., 2018; Zhang et al., 2019; An et al., 2022; Shan et al., 2020; Sun et al., 2023; Wang et al., 2023). Although important progress has been made in the genomics research of millet, the genetic transformation system of millet has not yet been established, which greatly limits the functional genomics research and biological breeding technology research and development of millet. The present invention aims to establish a genetic transformation system of millet based on mature seeds, so as to provide a base technology for the establishment of millet biological breeding technology. Summary of the invention

[0005] The purpose of the present invention is to obtain millet embryonic callus using mature embryos (seeds), and to use this embryonic callus to carry out efficient and stable Agrobacterium-mediated genetic transformation, thereby solving the key bottleneck problem that millet is difficult to carry out genetic transformation, establishing a large-scale millet genetic transformation system, and making millet truly develop into a model plant for functional gene research.

[0006] In order to achieve the above object, the present invention provides a method for obtaining millet embryonic callus, comprising the following steps:

[0007] (a1) dehulling and sterilizing mature millet seeds and inoculating them on an induction medium for induction culture to obtain primary callus;

[0008] (a2) selecting the primary callus tissue in better condition in (a1) for subculture, using the subculture medium as the subculture medium to obtain embryonic callus tissue;

[0009] (a3) selecting the pale yellow and dense embryonic callus in (a2) for differentiation and regeneration culture until green shoots are differentiated, so as to evaluate the regeneration ability of different millet seeds;

[0010] In step (a1), the solvent of the induction medium is water, and the solutes are MS salts, N6 vitamins, proline, inositol, 6-BA, aspartic acid, sucrose, 2,4-D and plant gel; in the induction medium, the final concentration of the MS salts is 2.5-7 g / L, the final concentration of the N6 vitamins is 0.5-3 ml / L, the final concentration of proline is 0.5-3 g / L, the final concentration of inositol is 0.01-2 g / L, the final concentration of 6-BA is 0.01-2 g / L, the final concentration of aspartic acid is 0.05-3 g / L, the final concentration of sucrose is 5-100 g / L, the final concentration of 2,4-D is 1-5 mg / L, and the final concentration of the plant gel is 1-12 g / L;

[0011] More preferably, the induction medium composition and final concentration are: 4.43 g / L MS salts (Cat. No.: M524), 1 ml / L N6 (Cat. No.: C149), 1.0 g / L proline, 0.1 g / L inositol, 0.1 mg / L 6-BA, 0.2 g / L aspartic acid, 30 g / L sucrose, 2 mg / L 2,4-D, 8.0 g / L phytagel, pH 5.7.

[0012] In step (a3), the solvent of the differentiation and regeneration medium used when the embryonic callus is subjected to differentiation and regeneration culture is water, and the solutes are MS salt, nicotinic acid, vitamin B6, vitamin B1, inositol, copper sulfate, sucrose, agar, 6-BA and carbenicillin; in the differentiation and regeneration medium, the final concentration of the MS salt is 2.5-7g / L, the final concentration of nicotinic acid is 0.5-3mg / L, the final concentration of vitamin B6 is 0.5-3mg / L, the final concentration of vitamin B1 is 1-10mg / L, the final concentration of inositol is 50-200mg / L, the final concentration of copper sulfate is 0.5-4mg / L, the final concentration of sucrose is 10-100g / L, the final concentration of agar is 1-12g / L, the final concentration of 6-BA is 1-3mg / L, and the final concentration of carbenicillin is 50-400mg / L.

[0013] More preferably, the composition and final concentration of the differentiation and regeneration medium are: 4.3 g / L MS salt (article number: M519), 1.0 mg / L nicotinic acid, 1.0 mg / L vitamin B6, 5.0 mg / L vitamin B1, 100 mg / L inositol, 1.25 mg / L copper sulfate, 30 g / L sucrose, 8.0 g / L agar, 2 mg / L 6-BA, 200 mg / L carbenicillin, pH 5.6

[0014] Preferably, the induction culture in step (a1) is carried out in dark culture at 28° C. for 20-30 days, and the subculture time in step (a2) is 10-15 days.

[0015] Preferably, in step (a3), the differentiation and regeneration culture is carried out according to a method comprising the following steps: inoculating the embryonic callus into the differentiation and regeneration medium and placing it in a 28°C tissue culture operation room with 16 hours of light / 8 hours of darkness for 4-8 weeks.

[0016] Furthermore, the present invention provides a method for genetic transformation of millet, comprising the following steps:

[0017] (b1) infecting the embryonic callus obtained by the above method with an Agrobacterium infection solution containing a target gene expression vector;

[0018] (b2) inoculating the infected embryonic callus onto a co-cultivation medium for co-cultivation, wherein the co-cultivation is divided into two stages, and obtaining co-cultivated callus after culturing;

[0019] (b3) inoculating the co-cultivated callus onto a screening medium for cultivation to obtain resistant callus;

[0020] (b4) inoculating the resistant callus onto a differentiation and regeneration medium for culturing to obtain regenerated shoots.

[0021] Preferably, in step (b1), the culture medium used for infection is an infection medium, the solvent of the culture medium is water, and the solutes are MS salts, B5 vitamins, sucrose, glucose, asparagine, casamino acids, cysteine, 2,4-dichlorophenoxyacetic acid and acetosyringone; in the infection medium, 0.2-2g / L MS salts, 1g / L B5 vitamins, 30-100g / L sucrose, 10-80g / L glucose, 0.5-3g / L asparagine, 0.5-3g / L casamino acids, 0.1-2g / L cysteine, 0.5-4mg / L2,4-dichlorophenoxyacetic acid, 150-300μM acetosyringone.

[0022] Preferably, in step (b2), in step (b2), the solvent of the co-cultivation medium in the first stage is water, and the solutes are MS salts, B5 vitamins, sucrose, glucose, asparagine, casamino acids, cysteine, 2,4-D, acetosyringone and agarose; in the co-cultivation medium, the final concentrations of the components are: 0.2-2 g / L MS salts, 1 g / L B5 vitamins, 30-100 g / L sucrose, 10-80 g / L glucose, 0.5-3 g / L asparagine, 0.5-3 g / L casamino acids, 0.1-2 g / L cysteine, 0.5-4 mg / L 2,4-D, 150-300 μM acetosyringone, 3-12 g / L agarose;

[0023] More preferably, the composition and final concentration of the components are: 0.44 g / L MS salt, 1 g / L vitamins, 68 g / L sucrose, 36 g / L glucose, 1 g / L asparagine, 1 g / L casamino acids, 0.2 g / L cysteine, 2 mg / L 2,4-D, 200 μM acetosyringone, 8 g / L agarose, and the pH value is 5.2.

[0024] The solvent of the second stage co-culture medium is water, and the solutes are MS salts, B5 vitamins, sucrose, glucose, asparagine, hydrolyzed casein, cysteine, 2,4-D, acetosyringone, agarose and carbenicillin; in the co-culture medium, the final concentrations of the components are: 0.2-2g / L MS salts, 1g / L B5 vitamins, 30-100g / L sucrose, 10-80g / L glucose, 0.5-3g / L asparagine, 0.5-3g / L hydrolyzed casein, 0.1-2g / L cysteine, 0.5-4mg / L 2,4-D, 150-300μM acetosyringone, 3-12g / L agarose, 150-350mg / L carbenicillin.

[0025] More preferably, the composition and final concentration of the components are: 0.44 g / L MS salt, 1 g / L B5 vitamin, 68 g / L sucrose, 36 g / L glucose, 1 g / L asparagine, 1 g / L hydrolyzed casein, 0.2 g / L cysteine, 2 mg / L 2,4-D, 200 μM acetosyringone, 8 g / L agarose, 250 mg / L carbenicillin, and the pH value is 5.2.

[0026] Preferably, in step (b3), the solvent of the screening medium is water, and the solutes are N6 vitamins, 2,4-D, sucrose, hydrolyzed casein, proline, inositol, 6-BA, phytagel, hygromycin and carbenicillin; in the screening medium, the final concentrations of the components are: 2-6 g / L N6, 1-3 mg / L 2,4-D, 20-80 g / L sucrose, 0.1-1 g / L hydrolyzed casein, 1-5 g / L proline, 0.05-0.5 g / L inositol, 0.05-0.5 g / L 6-BA, 3-12 g / L phytagel, 20-80 mg / L hygromycin, and 150-350 mg / L carbenicillin.

[0027] More preferably, in the screening culture medium, the 4.0 g / L N6, product number: C149, 2 mg / L 2,4-D, 30 g / L sucrose, 0.3 g / L hydrolyzed casein, 2.8 g / L proline, 0.1 g / L inositol, 0.1 g / L 6-BA, 8.0 g / L plant gel, 50 mg / L hygromycin, and 250 mg / L carbenicillin.

[0028] Preferably, in step (b4), the solvent of the differentiation and regeneration medium is water, and the solute contains MS salt, hydrolyzed casein, niacin, vitamin B6, thiamine hydrochloride (vitamin B1), inositol, copper sulfate, sucrose 8.0g / L, agar, 6-BA, naphthylacetic acid, and carbenicillin; in the differentiation and regeneration medium, the final concentrations of the components are: 2-6g / L MS salt, 0.5-2g / L hydrolyzed casein, 0.5-2mg / L niacin, 0.5-2mg / L vitamin B6, 2-8mg / L thiamine hydrochloride, 80-180mg / L inositol, 0.5-2mg / L copper sulfate, 20-70g / L sucrose, 5-12g / L agar, 1-4mg / L 6-BA, 0.1-0.5mg / L naphthylacetic acid, and 100-300mg / L carbenicillin.

[0029] More preferably, in the differentiation and regeneration medium, the vitamin-containing medium includes 4.3 g / L MS salt, 1.0 g / L hydrolyzed casein, 1.0 mg / L niacin, 1.0 mg / L vitamin B6, 5.0 mg / L thiamine hydrochloride (vitamin B1), 100 mg / L inositol, 1.25 mg / L copper sulfate, 30 g / L sucrose, 8.0 g / L agar, 2 mg / L 6-BA, 0.2 mg / L naphthylacetic acid, and 200 mg / L carbenicillin.

[0030] Preferably, in step (b1), the OD600 of the Agrobacterium infection solution used to infect callus tissue is 0.5-2.

[0031] Preferably, in step (b4), the co-cultivated callus is inoculated onto the screening medium, cultured in the dark at 28° C. for 10-14 days, and subcultured with the same screening medium every two weeks, for a total of 2-3 subcultures.

[0032] Furthermore, the present invention provides a protective culture medium or a set of culture medium.

[0033] The culture medium claimed in the present invention is any one of the culture medium in the above method.

[0034] The complete set of culture media claimed in the present invention is any two or more or all of the culture media used in the above method in combination.

[0035] Advantages and beneficial effects of the present invention: The present invention discloses that the present invention screened out "Hongmi (Getaihongmi, Hongmi)" as a germplasm with good embryonic induction rate and plant regeneration ability by evaluating the embryonic callus induction and plant regeneration ability of mature seeds of 13 millet genotypes. By optimizing the culture medium components, the embryonic callus of Hongmi suitable for genetic transformation was induced, the genetic transformation system of Hongmi mediated by Agrobacterium was established, and the transgenic plants with hygromycin as the screening marker were obtained. The present invention is of great significance for exploring the stress resistance functional genes of millet and establishing the biological breeding system of millet. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Millet genotypes that are easy to genetically transform were screened, and the callus induction rate, bud regeneration rate and albino rate of 13 millet germplasms were statistically analyzed; the quality of embryonic callus was represented by "+", "+++++" represented the best, and "+" represented the worst; the embryonic callus induction rate (the number of embryonic calli in the induction medium / the number of germinated seeds); 90 calli were cultured on the regeneration medium for 20 days to calculate the green seedling rate and white bud rate, respectively.

[0037] Figure 2 Establishment of the genetic transformation system of red ulmoides, a: 20-day-cultured red ulmoides callus; b: embryonic callus after subculture; c: co-culture with Agrobacterium infection (3 days); d: efficient transient expression of GFP in infected callus; e: callus on the first selection medium (10 days); f: the same callus as e showed strong GFP signal under ultraviolet light; gh: regenerated shoots under white light (g) and ultraviolet light (h); ij: wild type (left) and transgenic T0 plants (right) under white (i) and ultraviolet light (h); GFP fluorescence in leaf cross-section under UV light (j); kl: GFP in panicles under white (k) and UV light (l), wild type (left), transgenic (right); mn: florets of transgenic plants express GFP (right) compared with wild type florets under white light (m) and UV light (n); op: T1 seeds germinated under white (o) and UV light; Scale bars: 2 cm (a), 1 mm (bf and op) and 2 mm (g, h, k, l, m and n).

[0038] Figure 3 Identification of transgenic positive plants of Hong Mi, a: PCR amplification to detect GFP gene; b: Southern blotting experiment to detect the copy number of T0 plants. DETAILED DESCRIPTION

[0039] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials and reagents used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0040] The MS salt (item number: M524) used in the following examples is a product of Beijing Ximeijie Technology Co., Ltd.; N6 vitamin (item number: C149) is a product of Beijing Ximeijie Technology Co., Ltd.; and N6 salt (item number: C167) is a product of Beijing Ximeijie Technology Co., Ltd.

[0041] Millet germplasm was obtained from the National Crop Germplasm Bank of the Chinese Academy of Agricultural Sciences;

[0042] Vector pCAMBIA1305-GFP: pCAMBIA1305-GFP vector document: DOI: https: / / doi.org / 10.1105 / tpc.113.121376.

[0043] Example 1

[0044] (1) 13 portions of millet germplasm were shelled, wiped and disinfected with 75% alcohol, then disinfected with 10-12% (volume fraction) sodium hypochlorite for 10-12 minutes, and finally washed with sterile distilled water for 5 times, with the last wash lasting 40-50 minutes. All the above operations were completed in a sterile clean bench;

[0045] (2) Afterwards, wipe the surface moisture of the above disinfected seeds with sterile absorbent paper and place them in an induction medium. The induction medium composition is: 4.43 g / L MS salt (Product No.: M524), 1 ml / L N6 (Product No.: C149), 1.0 g / L proline, 0.1 g / L inositol, 0.1 mg / L 6-BA, 0.2 g / L aspartic acid, 30 g / L sucrose, 2 mg / L 2,4-D, 8.0 g / L phytagel, pH 5.7 (the concentration of each substance is the final concentration in the induction medium);

[0046] (3) After that, callus was induced at 28°C for 20-30 days, and callus with better condition was selected and continued to be placed on callus induction medium for 10-15 days. It was found that all 13 materials could induce primary callus tissue, but the induction frequency depended on the genotype. Among them, the callus induction rate of 3 germplasms: Hongmi, Qingyang Hongying and Jinshu 7 was higher than 40% ( Figure 1 ); about 40-90% of the induced callus continued to expand and grow into friable callus ( Figure 1 The proportion of embryonic callus was counted, and the quality of embryonic callus was graded from 1 to 5 according to texture, color and looseness ( Figure 1), and found that the embryonic callus quality of Hong Mi was the best, followed by Qingyang Hongying, and the embryonic callus induction rates were 56.14% (Hong Mi) and 50.39% (Qingyang Hongying) ( Figure 1 );

[0047] (4) Selection steps (1) Transfer the pale yellow and dense embryonic callus to the differentiation and regeneration medium, seal it and place it in a 28°C tissue culture operation room with 16 hours of light / 8 hours of darkness for 4-8 weeks until green shoots differentiate;

[0048] Differentiation and regeneration medium composition: 4.3g / LMS salt (item number: M519), 1.0mg / L niacin, 1.0mg / L vitamin B6, 5.0mg / L vitamin B1, 100mg / L inositol, 1.25mg / L copper sulfate, 30g / L sucrose, 8.0g / L agar 2mg / L 6-BA, 200mg / L carbenicillin, pH 5.6 (the concentrations of the above substances are the final concentrations in the differentiation and regeneration medium).

[0049] Example 2

[0050] (1) Agrobacterium-mediated genetic transformation experiment was performed using red embryonic callus induced for 25 days and subcultured for 12 days. Figure 2 a-2b), the pCAMBIA1305-GFP vector carrying the GFP reporter gene was used to transform Agrobacterium EHA105 and infect it, and the HPT gene was used as a marker gene to detect the optimal selection concentration of hygromycin. The 1305Ubi-GFP HPT vector was transferred into the Agrobacterium strain EHA105, and cultured overnight at a steady speed of 200 r / min in YEB medium (5 g / L beef extract, 5 g / L peptone, 1 g / L yeast extract, 5 g / L sucrose, 10 mM magnesium sulfate, pH = 7.0) until the optical density reached OD 600 = 1.0;

[0051] For Agrobacterium transformation, subcultured embryogenic calli were infected with Agrobacterium cells (OD600 = 0.5) in infection medium (components: 0.44 g / L MS salts, 1 g / L B5 vitamins, 68 g / L sucrose, 36 g / L glucose, 1 g / L asparagine, 1 g / L casamino acids, 0.2 g / L cysteine, 2 mg / L 2,4-dichlorophenoxyacetic acid, 200 μM acetosyringone, pH 5.2) for 30 min;

[0052] (2) Then transferred to co-culture medium (components: 0.44 g / L MS salts, 1 g / L B5 vitamins, 68 g / L sucrose, 36 g / L glucose, 1 g / L asparagine, 1 g / L casamino acids, 0.2 g / L cysteine, 2 mg / L 2,4-D, 200 μM acetosyringone, 8 g / L agarose, pH 5.2) and cultured at 22 °C in the dark for 72 h;

[0053] After 3 days of co-culture, GFP expression was observed. Figure 2 c-2d); the infected callus tissue was transferred to a co-cultivation medium supplemented with carbenicillin (components: 0.44 g / L MS salt, 1 g / L B5 vitamin, 68 g / L sucrose, 36 g / L glucose, 1 g / L asparagine, 1 g / L hydrolyzed casein, 0.2 g / L cysteine, 2 mg / L 2,4-D, 200 μM acetosyringone, 8 g / L agarose, 250 mg / L carbenicillin, pH 5.2) and cultured in a dark incubator at 28°C for 7 days;

[0054] (3) The co-cultivated callus tissue was transferred to a screening medium containing hygromycin (4.0 g / L N6, product number: C149, 2 mg / L 2,4-D, 30 g / L sucrose, 0.3 g / L hydrolyzed casein, 2.8 g / L proline, 0.1 g / L inositol, 0.1 g / L 6-BA, 8.0 g / L phytagel, 50 mg / L hygromycin, 250 mg / L carbenicillin). The callus tissue was cultured in the dark at 28°C for 10-14 days, and then subcultured twice using the same screening medium. Figure 2 It can be seen that green fluorescent protein is stably expressed in callus tissue, proving that the screening efficiency is high;

[0055] (4) After two rounds of screening with hygromycin (50 mg / L), it was confirmed that 50 mg / L hygromycin was sufficient to select cells containing GFP ( Figure 2 e-2f);

[0056] (5) Differentiate the GFP-positive callus on the regeneration medium ( Figure 2 g-2h), the resistant calli after screening and culture were transferred to differentiation and regeneration medium (4.3g / L MS salt, 1.0g / L hydrolyzed casein, 1.0mg / L nicotinic acid, 1.0mg / L vitamin B6, 5.0mg / L thiamine hydrochloride (vitamin B1), 100mg / L inositol, 1.25mg / L copper sulfate, 30g / L sucrose 8.0g / L, agar, 2mg / L6-BA, 0.2mg / L naphthaleneacetic acid, 200mg / L carbenicillin, pH 5.6), and cultured at 28°C with 16h light / 8h dark for 4-8 weeks to regenerate seedlings;

[0057] (6) The seedlings were transferred to a rooting medium (2.2 g / L MS salt, product number: M524, 30 g / L sucrose, 0.1 g / L inositol, 2.6 g / L phytagel, 30 mg / L hygromycin and 250 mg / L carbenicillin, 8 g / L phytagel, pH 5.6, the concentrations of the above substances are the final concentrations in the rooting medium) for rooting, cultured at 28°C, 16 h light, 8 h dark, for 3-4 weeks, and hardened in the culture room when the seedlings grew to 10-20 cm;

[0058] (7) The transgenic plants after hardening were transplanted into flower pots and cultured in a greenhouse. At the later stage of growth, leaves were taken for transgenic positive detection. Figure 2 i-2j)、Young spike( Figure 2 k-2l) and small flowers ( Figure 2 m-2n), and the seedlings germinated from T1 seeds could also stably express GFP fluorescence, indicating that the T-DNA vector can be stably inherited ( Figure 2 o-2p);

[0059] (8) PCR amplification of a 606 bp partial GFP fragment revealed that 22 of the 23 T0 plants were GFP-positive plants ( Figure 3 a). To further verify the accuracy of PCR and investigate the copy number of T0 plants, 10 T0 plants were randomly selected and subjected to Southern blotting experiments using two restriction enzymes (SacI and BamHI) and HPT as a probe. The results showed that four different events (PM1, PM11, PM15, and PM19) and three events (PM4, PM7, and PM8) were single copies, while PM2 and PM17 carried two and three copies, respectively ( Figure 3 b).

Claims

1. A method for obtaining millet embryonic callus, comprising the following steps: (a1) dehulling and sterilizing mature millet seeds and inoculating them on an induction medium for induction culture to obtain primary callus; (a2) selecting the primary callus tissue in better condition in (a1) for subculture, using the subculture medium as the subculture medium to obtain embryonic callus tissue; (a3) selecting the pale yellow and dense embryonic callus in (a2) for differentiation and regeneration culture until green shoots are differentiated, so as to evaluate the regeneration ability of different millet seeds; In step (a1), the solvent of the induction medium is water, and the solutes are MS salts, N6 vitamins, proline, inositol, 6-BA, aspartic acid, sucrose, 2,4-D and plant gel; in the induction medium, the final concentration of the MS salts is 2.5-7 g / L, the final concentration of the N6 vitamins is 0.5-3 ml / L, the final concentration of proline is 0.5-3 g / L, the final concentration of inositol is 0.01-2 g / L, the final concentration of 6-BA is 0.01-2 g / L, the final concentration of aspartic acid is 0.05-3 g / L, the final concentration of sucrose is 5-100 g / L, the final concentration of 2,4-D is 1-5 mg / L, and the final concentration of the plant gel is 1-12 g / L; In step (a3), the solvent of the differentiation and regeneration medium used when the embryonic callus is subjected to differentiation and regeneration culture is water, and the solutes are MS salt, nicotinic acid, vitamin B6, vitamin B1, inositol, copper sulfate, sucrose, agar, 6-BA and carbenicillin; in the differentiation and regeneration medium, the final concentration of the MS salt is 2.5-7g / L, the final concentration of nicotinic acid is 0.5-3mg / L, the final concentration of vitamin B6 is 0.5-3mg / L, the final concentration of vitamin B1 is 1-10mg / L, the final concentration of inositol is 50-200mg / L, the final concentration of copper sulfate is 0.5-4mg / L, the final concentration of sucrose is 10-100g / L, the final concentration of agar is 1-12g / L, the final concentration of 6-BA is 1-3mg / L, and the final concentration of carbenicillin is 50-400mg / L.

2. The method according to claim 1, characterized in that: The induction culture in step (a1) is carried out under dark conditions at 28° C. for 20-30 days, and the subculture time in step (a2) is 10-15 days.

3. The method according to claim 1, characterized in that: In step (a3), the differentiation and regeneration culture is carried out according to a method comprising the following steps: inoculating the embryonic callus into the differentiation and regeneration medium and placing it in a 28° C. tissue culture operation room with 16 hours of light / 8 hours of darkness for 4-8 weeks.

4. A method for genetic transformation of millet, comprising the following steps: (b1) infecting the embryonic callus obtained by any one of the methods described in claims 1 to 3 with an Agrobacterium infection solution containing a target gene expression vector; the culture medium used for the infection is an infection medium, the solvent of the culture medium is water, and the solutes are MS salts, B5 vitamins, sucrose, glucose, asparagine, casamino acids, cysteine, 2,4-dichlorophenoxyacetic acid and acetosyringone; in the infection medium, the final concentrations of the components are: 0.2-2 g / L MS salts, 1 g / L B5 vitamins, 30-100 g / L sucrose, 10-80 g / L glucose, 0.5-3 g / L asparagine, 0.5-3 g / L casamino acids, 0.1-2 g / L cysteine, 0.5-4 mg / L 2,4-dichlorophenoxyacetic acid, and 150-300 μM acetosyringone; (b2) inoculating the infected embryonic callus onto a co-cultivation medium for co-cultivation, wherein the co-cultivation is divided into two stages, and obtaining co-cultivated callus after culturing; The solvent of the co-culture medium in the first stage is water, and the solutes are MS salts, B5 vitamins, sucrose, glucose, asparagine, casamino acids, cysteine, 2,4-D, acetosyringone and agarose; in the co-culture medium, the final concentrations of the components are: 0.2-2 g / L MS salts, 1 g / L B5 vitamins, 30-100 g / L sucrose, 10-80 g / L glucose, 0.5-3 g / L asparagine, 0.5-3 g / L casamino acids, 0.1-2 g / L cysteine, 0.5-4 mg / L 2,4-D, 150-300 μM acetosyringone, 3-12 g / L agarose; The solvent of the second stage co-culture medium is water, and the solutes are MS salt, B5 vitamin, sucrose, glucose, asparagine, hydrolyzed casein, cysteine, 2,4-D, acetosyringone, agarose and carbenicillin; in the co-culture medium, the final concentrations of the components are: 0.2-2g / L MS salt, 1g / L B5 vitamin, 30-100g / L sucrose, 10-80g / L glucose, 0.5-3g / L asparagine, 0.5-3g / L hydrolyzed casein, 0.1-2g / L cysteine, 0.5-4mg / L 2,4-D, 150-300μM acetosyringone, 3-12g / L agarose, 150-350mg / L carbenicillin; (b3) inoculating the co-cultivated callus onto a screening medium for cultivation to obtain resistant callus; (b4) inoculating the resistant callus onto a differentiation and regeneration medium for culturing to obtain regenerated shoots.

5. The method according to claim 4, characterized in that: In step (b3), the solvent of the screening medium is water, and the solutes are N6 vitamin, 2,4-D, sucrose, hydrolyzed casein, proline, inositol, 6-BA, phytagel, hygromycin and carbenicillin; in the screening medium, the final concentrations of the components are: 2-6g / L N6, 1-3mg / L 2,4-D, 20-80g / L sucrose, 0.1-1g / L hydrolyzed casein, 1-5g / L proline, 0.05-0.5g / L inositol, 0.05-0.5g / L 6-BA, 3-12g / L phytagel, 20-80mg / L hygromycin, and 150-350mg / L carbenicillin.

6. The method according to claim 4, characterized in that: In step (b4), the solvent of the differentiation and regeneration medium is water, and the solutes are MS salt, hydrolyzed casein, nicotinic acid, vitamin B6, thiamine hydrochloride, inositol, copper sulfate, sucrose, agar, 6-BA, naphthylacetic acid, and carbenicillin; in the differentiation and regeneration medium, the final concentrations of the components are: 2-6 g / L MS salt, 0.5-2 g / L hydrolyzed casein, 0.5-2 mg / L nicotinic acid, 0.5-2 mg / L vitamin B6, 2-8 mg / L thiamine hydrochloride, 80-180 mg / L inositol, 0.5-2 mg / L copper sulfate, 20-70 g / L sucrose, 5-12 g / L agar, 1-4 mg / L 6-BA, 0.1-0.5 mg / L naphthylacetic acid, and 100-300 mg / L carbenicillin.

7. The method according to claim 4, characterized in that: In step (b1), the OD600 of the Agrobacterium infection solution used to infect callus tissue is 0.5-2.

8. The method according to claim 4, characterized in that: In step (b4), the co-cultivated callus is inoculated onto the screening medium, cultured in the dark at 28° C. for 10-14 days, and subcultured with the same screening medium every two weeks, for a total of 2-3 subcultures.

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