CN117204345B - A method for obtaining millet embryonic callus and transgenic plants - Google Patents
A method for obtaining millet embryonic callus and transgenic plants Download PDFInfo
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Abstract
本发明一种获得黍稷胚性愈伤组织的方法，包括如下步骤：(a1)将黍稷成熟种子脱壳消毒后接种于诱导培养基上进行诱导培养，获得初生愈伤组织；(a2)挑选(a1)中状态较好的初生愈伤组织继代培养，继代培养所用培养基为继代培养基，获得胚性愈伤组织；(a3)挑选(a2)中淡黄色且质密的胚性愈伤组织进行分化再生培养，直到分化出绿芽，以便评价不同黍稷种子的再生能力。通过优化培养基组分，诱导获得了适合遗传转化的红糜胚性愈伤组织，建立了基于农杆菌介导的红糜遗传转化体系，获得了以潮霉素作为筛选标记的转基因植株。本发明对挖掘黍稷抗逆功能基因与建立黍稷生物育种体系具有重要意义。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
The invention relates to the field of plant tissue culture and plant genetic engineering, in particular to a method for obtaining embryogenic callus of millet for genetic transformation and agrobacterium-mediated genetic transformation.
Background
Millet (Broomcorn Millet/Proso Millet, panicum miliaceum l.) belongs to the genus broomcorn (Panicum l.) of the family poaceae, annual herbaceous plants. Millet originated in northern regions of our country at the earliest and was one of the oldest crops so far, and had been a cultivation history of 10,000 years, spread to europe and around the world approximately 3,000 years ago, and planted in asia, africa, europe, america, etc. (Chai Yan et al, 2007; liu Minxuan et al, 2020; lu et al, 2009). pan millet was the staple food for ancient first people in China and is still one of the main food crops in northern arid and semiarid regions such as Shanxi, shaanxi, gansu, ningxia, inner Mongolia and Hebei (Cheng Bingwen et al, 2019). The millet has the characteristics of drought tolerance, salt and alkali tolerance, high temperature resistance, barren resistance, high concentration carbon dioxide resistance, short growth period, long amplitude and the like, is a stress-resistant pioneer crop and a main grain crop in arid and semiarid regions, is also an internationally recognized strategic reserve crop and environment-friendly crop for coping with climate warming and arid environments, and is also a 'disaster relief crop' (Fengli, 2016; blue and the like ,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, millet and the like, the water demand of the millet is minimum, and the grains can be produced only by the annual rainfall of 220-300 mm (Pardo et al, 2021, wu Jing and the like, 2023). The research of wheat, barley, corn, sorghum, millet and millet by Shanz, etc. for many years shows that the water utilization efficiency of the millet is highest, each liter of water of the millet can be consumed to synthesize 1.702g of dry matters, the next is 1.021 g and 0.7829g of the millet and the sorghum, respectively, 0.7829g, 0.6808 g and 0.5106g of barley, corn and wheat are respectively, when seeds germinate, the millet only needs to absorb 25 percent of water corresponding to the weight of the seeds to germinate, the water absorption amount of crops such as wheat (50%), corn (70%), sorghum (75%) and the like is less, in addition, the ear part of the millet in the maturity occupies 55 percent of the total weight of dry matters of plants, which indicates that the water utilization rate of the millet is extremely high (Wang Yutang, 2011, asparagus, 2019;Shantz et al, 1927;Ventura et al, 2020). the millet has nutritional and medicinal values, and the protein content is 8.6% -15.5%, which is obviously higher than that of rice, millet, sorghum rice, corn, barley and highland barley. The seeds also contain rich thiamine (vitamin B1), riboflavin (vitamin B2), yellow pigment and vitamin E, and the substances endow the millet with diversified health care functions. Millet dehulled millet has important medicinal values of tonifying middle-jiao and Qi, strengthening spleen and lung, removing heat and healing sores and the like and is mainly used for treating symptoms such as weakness of spleen and stomach. Millet proteins are gluten-free and have significant food application potential, so millet is also listed as one of the "future wisdom foods (Future Smart Food)" (sidtique et al, 2021). The global millet sowing area in 2018 was statistically about 600 ten thousand hm2, with the largest cultivated area being russia, followed by uk and china, and the chinese millet sowing area was about 53 ten thousand hm2 (Cheng Bingwen et al, 2019; wang et al, 2016).
At present, about 9,800 parts of millet genetic resources are collected and saved in a national germplasm library of China, the first place of the world is occupied, and meanwhile, related research on the millet in China also leaves the front of the world (Wang Guan and the like, 2015; wang Ruiyun, 2017). In 2019, the institute of science and technology of the Shanghai plant stress biology research center Zhang Heng topic and the cinnabar health topic group are combined with PacBio, illumina, hiC and a high-density genetic linkage map construction technology, a set of genome fine maps of millet Pm0390 (national institute of germplasm accession number 00000390) with the total length of 855Mb are obtained, 18 chromosomes (Contig N50= 0.36Mb,Scaffold N50 = 45.56 Mb) are provided, 55,930 protein coding genes are annotated, 99.3% of genes can be located on the chromosomes, and related researches are published on Nature Communication journal (Zou et al, 2019). Meanwhile, the journal also discloses the genome assembly work of Panicum H.H.Chen commercial variety Shang No. 4 from the group of the subject of China university of agriculture Lai Jincheng, and the study obtained high-quality genome sequences (Contig N50= 2.55Mb,Scaffold N50 =8.24 Mb), which were annotated with 63,671 protein-coding genes 14% more than Panicum Pm0390 (Shi et al, 2019). Team Shi Junpeng of Zhongshan university used 51 XPacBIO HiFi sequencing data and integrated the last version of the chyme No. 4 genome (Longmi _v1) to obtain a higher quality chyme No. 4 genome Longmi _v2. The upgraded Panicum miliaceum genome Longmi _v2 was approximately 846.0Mb,Contig N50 in size to approximately 26.2Mb, filling 525 gaps (Wang et al, 2023) left over from the genome Longmi _v1.
Restriction site-related DNA sequencing technology (RAD-seq) or specific focused amplified fragment sequencing technology (SLAF-seq) genotyping SNP markers have been used to correlate agronomic and seed traits of millet, greatly motivated genetic studies of millet (Boukail et al.,2021;Khound et al, 2022) since the release of the millet genome. Meanwhile, researchers analyze part of drought resistance, salt and alkali resistance, low nitrogen resistance and other adversity stress genes in the millet by adopting technologies such as graphic cloning, multi-group chemical combination analysis and the like, and lay a good foundation for analyzing the adversity resistance mechanism of the millet (Wang Ruiyun and the like, 2018; zhang et al, 2019; an et al, 2022; shan et al, 2020 Sun et al, 2023; wang et al, 2023). Although significant progress has been made in the study of millet genomics, this greatly limits the study of millet functional genomics in biological breeding technology development, since no genetic transformation system of millet has been established so far, the present invention aims to establish a genetic transformation system of millet based on mature seeds, providing a territorial technology for the establishment of millet biological breeding technology.
Disclosure of Invention
The invention aims to obtain embryogenic callus of millet by using mature embryo (seed), and can perform efficient and stable agrobacterium-mediated genetic transformation by using the embryogenic callus, thereby solving the key bottleneck problem that the millet is difficult to perform genetic transformation, and establishing a large-scale genetic transformation system of the millet so as to truly develop the millet into a functional gene research mode plant.
In order to achieve the above object, the present invention provides a method for obtaining embryogenic callus of Panicum Miliacei, comprising the steps of:
(a1) Husking and sterilizing mature seeds of millet, and inoculating the mature seeds to an induction culture medium for induction culture to obtain primary callus;
(a2) Selecting primary callus with better state in (a 1) for subculture, wherein a culture medium used for the subculture is a subculture medium, so as to obtain embryogenic callus;
(a3) Selecting the pale yellow and dense embryogenic callus of (a 2) for differentiation regeneration culture until green buds are differentiated so as to evaluate the regeneration capacity of different millet seeds;
In the step (a 1), the solvent of the induction culture medium is water, and solutes comprise MS salt, N6 vitamin, proline, inositol, 6-BA, aspartic acid, sucrose, 2,4-D and plant gel, wherein in the induction culture medium, the final concentration of the MS salt is 2.5-7g/L, the final concentration of the N6 vitamin is 0.5-3ml/L, the final concentration of the proline is 0.5-3g/L, the final concentration of the inositol is 0.01-2g/L, the final concentration of the 6-BA is 0.01-2g/L, the final concentration of the aspartic acid is 0.05-3g/L, the final concentration of the sucrose is 5-100g/L, the final concentration of the 2,4-D is 1-5mg/L, and the final concentration of the plant gel is 1-12g/L;
More preferably, the induction medium composition and final concentration are 4.43g/L MS salt (cat# M524), 1ml/L N6 (cat# C149), 1.0g/L proline, 0.1g/L inositol, 0.1 mg/L6-BA, 0.2g/L aspartic acid, 30g/L sucrose, 2 mg/L2, 4-D, 8.0g/L plant gel, pH 5.7.
In the step (a 3), the solvent of the differentiation regeneration culture medium adopted when the embryogenic callus is subjected to differentiation regeneration culture is water, and solutes are MS salt, nicotinic acid, vitamin B6, vitamin B1, inositol, copper sulfate, sucrose, agar, 6-BA and carbenicillin, wherein in the differentiation regeneration culture medium, the final concentration of the MS salt is 2.5-7g/L, the final concentration of the nicotinic acid is 0.5-3mg/L, the final concentration of the vitamin B6 is 0.5-3mg/L, the final concentration of the 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.
More preferably, the differentiation regeneration medium has a composition and final concentration of 4.3g/LMS salt (cat# 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 2 mg/L6-BA, 200mg/L carbenicillin, pH 5.6
Preferably, the induction culture is performed in step (a 1) under conditions of 28℃for 20 to 30 days in dark culture, and the subculture is performed in step (a 2) for 10 to 15 days.
Preferably, in step (a 3), the differentiation and regeneration culture is performed according to a method comprising the steps of inoculating the embryogenic callus into the differentiation and regeneration medium and placing in a 28℃tissue culture operating room for 16 hours of light/8 hours of darkness for 4-8 weeks.
Further, the invention provides a genetic transformation method of millet, comprising the following steps:
(b1) Impregnating embryogenic callus obtained by the method with agrobacterium infection liquid containing target gene expression vector;
(b2) Inoculating the infected embryogenic callus on a co-culture medium for co-culture, and co-culturing nutrients for 2 stages to obtain co-cultured callus after culture;
(b3) Inoculating the co-cultured callus to a screening culture medium for culture to obtain a resistant callus;
(b4) And inoculating the resistant callus to a differentiation regeneration culture medium for culture to obtain regenerated seedlings.
Preferably, in step (B1), the medium used for the infection is an infection medium, the solvent of the medium is water, the solutes are MS salt, B5 vitamin, sucrose, glucose, asparagine, casamino acid, cysteine, 2, 4-dichlorophenoxyacetic acid and acetosyringone, and in the infection medium, 0.2-2g/L MS salt, 1g/L B vitamin, 30-100g/L sucrose, 10-80g/L glucose, 0.5-3g/L asparagine, 0.5-3g/L casamino acid, 0.1-2g/L cysteine, 0.5-4mg/L2, 4-dichlorophenoxyacetic acid and 150-300 mu M acetosyringone are used.
Preferably, in the step (B2), the solvent of the first-stage co-culture medium is water, and solutes are MS salt, B5 vitamin, sucrose, glucose, asparagine, casein amino acid, cysteine, 2,4-D, acetosyringone and agarose, wherein the final concentration of each component in the co-culture medium is respectively 0.2-2g/L MS salt, 1g/L B vitamin, 30-100g/L sucrose, 10-80g/L glucose, 0.5-3g/L asparagine, 0.5-3g/L casein amino acid, 0.1-2g/L cysteine, 0.5-4 mg/L2, 4-D,150-300 mu M acetosyringone and 3-12g/L agarose;
More preferably, the components are composed and final concentrations of 0.44g/L MS salt, 1g/L vitamin, 68g/L sucrose, 36g/L glucose, 1g/L asparagine, 1g/L Casein amino acid, 0.2g/L cysteine, 2 mg/L2, 4-D, 200. Mu.M acetosyringone, 8g/L agarose, pH 5.2.
The second-stage co-culture medium comprises water as a solvent, MS salt, B5 vitamin, sucrose, glucose, asparagine, hydrolyzed casein, cysteine, 2,4-D, acetosyringone, agarose and carbenicillin as solutes, wherein the final concentration of each component in the co-culture medium is respectively 0.2-2g/L MS salt, 1g/L B 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, 0.5-4 mg/L2, 4-D,150-300 mu M acetosyringone, 3-12g/L agarose and 150-350mg/L carbenicillin.
More preferably, the components are composed and final concentrations of 0.44g/L MS salt, 1g/L B vitamin, 68g/L sucrose, 36g/L glucose, 1g/L asparagine, 1g/L hydrolyzed casein, 0.2g/L cysteine, 2 mg/L2, 4-D, 200. Mu.M acetosyringone, 8g/L agarose, 250mg/L carbenicillin, pH 5.2.
Preferably, in the step (b 3), the solvent of the screening culture medium is water, and the solute is N6 vitamin, 2,4-D, sucrose, hydrolyzed casein, proline, inositol, 6-BA, plant gel, hygromycin and carbenicillin, and the final concentration of each component in the screening culture medium is 2-6g/L N, 1-3 mg/L2, 4-D,20-80g/L sucrose, 0.1-1g/L hydrolyzed casein, 1-5g/L proline, 0.05-0.5g/L inositol, 0.05-0.5 g/L6-BA, 3-12g/L plant gel, 20-80mg/L hygromycin and 150-350mg/L carbenicillin respectively.
More preferably, in the screening medium, the 4.0g/L N, cat# C149,2mg/L2,4-D,30g/L sucrose, 0.3g/L hydrolyzed casein, 2.8g/L proline, 0.1g/L inositol, 0.1g/L6-BA,8.0g/L plant gel, 50mg/L hygromycin, 250mg/L carbenicillin.
Preferably, in the step (B4), the solvent of the differentiation regeneration culture medium is water, and the solute comprises MS salt, hydrolyzed casein, nicotinic acid, vitamin B6, thiamine hydrochloride (vitamin B1), inositol, copper sulfate, sucrose 8.0g/L, agar, 6-BA, naphthylacetic acid and carbenicillin, wherein the final concentration of each component in the differentiation regeneration culture medium is 2-6g/L MS salt, 0.5-2g/L hydrolyzed casein, 0.5-2mg/L nicotinic acid, 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, 1-4 mg/L6-BA, 0.1-0.5mg/L naphthylacetic acid and 100-300mg/L carbenicillin respectively.
More preferably, in the differentiation regeneration medium, the vitamin-containing 4.3g/L MS salt, 1.0g/L hydrolyzed casein, 1.0mg/L niacin, 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, 2 mg/L6-BA, 0.2mg/L naphthalene acetic acid, 200mg/L carbenicillin.
Preferably, in step (b 1), the OD600 of the agrobacterium infesting solution used to infest the callus is between 0.5 and 2.
Preferably, in step (b 4), the co-cultured calli are inoculated onto the selection medium, and are dark-cultured at 28 ℃ for 10-14 days, and are subjected to subculture with the same selection medium every two weeks, for a total of 2-3 times.
Still further, the present invention provides a protective medium or set of media.
The culture medium claimed by the invention is any one of the culture media in the above method.
The complete set of culture mediums claimed in the invention are used in combination by any 2 or more than 2 or all culture mediums in the method.
The invention has the advantages and beneficial effects that the invention discloses germplasm with better embryogenic induction rate and plant regeneration capability by evaluating embryogenic callus induction and plant regeneration capability of 13 mature seeds of Panicum miliaceum genotypes and screening red emulsion (Getaihongmi, hongmi). Through optimizing culture medium components, red chyme embryogenic callus suitable for genetic transformation is obtained through induction, an agrobacterium-mediated red chyme genetic transformation system is established, and a transgenic plant taking hygromycin as a screening mark is obtained. The invention has important significance for excavating the stress-resistant functional genes of the millet and establishing a millet biological breeding system.
Drawings
FIG. 1 shows the selection of the genotypes of Panicum Miliacei which is easy to be genetically transformed, the statistics of the induction rate, the shoot regeneration rate and the albino rate of 13 parts of Panicum Miliacei germplasm callus, the quality of embryogenic callus is represented by "+", "++ +" is best, "+" is worst, the induction rate of embryogenic callus (embryogenic callus number/germination seed number in induction medium) and 90 calli are cultured on regeneration medium for 20 days to count the green shoot rate and white shoot rate respectively.
FIG. 2 sets up a red chyme genetic transformation system, a, cultivated for 20 days of red chyme calli, b, embryogenic calli after subculture, c, agrobacterium infection co-cultivation (3 days), d, infection calli with high efficiency and transient expression of GFP, e, first selection of calli on medium (10 days), f, observation of the same calli under UV light showed strong GFP signals, g-h, regenerated shoots under white light (g) and UV light (h), i-j, leaf cross-section GFP fluorescence of wild type (left panel) and transgenic T0 plants (right panel) under white (i) and UV light (j), k-l, conical inflorescence under white (k) and UV light (l), wild type (left panel), transgenic (right panel), m-n, wild type floret under white light (m) and UV light (n), transgenic seed expression of GFP (right panel), o-p, germination ratio of T39 (o) and UV light (h), and p 2mm (5 mm-2, 5mm (b) and 53 mm (2 mm).
FIG. 3 identification of red chyme transgenic positive plants, a PCR amplification detection of GFP gene, b Southern blotting experiment detection of T0 plant copy number.
Detailed Description
The following examples are given for better understanding of the present invention, but are not limited thereto, and are conventional unless otherwise specified. The experimental materials and reagents used in the examples described below were purchased from conventional biochemical reagent stores unless otherwise specified.
The MS salt (cat No. M524) used in the examples described below is a product of Beijing, mejie technologies, inc., N6 vitamin (cat No. C149) is a product of Beijing, mejie technologies, inc., and N6 salt (cat No. C167) is a product of Beijing, mejie technologies, inc.
Millet germplasm is obtained from a national crop germplasm pool of China academy of agricultural sciences;
the vector pCAMBIA1305-GFP is described in the document pCAMBIA1305-GFP vector as DOI https:// doi.org/10.1105/tpc.113.121376.
Example 1
(1) Husking 13 parts of millet germplasm, wiping and sterilizing with 75% alcohol, sterilizing with 10-12% (volume fraction) sodium hypochlorite for 10-12min, washing with sterilized distilled water for 5 times, and washing for 40-50min. All the above operations are completed in a sterile super clean bench;
(2) Then wiping the surface water of the sterilized seeds with sterile absorbent paper, placing the sterilized seeds in an induction culture medium, wherein the composition of the induction culture medium comprises 4.43g/L MS salt (product number: M524), 1ml/L N6 (product number: C149), 1.0g/L proline, 0.1g/L inositol, 0.1 mg/L6-BA, 0.2g/L aspartic acid, 30g/L sucrose, 2 mg/L2, 4-D and 8.0g/L plant gel, and the pH value is 5.7 (the concentration of each substance is the final concentration in the induction culture medium);
(3) Then inducing the callus at 28 ℃ for 20-30 days, then selecting the callus with better state and continuously placing the callus in a callus induction culture medium for subculture for 10-15 days, finding that 13 parts of materials can induce and generate primary callus, but the induction frequency depends on genotype, wherein the induction rate of 3 parts of germplasm, namely red chyme, qingyang red hard and Jinshi No. 7 callus is higher than 40 percent (figure 1), and the induced callus of about 40-90 percent is continuously expanded and grows into friable callus (figure 1). Counting the proportion of embryogenic callus, classifying the mass of embryogenic callus into 1-5 grades according to the quality, color and porosity (figure 1), finding that the embryogenic callus of red paste has the best mass, and the embryogenic callus induction rate is 56.14 percent (red paste) and 50.39 percent (red paste) respectively (red paste) (figure 1);
(4) Transferring the embryogenic callus which is light yellow and is dense in quality in the selection step ⑴ to a differentiation regeneration culture medium, sealing, and placing the embryogenic callus in a 28 ℃ tissue culture operation room which is irradiated for 16 hours and dark for 8 hours for 4-8 weeks until green buds are differentiated;
The differentiation regeneration medium consists of 4.3g/LMS salt (product number: M519), 1.0mg/L nicotinic acid, 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 2 mg/L6-BA, 200mg/L carbenicillin and pH value of 5.6 (the concentrations of the above substances are all final concentrations in the differentiation regeneration medium).
Example 2
(1) Agrobacterium-mediated genetic transformation experiments were performed using 25 day and 12 day subculture red chymotrypsin calli (FIGS. 2a-2 b), agrobacterium EHA105 was transformed with pCAMBIA1305-GFP vector carrying GFP reporter gene and infested, HPT gene as marker gene, and the optimal selection concentration of hygromycin was detected. The 1305Ubi-GFP HPT vector was transferred into agrobacterium strain EHA105 and cultured overnight in yes medium (5 g/L beef extract, 5g/L peptone, 1g/L yeast extract, 5g/L sucrose, 10mM magnesium sulfate, ph=7.0) at a steady rotation speed of 200r/min until the optical density reached od600=1.0;
For Agrobacterium transformation, the subcultured embryogenic calli were infected with Agrobacterium cells (OD 600 = 0.5) for 30 min in infection medium (composition: 0.44g/L MS salt, 1g/LB5 vitamin, 68g/L sucrose, 36g/L glucose, 1g/L asparagine, 1g/L casamino acid, 0.2g/L cysteine, 2 mg/L2, 4-dichlorophenoxyacetic acid, 200. Mu.M acetosyringone, pH 5.2);
(2) Then transferred to a co-culture medium (composition: 0.44g/L MS salt, 1g/L B vitamin, 68g/L sucrose, 36g/L glucose, 1g/L asparagine, 1g/L casamino acid, 0.2g/L cysteine, 2 mg/L2, 4-D, 200. Mu.M acetosyringone, 8g/L agarose, pH 5.2), and 22℃dark culture for 72 hours;
GFP expression was found 3 days after co-cultivation (FIGS. 2C-2D), and the infected calli above were transferred to a carbenicillin-supplemented co-culture medium (composition: 0.44g/L MS salt, 1g/L B vitamin, 68g/L sucrose, 36g/L glucose, 1g/L asparagine, 1g/L hydrolyzed casein, 0.2g/L cysteine, 2 mg/L2, 4-D, 200. Mu.M acetosyringone, 8g/L agarose, 250mg/L carbenicillin, pH 5.2) and incubated in a 28℃dark incubator for 7 days;
(3) Transferring the co-cultured calli to a screening medium (4.0 g/L N, cat# C149,2 mg/L2, 4-D,30g/L sucrose, 0.3g/L hydrolyzed casein, 2.8g/L proline, 0.1g/L inositol, 0.1 g/L6-BA, 8.0g/L plant gel, 50mg/L hygromycin, 250mg/L carbenicillin.28 ℃ C.) containing hygromycin for 10-14 days, and then subculturing 2 times with the same screening medium as seen in FIG. 2, green fluorescent protein is stably expressed in the calli, demonstrating high screening efficiency;
(4) After two rounds of screening with Hygromycin (50 mg/L), it was confirmed that Hygromyin at 50mg/L was sufficient to select GFP-containing cells (FIGS. 2e-2 f);
(5) Differentiation of GFP-containing positive calli on regeneration medium (FIG. 2g-2 h), transfer of resistant calli after selection culture to differentiation regeneration medium (4.3 g/L MS salt, 1.0g/L hydrolyzed casein, 1.0mg/L niacin, 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 naphthalene acetic acid, 200mg/L carbenicillin, pH 5.6), light/8 hours dark culture at 28℃for 4-8 weeks for regeneration of seedlings;
(6) Transferring the young seedlings to rooting culture medium (2.2 g/L MS salt, commodity number: M524, 30g/L sucrose, 0.1g/L inositol, 2.6g/L plant gel, 30mg/L hygromycin and 250mg/L carbenicillin, 8g/L plant gel, pH value of 5.6, the concentrations of the above substances are all final concentrations in rooting culture medium), rooting, culturing in dark for 8 hours at 28 ℃ under 16 hours, culturing for 3-4 weeks, and hardening seedlings in culture when the young seedlings grow to 10-20 cm;
(7) Transplanting the transgenic plants after seedling hardening into a flowerpot, culturing in a greenhouse, and taking leaves in the later growth stage for transgenic positive detection. Observation revealed that GFP was expressed in leaf cross sections (FIGS. 2i-2 j), young ears (FIGS. 2k-2 l) and florets (FIGS. 2m-2 n) of transgenic plants, and that seedlings germinated on T 1 seeds were also stably expressing GFP fluorescence, indicating that the T-DNA vector was stably inherited (FIGS. 2o-2 p);
(8) The 22 out of 23T 0 plants was found to be positive plants containing GFP by PCR amplification of a 606bp partial GFP fragment (FIG. 3 a). To further verify the accuracy of the PCR and investigate the copy number of T 0 plants, 10T 0 plants were randomly selected and Southern blot experiments were performed using two restriction enzymes (SacI and BamHI) with HPT as probe. The results show that four different events (PM 1, PM11, PM15 and PM 19) and three events (PM 4, PM7 and PM 8) belong to a single copy, and that PM2 and PM17 carry two and three copies, respectively (fig. 3 b).
Claims (8)
1. A method for obtaining embryogenic callus of millet comprising the steps of:
(a1) Husking and sterilizing mature seeds of millet, and inoculating the mature seeds to an induction culture medium for induction culture to obtain primary callus;
(a2) Selecting primary callus with better state in (a 1) for subculture, wherein a culture medium used for the subculture is a subculture medium, so as to obtain embryogenic callus;
(a3) Selecting the pale yellow and dense embryogenic callus of (a 2) for differentiation regeneration culture until green buds are differentiated so as to evaluate the regeneration capacity of different millet seeds;
In the step (a 1), the solvent of the induction culture medium is water, and solutes comprise MS salt, N6 vitamin, proline, inositol, 6-BA, aspartic acid, sucrose, 2,4-D and plant gel, wherein in the induction culture medium, the final concentration of the MS salt is 2.5-7g/L, the final concentration of the N6 vitamin is 0.5-3ml/L, the final concentration of the proline is 0.5-3g/L, the final concentration of the inositol is 0.01-2g/L, the final concentration of the 6-BA is 0.01-2g/L, the final concentration of the aspartic acid is 0.05-3g/L, the final concentration of the sucrose is 5-100g/L, the final concentration of the 2,4-D is 1-5mg/L, and the final concentration of the plant gel is 1-12g/L;
In the step (a 3), the solvent of the differentiation regeneration culture medium adopted when the embryogenic callus is subjected to differentiation regeneration culture is water, and solutes are MS salt, nicotinic acid, vitamin B6, vitamin B1, inositol, copper sulfate, sucrose, agar, 6-BA and carbenicillin, wherein in the differentiation regeneration culture medium, the final concentration of the MS salt is 2.5-7g/L, the final concentration of the nicotinic acid is 0.5-3mg/L, the final concentration of the vitamin B6 is 0.5-3mg/L, the final concentration of the 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, wherein the induction culture is carried out in step (a 1) under a condition of 28℃for 20 to 30 days in dark culture, and the subculture is carried out in step (a 2) for 10 to 15 days.
3. The method according to claim 1, wherein in the step (a 3), the differentiation and regeneration culture is performed according to a method comprising the steps of inoculating the embryogenic callus into the differentiation and regeneration medium and placing the embryogenic callus in a 28℃tissue culture room with 16-hour light/8-hour darkness for 4-8 weeks.
4. A method for genetic transformation of millet comprising the steps of:
(b1) The embryogenic callus obtained by the method of any one of claims 1-3 is impregnated with agrobacterium infection liquid containing a target gene expression vector, the culture medium used for infection is an infection culture medium, the solvent of the culture medium is water, the solute is MS salt, B5 vitamin, sucrose, glucose, asparagine, casein amino acid, cysteine, 2, 4-dichlorophenoxyacetic acid and acetosyringone, and the final concentration of each component in the infection culture medium is respectively 0.2-2g/L MS salt, 1g/L B vitamin, 30-100g/L sucrose, 10-80g/L glucose, 0.5-3g/L asparagine, 0.5-3g/L casein amino acid, 0.1-2g/L cysteine, 0.5-4 mg/L2, 4-dichlorophenoxyacetic acid and 150-300 mu M acetosyringone;
(b2) Inoculating the infected embryogenic callus on a co-culture medium for co-culture, and co-culturing nutrients for 2 stages to obtain co-cultured callus after culture;
the first-stage co-culture medium comprises water as a solvent, wherein the solute comprises MS salt, B5 vitamin, sucrose, glucose, asparagine, casein amino acid, cysteine, 2,4-D, acetosyringone and agarose, and the final concentration of each component in the co-culture medium is respectively 0.2-2g/L MS salt, 1g/LB5 vitamin, 30-100g/L sucrose, 10-80g/L glucose, 0.5-3g/L asparagine, 0.5-3g/L casein amino acid, 0.1-2g/L cysteine, 0.5-4 mg/L2, 4-D,150-300 mu M acetosyringone and 3-12g/L agarose;
The second-stage co-culture medium comprises water as a solvent, wherein the solute comprises MS salt, B5 vitamin, sucrose, glucose, asparagine, hydrolyzed casein, cysteine, 2,4-D, acetosyringone, agarose and carbenicillin, and the final concentration of each component in the co-culture medium is respectively 0.2-2g/L MS salt, 1g/L B 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, 0.5-4 mg/L2, 4-D,150-300 mu M acetosyringone, 3-12g/L agarose and 150-350mg/L carbenicillin;
(b3) Inoculating the co-cultured callus to a screening culture medium for culture to obtain a resistant callus;
(b4) And inoculating the resistant callus to a differentiation regeneration culture medium for culture to obtain regenerated seedlings.
5. The method according to claim 4, wherein in the step (b 3), the solvent of the screening medium is water, the solute is N6 vitamins, 2,4-D, sucrose, hydrolyzed casein, proline, inositol, 6-BA, plant gel, hygromycin and carbenicillin, and the final concentration of each component in the screening medium is 2-6g/L N, 1-3 mg/L2, 4-D,20-80g/L sucrose, 0.1-1g/L hydrolyzed casein, 1-5g/L proline, 0.05-0.5g/L inositol, 0.05-0.5 g/L6-BA, 3-12g/L plant gel, 20-80mg/L hygromycin and 150-350mg/L carbenicillin, respectively.
6. The method according to claim 4, wherein in the step (B4), the solvent of the differentiation regeneration medium is water, the solute is MS salt, hydrolyzed casein, nicotinic acid, vitamin B6, thiamine hydrochloride, inositol, copper sulfate, sucrose, agar, 6-BA, naphthalene acetic acid, and carbenicillin, and the final concentration of each component in the differentiation regeneration medium is 2-6g/L MS salt, 0.5-2g/L hydrolyzed casein, 0.5-2mg/L nicotinic acid, 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, 1-4 mg/L6-BA, 0.1-0.5mg/L naphthalene acetic acid, and 100-300mg/L carbenicillin, respectively.
7. The method according to claim 4, wherein in step (b 1), the OD600 of the Agrobacterium tumefaciens solution used to infect the callus is between 0.5 and 2.
8. The method according to claim 4, wherein in the step (b 4), the co-cultured calli are inoculated onto the selection medium, and the co-cultured calli are subjected to a dark culture at 28 ℃ for 10 to 14 days, and are subjected to a total of 2 to 3 times of subculture with the same selection medium every two weeks.
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