CN117143907A

CN117143907A - Broom corn millet genetic transformation method

Info

Publication number: CN117143907A
Application number: CN202210565362.XA
Authority: CN
Inventors: 赖锦盛; 宋伟彬; 赵海铭; 白雨禾
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-05-23
Filing date: 2022-05-23
Publication date: 2023-12-01

Abstract

The application relates to a high-efficiency broom corn millet genetic transformation method. The applicant has repeatedly searched and tested, and optimized the multiple steps of the established broom corn millet genetic transformation method. Firstly, selecting mature seeds of plants as explants, wherein the sources of the mature seeds are not limited by seasons, and the acquisition period is short; secondly, on the basis of selecting seeds as explants, primary callus is selected for infection and genetic transformation of agrobacterium, so that the transformation efficiency is further improved, and the transformation method is optimized; finally, by adjusting the culture medium used in the callus culture process, the optimal culture medium combination is obtained, the transformation efficiency is further improved, and the transformation method is optimized. Thus, the efficient broom corn millet genetic transformation method is obtained.

Description

Broom corn millet genetic transformation method

Technical Field

The application relates to the technical field of biotechnology and plant bioengineering, in particular to a high-efficiency broom corn millet genetic transformation method.

Background

The transformation techniques that have been used at present are gene gun method, agrobacterium-mediated method, pollen tube channel method, PEG-mediated method, ovary injection method, ultrasonic method, electrode method, etc. Among the mainstream transformation methods of cereals are the gene gun method and the agrobacterium-mediated method. The gene gun bombardment method has the defects of relatively complex integration mode, relatively unstable passage of exogenous gene expression and easy technical limitation. The agrobacterium-mediated method has the advantages of capability of transforming larger DNA fragments, simpler operation, low cost, stable inheritance of the obtained transgenic successful plants and the like, although the agrobacterium-mediated method has the defect that a vector skeleton and even chromosome fragments can be transferred; therefore, agrobacterium-mediated transgene is one of the current methods of genetic transformation of cereals.

In agrobacterium-mediated genetic transformation, the establishment of a good receptor system is key to genetic transformation; currently, the receptors commonly used are: immature embryo, seed, stem node meristem, young spike, stem tip, root tip, etc. There are many restrictions on the acquisition of part of the explants, which can limit the routine transformation of the explants.

Broom corn millet (Panicum miliaceum l), also known as millet, belongs to the family of grasses, the heterologous 4-fold (2n=4×9=36), annual cereals. Is one of the traditional five cereals in China and is also one of the crops which are domesticated first by people in China. It is rich in multiple vitamins, amino acids and unsaturated fatty acids, and is beneficial to health after long-term consumption, and has important effect on improving the dietary structure of China; broom corn millet has a shorter growth cycle and better stress resistance, is used as a C4 crop, has higher carbon fixing capacity and higher water and fertilizer utilization capacity than a C3 crop, and is an ideal drought-resistant pioneer crop.

Broom corn millet has successfully completed whole genome sequencing in 2019 (team Lai Jincheng and Song Weibin of Chinese agricultural university), which also opens up functional genomics research of broom corn millet. However, there is no available broom corn millet genetic transformation technology system in the global scope so far, which makes the genetic improvement of broom corn millet by genetic transformation methods, especially genetic editing technology strategies, very limited.

Therefore, a genetic transformation system of broom corn millet is created, and the rapid increase of the yield and the improvement of the quality are developed through a gene editing technology system (CRISPR-Cas 12i and Cas12 j) of independent intellectual property rights, so that the method has great research significance in further improving and enriching the variety of food crops in China.

Disclosure of Invention

In order to solve the problems, the inventor of the application establishes a genetic transformation method of broom corn millet for the first time through a great deal of experiments. Furthermore, the applicant optimizes a plurality of steps of the established broom corn millet genetic transformation method through repeated fumbling, and the efficient broom corn millet genetic transformation method is obtained. Thus, the present application has been completed.

Accordingly, in a first aspect, the present application provides a method for genetic transformation of millet, the method comprising:

step (1): taking broom corn millet seeds as explants to obtain primary callus;

step (2): transforming target genes and hygromycin resistance genes into the primary callus obtained in the step (1) through agrobacterium;

step (3): putting the product obtained in the step (2) into a co-culture medium for co-culture;

step (4): placing the product obtained in the step (3) into an induction culture medium for culture;

step (5): placing the product obtained in the step (4) into a screening culture medium containing 50mg/L hygromycin to screen callus containing hygromycin resistance genes;

step (6): placing the product obtained in the step (5) into a pre-differentiation culture medium to enable the callus to bud;

step (7): placing the product obtained in the step (6) into a differentiation medium to root the callus;

step (8): placing the product obtained in the step (7) into a seedling culture medium so as to enable seedlings of the callus to grow.

In certain embodiments, the amino acid sequence of the gene of interest is shown in SEQ ID NO. 2.

In certain embodiments, the hygromycin resistance gene has an amino acid sequence as set forth in SEQ ID NO. 1.

In certain embodiments, in step (3), the co-culture medium has a ph=5.4, which comprises or consists of MS basal medium supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 50mg/L inositol, 0.1mg/L thiamine hydrochloride, 30g/L sucrose and 2mg/L2,4-D and 0.5mg/L kinetin; and 200. Mu.M/L acetosyringone was added after sterilization.

In certain embodiments, in step (3), the co-culture medium is a solid co-culture medium. In certain embodiments, the solid co-culture medium further comprises 3g/LPhytagel ^TM 。

In certain embodiments, the solid co-culture medium has a ph=5.4, which comprises or is supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 50mg/L inositol, 0.1mg/L thiamine hydrochloride, 30g/L sucrose and 2mg/L2,4-D,0.5mg/L kinetin, 3g/L Phytagel from MS minimal medium ^TM Composition; and 200. Mu.M/L acetosyringone was added after sterilization.

In certain embodiments, in step (4), the induction medium has a ph=5.8, which comprises or is supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin and 3g/LPhytagel from MS minimal medium ^TM Composition; and, after sterilization, 2mg/L AgNO is added ₃ 。

In certain embodiments, in step (5), the screening medium has a ph=5.8, which comprises or is supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin and 3g/LPhytagel from MS minimal medium ^TM Composition; and, after sterilization, 2mg/L AgNO is added ₃ 50mg/L hygromycin, and 150mg/L Timentin.

In certain embodiments, at step (iv)(6) Wherein the pre-fractionation medium has a pH=5.8, comprising or supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose, 0.2 mg/L2,4-D, 3 mg/L6-BA and 3g/LPhytagel from MS minimal medium ^TM Composition; and 150mg/L Tintin was added after sterilization.

In certain embodiments, in step (7), the differentiation medium has a ph=5.8, which comprises or is supplemented with 1mg/L d-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose and 3g/L Phytagel from MS minimal medium ^TM Composition; and 150mg/L Tintin was added after sterilization.

In certain embodiments, in step (8), the pH of the seedling medium = 5.8, which comprises or consists of 1/2MS minimal medium supplemented with 0.25mg/L pyridoxine hydrochloride, 0.25mg/L niacin, 100mg/L inositol, 0.05mg/L thiamine hydrochloride, 15g/L sucrose, 1mM isoleucine and 8g/L agar; and 100mg/L Tintin is added after sterilization; in certain embodiments, the methods described above use one or more of the media described above. In certain preferred embodiments, the methods described above use a combination of 6 media as described above.

In certain embodiments, the 1/2MS minimal medium comprises 2.165g/L MS salts.

In certain embodiments, the MS minimal medium comprises 4.33g/L MS salts.

In certain embodiments, the methods described above use one or more of the media described above. In certain preferred embodiments, the methods described above use a combination of all media described above.

In certain embodiments, the method as described above, wherein, in step (1), the seeds of broom corn millet are used as explants and inoculated on induction medium to obtain primary calli.

In certain embodiments, as described above, in step (1), mature seeds of broom corn millet are sterilized, palea and lemma are removed, sterilized with 75% alcohol for two minutes, and rinsed three to four times with sterilized deionized water for 2 minutes each; the sterilized and washed seeds were placed on sterile filter paper until they were thoroughly dried, then the seeds were inoculated on an induction medium, and after culturing for 20 days at 28℃in the dark, they were transferred to a fresh induction medium until white yellowish primary calli were isolated.

In certain embodiments, a method as described above, wherein, in step (2), the expression vector containing the gene of interest and the hygromycin resistance gene is infected with primary callus by agrobacterium tumefaciens, such that the gene of interest and the hygromycin resistance gene are integrated into the genome of the primary callus.

In certain embodiments, the strain of agrobacterium tumefaciens is EHA105.

In certain embodiments, 300 μl of agrobacterium tumefaciens EHA105 bacterial fluid containing the vector is pipetted into 25ml LB and 50mg/L rifampicin and 100mg/L kanamycin are added and cultured overnight at 28 ℃; then centrifuged for 22min, the supernatant discarded and resuspended in liquid co-culture medium to od600=1.8; incubating at 28 ℃ until the OD600 = 2.0-3.0, and placing on ice for standby to obtain agrobacterium tumefaciens bacteria liquid; wherein the vector contains a gene of interest and a hygromycin resistance gene.

In certain embodiments, a method as described above, wherein, in step (3), the embryogenic callus obtained in step (2) is divided into small pieces of 2mm-3mm, picked up and placed in a co-culture medium containing a liquid, placed on ice for pre-cooling for 20min, then placed in the agrobacterium tumefaciens bacterial solution, and incubated for half an hour at 26 ℃; the calli were then transferred to solid co-culture medium at 22℃for 3 days in the dark.

In certain embodiments, the method as described above, wherein, in step (4), the induction medium further comprises 150mg/L Timentin.

In certain embodiments, in step (4), the co-cultured calli are transferred to induction medium containing 150mg/L Timentin and dark cultured at 28℃for 15 days.

In certain embodiments, the method as described above, wherein, in step (5), the product obtained in step (4) is placed in a screening medium containing 50mg/L hygromycin, and dark-cultured at 28℃for 7-10 days to screen calli containing hygromycin resistance gene.

In certain embodiments, the method as described above, wherein, in step (6), the well-grown callus obtained after the screening of step (5) is inoculated into a pre-differentiation medium, and the pre-differentiation medium is transferred to an illumination culture room for culturing for 14 days, and a layer of gauze is covered thereon to prevent the illumination from being too strong.

In certain embodiments, the method as described above, wherein, in step (7), the differentiated, greenish callus obtained in step (6) is transferred to a differentiation medium to allow the callus to root.

In certain embodiments, a method as described above, wherein, in step (8), seedlings with leaf lengths exceeding 3cm are transferred into a seedling medium to allow the seedlings of the callus to grow.

Definition of terms

In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present application, definitions and explanations of related terms are provided below.

As used herein, the term "callus" refers to new tissue formed by cutting out a portion of an organ, tissue or seed of a plant, and properly culturing the tissue. Callus did not differentiate, but only a mass of parenchyma cells. Generally, under the conditions of proper illumination, temperature, certain nutrients, hormones and the like, the callus can differentiate and produce various organs and tissues of the plant, so that a complete plant can be developed.

As used herein, the term "primary callus" refers to tissue that is first formed after a portion of a plant's organ, tissue or seed has been excised, suitably cultured.

As used herein, the term "secondary calli" refers to tissues that have been formed by the first callus, then excised again to form a portion, and then cultured as appropriate to form the second callus.

As used herein, the term "tertiary secondary callus" refers to a tissue formed by the secondary callus, after which a portion is excised again, cultured appropriately, and formed the third time.

As used herein, the term "MS medium" refers to a medium designed by Murashige and Skoog in 1962 for tobacco cell culture, which is characterized by higher concentration of inorganic salts and ions, can meet the nutritional and physiological needs of plant cells, and is a relatively common medium in plant callus culture.

In certain embodiments, the MS medium comprises the basic components that make up the medium, e.g., sucrose, agar.

In certain embodiments, the MS medium further comprises inorganic salts and ions required for plant growth, for example, ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, potassium iodide, boric acid, zinc sulfate, copper sulfate, or iron sulfate.

In certain embodiments, the MS medium further comprises an MS salt.

Advantageous effects of the application

Compared with the prior art, the application establishes the genetic transformation method of millet for the first time. Further, the applicant has repeatedly searched for optimizing a plurality of steps of the established broom corn millet genetic transformation method. Firstly, selecting mature seeds of plants as explants, wherein the sources are rich, and the acquisition period is short; secondly, on the basis of selecting seeds as explants, primary callus is selected for infection of agrobacterium, so that the transformation efficiency is further improved, and the transformation method is optimized; finally, by adjusting the culture medium used in the callus culture process, the optimal culture medium combination is obtained, the transformation efficiency is further improved, and the transformation method is optimized. Thus, the efficient broom corn millet genetic transformation method is obtained.

Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present application and are not to be construed as limiting the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 is a schematic representation of a binary vector T-DNA for genetic transformation of broom corn millet in example 2, which contains hygromycin resistance gene (hyg R) and green fluorescent protein Gene (GFP).

FIG. 2 is a flow chart of Agrobacterium-mediated genetic transformation of millet in example 2, wherein FIG. 2a is calli after three days of co-culture (open field on the left, fluorescent field on the right); FIG. 2b shows calli after hygromycin screening (bright field on the left, fluorescent field on the right); FIG. 2c is calli starting pre-differentiation (bright field on the left, fluorescent field on the right); FIG. 2d shows calli that begin to differentiate (bright field on the left, fluorescent field on the right); FIG. 2e shows positive seedlings to be transplanted to soil (bright field on the left, fluorescent field on the right); FIG. 2f shows harvested T1 generation seeds (bright field left and fluorescent field right).

FIG. 3 is a graph showing the results of PCR identification of transgenic broom corn millet plants in example 3, wherein M represents a DNA marker; w represents a non-transgenic wild type WT broom corn millet plant; CK-represents the negative control (no DNA template) of the PCR system; 1-9 respectively represent different converted broom corn millet plants; ck+ represents a transformation vector plasmid.

FIG. 4 shows the results of a concentration gradient experiment for hygromycin selection as used in example 3, wherein FIG. 4a (bright field), 4b (fluorescent field) is hygromycin-free treatment medium (normal callus left, positive transformed callus right); FIG. 4c (open field), 4d (fluorescent field) 30mg/L hygromycin treatment medium (left normal callus, right positive transformed callus); FIG. 4e (open field), 4f (fluorescent field) 50mg/L hygromycin treatment medium (left normal callus, right positive transformed callus).

Sequence information

The information of the partial sequences to which the present application relates is provided in table 1 below.

Table 1: description of the sequence

Detailed Description

The application will now be described with reference to the following examples, which are intended to illustrate the application, but not to limit it.

The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present application, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel (f.m. ausubel) et al, (1987)); series (academic publishing company) of methods in enzymology (METHODS IN ENZYMOLOGY): PCR2: practical methods (PCR 2: A PRACTICAL APPROACH) (M.J. MaxPherson (M.J. MacPherson), B.D. Thoms (B.D. Hames) and G.R. Taylor (G.R. Taylor) editions (1995)), and animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. French Lei Xieni (R.I. Freshney) editions (1987)).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the application by way of example and are not intended to limit the scope of the application as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 obtaining broom corn millet callus

To establish an efficient broom corn millet conversion system, the inventor of the application usesBroom corn milletExplants used for establishing a transformation system are selected from immature embryos, seeds, stem node meristems, young spikes, stem tips and root tips. Among them, there are many restrictions on the acquisition of part of the explants, so that the applicant selects mature seeds of broom corn millet as the explants, and this method can obtain a large amount of explants at any time in the year and be used for preparing callus for transformation.

On the basis of selecting seeds as explants, the applicant found that different generations of calli of the seeds have a large transformation rate difference. Thus, primary, secondary and tertiary calli were prepared for comparison of the subsequent transformation efficiencies in this example.

Preparation of primary callus: sterilizing mature broom corn millet seeds, sterilizing the mature broom corn millet seeds without the lemma and the No. four seed of the Longqiu of the palea with 75% alcohol for two minutes and flushing three to four times (2 minutes each) with sterilized deionized water. Placing sterilized and cleaned broom corn millet mature seeds on sterile filter paper for 15min until the broom corn millet mature seeds are thoroughly dried, inoculating the seeds on a Callus Induction Medium (CIM), culturing for 20 days under a dark condition (28 ℃) and transferring the seeds on a fresh CIM, and separating white yellowish callus after 10-15 days.

Preparation of secondary subculture callus: and transferring a better part of the primary callus which is successfully induced to a new CIM culture medium, and culturing for 14 days to separate secondary callus.

Preparation of three times of subculture calli: and selecting a better part of the secondary subculture callus after 14 days to transfer to a new CIM culture medium, and sorting out the tertiary subculture callus after 14 days of culture.

Wherein, CIM (pH=5.8) of the seed callus induction culture medium is MS basic culture medium added with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin and 3g/L Phytagel ^TM (purchased from Sigma Co., ltd., product number P8169-1 KG); after sterilization, 2mg/L AgNO is added ₃ . Wherein the MS minimal medium contains 4.33g/L MS salt (Murashige&Skoog Basal Salt Mixture from the company PHYTOTECH LABS under the accession number M524).

Example 2 obtaining transgenic broom corn millet callus and plants

FIG. 2 is a flowchart of Agrobacterium-mediated genetic transformation of millet in this example, wherein FIG. 2a is calli after three days of co-cultivation (open field on the left, fluorescent field on the right); FIG. 2b shows calli after hygromycin screening (bright field on the left, fluorescent field on the right); FIG. 2c is calli starting pre-differentiation (bright field on the left, fluorescent field on the right); FIG. 2d shows calli that begin to differentiate (bright field on the left, fluorescent field on the right); FIG. 2e shows positive seedlings to be transplanted to soil (bright field on the left, fluorescent field on the right); FIG. 2f shows harvested T1 generation seeds (bright field left and fluorescent field right). The specific experimental steps are as follows:

1) Agrobacterium tumefaciens EHA105 bacterial liquid containing a 35s-GFP gene and hygromycin resistance gene (hyg R) binary vector (figure 1) stored at-80 ℃ is sucked up to 300 mu L and added into 25ml of LB liquid medium added with 50mg/L rifampicin and 100mg/L kanamycin, and centrifuged at 3500R for 22min after overnight culture at 28 ℃ by a shaker (200 rpm), the supernatant is discarded, resuspended to OD600 = 1.8 by liquid co-culture medium (CoIM), incubated to OD600 = 2.0-3.0 at 28 ℃ and 180R, and placed on ice for later use.

2) The three embryogenic calli obtained in the example 1 are respectively divided into small pieces (2 mm-3 mm), picked up and put into a 50ml centrifuge tube containing liquid CoIM, placed on ice for precooling for 20min, and then subjected to incubation at 26 ℃ and 100r for half an hour in the invasion solution prepared in the step (1); the calli were then transferred to solid CoIM and incubated for 3 days at 22℃under dark conditions.

3) Transferring the callus after co-cultivation in the step (2) to CIM added with 150mg/L Tintin, and performing dark cultivation at 28 ℃ for 15 days, and then transferring the callus to SM, performing dark cultivation at 28 ℃ for 7-10 days.

4) Inoculating the callus with good growth condition screened in the step (3) to a pre-differentiation culture medium (PRM), and transferring the culture medium to an illumination culture chamber (26+/-2 ℃;16h light/8h dark), a layer of gauze is covered on the culture medium to prevent the illumination from being too strong, the differentiated and greenish callus is transferred to a differentiation medium (RM) after fourteen days, the differentiated callus is induced to root, seedlings with better growth condition (seedlings with the leaf length exceeding 3 cm) in the medium are transferred into PM after every 15-20 days, and the rest tissues are transferred into a new RM medium.

5) Transplanting the seedlings which grow to more than 15cm after being transplanted into the RM culture medium in the step (4) into soil, and harvesting the T1 generation seeds about 4 months.

The agrobacterium tumefaciens EHA105 carries a binary vector containing hygromycin resistance gene (hyg R) and green fluorescent protein Gene (GFP).

The coculture medium CoIM (pH=5.4) was added with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 50mg/L inositol, 0.1mg/L thiamine hydrochloride, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin to MS minimal medium, and 3g/L Phytagel was added to the solid coculture medium ^TM (200. Mu.M/L acetosyringone after sterilization).

The resistant callus screening culture medium SM (pH=5.8) is prepared by adding 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin and 3g/L Phytagel to MS basic culture medium ^TM (after sterilization +2mg/L AgNO3, +50mg/L hygromycin, +150mg/L Tintin (which is a novel gram-negative antibiotic, acting to inhibit Agrobacterium growth, purchased from Solarbio Co., under the designation T8660)).

The pre-differentiation culture medium PRM (pH=5.8) is prepared by adding 1 mg/Ld-biotin, 0.5mg/L pyridoxine hydrochloride and 0.5mg/L smoke to MS basic culture mediumAcid, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose, 0.2 mg/L2,4-D, 3 mg/L6-BA, 3g/L Phytagel ^TM (after sterilization +150 mg/LTimentin).

The differentiation medium RM (pH=5.8) is prepared by adding 1mg/L d-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose and 3g/L LPhytagel into MS basic culture medium ^TM (150 mg/L Tintin after sterilization).

The seedling culture medium PM (pH=5.8) is 1/2MS minimal medium added with 0.25mg/L pyridoxine hydrochloride, 0.25mg/L nicotinic acid, 100mg/L inositol, 0.05mg/L thiamine hydrochloride, 15g/L sucrose, 1mM isoleucine, 8g/L agar (after sterilization +100 mg/LTimentin). Wherein the 1/2MS minimal medium contained 2.165g/L MS salt (Murashige & Skoog Basal Salt Mixture, available from PHYTOTECH LABS Co., ltd., cat. M524).

Example 3 molecular characterization of transgenic broom corn millet plants obtained by hygromycin resistance screening

In this example, the transformation efficiencies of the calli and plants obtained in example 2 were respectively examined by PCR amplification methods as follows:

1. transformation efficiency of callus

DNA is extracted from 2-5mm callus after about 10 days of screening culture by a CTAB method, and PCR amplification of exogenous gene (Hyg R) is performed, so that the method can be used for identifying positive transgenic broom corn millet callus.

The PCR primers for detecting the exogenous gene (Hyg R) are 35s-Hyg-F and 35s-Hyg-R (the nucleotide sequences are shown as SEQ ID NO:3 and SEQ ID NO: 4).

The amplification conditions for PCR were: 95 ℃ for 5min; then, 33 cycles were performed, each cycle being conditioned. 95 ℃ for 30s;58 ℃ for 30s;72 ℃,1min for 30s; and finally, at 72 ℃ for 10min.

Successful transformation of each callus (hyg R gene identified) and successful expression of GFP gene (visible as distinct fluorescence under a fluorescent field) was considered successful, and each callus was able to successfully transform and differentiate into viable seedlings, considered a successful transformation event.

2. Transformation efficiency of plants

And (3) taking transgenic broom corn millet plants grown in a greenhouse for 1 month, taking leaves with the top of 1cm, extracting DNA by a CTAB method, and carrying out PCR amplification of exogenous genes (Hyg R) to identify positive transgenic broom corn millet plants.

The genetic transformation efficiency of the broom corn millet is achieved through the number of successful transformation events (the number of calli which can be successfully transformed and differentiated to contain exogenous genes): the number of callus blocks before the infection of agrobacterium is calculated.

3. Results

The PCR identification junction of the transgenic broom corn millet plant in the embodiment is shown in fig. 3, wherein M represents a DNA marker; w represents a non-transgenic wild type WT broom corn millet plant; CK-represents the negative control (no DNA template) of the PCR system; 1-9 respectively represent different converted broom corn millet plants; ck+ represents a transformation vector plasmid.

TABLE 1 relationship between callus number of times and transformation rate

Experimental results show that the primary callus is more suitable for the broom corn millet transformation system established by the application, and the transformation efficiency is obviously better than that of the secondary callus and the tertiary callus.

EXAMPLE 4 callus culture

From the results of the above examples (Table 1), the transformation rate was greatly reduced from callus to final emergence. Therefore, the applicant also compares the culture methods of various calli in the process of establishing the broom corn millet conversion system, and obtains an optimized broom corn millet conversion system.

The present application was carried out in a number of parallel experiments according to the method described in examples 1-3, except that in the procedure of example 2, different media were used to treat the calli, as follows:

1. first experiment group

Co-culture Medium CoIM (pH=5.4) 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 50mg/L inositol, 0.1mg/L thiamine hydrochloride, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin were added to MS minimal medium, and 3g/LPhytagel was added to solid co-culture medium ^TM (200. Mu.M/L acetosyringone after sterilization).

Resistant callus screening Medium SM (pH=5.8) 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin, 3g/L Phytagel were added to MS minimal medium ^TM (after sterilization +2mg/LAgNO3, +50mg/L hygromycin, +150mg/L Tintin).

Pre-fractionation Medium PRM (pH=5.8) 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose, 0.2 mg/L2,4-D, 3 mg/L6-BA, 3g/L Phytagel were added to MS minimal medium ^TM (after sterilization +150 mg/LTimentin).

Differentiation Medium RM (pH=5.8) 1mg/L d-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose, 3g/LPhytagel were added to MS minimal medium ^TM (150 mg/L Tintin after sterilization).

Seedling medium PM (ph=5.8) was 1/2MS minimal medium supplemented with 0.25mg/L pyridoxine hydrochloride, 0.25mg/L niacin, 100mg/L inositol, 0.05mg/L thiamine hydrochloride, 15g/L sucrose, 1mM isoleucine, 8g/L agar (after sterilization +100mg/L Timentin).

2. Second experiment group

The experimental group was intended to investigate the effect of hygromycin content in the SM medium on callus, and therefore the method of the experimental group was identical to the previous experimental group, except that three different SM media were used, the specific formulation was as follows:

(1) Formula I: SM (pH=5.8) 1mg/L D-Biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin, 3g/L Phytagel were added to MS minimal medium ^TM (after sterilization +2mg/L AgNO3, +150mg/L Tintin).

(2) And the formula II: SM (pH=5.8) 1mg/L D-Biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin, 3g/L Phytagel were added to MS minimal medium ^TM (after sterilization +2mg/L AgNO3, +30mg/L hygromycin, +150mg/L Tintin).

(3) And the formula III: SM (pH=5.8) 1mg/L D-Biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin, 3g/L Phytagel were added to MS minimal medium ^TM (after sterilization +2mg/L AgNO3, +50mg/L hygromycin, +150mg/L Tintin).

The results of the second experimental group are shown in fig. 4, fig. 4a (bright field), 4b (fluorescent field) for hygromycin-free SM medium (normal callus on the left and positive transformed callus on the right); FIG. 4c (open field), results for SM medium (left plain callus, right positive transformed callus) at 30mg/L hygromycin for 4d (fluorescent field); FIG. 4e (open field), results for SM medium (left plain callus, right positive transformed callus) at 50mg/L hygromycin for 4f (fluorescent field).

As can be seen from FIG. 4, when 50mg/L hygromycin was contained in the SM medium, the growth of the calli was optimal, and at this concentration, positive calli were seen to grow well, and negative calli were essentially brown and dead.

3. Third experimental group

The experimental group was intended to investigate the effect of RM medium on calli, and therefore the method of the experimental group was identical to the previous experimental group, the only difference being that different RM media were used, the specific formulation being: MS minimal medium added 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose, 0.2 mg/L2,4-D, 3 mg/L6-BA, 3g/L Phytagel ^TM (150 mg/L Tintin after sterilization).

Results: compared with the first experimental group, the callus of the third experimental group using different RM culture medium can not root about 80% of the callus after differentiation and budding, and finally whiten and die, so that the conversion rate is greatly reduced.

4. Fourth experiment group

The experimental group was intended to investigate the effect of the CoIM medium on callus, and therefore the method of the experimental group was identical to the previous experimental group, except that different CoIM media were used, and the specific formulation was as follows: 0.44g/LMS salt, 1 XB 5 vitamin, 68g/L sucrose, 36g/L glucose, 1g/L asparagine, 1g/L casein hydrolysate, 0.2g/L cysteine, 2mg/L2,4-D, (200. Mu.M acetosyringone added after sterilization) (pH=5.2).

Results: callus of the fourth experimental group using a different CoIM medium failed to be transformed successfully, as compared to the first experimental group.

In summary, from the experimental results of this example, it can be seen that the combination of media used in the method of the present application can achieve higher transformation efficiency and better transformation effect than other combinations of media.

Although specific embodiments of the application have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the application is intended to be within the scope of the application. The full scope of the application is given by the appended claims together with any equivalents thereof.

SEQUENCE LISTING

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Claims

1. A method for genetic transformation of broom corn millet, the method comprising:

step (1): taking broom corn millet seeds as explants to obtain primary callus;

step (8): placing the product obtained in the step (7) into a seedling culture medium so as to enable seedlings of the callus to grow;

preferably, the amino acid sequence of the target gene is shown as SEQ ID NO. 2;

preferably, the amino acid sequence of the hygromycin resistance gene is shown as SEQ ID NO. 1.

2. The method of claim 1, having one or more features selected from the group consisting of:

(1) In step (3), the co-culture medium has a ph=5.4, which comprises or consists of MS minimal medium supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 50mg/L inositol, 0.1mg/L thiamine hydrochloride, 30g/L sucrose and 2mg/L2,4-D and 0.5mg/L kinetin; and 200 mu M/L acetosyringone is added after sterilization;

preferably, the co-culture medium is a solid co-culture medium; preferably, the solid co-culture medium further comprises 3g/L Phytagel ^TM ；

(2) In step (4), the induction medium has a ph=5.8, which comprises or is supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin and 3g/L Phytagel from MS minimal medium ^TM Composition; and, after sterilization, 2mg/L AgNO is added ₃ ；

(3) In step (5), the screening medium has a ph=5.8, which comprises or is supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 50mg/L inositol, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 30g/L sucrose, 2mg/L2,4-D,0.5mg/L kinetin and 3g/L Phytagel from MS minimal medium ^TM Composition; and, after sterilization, 2mg/L AgNO is added ₃ 50mg/L hygromycin, and 150mg/L Timentin;

(4) In step (6), the pre-fractionation medium has a ph=5.8, which comprises or is supplemented with 1mg/L D-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose, 0.2 mg/L2,4-D, 3 mg/L6-BA and 3g/L Phytagel from MS minimal medium ^TM Composition; and 150mg/L Tintin is added after sterilization;

(5) In step (7), the differentiation medium has a ph=5.8, which comprises or is supplemented with 1mg/L d-biotin, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L niacin, 300mg/L casein hydrolysate, 0.1mg/L thiamine hydrochloride, 0.6mg/L copper sulfate, 15g/L sucrose, 15g/L glucose and 3g/L Phytagel ^TM Composition; and 150mg/L Tintin is added after sterilization;

(6) In step (8), the ph=5.8 of the seedling medium comprising or consisting of 1/2MS minimal medium supplemented with 0.25mg/L pyridoxine hydrochloride, 0.25mg/L niacin, 100mg/L inositol, 0.05mg/L thiamine hydrochloride, 15g/L sucrose, 1mM isoleucine and 8g/L agar; and 100mg/L Tintin is added after sterilization;

preferably, the 1/2MS minimal medium comprises or consists of 4.33g/L MS salts;

preferably, the MS minimal medium comprises or consists of 2.165g/L MS salts.

3. The method of claim 1 or 2, wherein, in step (1), seeds of broom corn millet are used as explants and inoculated on induction medium to obtain primary calli;

preferably, in the step (1), mature seeds of broom corn millet are sterilized, lemma and palea are removed, sterilized with 75% alcohol for two minutes, and rinsed three to four times with sterilized deionized water for 2 minutes each; the sterilized and washed seeds were placed on sterile filter paper until they were thoroughly dried, then the seeds were inoculated on an induction medium, and after culturing for 20 days at 28℃in the dark, they were transferred to a fresh induction medium until white yellowish primary calli were isolated.

4. A method according to any one of claims 1 to 3, wherein in step (2) an expression vector containing a gene of interest and a hygromycin resistance gene is infected with primary callus by agrobacterium tumefaciens such that the gene of interest and hygromycin resistance gene are integrated into the genome of the primary callus;

preferably, the strain of agrobacterium tumefaciens is EHA105;

preferably, 300. Mu.L of Agrobacterium tumefaciens EHA105 bacterial liquid containing the vector is pipetted into 25ml LB and 50mg/L rifampicin and 100mg/L kanamycin are added and cultured overnight at 28℃before step (2); then centrifuged for 22min, the supernatant discarded and resuspended in liquid co-culture medium to od600=1.8; incubating at 28 ℃ until the OD600 = 2.0-3.0, and placing on ice for standby to obtain agrobacterium tumefaciens bacteria liquid; wherein the vector contains a gene of interest and a hygromycin resistance gene.

5. The method according to any one of claims 1 to 4, wherein, in step (3), the embryogenic callus obtained in step (2) is divided into small pieces of 2mm to 3mm, picked up and placed in a co-culture medium containing a liquid, placed on ice for pre-cooling for 20min, then placed in the agrobacterium tumefaciens bacteria solution, and incubated for half an hour at 26 ℃; the calli were then transferred to solid co-culture medium at 22℃for 3 days in the dark.

6. The method of any one of claims 1-5, wherein in step (4), the induction medium further comprises 150mg/L Timentin;

preferably, in step (4), the co-cultured calli are transferred to induction medium containing 150mg/L Timentin and dark cultured at 28℃for 15 days.

7. The method according to any one of claims 1 to 6, wherein, in step (5), the product obtained in step (4) is placed in a screening medium containing 50mg/L hygromycin and is dark-cultured at 28℃for 7 to 10 days to screen calli containing a hygromycin resistance gene.

8. The method according to any one of claims 1 to 7, wherein, in step (6), the selected well-grown callus obtained in step (5) is inoculated into a pre-differentiation medium, and the pre-differentiation medium is transferred to an illumination culture room for culturing for 14 days, and a gauze layer is covered on the pre-differentiation medium to prevent the illumination from being too strong.

9. The method according to any one of claims 1 to 8, wherein, in step (7), the differentiated green-turning callus obtained in step (6) is transferred to a differentiation medium to root the callus.

10. The method according to any one of claims 1 to 9, wherein in step (8) seedlings with leaf lengths exceeding 3cm are transferred into a seedling medium to allow the seedlings of the calli to grow.