CN107142277B - High-efficiency genetic transformation method for indica rice - Google Patents
High-efficiency genetic transformation method for indica rice Download PDFInfo
- Publication number
- CN107142277B CN107142277B CN201710512324.7A CN201710512324A CN107142277B CN 107142277 B CN107142277 B CN 107142277B CN 201710512324 A CN201710512324 A CN 201710512324A CN 107142277 B CN107142277 B CN 107142277B
- Authority
- CN
- China
- Prior art keywords
- callus
- days
- agrobacterium
- culture medium
- inositol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a high-efficiency indica rice genetic transformation method, which comprises the following steps: inducing the callus of mature embryo of indica rice, pre-culturing the callus, culturing agrobacterium tumefaciens, transforming the agrobacterium tumefaciens mediated, co-culturing the agrobacterium tumefaciens and the callus, screening the resistant callus, differentiating and rooting the regenerated seedling; the method is simple, convenient and efficient, the obtained callus has good quality, is suitable for transformation, the transformation operation process is simple and convenient, indica rice positive transformation plants can be obtained after about 60 days by transforming the indica rice type two-line sterile line 628S and the indica rice type two-line restorer line 2537, the transformation efficiency is up to more than 35%, the transformation rate is high, and the genetic transformation efficiency of indica rice is improved.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture and transgenic engineering, and particularly relates to a high-efficiency genetic transformation method of indica rice, which is suitable for inducing and subculturing calluses of mature seeds of indica rice and genetic transformation methods of agrobacterium-mediated embryonic calluses.
Background
Rice (Oryza sativa L.) is one of the most important food crops in China and is divided into two major subspecies, namely indica rice and japonica rice, wherein the indica rice is the largest cultivated rice type in China, and the cultivated area accounts for about 74% of the area of rice in China. The transgenic technology is utilized to improve the rice variety, and the cultivation of a new variety with high yield, high quality, multiple resistance and wide adaptability is the development direction of rice breeding in the future while guaranteeing the global grain safety. Therefore, genetic transformation of rice, especially genetic transformation of indica rice, plays an increasingly important role in variety improvement, gene function detection and mutant creation, and establishment of an efficient and stable indica rice genetic transformation system is a precondition for research in the fields of molecular breeding, functional genomics and the like.
In recent years, the rice genetic transformation technology makes an important breakthrough, and a rapid, efficient and stable genetic transformation system is mostly established in japonica rice. On the basis, many exogenous genes with important traits such as insect resistance, disease resistance, stress resistance, quality improvement, development regulation, high-efficiency utilization of nutrition and the like have been transferred into rice. However, for indica rice with a large planting area, the tissue culture characteristics are generally poor due to the difference of genotypes, and the transformation difficulty is obviously higher than that of japonica rice. In recent years, some success is achieved by utilizing agrobacterium tumefaciens mediated transformation to transform indica rice, but a stable and efficient genetic transformation system like japonica rice is not established yet.
Research has shown that the agrobacterium-mediated gene transformation efficiency is related to factors such as callus state, culture medium, infection mode and the like of receptor genotype. Therefore, by optimizing parameters of various stages of indica rice genetic transformation, a high-efficiency indica rice genetic transformation system is expected to be established.
Disclosure of Invention
The invention aims to provide a high-efficiency indica rice genetic transformation method, the callus obtained by the method has good quality, is suitable for transformation, the transformation operation process is simple and convenient, the transformation rate is high, 2 indica rice varieties tested are successful, and the transformation efficiency is between 35 and 40 percent.
The invention is realized by the following technical scheme:
a high-efficiency genetic transformation method of indica rice comprises the following steps:
(1) induction of callus
Selecting mature and complete indica rice grains, sterilizing with 75% alcohol for 3 min, soaking in 25% (V/V) sodium hypochlorite solution, and sterilizing in a shaking table at 150rpm and 25 deg.C for 20 min; washing the sterilized seeds with sterile water on an ultra-clean workbench for 4-5 times, transferring to sterile filter paper, sucking water, inoculating appropriate amount of seeds to NB culture medium, culturing at 28-30 deg.C under illumination for 8-10 days, and inducing callus;
(2) preculture of callus
Selecting compact-structure bright-color granular callus from the callus in the step (1), transferring the callus into a pre-culture medium, and culturing for 3 days at 28-30 ℃ for transformation;
(3) agrobacterium culture and Agrobacterium-mediated transformation
Transferring a plant expression vector containing a target gene into agrobacterium tumefaciens EHA105 by an electric excitation transformation method, selecting a positive strain, marking on a YEB resistant plate, carrying out dark culture at 28 ℃ for 3 days, scraping the agrobacterium tumefaciens with the size of rice grains by using an inoculating needle, shaking and suspending the agrobacterium tumefaciens in a 150ml triangular flask filled with 70ml of agrobacterium tumefaciens staining solution, placing the triangular flask in a shaking table at 150rpm and 28 ℃ for 10 minutes, then soaking the indica rice callus cultured for 3 days by illumination in the step (2) in the agrobacterium tumefaciens staining solution, and shaking the indica rice callus on the shaking table at 150rpm and 28 ℃ for 10 minutes;
(4) co-culture of Agrobacterium and callus
Placing the soaked indica rice calluses in the step (3) on sterile filter paper for drying, then transferring the calluses to a co-culture medium, pre-filling a piece of sterile filter paper on a co-culture medium flat plate, wetting the co-culture medium flat plate by using 1ml of agrobacterium tumefaciens soaking liquid, removing bubbles, and carrying out dark culture at 28 ℃ for 3 days;
(5) selection of resistant callus
Placing the callus cultured for 3 days in the step (4) in a 250ml triangular flask, and washing with sterile water for 4-5 times until a washing solution is clear and transparent; then adding a 500mg/L carbenicillin solution for filtration and sterilization to wash the callus, placing the callus on a shaking table at 150rpm at room temperature for shaking and washing for 15min, then placing the callus on sterile filter paper to absorb water, inoculating the callus on a screening culture medium containing a corresponding screening agent for screening, and culturing the callus under the conditions of 28 ℃ and illumination for 20-22 days, and carrying out subculture once in the middle until resistant callus grows out;
(6) differentiation of resistant callus
Transferring the resistant callus obtained after screening in the step (5) to a differentiation culture medium containing a screening agent, and culturing for 18-23 days at 28 ℃ under illumination, wherein the intermediate subculture is performed once to regenerate green seedlings;
(7) rooting of regenerated plantlets
Transferring the seedling grown on the differentiation culture medium in the step (6) into a rooting culture medium containing a screening agent, and culturing for 10-12 days at 30 ℃ under illumination to obtain a regenerated seedling.
The NB medium in the step (1) comprises the following components: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, glutamine 0.5g/L, acid hydrolyzed casein 0.5g/L, proline 2.878g/L, cane sugar 30.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L and pH 5.8.
The pre-culture medium in the step (2) comprises the following components: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, glutamine 0.5g/L, acid hydrolyzed casein 0.5g/L, proline 2.878g/L, cane sugar 30.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L and acetosyringone 0.1mM, and pH 5.8.
The agrobacterium tumefaciens staining solution in the step (3) comprises the following components: AA inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 0.5g/L, sucrose 68.5g/L, glucose 36.0g/L, glutamine 0.9g/L, asparagine 0.3g/L and arginine 0.176g/L, and the pH value is 5.2; 0.1mM acetosyringone is added before use.
The components of the co-culture medium in the step (4) are as follows: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 0.3g/L, cane sugar 30.0g/L, glucose 10.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L, pH 5.2, and acetosyringone 0.1mM after sterilization.
The screening medium in the step (5) comprises the following components: n is a radical of6Inorganic salt and organic matter + inositol 0.1g/L + acid hydrolyzed casein 0.5g/L + proline 2.878g/L + glutamine 0.3g/L + sucrose 30.0g/L + phytohemagglutinin 4.0g/L +2,4-D2.0mg/L, pH 5.8; after sterilization, adding carbenicillin 400mg/L + hygromycin 30mg/L or Basta4 mg/L.
The components of the differentiation medium in the step (6) are as follows: MS inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 2.0g/L, proline 0.3g/L, cane sugar 30.0g/L, sorbitol 30g/L, phytohemagglutinin 4.0g/L, NAA0.02mg/L, Kinetin2mg/L and pH 5.8; after sterilization, adding carbenicillin 400mg/L + hygromycin 30mg/L or Basta4 mg/L.
The rooting medium in the step (7) comprises the following components: MS inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 2.0g/L, proline 0.3g/L, cane sugar 30.0g/L, phytohemagglutinin 4.0g/L and pH 5.8; after sterilization, adding 400mg/L carbenicillin.
The invention has the advantages that:
the invention carries out agrobacterium EHA105 mediated transformation on 2 indica rice representative varieties indica type two-line sterile line 628S and indica type two-line restorer line 2537, and the transformation efficiency is high and is more than 35 percent; the callus induced by the method is good in state and suitable for transformation, and the whole transformation operation process is short in period.
Drawings
The invention is further explained below with reference to the drawings and the embodiments.
FIG. 1 shows callus induced by mature embryos of indica two-line restorer line 2537(a) and indica two-line sterile line 628S (b) of the present invention;
FIG. 2 shows pre-culture of callus of indica type two-line restorer 2537(a) and indica type two-line sterile 628S (b) of the present invention;
FIG. 3 shows the callus of the indica two-line restorer line 2537(a) and the indica two-line sterile line 628S (b) of the present invention screened for 10 days;
FIG. 4 shows calluses of indica two-line restorer 2537(a) and indica two-line sterile 628S (b) differentiated for 10 days in the present invention;
FIG. 5 shows the rooting culture of the seedlings of the indica two-line restorer 2537(a) and the indica two-line sterile 628S (b) of the present invention after differentiation;
FIG. 6 shows part T of the restorer line 2537(a) and sterile line 628S (b) of indica two lines0GUS staining and identifying the leaves of the generation plants;
FIG. 7 shows part T of the restorer line 2537(a) and sterile line 628S (b) of indica two lines0PCR detection electrophoresis picture of plant hygromycin gene.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
(1) Induction of callus
Selecting mature and complete indica rice variety indica type two-line restorer line 2537 rice grains, sterilizing with 75% alcohol for 3 min, soaking in 25% (V/V) sodium hypochlorite solution, and sterilizing in a shaking table at 150rpm and 25 deg.C for 20 min; washing the disinfected seeds for 5 times by using sterile water on a superclean bench, then sucking water by using sterile filter paper, and inoculating the seeds to an NB culture medium; callus was induced by light culture at 28-30 ℃ for 8 days (FIG. 1a), wherein NB medium components were: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, glutamine 0.5g/L, acid hydrolyzed casein 0.5g/L, proline 2.878g/L, cane sugar 30.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L and pH 5.8.
(2) Preculture of callus
Selecting compact-structure bright-color granular callus, transferring the callus into a pre-culture medium, and culturing the callus in light at 28-30 ℃ for 3 days for transformation (figure 2a), wherein the pre-culture medium comprises the following components: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, glutamine 0.5g/L, acid hydrolyzed casein 0.5g/L, proline 2.878g/L, cane sugar 30.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L and acetosyringone 0.1mM, and pH 5.8.
(3) Agrobacterium culture and Agrobacterium-mediated transformation
Transferring a plant expression vector containing a target gene into EHA105 by an electric excitation transformation method, streaking a transformed positive EHA105 agrobacterium strain on a YEB resistant plate, carrying out dark culture at 28 ℃ for 3 days, scraping the agrobacterium with the size of rice grains by using an inoculating needle, oscillating and suspending the agrobacterium in a 150ml triangular flask filled with 70ml of agrobacterium staining solution, and placing the flask on a shaking table at 150rpm and 28 ℃ for 10 minutes; meanwhile, the callus after 3 days of pre-culture is soaked in the agrobacterium tumefaciens staining solution and shaken for 10 minutes on a shaking table with 150rpm and 28 ℃; wherein, the components of the agrobacterium tumefaciens staining solution are as follows: AA inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 0.5g/L, sucrose 68.5g/L, glucose 36.0g/L, glutamine 0.9g/L, asparagine 0.3g/L and arginine 0.176g/L, and the pH value is 5.2; 0.1mM acetosyringone is added before use.
(4) Co-culture of Agrobacterium and callus
The impregnated callus was placed on sterile filter paper and water was blotted for use. A piece of sterile filter paper is padded on a co-culture medium flat plate, then 1ml of agrobacterium tumefaciens staining solution is used for wetting, air bubbles are removed, then the callus with water absorbed in advance is transferred to a co-culture medium, and dark culture is carried out for 3 days at 28 ℃; wherein the components of the co-culture medium are as follows: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 0.3g/L, sucrose 30.0g/L, glucose 10.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L, pH 5.2, and acetosyringone 0.1mM after sterilization.
(5) Selection of resistant callus
The callus after 3 days of co-culture was placed in a 250ml Erlenmeyer flask and washed 4 times with sterile water until the wash was clear and transparent. Adding a 500mg/L carbenicillin solution for filtration and sterilization to wash the callus, placing the callus on a shaking table at 150rpm at room temperature for shaking and washing for 15min, placing the callus on sterile filter paper for absorbing water, and inoculating the callus on a screening culture medium containing a corresponding screening agent for screening; the selection culture conditions were 28 ℃, light culture for 20 days, and subculture every 10 days until resistant callus was grown (fig. 3a), wherein the selection medium components were: n is a radical of6Inorganic salt and organic matter + inositol 0.1g/L + acid hydrolyzed casein 0.5g/L + proline 2.878g/L + glutamine 0.3g/L + sucrose 30.0g/L + phytohemagglutinin 4.0g/L +2,4-D2.0mg/L, pH 5.8; after sterilization, adding carbenicillin 400mg/L + hygromycin 30mg/L or Basta4 mg/L.
(6) Differentiation of resistant callus
Transferring the screened resistant callus onto a differentiation medium containing a screening agent, culturing for 20 days at 28 ℃ under light, and subculturing every 10 days to regenerate green seedlings (figure 4a), wherein the differentiation medium comprises the following components: MS inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 2.0g/L, proline 0.3g/L, cane sugar 30.0g/L, sorbitol 30g/L, phytohemagglutinin 4.0g/L, NAA0.02mg/L, Kinetin2mg/L and pH 5.8; after sterilization, adding carbenicillin 400mg/L + hygromycin 30mg/L or Basta4 mg/L.
(7) Rooting of regenerated plantlets
Transferring the seedlings grown on the differential culture into a rooting culture medium containing a screening agent, and culturing for 10 days at 30 ℃ under illumination to obtain regenerated seedlings; wherein, the rooting medium comprises the following components: MS inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 2.0g/L, proline 0.3g/L, cane sugar 30.0g/L, phytohemagglutinin 4.0g/L and pH 5.8; after sterilization, adding 400mg/L carbenicillin.
Example 2
(1) Selecting mature and complete indica rice 628S rice grains, sterilizing with 75% alcohol for 3 min, soaking in 25% (V/V) sodium hypochlorite solution, and sterilizing in a shaker at 25 deg.C and 150rpm for 20 min. The sterilized seeds were washed 5 times with sterile water on a clean bench, then blotted dry with sterile filter paper, and then inoculated into NB medium. Culturing at 28-30 deg.C for 10 days under illumination to induce callus (FIG. 1 b); the NB medium comprises the following components: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, glutamine 0.5g/L, acid hydrolyzed casein 0.5g/L, proline 2.878g/L, cane sugar 30.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L and pH 5.8. .
(2) Preculture of callus
Selecting compact-structured, bright-colored and granular callus, transferring the callus into a pre-culture medium, and culturing the callus under illumination at 28-30 ℃ for 3 days for transformation (figure 2 b); the pre-culture medium comprises the following components: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, glutamine 0.5g/L, acid hydrolyzed casein 0.5g/L, proline 2.878g/L, cane sugar 30.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L and acetosyringone 0.1mM, and pH 5.8.
(3) Agrobacterium culture and Agrobacterium-mediated transformation
The plant expression vector containing the target gene is transferred into the EHA105 by an electric excitation transformation method. Streaking the transformed positive EHA105 strain on a YEB resistant plate, carrying out dark culture at 28 ℃ for 3 days, scraping the agrobacterium with the size of rice grains by using an inoculating needle, oscillating and suspending the agrobacterium in a 150ml triangular flask filled with 70ml of agrobacterium staining solution, and shaking the flask on a shaking table with the rotation speed of 150rpm and the temperature of 28 ℃ for 10 minutes; simultaneously, soaking the indica rice callus after pre-culturing for 3 days in the agrobacterium tumefaciens dip-dyeing solution, and shaking for 10 minutes on a shaking table with 150rpm and 28 ℃; the components of the agrobacterium tumefaciens dip dyeing solution are as follows: AA inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 0.5g/L, sucrose 68.5g/L, glucose 36.0g/L, glutamine 0.9g/L, asparagine 0.3g/L and arginine 0.176g/L, and the pH value is 5.2; 0.1mM acetosyringone is added before use.
(4) Co-culture of Agrobacterium and callus
The impregnated callus was placed on sterile filter paper and water was blotted for use. A piece of sterile filter paper is padded on a co-culture medium flat plate, then 1ml of agrobacterium tumefaciens staining solution is used for wetting, air bubbles are removed, then the callus with water absorbed in advance is transferred to a co-culture medium, and dark culture is carried out for 3 days at 28 ℃; the components of the co-culture medium are as follows: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 0.3g/L, sucrose 30.0g/L, glucose 10.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L, pH 5.2, and acetosyringone 0.1mM after sterilization.
(5) Selection of resistant callus
The callus after 3 days of co-culture was placed in a 250ml Erlenmeyer flask and washed 4 times with sterile water until the wash was clear and transparent. Adding filter-sterilized carbenicillin solution 500mg/L to wash the callus, placing the callus on a shaking table at 150rpm for shaking and washing for 15min at room temperature, placing the callus on sterile filter paper to suck water, and inoculating the callus on a screening culture medium containing a corresponding screening agent for screening. The selection culture conditions were 28 ℃ and light culture for 22 days, and subculture every 11 days until resistant callus was grown (FIG. 3 b); the screening medium comprises the following components: n is a radical of6Inorganic salt and organic matter + inositol 0.1g/L + acid hydrolyzed casein 0.5g/L + proline 2.878g/L + glutamine 0.3g/L + sucrose 30.0g/L + phytohemagglutinin 4.0g/L +2,4-D2.0mg/L, pH 5.8; after sterilization, adding carbenicillin 400mg/L + hygromycin 30mg/L or Basta4 mg/L.
(6) Differentiation of resistant callus
Transferring the screened resistant callus to a differentiation medium containing a screening agent, culturing for 18 days at 28 ℃ under illumination, and subculturing once every 9 days to regenerate green seedlings (figure 4 b); the components of the differentiation medium are as follows: MS inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 2.0g/L, proline 0.3g/L, cane sugar 30.0g/L, sorbitol 30g/L, phytohemagglutinin 4.0g/L, NAA0.02mg/L, Kinetin2mg/L and pH 5.8; after sterilization, adding carbenicillin 400mg/L + hygromycin 30mg/L or Basta4 mg/L.
(7) Rooting of regenerated plantlets
Transferring the seedling grown on the differentiation culture into a rooting culture medium containing a screening agent, and culturing for 12 days at 30 ℃ under illumination to obtain a regenerated seedling (figure 5 b); the rooting medium comprises the following components: MS inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 2.0g/L, proline 0.3g/L, cane sugar 30.0g/L, phytohemagglutinin 4.0g/L and pH 5.8; after sterilization, adding 400mg/L carbenicillin.
In example 1, the present invention transformed calluses of two-line restorer 2537 of indica rice variety, screened for hygromycin resistance in 63 transformed calluses, and subjected to GUS staining (fig. 6a) and PCR detection (fig. 7a) on leaves of T0 generation plants to finally obtain 24 positive transgenic plants, wherein the transformation efficiency of the 24 positive transgenic plants reaches 38.1%.
In example 2, the present invention transformed the 628S callus of the indica two-line sterile line, screened 54 transformed calli for hygromycin resistance, and performed GUS staining (fig. 6b) and PCR detection (fig. 7b) on the leaves of T0 generation plants, and finally obtained 20 positive transgenic plants with transformation efficiency up to 37.04%.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (1)
1. A high-efficiency genetic transformation method of indica rice is characterized by comprising the following steps:
step 1: induction of callus
Selecting mature and complete indica rice grains, sterilizing with 75% alcohol for 3 min, soaking in 25% (V/V) sodium hypochlorite solution, and sterilizing in a shaking table at 150rpm and 25 deg.C for 20 min; washing the sterilized seeds with sterile water on an ultra-clean workbench for 4-5 times, transferring to sterile filter paper, sucking water, inoculating appropriate amount of seeds to NB culture medium, culturing at 28-30 deg.C under illumination for 8-10 days, and inducing callus;
step 2: preculture of callus
Selecting compact-structure bright-color granular callus from the callus in the step 1, transferring the callus into a pre-culture medium, and culturing the callus under illumination at 28-30 ℃ for 3 days for transformation;
and step 3: agrobacterium culture and Agrobacterium-mediated transformation
Transferring a plant expression vector containing a target gene into an agrobacterium strain EHA105 by an electric excitation transformation method, selecting a positive strain, marking on a YEB resistant flat plate, carrying out dark culture at 28 ℃ for 3 days, scraping the agrobacterium with the size of rice grains by using an inoculating needle, shaking and suspending the agrobacterium in a 150ml triangular flask filled with 70ml of agrobacterium staining solution, shaking for 10 minutes on a shaking table at 150rpm and 28 ℃, soaking the indica rice callus cultured for 3 days in the step 2 in illumination in the agrobacterium staining solution, and shaking for 10 minutes on the shaking table at 150rpm and 28 ℃;
and 4, step 4: co-culture of Agrobacterium and callus
Placing the impregnated indica rice calluses in the step 3 on sterile filter paper for drying, then transferring the calluses to a co-culture medium, pre-filling a piece of sterile filter paper on a co-culture medium flat plate, wetting by using 1ml of agrobacterium tumefaciens impregnation liquid, removing bubbles, and carrying out dark culture at 28 ℃ for 3 days;
and 5: selection of resistant callus
Placing the callus cultured for 3 days in the step 4 in a 250ml triangular flask, and washing with sterile water for 4-5 times until the washing liquid is clear and transparent; then adding a 500mg/L carbenicillin solution for filtration and sterilization to wash the callus, placing the callus on a shaking table at 150rpm at room temperature for shaking and washing for 15min, then placing the callus on sterile filter paper to absorb water, inoculating the callus on a screening culture medium containing a corresponding screening agent for screening, and culturing the callus under the conditions of 28 ℃ and illumination for 20-22 days, and carrying out subculture once in the middle until resistant callus grows out;
step 6: differentiation of resistant callus
Transferring the resistant callus obtained by screening in the step 5 onto a differential medium containing a screening agent, and culturing for 18-23 days at 28 ℃ under illumination, wherein the subculture is performed once to regenerate green seedlings;
and 7: rooting of regenerated plantlets
Transferring the seedlings growing on the differentiation culture medium in the step 6 into a rooting culture medium containing a screening agent, and culturing for 10-12 days at 30 ℃ under illumination to obtain regenerated seedlings;
the NB medium in the step 1 comprises the following components: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, glutamine 0.5g/L, acid hydrolyzed casein 0.5g/L, proline 2.878g/L, sucrose 30.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L, and pH 5.8;
the pre-culture medium in the step 2 comprises the following components: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, glutamine 0.5g/L, acid hydrolyzed casein 0.5g/L, proline 2.878g/L, sucrose 30.0g/L, agar powder 8.0g/L, 2,4-D2.0mg/L, acetosyringone 0.1mM and pH 5.8;
the agrobacterium tumefaciens staining solution in the step 3 comprises the following components: AA inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 0.5g/L, sucrose 68.5g/L, glucose 36.0g/L, glutamine 0.9g/L, asparagine 0.3g/L and arginine 0.176g/L, and the pH value is 5.2; adding 0.1mM of acetosyringone before use;
the components of the co-culture medium in the step 4 are as follows: n is a radical of6Inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 0.3g/L, sucrose 30.0g/L, glucose 10.0g/L, agar powder 8.0g/L +2,4-D2.0mg/L, pH 5.2, and acetosyringone 0.1mM is added after sterilization;
the screening medium in the step 5 comprises the following components: n is a radical of6Inorganic salt and organic matter + inositol 0.1g/L + acid hydrolyzed casein 0.5g/L + proline 2.878g/L + glutamine 0.3g/L + sucrose 30.0g/L + phytohemagglutinin 4.0g/L +2,4-D2.0mg/L, pH 5.8; after sterilization, adding carbenicillin 400mg/L and hygromycin 30mg/L or Basta4 mg/L;
the differentiation medium in step 6 comprises the following components: MS inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 2.0g/L, proline 0.3g/L, cane sugar 30.0g/L, sorbitol 30g/L, phytohemagglutinin 4.0g/L, NAA0.02mg/L, Kinetin2mg/L and pH 5.8; after sterilization, adding carbenicillin 400mg/L and hygromycin 30mg/L or Basta4 mg/L;
the rooting medium in the step 7 comprises the following components: MS inorganic salt and organic matter, inositol 0.1g/L, acid hydrolyzed casein 2.0g/L, proline 0.3g/L, cane sugar 30.0g/L, phytohemagglutinin 4.0g/L and pH 5.8; after sterilization, adding 400mg/L carbenicillin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710512324.7A CN107142277B (en) | 2017-06-29 | 2017-06-29 | High-efficiency genetic transformation method for indica rice |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710512324.7A CN107142277B (en) | 2017-06-29 | 2017-06-29 | High-efficiency genetic transformation method for indica rice |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107142277A CN107142277A (en) | 2017-09-08 |
| CN107142277B true CN107142277B (en) | 2020-07-03 |
Family
ID=59784513
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710512324.7A Expired - Fee Related CN107142277B (en) | 2017-06-29 | 2017-06-29 | High-efficiency genetic transformation method for indica rice |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107142277B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277233A (en) * | 2017-12-29 | 2018-07-13 | 青岛袁策生物科技有限公司 | The method for inducing and cultivating of Mature Embryos of Rice callus |
| CN108315347A (en) * | 2018-04-20 | 2018-07-24 | 刘寒冬 | A kind of rice transformation method |
| CN110607323A (en) * | 2019-09-24 | 2019-12-24 | 四川育良生物科技有限公司 | Agrobacterium tumefaciens-mediated rice genetic transformation method |
| CN113637701B (en) * | 2021-09-10 | 2023-04-07 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Method for establishing wild rice genetic transformation system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229950A (en) * | 2011-06-03 | 2011-11-02 | 湖南大学 | Rapid and high-efficiency transgenic method for indica rice |
| CN102943090A (en) * | 2012-10-19 | 2013-02-27 | 湖北省农业科学院 | Method for high efficiency regeneration and genetic transformation of indica rice |
| CN106609254A (en) * | 2015-10-26 | 2017-05-03 | 中国种子集团有限公司 | Preparation method of transgenic glyphosate-resistant indica type rice |
-
2017
- 2017-06-29 CN CN201710512324.7A patent/CN107142277B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229950A (en) * | 2011-06-03 | 2011-11-02 | 湖南大学 | Rapid and high-efficiency transgenic method for indica rice |
| CN102943090A (en) * | 2012-10-19 | 2013-02-27 | 湖北省农业科学院 | Method for high efficiency regeneration and genetic transformation of indica rice |
| CN106609254A (en) * | 2015-10-26 | 2017-05-03 | 中国种子集团有限公司 | Preparation method of transgenic glyphosate-resistant indica type rice |
Non-Patent Citations (1)
| Title |
|---|
| Highly efficientAgrobacterium -mediated transformation of suspension-cultured cell clusters of rice ( Oryza sativa L.);Kenjirou Ozawa et al.;《Plant Science》;20100630;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107142277A (en) | 2017-09-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107142277B (en) | High-efficiency genetic transformation method for indica rice | |
| CN102943090B (en) | Method for high efficiency regeneration and genetic transformation of indica rice | |
| CN108456690B (en) | Efficient genetic transformation and rapid identification method for brassica napus | |
| CN108338073A (en) | A kind of method for tissue culture of capsicum | |
| CN104883874B (en) | The directly or indirectly organ of Jatropha plant occurs | |
| CN114058640A (en) | Efficient agrobacterium-mediated sugarcane genetic transformation method | |
| CN105063086B (en) | A kind of molecular breeding method for quickly obtaining a large amount of transgenosis sedum lineare new varieties | |
| CN107475287B (en) | Eggplant genetic transformation method | |
| CN102191269B (en) | Non tissue culture gene transferring method by using half of peanut seed as acceptor | |
| Chauhan et al. | Wheat genetic transformation using mature embryos as explants | |
| CN107190019B (en) | A kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus | |
| WO2015099674A1 (en) | Sugarcane regeneration and transformation methods | |
| CN107012162B (en) | Agrobacterium-mediated cotton embryo tip rapid transformation method | |
| CN107006374B (en) | A kind of method of cell suspension culture of rice fast culture | |
| Corredoira et al. | Chestnut, European (Castanea sativa) | |
| CN104017823B (en) | Screening method for fast and effectively reducing false positive rate of peanut genetic transformation plant | |
| KR20100026019A (en) | Method to derive regeneration from callus of leymus chinensis trin and culture medium thereof | |
| CN108486150A (en) | A kind of Cotton Transformation method | |
| CN114568309A (en) | Peanut tissue culture method and peanut genetic transformation method | |
| CN102124947B (en) | Efficient transgene method for inducing caespitose shoots of soybean at high frequency | |
| CN106636196B (en) | A kind of peanut method of optimization, efficient mediated by agriculture bacillus | |
| CN110150150B (en) | Peony molecular breeding method | |
| CN106480087B (en) | Soybean genetic transformation method for vacuum-assisted agrobacterium infection | |
| Khan et al. | Agrobacterium-mediated genetic transformation of two varieties of Brassica: optimization of protocol | |
| CN104031938B (en) | A kind of genetic transforming method that exogenous gene importing is closed grain husk pollination rice variety |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200703 Termination date: 20210629 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |