WO2023005160A1 - Procédé de transformation génétique de plantes graminées - Google Patents

Procédé de transformation génétique de plantes graminées Download PDF

Info

Publication number
WO2023005160A1
WO2023005160A1 PCT/CN2022/071299 CN2022071299W WO2023005160A1 WO 2023005160 A1 WO2023005160 A1 WO 2023005160A1 CN 2022071299 W CN2022071299 W CN 2022071299W WO 2023005160 A1 WO2023005160 A1 WO 2023005160A1
Authority
WO
WIPO (PCT)
Prior art keywords
genetic transformation
cup
plants
leaf
seedlings
Prior art date
Application number
PCT/CN2022/071299
Other languages
English (en)
Chinese (zh)
Inventor
蒋立希
朴学成
Original Assignee
浙江大学
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 浙江大学 filed Critical 浙江大学
Publication of WO2023005160A1 publication Critical patent/WO2023005160A1/fr

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

Definitions

  • the invention relates to the field of agricultural biotechnology, in particular to a method for genetic transformation of gramineous plants.
  • Plant genetic transformation is an important technological advancement in modern science, which not only promotes human understanding of plant physiology and development, but also ushers in a new era of crop genetic improvement.
  • efficient transformation and regeneration remains a great challenge for many crops.
  • the method of plant genetic transformation mainly contains following several kinds: (1) based on the plant genetic transformation technology mediated by Agrobacterium, (2) based on the plant genetic transformation technology mediated by particle gun plasmid bombardment, (3) based on Bioactive beads (calcium alginate beads) mediated plant genetic transformation, (4) plant genetic transformation based on pollen and pollen growth channel, (5) plant transformation technology based on silicon carbide filament, (6) based on Plant genetic transformation mediated by electroporation, (7) plant genetic transformation technology mediated by microinjection, (8) genetic transformation method based on regulation of apical meristem development.
  • the CRISPR-Cas system is an RNA-guided adaptive immune response evolved in prokaryotic bacteria and archaea to defend against foreign genetic factors such as viruses, transposons, and plasmids.
  • CRISPR/Cas9-mediated genome editing has been performed in many model plants as well as rice, tobacco, wheat, barley, sorghum bicolor, corn, corn, eggplant, potato, rapeseed, soybean, lettuce, cucumber, citrus, Populus tomentosa, honeysuckle, etc.
  • CRISPR/Cas9-mediated genome editing technology also depends on subsequent genetic transformation technology including tissue culture, and tissue culture is extremely difficult for many species.
  • Plant genetic transformation is the application of recombinant DNA technology or gene editing technology, through cell tissue culture technology or germplasm system transformation technology, to purposefully insert foreign genes or DNA fragments into the recipient plant genome/or to target the recipient genome Editing, the technique of obtaining new plants with modified genomes through meiosis. Whether it is gene editing technology based on CRISPR/Cas9 or other nucleases, or transgenic technology based on DNA recombination, plant genetic transformation (transformation) is one of the indispensable key steps to achieve the purpose of gene editing or transgenic.
  • the current prevailing technology is to infect (or bombard with gene gun DNA plasmid) embryos, hypocotyls, cotyledon explants, apical meristems, etc. Tissues with strong meristematic ability, such as tissues, are then cultured to obtain (transgenic or gene-edited) regenerated shoots.
  • the current method has the following limitations: (1) The tissue culture technique is highly dependent on the species and genotype, and the transformation efficiency of the indica subspecies, barley and wheat in rice is very low; The requirements are very strict, and the configuration of the medium, the pretreatment of plant tissues, dark culture/light culture, and the replacement of culture vessels will be polluted if you are not careful; (3) The construction cost and maintenance cost of the tissue culture space are both high and low. Higher, higher energy consumption; (4) the tissue culture process is more time-consuming, through tissue culture to obtain seedlings and flowering and setting seeds, which is 3-6 months longer than normal seed germination to flowering and setting seeds.
  • the application aims at the above-mentioned deficiencies existing in the prior art, provides a kind of new plant genetic transformation technology, overcomes four kinds of crops genetic transformation technology difficulty such as gramineous food crops (rice, corn, wheat, barley etc.) Low), long period, and dependent on species and genotype disadvantages, save the tissue culture step in the genetic transformation process, create a simple and easy to operate, do not depend on genotype, low cost, high efficiency Gramineae crop genetic transformation method .
  • a method for genetic transformation of gramineous plants comprising the following steps:
  • step 2 2) adding the Agrobacterium tumefaciens bacterium liquid with the recombinant vector for gene editing to the cup-shaped structure in step 1);
  • the gramineous plants are rice, corn, wheat, barley, rye, triticale, sorghum, sugarcane and the like. Since the method for genetic transformation of gramineous plants in the present application is to pull out the first blade after the first blade grows after the seed germinates, a cup-shaped structure is formed in the leaf sheath at this time, and since all gramineous plants have a similar structure, , can be generalized and applied to all grasses.
  • step 1) the first leaf grows when the seed germinates for 3 to 5 days, and then the first leaf is pulled out.
  • the vector used for genetic transformation is a binary vector.
  • the binary vector can be a commonly used plasmid for transgenic Agrobacterium, such as plasmid pTX172.
  • Conventional methods in the prior art can be used for the construction of the recombinant vector and the method for transferring the recombinant vector into Agrobacterium.
  • the amount of bacterial solution can be determined according to different types of plants and culture conditions.
  • the amount of bacterial solution added to the cup-shaped structure in step 1) is 15-30 ⁇ L. More preferably, in step 2), the bacterial liquid is added to the cup-shaped structure in step 1) until it is full. Add as much culture liquid as possible to the cup structure, which can improve the transformation efficiency.
  • the seed germination stage of step (1) is carried out under dark conditions or under light conditions.
  • This application is based on the method of genetic transformation. Since the Agrobacterium liquid for transgenesis is added after the plant seeds germinate and grow the first leaf, during the subsequent plant growth process, the meristems will grow from the top of the shoot tip at the bottom of the cup-shaped structure. Cells in stems and leaves newly grown from tissues can be easily transformed, but not all of them can be genetically modified. Therefore, the plants obtained after continued cultivation may be a kind of chimera. If it is desired to obtain homozygous (homozygote) or heterozygous (heterozygote) with the same genetic background, the selection of populations can be segregated by genetic inheritance of offspring.
  • What this application describes is a kind of genetic transformation technology that uses the anatomical structure characteristics of Gramineae crop seedling stage to directly process the apical meristem in the seedling stage.
  • the characteristics of the anatomical structure of corn, wheat, barley, sorghum, rye, triticale, etc.) crops directly infect the plant apical meristem (SAM) with Agrobacterium liquid, no need to go through the tissue culture stage, and independent of genotype Genetic transformation technology, low cost, high efficiency, simple and easy.
  • Figure 1 shows the sequence construction between the left border and the right border of the vector used for gene editing. It shows the positional relationship between the core elements of the vector, PolII promoter, Cas9 nucleic acid protein gene, PolyA, ribozyme cleavage sequence, sgRNA1, sgRNA2, and ribozyme termination sequence. Arrows indicate the position of the ribozyme cleavage sequence; RZ, Ribozyme terminator, indicate the ribozyme termination sequence.
  • Fig. 2 shows the rice seedlings whose OsPDS gene was edited after the genetic transformation treatment by the ICCU method.
  • A japonica rice variety Nipponbare;
  • B indica rice variety Kasalath. Albino seedlings are indicated by white arrows.
  • Figure 3 shows the status of editing the target region of the rice PDS gene (Nipponbare), where the number after # represents the code of the transformant; a1-a18 represents the allelic change created by gene editing, and A0 represents the original allelic base of Nipponbare Sequence; Ho, means that the two DNA strands are homozygous; Bi, means that both DNA strands are edited and a new allele type appears; He, means that the two DNA strands are heterozygous; minus sign (-) means The number of bases lost, plus sign (+) represents the number of bases increased. The same below.
  • FIG. 4 shows the status of the target region of the rice PDS gene being edited (kasaras).
  • Fig. 5 is the maize seedling (CML322) whose ZmPDS gene was edited after the genetic transformation treatment by the ICCU method.
  • the white solid line arrow shows the albino seedlings of the whole plant. Such seedlings gradually die due to the inability to carry out photosynthesis.
  • the white dotted line arrow shows the chimeric plants with white leaves.
  • Fig. 6 shows the status of the edited target region of the maize PDS gene (B73).
  • Fig. 7 shows the edited target region of the maize PDS gene (CML322).
  • Fig. 8 shows the wheat seedlings (Chinese spring) whose TaPDS gene was edited after the genetic transformation treatment by the ICCU method.
  • the white solid line arrows show the albino seedlings of the whole plant, and such seedlings gradually die due to the inability to carry out photosynthesis.
  • the 2 plants on the left are chimeric plants with partially white flower leaves.
  • Figure 9 shows the status of the target region of the wheat PDS gene being edited (Chinese Spring).
  • Figure 10 shows the status of editing the target region of the wheat PDS gene (Yangmai 16).
  • Fig. 11 shows the barley seedlings (Zheda 9, ZU9) whose HvPDS gene was edited after the genetic transformation treatment by the ICCU method.
  • the black solid line arrows indicate albino seedlings, which gradually die due to the inability to carry out photosynthesis.
  • Figure 12 shows the status of the target region of the barley PDS gene being edited (Golden Hope, GP).
  • Figure 13 shows the edited status of the target region of the barley PDS gene (Zheda No. 9).
  • the left and right seedlings in each figure in Fig. 14 show the state of the seedlings under light culture and dark culture respectively.
  • the dark culture (the seedling on the right) forms a larger cup-shaped space than the light culture (the left seedling) and can carry a larger volume Agrobacterium infection solution.
  • 1 the rice seedlings 5 days after the seeds absorbed water and germinated; 2: gently pull out the first leaf (bud) from the leaf sheath by hand or tools, and after the leaves are pulled out, a cup-shaped space is formed in the leaf sheath; 3: use drops About 5 microliters (left) and 25 microliters (right) of Agrobacterium infection solution were dripped into the "cup-shaped" structural space formed in the leaf sheath after pulling out the first leaf; 4: After injection of Agrobacterium infection solution Within 1-3 days, maintain a certain volume of bacterial liquid in the "cup-shaped” container, so that the Agrobacterium bacterial liquid drips through the "cup bottom” tissue, penetrates into the meristem cells at the top of the shoot tip, and effectively infects SAM; 5 : After 5 days, move to an artificial incubator for cultivation under normal conditions (ie 28° C. under light conditions for 16 hours, and 25° C. under dark conditions for 8 hours).
  • the rice variety is kashalash.
  • the Agrobacterium bacterial liquid penetrates into the "cup bottom” tissue, invades the shoot tip SAM, and effectively infects the SAM.
  • the SAM tissue and the Agrobacterium bacterial fluid were co-cultured under light or dark conditions (the effect of the dark condition is better).
  • the transformed SAMs differentiated into new green shoots and leaves. Collect about 14-21 days of leaves (from the date of germination) to sequence the target gene (hereinafter, PDS gene as an example).
  • results show that, depending on the species and variety, 20-80% of the new leaves of a single plant are extracted The bases of single or double strands of DNA are effectively genetically modified. Subsequently, the single seedling whose genome has been modified is transplanted to a culture medium (such as soil or culture medium) for planting, and after the seedling grows and enters reproductive growth, seeds of the T2 generation are produced.
  • a culture medium such as soil or culture medium
  • Agrobacterium bacterial liquid centrifuge the Agrobacterium bacterial liquid at 4000 rpm, take out the precipitated Agrobacterium cells with a medicine spoon, and dissolve them in 30 ml of 1/2MS culture medium (containing 30 microliters of DMSO with a concentration of 10 mg Acetosyringone (Acetosyringone) per milliliter, carefully resuspend the Agrobacterium cells with a disposable sterilized pipette. The processed bacterial solution is used for the following treatment within 2 hours.
  • 1/2MS culture medium containing 30 microliters of DMSO with a concentration of 10 mg Acetosyringone (Acetosyringone) per milliliter
  • Seedling SAM was co-cultured with Agrobacterium in dark condition. After 3-5 days, move to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.). About 15 days later, select half a leaf from each seedling to extract a small amount of DNA, and then use it for the detection of the target gene sequence; after sequencing, eliminate the target gene that has not been genetically modified (not edited or the target fragment has not been inserted into the plant genome ) negative plants, transplant the positive plants to common media (soil or other culture media) and carefully cultivate them.
  • the seedlings enter reproductive growth through vegetative growth, and the seeds of the T2 generation plants are harvested after the plants mature.
  • T0 generation the generation in which the genomic DNA sequence of the vegetative organ has not been modified
  • T1 generation the generation in which the genomic DNA sequence of the vegetative organ has been modified
  • T2 generation the seeds harvested from the T1 generation plants produce T2 generation plants, and the T2 generation population can be isolated to homozygous mutants.
  • the seedlings of rice, corn, wheat, barley, etc. are treated with the Agrobacterium liquid carrying the target gene target sequence or insert vector.
  • ICCU method genetic transformation method using endogenous cup-shaped structure
  • PDS phytoene desaturase
  • the reason why this gene is taken as an example is because the homozygous mutant of this gene presents the phenomenon of albino seedlings, which is very easy to be observed intuitively.
  • genomic DNA from the leaves of seedlings about 14-21 days after germination, and sequenced them to understand the genetic modification of target genes.
  • Example 1 Editing effect of ICCU method on rice PDS gene
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: EHA105.
  • Target gene OsPDS (LOC_Os03g08570).
  • Figure 1 shows the positional relationship between the core elements of the molecular construction Pol II promoter, Cas9 nucleic acid protein gene, PolyA, ribozyme cleavage sequence, sgRNA1, sgRNA2, and ribozyme termination sequence.
  • Base sequence design of sgRNA1 and sgRNA2 the DNA base sequence transcribed into sgRNA1 is as follows: AGTTGCTTCAGCATGGATAC; the base sequence of sgRNA2 is as follows: CGGGACAACTTCCTACTCAT.
  • the sgRNA1 and sgRNA2 sequences correspond to the positions on LOC_Os03g08570 separated by 50 bases.
  • a "cup-shaped" container structure is formed in the coleoptile, and several layers of residual cells between the cup-shaped space and the SAM tissue form the "cup bottom" of the cup-shaped structure.
  • (c) Using a pipette, inject about 25 microliters (prepared in step (6)) of the Agrobacterium infection solution into the "cup" structure space. The injection process can be performed under a stereomicroscope.
  • Seedlings were cultured under dark conditions. After about 3 days, move to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.).
  • Leaf color of T1 generation rice seedlings treated by ICCU method under dark conditions According to the above method, we used ICCU method to treat 60 late rice varieties Nipponbare and 60 early rice varieties Kasalath seedling. Among the 60 Nipponbare rice plants, we found 4 seedlings with white flowers, 5 seedlings with light green leaves, and 51 seedlings with completely normal color. After ICCU treatment, some Nipponbare plants are shown in Figure 2A. Among the 60 Kasaras rice plants, we found 5 seedlings with white flowers, 8 seedlings with light green leaves, and 47 seedlings with completely normal color. After ICCU treatment, some of the Kasaras plants were as follows: Figure 2B.
  • Example 2 The editing effect of the ICCU method on the corn PDS gene
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: GV3101.
  • Figure 1 schematically shows the positional relationship between the core elements of the molecular construction, the Pol II promoter, the Cas9 nucleic acid protein gene, PolyA, the ribozyme cleavage sequence, sgRNA1, sgRNA2, and the ribozyme termination sequence.
  • the base sequence design of sgRNA1 and sgRNA2 is as follows: AGTTGCTCAACAATGGACAC; the base sequence of sgRNA2 is as follows: TAGACATCCTGCCTTGCAGG; the corresponding positions of sgRNA1 and sgRNA2 sequences on the maize PDS gene are separated by 50 bases.
  • Infection treatment of corn SAM by Agrobacterium bacterium liquid (a) select corn seeds with germination ability, soak the seeds in clear water for 1 hour, and first use 70% alcohol to disinfect the seed epidermis for 1 minute , wash 3 times with clear water, then soak the seeds with 3% H2O2 or 3% sodium hypochlorite solution for 30 minutes, wash the treated seeds 5 times with sterile clear water, then soak them in sterile clear water for 3 hours. (b) Germinate the seeds under dark conditions. After 4 days, treat the seeds to germinate and grow the first leaf sprout, pull out the first leaf sprout by hand or other tools. The pull-out process should not damage the SAM as much as possible, and minimize the residual tissue remaining on the upper layer of the SAM.
  • a "cup-shaped" container structure is formed in the coleoptile, and several layers of residual cells between the cup-shaped space and the SAM tissue form the "cup bottom" of the cup-shaped structure.
  • (c) Using a pipette, inject about 30 microliters (prepared in step (6)) of the Agrobacterium infection solution into the "cup" structure space. The injection process can be performed under a stereomicroscope or a magnifying glass.
  • Seedlings were cultured under dark conditions. After 4 days, they were transferred to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.).
  • Leaf color of T1 maize seedlings treated by ICCU method under dark conditions According to the above method, we treated a total of 50 B73 and 50 CML322 seedlings. Among the 50 B73 plants, we found 6 seedlings with white flowers, 9 seedlings with light green leaves, and 35 seedlings with completely normal color. Among the 50 CML322 plants, we found 3 seedlings with white flowers, 8 seedlings with light green leaves, and 39 seedlings with completely normal color. After ICCU treatment, some of the seedling plants of CML322 are shown in Figure 5.
  • Example 3 Editing effect of ICCU method on wheat PDS gene
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: EHA105.
  • Target genes TraesCS4A02G004900.1 (chromosome 4A: 3133866-3141315), TraesCS4B02G300100.1 (chromosome 4B: 586574959-586580910), TraesCS4B02G300100.2 (chromosome 4D: 468262170-468267694).
  • Figure 1 schematically shows the positional relationship between the core elements of the molecular construction, the Pol II promoter, the Cas9 nucleic acid protein gene, PolyA, the ribozyme cleavage sequence, sgRNA1, sgRNA2, and the ribozyme termination sequence.
  • the base sequence design of sgRNA1 and sgRNA2 is as follows: TTGTTTGCCAAGATTTTCCA; the base sequence of sgRNA2 is as follows: GAGGCAAGAGATGTGTTGGG; sgRNA1 and sgRNA2 sequences correspond to the positions on the wheat PDS gene with an interval of 50 bases.
  • Infection treatment of Agrobacterium bacterium liquid to wheat SAM (a) select corn seeds with germination ability, the seeds are absorbed in clear water for 1 hour, and after 1 minute the epidermis of the seeds is sterilized with 70% alcohol, Wash 3 times with clear water, then soak the seeds with 3% H2O2 or 3% sodium hypochlorite solution for 20 minutes, wash the treated seeds 5 times with sterile clear water, then soak them in sterile clear water for 3 hours. (b) Germinate the seeds under dark conditions. After 5 days, treat the seeds to germinate and grow the first leaf sprout, pull out the first leaf sprout by hand.
  • a "cup-shaped" container structure is formed in the coleoptile, and several layers of residual cells between the cup-shaped space and the SAM tissue form the "cup bottom" of the cup-shaped structure.
  • (c) Using a pipette, inject about 25 microliters (prepared in step (6)) of the Agrobacterium infection solution into the "cup" structure space. The injection process can be performed under a stereomicroscope.
  • Seedlings were cultured under dark conditions. After 3 days, they were transferred to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.).
  • Leaf color of T1 generation wheat seedlings treated by ICCU method under dark conditions According to the above method, we treated a total of 35 Chinese spring and 35 Yangmai 16 seedlings. Among the 30 Chinese spring plants, we found 3 seedlings with white flowers, 6 seedlings with light green leaves, and 26 seedlings with completely normal color. Among the 35 Yangmai 16 plants, we found 1 seedling with white flowers, 6 seedlings with light green leaves, and 28 seedlings with completely normal color. After ICCU treatment, some of the seedlings of Chinese spring after treatment are shown in Figure 8 Show.
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: EHA105.
  • Figure 1 schematically shows the positional relationship between the core elements of the molecular construction, the Pol II promoter, the Cas9 nucleic acid protein gene, PolyA, the ribozyme cleavage sequence, sgRNA1, sgRNA2, and the ribozyme termination sequence.
  • Base sequence design of sgRNA1 and sgRNA2 the base sequence of sgRNA1 is as follows: TTGTTTGCCAAGATTTTCCA; the base sequence of sgRNA2 is as follows: GAGGCAAGAGATGTGTTGGG; the positions of sgRNA1 and sgRNA2 sequences on the barley PDS gene are separated by 50 bases.
  • a "cup-shaped" container structure is formed in the coleoptile, and several layers of residual cells between the cup-shaped space and the SAM tissue form the "cup bottom" of the cup-shaped structure.
  • (c) Using a pipette gun, inject about 20 microliters (prepared in step (6)) of the Agrobacterium infection solution into the "cup" structure space. The injection process can be performed under a stereomicroscope or a magnifying glass.
  • Seedlings were cultured under dark conditions. After 5 days, they were transferred to an artificial incubator for cultivation under normal conditions (ie, 16 hours under light conditions at 28° C. and 8 hours under dark conditions at 25° C.).
  • Leaf color of T1 generation barley seedlings treated by ICCU method under dark conditions According to the above method, we treated a total of 35 Golden Hope and 35 Zheda No. 9 seedlings. Among the 35 plants of Golden Hope, we found 4 seedlings with white flowers, 7 seedlings with light green leaves, and 24 seedlings with completely normal color. Among the 35 Zheda No. 9 plants, we found 0 seedlings with white flowers, 4 seedlings with light green leaves, and 31 seedlings with completely normal color. After ICCU treatment, some of the seedling plants of Zheda No. 9 are shown in Figure 11 .
  • Embodiment 5 Comparison of cup-shaped structure and genetic transformation effect formed by ICCU method light culture and dark culture treatment of germinated seeds
  • Vector and Agrobacterium strain vector, pTX172; Agrobacterium strain: EHA105.
  • Target gene OsPDS (Os03g08570).
  • Figure 1 shows the positional relationship between the core elements of the molecular construction Pol II promoter, Cas9 nucleic acid protein gene, PolyA, ribozyme cleavage sequence, sgRNA1, sgRNA2, and ribozyme termination sequence.
  • Base sequence design of sgRNA1 and sgRNA2 the base sequence of sgRNA1 is as follows: AGTTGCTTCAGCATGGATAC; the base sequence of sgRNA2 is as follows: CGGGACAACTTCCTACTCAT. The positions on Os03g08570 corresponding to the sequences of sgRNA1 and sgRNA2 are separated by 50 bases.
  • Infection treatment of rice SAM by Agrobacterium bacterium liquid (a) select rice seeds with germination ability, soak the seeds in clear water for 1 hour, first treat the seed epidermis with 70% alcohol for 1 minute, and use Clean water 3 times, then use 3% H2O2 or 3% sodium hypochlorite solution to carry out 20 minutes to seed, the seed after treatment is washed 5 times with sterile water, then soak in sterile water for 3 hours. (b) The seeds were germinated under light and dark conditions, respectively. After 5 days, treat the seeds to germinate and grow the first leaf sprout, pull out the first leaf sprout by hand or other tools.
  • the pull-out process should not damage the SAM as much as possible, and minimize the residual tissue remaining on the upper layer of the SAM.
  • "cup-shaped" container structures of different sizes are formed in the leaf sheath (coleoptile).
  • (c) Using a pipette gun, inject about 25 microliters (prepared in step (6)) of the Agrobacterium infection solution into the darkened "cup-shaped" structure space, inject about 15 microliters (prepared in step (6) The Agrobacterium infection solution prepared in (6) was injected into the light-treated "cup-shaped" structural space. The injection process can be performed under a stereomicroscope or a magnifying glass.
  • (d) Continue culturing the seedlings under light or dark conditions respectively.
  • Table 1 The comparison of the final transformation efficiency of germinated seeds under light or dark culture treatment

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne un procédé de transformation génétique de plantes graminées. Le procédé comprend les étapes suivantes : (1) l'arrachage d'une première feuille après la germination d'une graine de plante graminée et la croissance de la première feuille, de manière à former une structure en forme de coupelle dans la gaine au moment considéré ; (2) l'ajout d'un liquide constitué d'Agrobacterium tumefaciens possédant un vecteur recombiné pour l'édition génique à la structure en forme de coupelle dans l'étape (1) ; et (3) la poursuite de la culture de celle-ci pour obtenir une plante transgénique qui est au moins partiellement éditée par le gène. Selon le procédé, un liquide bactérien constitué d'Agrobacterium est directement utilisé pour infecter le méristème apical des pousses (SAM) de la plante sur un corps vivant (corps nutritif) au stade de plantule, basé sur les caractéristiques de la structure anatomique des cultures de graminées. Le procédé ne nécessite pas d'étape de culture tissulaire et ne dépend pas d'une technologie de transformation génétique de génotype.
PCT/CN2022/071299 2021-07-28 2022-01-11 Procédé de transformation génétique de plantes graminées WO2023005160A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110861664.7 2021-07-28
CN202110861664.7A CN113604497B (zh) 2021-07-28 2021-07-28 一种禾本科植物的遗传转化方法

Publications (1)

Publication Number Publication Date
WO2023005160A1 true WO2023005160A1 (fr) 2023-02-02

Family

ID=78305934

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/071299 WO2023005160A1 (fr) 2021-07-28 2022-01-11 Procédé de transformation génétique de plantes graminées

Country Status (2)

Country Link
CN (1) CN113604497B (fr)
WO (1) WO2023005160A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117487847A (zh) * 2023-10-31 2024-02-02 中国热带农业科学院橡胶研究所 一种获得橡胶树纯合基因编辑植株的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113604497B (zh) * 2021-07-28 2023-03-24 浙江大学 一种禾本科植物的遗传转化方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7494787A (en) * 1986-06-30 1988-01-07 University Of Toledo, The Process for transforming gramineae and the products thereof
US5187073A (en) * 1986-06-30 1993-02-16 The University Of Toledo Process for transforming gramineae and the products thereof
CN1305005A (zh) * 2001-02-26 2001-07-25 山东大学 一种转化大粒种子植物的方法及其应用
WO2014029044A1 (fr) * 2012-08-22 2014-02-27 河北省农林科学院遗传生理研究所 Procédé de transformation génétique de la monocotylédone pour envahir les points de croissance des bourgeons de semences minimalement et pleinement
CN113604497A (zh) * 2021-07-28 2021-11-05 浙江大学 一种禾本科植物的遗传转化方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103205455B (zh) * 2012-01-11 2014-12-31 山东省农业科学院作物研究所 利用幼穗拯救获得转基因小麦的方法
CN105420257A (zh) * 2015-12-10 2016-03-23 山东大学 玉米分泌型磷脂酶A2基因ZmsPLA2-2在改变植物抗旱性中的应用
WO2020005933A1 (fr) * 2018-06-28 2020-01-02 Pioneer Hi-Bred International, Inc. Procédés de sélection de plantes transformées

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7494787A (en) * 1986-06-30 1988-01-07 University Of Toledo, The Process for transforming gramineae and the products thereof
US5187073A (en) * 1986-06-30 1993-02-16 The University Of Toledo Process for transforming gramineae and the products thereof
CN1305005A (zh) * 2001-02-26 2001-07-25 山东大学 一种转化大粒种子植物的方法及其应用
WO2014029044A1 (fr) * 2012-08-22 2014-02-27 河北省农林科学院遗传生理研究所 Procédé de transformation génétique de la monocotylédone pour envahir les points de croissance des bourgeons de semences minimalement et pleinement
CN113604497A (zh) * 2021-07-28 2021-11-05 浙江大学 一种禾本科植物的遗传转化方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HE, CONGFEN ET AL.: "Genetic transformation of aloe barbadensis Miller by agrobacterium tumefaciens", JOURNAL OFGENETICS AND GENOMICS, vol. 34, no. 12, 31 December 2007 (2007-12-31), XP022937560, ISSN: 1053-1060, DOI: 10.1016/S1673-8527(07)60120-9 *
LIU FANG, LV WEI-TAO, CUI DE-CAI, ZHAO TAN-FANG: "Transformation of Wheat with Anti-Phospholipase Dγ Gene", LETTERS IN BIOTECHNOLOGY, JUNSHI YIXUE KEXUEYUAN, CN, vol. 17, no. 2, 31 March 2006 (2006-03-31), CN , pages 189 - 191, XP093028925, ISSN: 1009-0002 *
LIU TAO, WU BAOWEI; LI ZHIJIE; LIU ZHENLAN; WANG QIAN; ZHAO HUIXIAN: "Identification and Functional Analysis of miR5062 Target Genes in Wheat (Triticum aestivum)", JOURNAL OF TRITICEAE CROPS, vol. 41, no. 1, 1 January 2021 (2021-01-01), pages 1 - 10, XP093028924, ISSN: 1009-1041, DOI: 10.7606/j.issn.1009-1041.2021.01.01 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117487847A (zh) * 2023-10-31 2024-02-02 中国热带农业科学院橡胶研究所 一种获得橡胶树纯合基因编辑植株的方法

Also Published As

Publication number Publication date
CN113604497A (zh) 2021-11-05
CN113604497B (zh) 2023-03-24

Similar Documents

Publication Publication Date Title
Cao et al. Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture
JP2018503392A (ja) 遺伝子一過性発現により完全な植物において部位特異的改変を行うための方法
CN101948867A (zh) 一种农杆菌介导的小油桐基因转化方法
WO2023005160A1 (fr) Procédé de transformation génétique de plantes graminées
CN102154364A (zh) 一种根癌农杆菌介导的甘蔗遗传转化方法
WO2022135246A1 (fr) Gène r pour commander l'appariement du soja-rhizobium, protéine et son utilisation
US20210269816A1 (en) Method of Obtaining Multileaflet Medicago Sativa Materials by Means of MsPALM1 Artificial Site-Directed Mutants
CN107338230B (zh) OsMPK11蛋白及其编码基因在调控植物抗旱性中的应用
JP2000510325A (ja) 植物性材料の繁殖および／または選択方法
CN109735538B (zh) 一种提高森林草莓叶片再生效率的载体及其制备方法和应用
CN107805632A (zh) OsMKK6蛋白及编码基因在调控植物种子发育中的应用
US20210171973A1 (en) Method of Obtaining Multileaflet Medicago Sativa Materials by Means of MsPALM1 Artificial Site-Directed Mutants
JP4463456B2 (ja) 葉柄移植片を使用する綿の高効率アグロバクテリア菌仲介形質転換
CN106811471A (zh) 水稻spl7基因在调控株型中的应用
CN115605600A (zh) 未成熟花序分生组织编辑
CN107805633A (zh) OsMPK4蛋白及编码基因在调控植物种子发育中的应用
CN107338231A (zh) OsMPK21-1蛋白及其编码基因在调控植物抗旱性中的应用
CN114350673B (zh) 一种调控种子活力的水稻kob1基因及其调控方法
CN113528535B (zh) 一种提高植物抵抗逆境胁迫能力的孤基因PpDRO及应用
JP4228044B2 (ja) シバ属植物の再分化植物体及び形質転換植物体
CN108486112A (zh) 一种具有花药组织特异性的启动子
JP2008259497A (ja) アグロバクテリウムを介したダイズ国内品種の形質転換体作出方法並びに形質転換体当代及び後代の種子を短期間で獲得する方法
CN104513831B (zh) 一种促进植物生长的方法
CN102229946B (zh) 一种改良的农杆菌介导的小麦幼穗转化方法及其应用
CN110951771A (zh) 春兰miR390a在控制植物根系发育中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22847781

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE