WO2018177446A2 - Plantas de soya que comprenden el evento transgenico cigbdt-def1 o cigbis-def5 - Google Patents
Plantas de soya que comprenden el evento transgenico cigbdt-def1 o cigbis-def5 Download PDFInfo
- Publication number
- WO2018177446A2 WO2018177446A2 PCT/CU2018/050002 CU2018050002W WO2018177446A2 WO 2018177446 A2 WO2018177446 A2 WO 2018177446A2 CU 2018050002 W CU2018050002 W CU 2018050002W WO 2018177446 A2 WO2018177446 A2 WO 2018177446A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- event
- cigbis
- cigbdt
- def1
- def5
- Prior art date
Links
Classifications
-
- 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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
- A01H6/542—Glycine max [soybean]
-
- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
-
- 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
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/10—Seeds
-
- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
- C12N15/8275—Glyphosate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Definitions
- the present invention relates to the branch of plant biotechnology, specifically with the protection of soybeans against diseases caused by fungi and oomycetes, in commercial genotypes of that crop.
- the invention relates to two new transgenic events, which result from the genetic transformation of two soybean genotypes.
- the events were designated as CIGBDt-Defl and CIGBIs-Def5.
- Plants are constantly exposed to different pathogens present in nature. As a consequence, they have developed a complex defense system to protect themselves against attack. These defense systems can be classified, according to the moment of their expression, in constitutive ones, if they are present during the whole life cycle of the plant; and induced, if its expression is evidenced in greater proportion in the presence of a pathogen.
- One of these mechanisms is the plant defenses. These compounds are small cationic peptides of 45 to 54 amino acid residues, with disulfide bridges in the cisterns that compose them (Zhu et al., Cell Mol. Life Sci. (2015) 62: 2257-2269).
- Plant defensins were first described in wheat and barley seeds, and their role in the defense system of this type of organism has been well characterized. They are distributed in correspondence to their role in the biological system and to the expression profile, according to the mode of action of the peptide (from Beer and Vivier, BMC Res. Notes (201 1) 4: 459).
- Soy plants are affected by various diseases, which negatively affect yields and seed quality, causing total loss of production.
- the most important diseases are those caused by fungi, due to the magnitude of the damage they cause.
- For conventional programs to improve soybean cultivation it has been extremely difficult to obtain commercial genotypes that show resistance to the main fungal diseases that affect this crop.
- the genus Colletotrichum is of great importance, as it is the causative agent of anthracnose, a typical disease of tropical and subtropical countries, which appears from the initial stages of plant development. This disease causes great effects on the quality of the grains, causing the death of seedlings, due to the absence of symptoms in the seeds when the infection is milder. In this way, the fungus can cause deterioration in the seeds and systemic infection in adult plants, and the damage is exacerbated when rains occur, the population density is excessive, or the harvest is delayed, which affects yields (Gally et al., Integrated Pest Management and Agroecology (2006) 78: 86-90).
- End-of-cycle diseases of soybeans are the most frequent, and can cause damage of approximately 8% to 10% of the yield, with a maximum of up to 30%. Symptoms vary depending on the disease present, and manifest in intermediate and advanced reproductive states. The ones that stand out the most are the decrease in the healthy leaf area, the premature defoliation of the plants, the anticipated maturity of the crop, and the decrease in the quality of the seeds.
- the diseases that form the so-called "end-of-cycle complex” are the blight of the stem and purple spot of the seed (caused by Cercospora kikuchii), frog eye spot (caused by Cercospora sojina), ringed spot (caused by Corynespora cassiicola), brown spot (caused by Septoria glycines), sheath blight and stem (caused by Phomopsis sojae), mildew (caused by Peronospora manshurica), leaf spot by alternation (caused by Alternar ⁇ a spp.), anthracnose (caused by Colletotrichum truncatum) and Asian rust, among others (Carmona et al., Phytophatologica (201 1) 37: 134-139).
- Phakopsora pachyrhizi is the causative agent of Asian soybean rust (Torres et al., Pesq. Agropec. Bras (2012) 38: 1053-1057). This disease is considered the most devastating that affects the crop, since it affects early defoliation and the affectation of the main components of yield, and causes losses of between 10 and 90% of production (Ribeiro et al., Genetics and Molecular Biology (2008) 31: 98-105). It was detected in the Americas in 2001, and has caused great losses to soy production in South America. In Cuba, the presence of Asian rust was detected in 2010 (Pérez et al., Plant Pathology (201 1) 59: 803) and spread through different soy producing provinces.
- rust in soybeans Two types have been identified. In addition to Asian rust, there is the American rust of soybeans, caused by P. meibomiae. The structures that serve to differentiate between these rust species are telospores. However, these are difficult to find, due to the impossibility of in vitro culture of both pathogens. For correct identification, molecular techniques, such as polymerase chain reaction (PCR) with oligonucleotides specific for P. pachyrhizi, can be used. The presence of Asian rust in the field can be determined by the use of immunochromatographic diagnostic strips, these are capable of detecting the presence of the pathogen in the initial stages of infection, and are designed to be used in foliar tissue.
- PCR polymerase chain reaction
- Hc-AFP1 -4 defensins isolated from the Heliophila coronopifolia species, showed inhibitory activity in vitro against Botrytis cinerea and Fusarium solani.
- Defensins Rs-AFP1 and Rs-AFP2 are strongly induced in radish leaves after infection with Alternar ⁇ a brassicola, showing "in vitro" antifungal activity.
- Glyphosate disrupts the synthesis of aromatic amino acids in plants, through the competitive inhibition of the enzyme 5-enolpiruvil shikimato 3-phosphate synthetase (Funke, et al., PNAS (2006) 103 (35): 13010-5).
- the presence of the herbicide in the meristems of the plants inhibits growth, and causes their death.
- transgenic soy plants that contain the cp4epsps gene (whose sequence is deposited in the NCBI GenBank databank, with accession no .: AB209952.1) continue the synthesis of aromatic amino acids in the presence of the herbicide.
- the present invention solves the problem set forth above, by providing a soybean plant, or a part of said plant, which is characterized in that it comprises the CIGBDt-Def1 transgenic event or the CIGBIs-Def5 transgenic event.
- Soybeans representative of both events were deposited in the National Collections of Industrial, Food and Marine Bacteria (NCIMB) of the United Kingdom, an institution recognized as an International Depository Authority.
- the representative seeds of the CIGBDt-Def1 event were deposited under the accession number NCIMB 42724, and the representative seeds of the CIGBIs-Def5 event were deposited under the accession number NCIMB 42725.
- Such transgenic events were obtained by genetic engineering, and so singular confer self-fungicidal activity to the plant that contains them.
- the transgenic plant is able to avoid the negative effect caused by fungi and pathogenic oomycetes in the cultivation of soy.
- the two transgenic events of the invention express the defense gene nmdef02 and the gene cp4epsps, responsible for glyphosate herbicide resistance, and were generated by the Agrobacterium method or by biolistics.
- "soybean plant” refers to all plants of the species Glycine max, and includes all soybean varieties of commercial interest, also containing all parts of the plant.
- CIGBDt-Def1 is also referred to as CIGB-DtDEF1, DTDefl, DtDEFI and DtDefl.
- CIGBIs-Def5 is also referred to as CIGB-lsDEF5, lsDEF5 and lsDef5.
- DNA deoxyribonucleic acid
- the CIGBDt-Def1 and CIGBIs-Def5 events were obtained from two conventional soybean varieties: Dt84 e lncasoy36 (Is36).
- the expression of the defensin encoded by the nmdef02 gene is important for protection against phytopathogenic fungi associated with the crop, which limit both production levels and grain quality. Plants comprising these transgenic events tolerate up to five times the effective field dose of the non-selective herbicide glyphosate, which allows the use of this herbicide for weed control under production conditions.
- the invention reveals a soybean plant that is characterized in that it is the progeny of any generation of a soybean plant comprising the CIGBDt-Def1 event or the CIGBIs-Def5 event.
- the invention provides a soybean plant that is characterized in that it results from a crossing of a soybean plant comprising the transgenic event CIGBDt-Def1 or CIGBIs-Def5 with another non-transgenic soybean plant.
- transgenic or transgenic includes any line, plant, cell or event that has been modified or modified by the presence of a heterologous nucleic acid, which includes those originally modified transgenic, as well as those created by sexual crossings or asexual propagation of the event. initially regenerated.
- progeny refers to plants produced by a sexual cross (for example, backcrossing, self-crossing or cross-linking) between a plant comprising the CIGBDt-Def1 and CIGBIs-Def5 events and other commercial soybean genotype.
- the term "part of the plant” includes plant cells, plant organs, plant protoplasts, plant cell tissue cultures, from which plants can be regenerated, plant corns, seedlings and plant cells. intact in plants.
- the term “part of the plant” also includes embryos, pollen, ovules, seeds, seed pods, leaves, flowers, branches, stems, roots, root apices, anthers, cotyledons, hypocotyls, and the like.
- the term “grains” means the mature seed produced by commercial growers for purposes other than growth or reproduction of the species.
- the invention relates to a part of the plant comprising the CIGBDt-Def1 event or the CIGBIs-Def5 event, where the part can be a root, bud, leaf, pollen, ovule, flower or cell.
- the invention also provides a seed of a soy plant comprising the event CIGBDt-Def1 or CIGBIs-Def5.
- transgenic line refers to a plant whose genome has been modified, by genetic engineering, to introduce one or more new genes from another unrelated plant, or of a different species; or to modify the function of a proper gene. As a consequence of the insertion or modification of the gene, the transgenic line shows a new characteristic, which is transmitted regularly to the offspring, from a certain clonal generation.
- soy plants can acquire a new phenotype, in addition to what they already have with the events of the invention, and for this purpose the transformation of plant material or explants derived from the plants comprising the events can be used.
- These characteristics, in addition to the reference events, can be acquired by any of the available genetic transformation methods.
- transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in a genetically stable inheritance.
- Plants comprising the CIGBDt-Def1 or CIGBIs-Def5 events can be used as a source of plant material in transformation methods, for introduce heterologous nucleic acid molecules to new soy events. Transformation vectors can be prepared to introduce other genes of interest, and correspondingly achieve other lines with different characters introduced into the same plant,
- a "transgenic event” is produced by transformation of plant cells with a genetic construct that contains heterologous DNA, including a nucleic acid expression cassette comprising a transgene of interest, the regeneration of a population of plants that result from the insertion of the transgene in the genome of the plant, and the selection of a particular regenerated plant.
- the event is characterized by the location of the insertion in a particular genome and phenotypically by the expression of the transgenes.
- an event is a segment of DNA that is part of the genetic makeup of a plant.
- transgenic events comprise two transgenes.
- the invention provides a soy product that is produced from a plant or a soybean seed that comprises the CIGBDt-Def1 or CIGBIs-Def5 event.
- the product consists of flour, flakes, oil or a product for human or animal feed.
- Another aspect of the invention is a method for the production of a soybean plant resistant to glyphosate herbicide and diseases caused by fungi or oomycetes where the CIGBDt-Def1 or CIGBIs-Def5 event is introduced into the genome of said plant.
- soy disease is Asian rust, caused by P. pachyrhizi.
- plants resistant to Asian rust are those plants of a line where the presence of symptoms of this disease does not exceed 50% of the plants under conditions of natural infection.
- said method comprises the crossing of a soybean plant comprising the CIGBDt-Def1 event or the CIGBIs-Def5 event with another soybean plant, and the selection of the progeny comprising one of these events.
- the object of the present invention is also a set of reagents for the detection of nucleic acid corresponding to the CIGBDt-Def1 or CIGBIs-Def5 event, in a sample of genomic soy nucleic acid, wherein said set comprises at least one pair of oligonucleotides, for acid fragment amplification nucleic corresponding to the binding regions between the nucleic acid of a soybean plant and the nucleic acid corresponding to the CIGBDt-Def1 or CIGBIs-Def5 event.
- pairs of useful oligonucleotides or primers including a primer that overlaps with the junction point between the insert DNA and the 5 'end of the flanking DNA; or the insert DNA and the 3 'end of the flanking DNA.
- the methods for designing, obtaining and employing DNA probes or primers in the present invention are known in molecular biology.
- the pairs of PCR primers can be derived from a known sequence, for example, by using computer programs intended for this purpose, such as the PCR primer analysis tool in version 1 1 of the NTI Vector (Thermo Fisher Scientific).
- the invention provides a method for diagnosing the CIGBDt-Def1 event or the CIGBIs-Def5 event in a soy sample comprising analyzing said sample to detect the presence of nucleic acid or protein corresponding to the CIGBDt-Def1 or CIGBIs event. -Def5.
- sample includes any fraction containing nucleic acids or polypeptides and is obtained from a plant, plant material or products, such as animal feed or fresh or processed products derived from plant material.
- Example 3 shows a method for the identification of nucleic acids of the events of the invention in a biological sample.
- This method comprises the preparation of a mixture of a biological sample and a pair of nucleic acid complementary primers, capable of amplifying specific regions of the nucleic acid molecule of the CIGBIs-Def5 or CIGBDt-Def1 events.
- such events can be detected or identified by the detection of the polypeptide or a defensin peptide, expressed in soybean plants. Any method can be used in the detection of the polypeptide or peptide.
- antibodies produced against defensin can be used, and any known immunochemical method, such as ELISA, Immunoblot, Dot Blot, can be used.
- the diagnosis of the CIGBDt-Def1 event or of the CIGBIs-Def5 event, in a soy sample is characterized in that a set of reagents comprising at least one pair of oligonucleotides is used for amplification of the fragments of nucleic acid corresponding to the binding regions between the nucleic acid of a soybean plant and the nucleic acid corresponding to the CIGBDt-Def1 or CIGBIs-Def5 event.
- the invention also provides a method for increasing the yield of a soybean crop in the field, which comprises sowing seeds of a soybean plant comprising the CIGBDt-Def1 or CIGBIs-Def5 event, establishing the soybean crop in the field, and treating the field with an effective amount of the herbicide glyphosate to control weeds.
- a materialization of the invention said method is characterized in that the field is treated between the phases of culture V1 and R4. Methods of establishing soybean cultivation and herbicide application are well known to those skilled in this field of the art.
- Soy plants that comprise the CIGBDt-Def1 or CIGBIs-Def5 event can be used in an breeding program, using cross-breeding or hybridization methods to reproduce these events in soybean plants of other genotypes. Such breeding methods can be used to produce soybean plants, for example, for use in commercial production in different geographical regions, or to produce additional soybean breeding populations.
- the plants carrying the events of the invention can be used in breeding programs, using breeding methods, to produce soy plants with additional characteristics of interest, which are combined with glyphosate herbicide tolerance and fungal resistance (also referred to as "stacked features").
- an additional feature may be resistance to additional herbicides, such as glufosinate, and the combination with other traits. agronomically important, including resistance to other diseases and pathogens (for example, Bt genes), as well as the improvement of traits related to the quantity and quality of oils and proteins present in soybeans.
- These combinations of desired characteristics (stacked) can be created by any method, including, but not limited to, plant breeding by any known methodology, or by genetic transformation.
- the nucleic acid sequences typical of the CIGBDt-Def1 or CIGBIs-Def5 event can be combined at any time, and in any order. Traits can be introduced simultaneously, by co-transformation with the nucleic acid molecules of interest, provided by any combination of transformation cassettes. For example, if two additional sequences are introduced, the two sequences may be contained in separate (trans) transformation cassettes or contained in the same transformation cassette (cs). The expression of the sequences can be regulated by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that suppresses the expression of a nucleic acid molecule of interest. This can be adjusted with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of features in the plant. It is further recognized that nucleic acid molecules can be stacked at a desired genomic location, using a site-specific recombination system.
- FIG. 1 Map of plasmid pCP4EPSPS-DEF used for the transformation of meristematic soy tissues using Agrobacterium and biolistics.
- FIG. 1 Immunodetection of the Roundup Ready® CP4EPSPS protein.
- the four strips to the right correspond to transgenic soy lines.
- the strip to the left corresponds to the control of unprocessed soy.
- the upper line functions as a positive control of the strip and the lower line indicates reactivity with an antiCP4EPSPS antibody.
- Figure 3 Products of the PCR reaction from genomic DNA from soybean plants transformed with plasmid pCP4EPSPS-DEF and non-transgenic control. Amplification of a 140 bp sequence corresponding to the nmdef02 gene.
- C + Plasmid pCP4EPSPS-DEF.
- M Molecular weight marker (Promega).
- Lanes DTDefl, DT3, DT4, DT5, DT16, DT17, DT18 and lsDef5 transgenic soy lines.
- C- non-transformed plant.
- Figure 4 Representation of fragment of plasmid pCP4EPSPS-DEF containing the two restriction sites of the EcoRV enzyme (5.3 Kb) and the fragment of the cp4epsps gene used as a probe (886 bp).
- FIG. 6 Relative expression of the nmdef02 gene in transgenic soy plants transformed with plasmid pCP4EPSPS-DEF.
- the quantitative polymerase chain reaction with reverse transcriptase was used to measure transcript levels of the nmdef02 defensin gene, compared to the constitutive expression of the endogenous actin of the non-transformed Dt84 control.
- Transgenic lines DtDefl, Dt3, Dt4, Dt5, Dt6 and lsDef5.
- FIG. 7 Relative expression of the nmdef02 gene in transgenic soy plants of the T3 generation. The bars represent the average of the results obtained from three replicates for p ⁇ 0.0001.
- the qRT-PCR was used to measure the transcript levels of the nmdef02 defensin gene, compared to the constitutive expression of the endogenous actin of the non-transformed control.
- Transgenic lines DtDefl, Dt3, Dt4, Dt5, Dt6 and lsDef5.
- FIG. 8 Quantification of the P. pachyrhizi fungus biomass present in the transgenic soybean lines DtDefl, Dt3, Dt4, Dt5 and Dt6, and in the non-transformed control Dt84. Quantification was done using qRT-PCR.
- Figure 9 Incidence of Asian rust in soybean plants planted in an experimental plot, expressed as a percentage of affected plants.
- Transgenic lines DtDefl, Dt3, Dt6 and lsDef5.
- Untransformed controls DT84 (NT) and LS36 (NT).
- FIG 10. Incidence of Asian rust in transgenic soy plants and in non-transformed controls Dt84 (NT) and ls36 (NT). The percentage of plants affected by the fungus P. pachyrhizi is shown in several transgenic lines.
- Figure 11. Evaluation of the severity of Asian rust in transgenic soy plants that express the nmdef02 and cp4epsps genes, and the non-transformed control (Dt84), sown in the field.
- FIG. 13 Quantification of P. pachyrhizi biomass in soybean plants of different transgenic lines and of the non-transformed control Dt84 (NT). Quantification was performed using qRT-PCR.
- Figure 14 Products of the PCR reaction from DNA from soybean plants (DtDefl Line) and non-transgenic control, with primers designed in regions of the soybean genome adjacent to the sites identified in the bioinformatic analysis. Amplification of a 344 bp sequence corresponding to site 8002360 and a 273 bp sequence corresponding to site 8334726 in the unprocessed soybean plant. Lanes: 1 Kb: Molecular weight marker (Promega), DTDef 1: DTDefl transgenic soybean line. Dt84: unprocessed soybean plant. B: White.
- Figure 15 Products of the PCR reaction from DNA of plants of the CIGBDtDefl transgenic line and of the non-transgenic control, obtained with the combination of soy primers with primers of the cp4epsps gene in the pCP4EPSPS-Def vector ( Figure 1). Amplification of a sequence of 1000 bp at site 8002360 (A) and 900 bp at site 8334726 (B), for the transgenic event CIGBDtDefl. Lanes: 1 Kb: Molecular weight marker (Promega). DTDefl: Transgenic soy line. Dt84: unprocessed soybean plant. B: White.
- Figure 16 Products of the PCR reaction from DNA from the CIGBDtDefl transgenic line and from the non-transgenic control, obtained with the combination of soy primers with nmdef02 gene primers in the pCP4EPSPS-Def vector ( Figure 1). Amplification of a 2000 bp sequence at site 8002360 (A) and 300 bp at site 8334726 (B), for the CIGBDtDefl transgenic event. Lanes: 1 Kb: Molecular weight marker (Promega). DTDefl: Transgenic soy line. Dt84: unprocessed soybean plant. B: White.
- Embryogenic axes were obtained from disinfected seeds of varieties Dt84 and Is36, from the National Institute of Agricultural Sciences. The seeds were disinfested by incubation for one minute in 70% ethanol, and then in a solution of 10% hydrogen peroxide (v / v), for 7 hours, and after successive rinses with sterile distilled water. The disinfected seeds were left at rest, immersed in sterile distilled water, for 28 hours, at room temperature and in the dark. The seed bark was removed on a Petri dish, and the cotyledons were separated to extract the embryo, which was used as an explant in the soybean transformation and regeneration experiments.
- the BAP / IBA combination in the culture medium favored the regeneration of outbreaks, from the apical zone of the meristematic axis, but caused an abundant formation of calluses in the area of the radicle. Therefore, the response of the explants was evaluated in a regeneration medium without auxin (variant B). The results obtained in the regeneration of outbreaks were similar in variant A and B, however, a higher frequency of regeneration and a greater number of outbreaks in the variant without IBA were achieved.
- GUS ⁇ -glucuronidase histochemical test confirmed the presence of enzymatic activity in 56 of the 100 explants, after 48 h, in an induction medium with 5 mg of BAP / L.
- the transient expression of GUS was evident in regions characterized by a strong capacity for division, such as the apical zone of the embryonic axes and the tip of the radicle.
- the apical region of the embryogenic axes was bombarded with the plasmid pCP4EPSPS (Soto et al., Plant Cell Tissue & Organ Culture (2017) 128: 187-196), and transgenic plants were obtained from soybeans that carry the cp4epsps gene.
- Outbreak induction occurred after 48 h, in 5 mg of BAP / L, and the explants were subcultured to MSB5 medium with 25 mg of glyphosate / L.
- the negative control in MSB5 with selection did not show any morphogenesis, and the explants maintained their green coloration.
- the development of well-defined outbreaks (2 cm) was observed after 10 days in MSB5 without selection.
- T-Border (right): Right edge; P35S: Promoter corresponding to the cauliflower mosaic virus (CaMV 35S); cp4epsps: Glyphosate herbicide resistance gene; t35S: Terminator corresponding to the CaMV 35S; Defensin: nmdef02 defensin gene for fungal resistance; TMVomega: Leading sequence of tobacco mosaic virus (TMV); tnos: Nopaline synthase terminator; T-DNA (left): Left border; Kanamycin (R): Gen npt II for kanamycin resistance; pBR322 originated: Origin of replication of plasmid pMB1; pVS1: Origin of replication derived from Pseudomonas aeruginosa.
- the embryogenic axes of the Is36 cultivar were used for transformation via Agrobacter ⁇ um tumefaciens, for which strain LBA4404 was transformed with plasmid pCP4EPSPS-DEF ( Figure 1).
- the procedure consisted of the culture of the bacterium at an optical density at 620 nm of 0.8; and later the infection of the explants, during 30 minutes, in the presence of 200 ⁇ of acetosiringone.
- the explants were co-cultured in dark conditions, for 48 hours, and transferred to MSB5 medium with 25 mg glyphosate / L.
- the defined shoots that emerge from the infested explants were grown in medium without selection, to achieve their rooting. Rooted shoots were adapted under natural conditions, for their molecular and biological characterization.
- genomic DNA was isolated from young leaves of transgenic plants (T2 generation) and from the non-transformed control. All glyphosate-tolerant transgenic lines (and positive for the CP4EPSPS protein immunodetection kit) were analyzed by PCR, to confirm the presence of the nmdef02 gene. In this analysis, a 140 bp signal was obtained, which corresponds to the expected size to confirm the presence of said gene, both in the seven lines derived from the DT genotype, and in those derived from the Is36 genotype, where the lsDEF5 line was selected . The results of this analysis are shown in Figure 3. In the non-transformed plants, used as a negative control, no amplification reaction of the defensin gene was detected.
- the genomic DNA of the transgenic plants of that generation, and of the non-transformed control was digested with the EcoRV enzyme.
- This enzyme has two restriction sites in plasmid pCP4EPSPS-DEF, as shown in Figure 4.
- the digested genomic DNA was analyzed, by Southern blot, to determine the integration of the plasmid into transgenic soy events.
- the results of the Southern blot are shown in Figure 5, where the signals corresponding to the region of the plasmid between the two sites that are recognized by the EcoRV enzyme are visualized.
- the analysis of the relative expression of the defense gene nmdef02 was performed at the transgenic lines (DtDefl; Dt3; Dt4; Dt5; Dt6 e lsDef5), of the T2 generation, using qRT-PCR, to select those with the highest expression for field trials
- the lines evaluated showed different levels of defense gene expression, as shown in Figure 6. It was shown that the accumulation levels of the nmdef02 gene transcript were higher in the DtDefl and lsDef5 lines, followed by Dt3 and Dt6, by what were chosen for the field evaluation, against fungal pathogens. It was found in this analysis that these lines were significantly different from the rest of the lines and the non-transgenic control (p ⁇ 0.0001).
- the response of transgenic plants to glyphosate was evaluated in greenhouse conditions and in the field.
- the application of glyphosate herbicide in greenhouse conditions was carried out in transformed plants, 15 days after its adaptation.
- the herbicide was used at a final dose of 3.5 L / ha, and was applied by a manual sprayer with a capacity of 25 L.
- Two or three days after application all herbicide sensitive plants showed symptoms of chlorosis, and after 7 days they died. However, tolerant plants maintained their green coloration.
- the behavior of the transformed plants against the herbicide was similar in both soybean genotypes. All the plants of the non-transformed control were sensitive to the herbicide.
- the transgenic plants resistant to the herbicide had a growth and development similar to the non-transformed control, which was not subject to the application of the herbicide.
- the herbicide tolerance percentage data in the three generations evaluated (T1, T2 and T3) are shown in Table 2.
- the evaluation of herbicide tolerance in greenhouse conditions showed a group of transgenic lines that presented 80% or more of the plants tolerant to herbicide in the T1 generation.
- the Is19 and Is20 lines were the ones with the highest number of glyphosate sensitive plants, so they were not evaluated in the following generations. From the T2 generation, 100% herbicide tolerance was observed for DtDefl and Dt5, achieving homozygosis in relation to the cp4epsps gene.
- the rest of the events evaluated showed tolerance to the herbicide in the T3 generation, and 100% herbicide resistant plants in the T3 generation were achieved in the lsDef5 line.
- Example 5 Evaluation of Fusarium infection in transgenic soy lines.
- necrosis covered 75% of the leaf, after 15 days. At 25 days, 100% of the leaves were necrotic, and became translucent in the control. On the contrary, in the leaves of genetically transformed plants these symptoms were not observed with that level of affectation so advanced. Events Dt3 and Dt4 showed a nerval chlorosis in the leaves, 10 days after inoculation, and a slight necrosis in the central nerve. These lesions covered less than 12% of the total leaf area in both lines. In contrast, in the DtDefl line there was no involvement by the fungus Fusarium sp., And all the leaves they maintained their intense green coloration. The infection rate observed in some of the lines evaluated in this experiment is shown in Table 3.
- Table 3 Index of infection of the leaves of transgenic soybean plants and of the non-transformed control inoculated with Fusar ⁇ um sp.
- the three evaluated lines were classified as resistant, due to their low infection rate (0.025-1, 20).
- the non-transformed control was classified as highly susceptible, due to its high infection rate (33,1), as shown in Table 3.
- Example 6 Response of the transgenic lines against natural infection by Phakopsora pachyrhizi in mesh house conditions.
- the presence of Asian rust disease was confirmed with the strips of the Envirologix QuickStix® immunodetection kit, for the detection of Phakopsora pachyrhizi. This test was positive in the plants of the non-transformed control and of the transformed lines, although in the transgenic plants few symptoms were observed in the leaves.
- immunodetection the 2.5 cm diameter portion of the sample is taken and placed in a mesh bag. The sample is rubbed across the surface of the mesh bag, and 5 mL of extraction buffer is added. Finally, 200 ⁇ of the solution is transferred to an eppendorf tube and a strip of the kit is placed inside it for 10 minutes. The presence of a second signal, at the bottom of the strip, indicates the presence of Asian rust in the analyzed sample.
- the evaluated lines were harvested, and an agronomic evaluation was made, where different parameters related to performance were considered. In the agronomic evaluation, it was confirmed that the transgenic plants did not show affectations in any of their organs, and their phenotypic characteristics are similar to those of the control. The parameters evaluated were similar between the five transgenic lines, with the exception of the number and weight of the seeds, which was higher in the DtDefl, Dt4 and Dt5 lines. There was a reduction in the yields of Dt84 control, when compared to three of the transgenic lines, as seen in Table 4.
- Example 7 Response of transgenic lines against natural infection by Phakopsora pachyrhizi under field conditions.
- transgenic soy lines which express the defensin gene and the gene responsible for glyphosate tolerance
- the transgenic lines DtDefl, Dt3 and Dt6, and the non-transformed control (Dt84) were evaluated, as well as the lsDef5 line with their respective control (Is36).
- the first symptoms of Asian rust involvement were observed in the non-transgenic control, when the plants were in the grain filling phase. The presence of the disease was confirmed with the Envirologix QuickStix ® immunodetection kit for the detection of Asian rust.
- the lsDef5 line showed a low affectation by rust in plants, of less than 5%, compared with more than 80% of affected plants in the Is36 genotype, used as a non-transformed control of the experiment.
- the plants were harvested and different parameters related to yield were evaluated. These were superior, with statistical significance, in the DtDefl line with respect to the non-transformed control and the rest of the transgenic lines evaluated in this experiment.
- the incidence of the disease was quantified as the percentage of affected plants, where the transgenic lines had some degree of involvement by Asian rust. These showed lower incidence values than the non-transformed control, as shown in Figure 10.
- the DtDefl, Dt12, Dt16, Dt17 and lsDef5 lines were the ones with the lowest incidence percentage.
- the severity of the disease was analyzed using the scale proposed in 2006, by Ploper et al. (Ploper et al., Advance agroindustrial-Agro-Industrial Experimental Station Obispo Colombres (2006) 27: 35-37). For this, leaflets were sampled from the basal, middle and apical areas of the plants. The results shown in Figure 1 1 indicate that the Severity values, expressed in% affectation, were higher in the basal zone than in the middle zone, and these in turn are greater than those in the apical zone. All the transgenic lines showed a lower severity of the disease than the Dt84 control, which had more than 40% involvement due to rust in the leaves of the basal area. The plants of the DtDefl, Dt17 and Dt18 lines showed less involvement by Asian rust; of them, the DtDefl line had less than 8% of affected leaves in the basal area, with values below 5% in the middle and apical areas.
- the plants used as a negative control had a 100% incidence of this disease, which demonstrates the high susceptibility of this genotype to Asian rust.
- the transgenic plants of the susceptible lines evaluated had an incidence of rust that ranged between 58-97%.
- the plants of the resistant transgenic lines had an incidence of 16-35%.
- the leaves of the transgenic plants maintained their green coloration, despite the incidence of the pathogen, unlike the non-transgenic control, which showed chlorosis in their leaves, followed by a generalized defoliation.
- Table 5 shows some results of the evaluation of the harvest of the plants, there was a high difference between the transgenic lines and the control. Defoliation, greater than 20% in the stages near the filling of the grain (R3 to R5), caused an impact on yields in the non-transformed controls.
- each fragment of the library is subject to 2 readings.
- ITIS uses the bwa program (version 0.7.7) to align the readings to the genome and the vector.
- the analysis of the sequences mapped to the vector indicated that a large part of them have little coverage, with the exception of those located on chromosome 2. For this reason, it was decided to continue the analysis in the readings mapped on said chromosome and in the vector (which is represented in the Figure 1 ).
- the transgene insertion at sites 8002360 and 8334726 was validated by PCR, with primers designed in the soybean genome regions adjacent to the sites identified in the bioinformatic analysis.
- the primer sequences were: 3 ' -AAGCGGCAAGTCAATCGTGTCG-5 ' (SEQ ID No.1) and 5 ' -CTGAATCCCTAC ATTGCGATTCTCG-3 ' (SEQ ID No.2), whose combination generated a band at Size of 344 bp with soy DNA without genetic modification, as shown in Figure 14.
- the primer sequences were: 3 ' -ATGTGCAATAATTCCTTCTTCG-5 ' (SEQ ID No.3) and 5 ' - CGAAACACGAATCACGAAGC-3 ' (SEQ ID No.4), this combination generated a band at the size of 273 bp with the soy DNA without genetic modification ( Figure 14). In the transgenic line no band was amplified, when performing the procedure with both pairs of oligonucleotides. The results obtained confirm the insertion of the transgene at sites 8002360 and 8334726, both on chromosome 2 of soy.
- primers designed from the soybean genome sequence were combined with primers in the cp4epsps and nmdef02 genes, both in the pCP4EPSPS-Def vector (shown in Figure 1).
- the combination of primers (Forward and Reverso) in soybeans with primers in the cp4epsps (5 ' - GGATTTCAGCATCAGTGGCTACAGC-3 ' (SEQ ID No.5)) and (3 ' - GCGGGTTGATGACTTCGATGTCG-5 ' (SEQ ID No.6) gene ) confirmed the presence of the transgene at sites 8002360 ( Figure 15A) and 8334726 ( Figure 15B), for the CIGBDtDefl transgenic event.
- Figure 15A shows the amplification of a 1000 bp sequence at site 8002360
- Figure 16B shows the amplification of a 900 bp sequence at site 8334726, for the transgenic event CIGBDtDefl.
- These amplified regions correspond to nucleotide sequences comprising both plant nucleotides and the introduced transgene.
- Figure 16A shows the amplification of a 2000 bp sequence at site 8002360
- Figure 16B shows the amplification of a 300 bp sequence at site 8334726, for the CIGBDtDefl transgenic event.
- These amplified regions correspond to nucleotide sequences comprising both plant nucleotides and the introduced transgene. This demonstrates the insertion of the transgene in the DtDef 1 event.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Physiology (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Harvesting Machines For Specific Crops (AREA)
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201880035654.4A CN110914436B (zh) | 2017-03-31 | 2018-03-29 | 包含转基因事件CIGBDt-Def1或CIGBIs-Def5的大豆植物 |
MX2019011633A MX2019011633A (es) | 2017-03-31 | 2018-03-29 | Plantas de soya que comprenden el evento transgenico cigbdt-def1 o cigbis-def5. |
BR112019020260-9A BR112019020260A2 (pt) | 2017-03-31 | 2018-03-29 | Planta de soja ou uma parte da dita planta, semente de uma planta de soja, produto de soja, métodos para a produção de uma planta de soja, para o diagnóstico do evento cigbdt-def1 ou do evento cigbis-def5 em uma amostra de soja e para aumentar o rendimento de uma colheita de soja no campo, e, kit de reagentes. |
CA3057889A CA3057889A1 (en) | 2017-03-31 | 2018-03-29 | Soy plants comprising the transgenic event cigbdt-def1 or cigbis-def5 |
US16/498,475 US11917966B2 (en) | 2017-03-31 | 2018-03-29 | Soy plants comprising the transgenic event CIGBDT-DEF1 or CIGBIS-DEF5 |
EP18725387.7A EP3604545A2 (en) | 2017-03-31 | 2018-03-29 | Soy plants comprising the transgenic event cigbdt-def1 or cigbis-def5 |
RU2019134559A RU2019134559A (ru) | 2017-03-31 | 2018-03-29 | РАСТЕНИЯ СОИ, СОДЕРЖАЩИЕ ТРАНСГЕННОЕ СОБЫТИЕ CIGBDt-DEf1 ИЛИ CIGBIs-DEf5 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CU2017-0042 | 2017-03-31 | ||
CU2017000042A CU24471B1 (es) | 2017-03-31 | 2017-03-31 | Método para la producción de plantas de soya resistentes a glifosato y a enfermedades causadas por hongos y oomicetos |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2018177446A2 true WO2018177446A2 (es) | 2018-10-04 |
WO2018177446A3 WO2018177446A3 (es) | 2018-11-15 |
Family
ID=62196279
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CU2018/050002 WO2018177446A2 (es) | 2017-03-31 | 2018-03-29 | Plantas de soya que comprenden el evento transgenico cigbdt-def1 o cigbis-def5 |
Country Status (10)
Country | Link |
---|---|
US (1) | US11917966B2 (es) |
EP (1) | EP3604545A2 (es) |
CN (1) | CN110914436B (es) |
AR (1) | AR111206A1 (es) |
BR (1) | BR112019020260A2 (es) |
CA (1) | CA3057889A1 (es) |
CU (1) | CU24471B1 (es) |
MX (1) | MX2019011633A (es) |
RU (1) | RU2019134559A (es) |
WO (1) | WO2018177446A2 (es) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AR123378A1 (es) * | 2020-08-31 | 2022-11-23 | Basf Se | Mejora del rendimiento |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MX346994B (es) * | 2010-10-12 | 2017-04-06 | Monsanto Technology Llc | Planta y semilla de soja correspondiente al evento transgénico mon87712 y métodos para su detección. |
KR20140135576A (ko) * | 2013-05-16 | 2014-11-26 | 한국표준과학연구원 | 유전자 변형체의 검정을 위한 표준 플라스미드, 이를 이용한 분석 방법 및 검정용 키트 |
-
2017
- 2017-03-31 CU CU2017000042A patent/CU24471B1/es unknown
-
2018
- 2018-03-29 EP EP18725387.7A patent/EP3604545A2/en active Pending
- 2018-03-29 CN CN201880035654.4A patent/CN110914436B/zh active Active
- 2018-03-29 CA CA3057889A patent/CA3057889A1/en active Pending
- 2018-03-29 WO PCT/CU2018/050002 patent/WO2018177446A2/es unknown
- 2018-03-29 US US16/498,475 patent/US11917966B2/en active Active
- 2018-03-29 BR BR112019020260-9A patent/BR112019020260A2/pt unknown
- 2018-03-29 RU RU2019134559A patent/RU2019134559A/ru not_active Application Discontinuation
- 2018-03-29 MX MX2019011633A patent/MX2019011633A/es unknown
- 2018-04-03 AR ARP180100831A patent/AR111206A1/es unknown
Non-Patent Citations (15)
Title |
---|
CARMONA ET AL., PHYTOPHATOLOGICA, vol. 37, 2011, pages 134 - 139 |
DE BEER Y VIVIER, BMC RES. NOTES, vol. 4, 2011, pages 459 |
FUNKE ET AL., PNAS, vol. 103, no. 35, 2006, pages 13010 - 13015 |
GALLY ET AL., MANEJO INTEGRADO DE PLAGAS Y AGROECOLOGÍA, vol. 78, 2006, pages 86 - 90 |
MURSHIGE; SKOOG, PLANT PHYSIOL, vol. 15, 1962, pages 473 - 479 |
NTUI ET AL., PLANT CELL REPORT, vol. 29, 2010, pages 943 - 954 |
PANDEY ET AL., MOLECULAR PLANT-MICROBE INTERACTIONS, vol. 24, 2010, pages 194 - 206 |
PÉREZ ET AL., PLANT PATHOLOGY, vol. 59, 2011, pages 803 |
PLOPER ET AL., AVANCE AGROINDUSTRIAL-ESTACIÓN EXPERIMENTAL AGRO-INDUSTRIAL OBISPO COLOMBRES, vol. 27, 2006, pages 35 - 37 |
PORTIELES ET AL., PLANT BIOTECHNOLOGY JOURNAL, vol. 8, 2010, pages 678 - 690 |
RIBEIRO ET AL., GENETICS AND MOLECULAR BIOLOGY, vol. 31, 2008, pages 98 - 105 |
SCONYERS ET AL., PLANT DISEASE, vol. 90, 2006, pages 972 - 972 |
SOTO ET AL., PLANT CELL TISSUE & ORGAN CULTURE, vol. 128, 2017, pages 187 - 196 |
TORRES ET AL., PESQ. AGROPEC. BRAS, vol. 38, 2012, pages 1053 - 1057 |
ZHU ET AL., CELL MOL.LIFE SCI., vol. 62, 2015, pages 2257 - 2269 |
Also Published As
Publication number | Publication date |
---|---|
US11917966B2 (en) | 2024-03-05 |
WO2018177446A3 (es) | 2018-11-15 |
EP3604545A2 (en) | 2020-02-05 |
CN110914436A (zh) | 2020-03-24 |
CU24471B1 (es) | 2020-02-04 |
BR112019020260A2 (pt) | 2020-05-12 |
MX2019011633A (es) | 2019-11-18 |
AR111206A1 (es) | 2019-06-12 |
CA3057889A1 (en) | 2018-10-04 |
RU2019134559A (ru) | 2021-04-30 |
CN110914436B (zh) | 2023-06-23 |
CU20170042A7 (es) | 2018-11-06 |
US20230135233A1 (en) | 2023-05-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20090044297A1 (en) | Transgenic plants with enhanced agronomic traits | |
CN112831584B (zh) | 转基因玉米事件lp007-2及其检测方法 | |
CN112852801B (zh) | 转基因玉米事件lp007-1及其检测方法 | |
US20220145314A1 (en) | Transgenic plants with enhanced traits | |
US20090049573A1 (en) | Transgenic plants with enhanced agronomic traits | |
BRPI0413917B1 (pt) | ácido nucleico ahas de arroz mutagenizado não transgênico, polipeptídeo ahas isolado, e, método de controle de ervas daninhas dentro da vizinhança de uma planta de arroz | |
JP5652799B1 (ja) | ダイズ第3番染色体に座上する耐塩性を制御する遺伝子qNaCl3とその利用法 | |
US10844390B2 (en) | Root-preferential and stress inducible promoter and uses thereof | |
US20190136247A1 (en) | Flowering time-regulating genes and related constructs and applications thereof | |
US20130145493A1 (en) | Transgenic Plants with Enhanced Agronomic Traits | |
US9926573B2 (en) | Glyphosate-tolerant gene and use thereof | |
CN113151533B (zh) | 转基因玉米事件lp007-6及其检测方法 | |
US10550403B2 (en) | Transgenic plants with enhanced traits | |
US20240102037A1 (en) | Transgenic plants with enhanced traits | |
US9322031B2 (en) | Transgenic plants with enhanced agronomic traits | |
ES2376003T3 (es) | Uso de trehalosa-6-fosfato sintasa para modular el crecimiento vegetal. | |
US20230151382A1 (en) | Plant pathogen effector and disease resistance gene identification, compositions, and methods of use | |
BR102016021980A2 (pt) | Genetically modified plants for improving cultural performance | |
WO2018177446A2 (es) | Plantas de soya que comprenden el evento transgenico cigbdt-def1 o cigbis-def5 | |
US20190023753A9 (en) | Transgenic Plants with Enhanced Traits | |
BR112020003918A2 (pt) | composições e métodos para expressar transgenes usando elementos reguladores de genes ab de ligação de clorofila | |
ES2902959T3 (es) | Planta restauradora | |
ES2291208T3 (es) | Procedimiento para transmitir resistencia bnyvv a plantas de remolacha azucarera. | |
Serrat Gurrera | Applied biotechnology to improve Mediterranean rice varieties= Biotecnologia aplicada a la millora de varietats d’arròs mediterrànies | |
Lokuge | Tissue culture, genetic transformation and cold tolerance mechanisms in cold-hardy palms |
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: 18725387 Country of ref document: EP Kind code of ref document: A2 |
|
ENP | Entry into the national phase |
Ref document number: 3057889 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112019020260 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 2018725387 Country of ref document: EP Effective date: 20191031 |
|
ENP | Entry into the national phase |
Ref document number: 112019020260 Country of ref document: BR Kind code of ref document: A2 Effective date: 20190927 |