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  • 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
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  • 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
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  • 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
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• Agrobacterium as well as transgenic plants or parts thereof, which were produced by the improved method.
• Triticum aestivum Products of plants of the genus Triticum such as wheat (Triticum aestivum) are one of the most important raw materials and play a key role as staple food in large parts of the world. Nevertheless, in the last fifty years, for example, the progress achieved by conventional breeding has fallen in the wheat
• marker genes provide a way to distinguish the transgenic from the non-transgenic plants.
• the application allows the selection with marker genes a more efficient transformation, or makes a
• the selection marker Since the selection marker is needed only during the ' iro-phase in the transgenic plant, it later fulfills no function in the plant and is therefore superfluous. However, since the number of available selection markers is limited, the presence of the no longer required selection marker makes it difficult to subsequently super-transform the already transgenic plant with a second goi. Stacking of multiple genes by sequential transformation is thus limited and limited by the number of different selection markers that are available for the particular plant species.
• selection marker is associated with a considerable work, cost and time.
• nucleases e.g., zinc finger nucleases. Such nucleases must do so by crossing with a nuclease
• the plants can be transformed with two T-DNAs, with one T-DNA carrying the goi and the other T-DNA carrying the selection marker.
• the plants In about 30% to 50% of the generated, transgenic plants, it then comes to the integration of the two T-DNAs in one Cell, but at different locations of the genome. This allows meiosis to segregate the selection marker and the goi in the next generation.
• identification of selection marker-free plants is only in the first
• the transformed explant usually undergoes several selection steps in the callus phase. During this selection phase takes place through the presence of an antibiotic or a herbicide, an enrichment of transgenic cells in the callus, which carry the corresponding resistance gene, that is transgenic. Non-transgenic cells are inhibited in their growth and die off, which significantly increases the likelihood that, in particular, transgenic shoots will regenerate from the selected callus.
• Faize et al. (2010) that during the transformation process of apricot the proportion of transgenic tissue in apricot shoots can be increased by multiple subculturing on selective medium and thus a chimeric character of the shoots can be reduced or eliminated by applying the selection.
• the selection steps are lacking, there is a clear risk that the non-transgenic shoots will be superior to those from transgenic cells during regeneration. It is believed that the transformed cells through the one
• Transgen is not passed on to the next generation.
• WO 2008/028121 describes the preparation of selection marker-free maize plants which can be generated without the use of a selection. While the authors suggest applying the disclosed method to other Poaceae such as wheat, the examples presented are limited to the production of transgenic maize plants only. In addition, while the authors suggest that the maize plants produced should preferably not be chimeric, no experimental data is provided on the inheritance of the transgene to the next generation, so that it can not be ruled out that a large part of the transgenic maize lines produced are chimeric , EP 2 274 973 likewise describes the production of transgenic monocotyledonous plants, in particular maize and rice plants, by means of / Agrobacter / Um-mediated transformation, in which no selection step is used. For corn it is clearly shown that a not inconsiderable number
• Chimeric plants is created, which must be identified and sorted out consuming.
• the proportion of chimeric starting transformants was> 50% of the obtained transgenic shoots. Only less than 20% of the generated transgenic plants were not chimeric (uniform) at all.
• the number of transformants with chimeric character is many times higher than in the transformation with corresponding selection steps. For example, in Coussens et al. (2012) showed that at the
• EP 2 460 402 A1 discloses a particularly efficient process for the transformation of wheat cells by means of Agrobacterium tumefaciens, which in regeneration is intended to enable yields of 70% and more transgenic lines per isolated initial explant.
• the transformation protocol used here always involves the use of the selection markers hygromycin phosphotransferase (hpt) or phosphinotricin acetyltransferase (PAT / bar).
• the present invention has been made in light of the above-described prior art, and it is an object of the present invention to provide a Rhizobiaceae-mediated method for producing a transgenic plant of the genus Triticum which does not require marker gene-based selection and undesirable ones described above Effects minimized or only to a small extent shows.
• a further object of the present invention is a process for producing a transgenic plant of the genus Triticum, which is superior from previous methods both from an economic and a regulatory point of view.
• a method for producing a transgenic plant of the genus Triticum comprising the steps of (a) transforming at least one cell of a plant of the genus Triticum with a genetic
• Step (a) to step (b) does not select a transformed cell from (a) based on a property mediated by the genetic component or a part thereof.
• a bacterial cell of the family Rhizobiaceae is a bacterial cell of the genus Agrobacterium and more preferably a bacterial cell of the species
• Bacterial cell the genetic component on a vector, in particular on a binary vector, a super binary vector or a vector of a cointegrative
• the genetic component is a nucleic acid molecule, in particular a DNA molecule or a recombinant DNA, and comprises at least the gene of interest.
• the genetic component may comprise a regulatory sequence, an intron, a recognition sequence for an RNA molecule, a DNA molecule or a protein or a 5 'or 3' UTR (untranslated region).
• the transformation in step (a) may be carried out under conditions which permit successful infection of at least one cell of an explant of the plant of the genus Triticum with a bacterial cell of the family Rhizobiaceae.
• transformation conditions are known to those skilled in the art (Cheng et al., 1997).
• the explant used in step (a) is an embryonic tissue, in particular radicle, embryo axis, scutellum or germ, or a part thereof and constitutes part of an immature embryo or mature seed (EP 0 672 752 B1).
• other suitable tissues are also known which can be successfully used for transformation of plants of the genus Triticum such as wheat (Shrawat and Lörz (2006)).
• regeneration of a transgenic plant of the genus Triticum from at least one transformed cell from (a) in step (b) also means the regeneration of a plant from a transformed cell which has emerged from at least one transformed cell from (a) by cell division, for example in the course of the formation of a callus, which transforms into somatic embryos, to then lead to the shoot regeneration.
• Various techniques for the regeneration of a plant of the genus Triticum are known to those skilled in the art. Regeneration can be done, for example, from immature embryos (Vasil et al., 1993). Another possibility for regeneration results from anthers or microspores (eg Maluszynski et al., 2003).
• wheat plants have already been regenerated from flower tissue (Amoah et al., 2001) and from callus of mature embryos (Wang et al , 2009)
• a transformed cell from (a) may also mean a transformed cell which has emerged from at least one transformed cell from (a) by cell division.
• no selection based on a property mediated by the genetic component or a portion thereof is not selection based on herbicidal or antibiotic resistance.
• a herbicide resistance can be achieved, for example, by the expression of the phosphinotricin acetyltransferase from Streptomyces hygroscopicus or Streptomyces viridochromogenes, which confers resistance to the herbicide phosphinotricin or bialaphos (De Block et al., 1987).
• Another herbicide resistance, the resistance to the drug glyphosate can be achieved by the overexpression of 5-enolpyruvylshikimate-3-phosphate synthase. Usually, this enzyme is used which is insensitive to glyphosate (Comai et al., 1983).
• Pyrimidinyl (thio) benzoates can be achieved by expression of a mutagenized form of the enzyme acetolactate synthase (ALS). Different mutations lead to a resistance to the different herbicides. An overview of commonly used herbicide resistance can be found in Tuteja et al (2012), Kraus (2010) or Shrawat and Lörz (2006). Antibiotic resistance can be achieved by expression of bacterial genes that inactivate the antibiotic used by transferring a phosphate or acetyl group. Examples of these are neomycin phosphotransferase (npt), which has a
• hygromycin phosphotransferase is used, which mediates resistance to the antibiotic hygromycin B.
• Antibiotic resistance used in plant transformation can be found in Tuteja et al (2012), Kraus (2010) or Shrawat and Lörz (2006).
• Anthocyanins or other plant dyes by the expression of certain
• PMI Phosphomannose isomerase
• step (b) of the method according to the invention not only transgenic plants but also transgenic or chimeric plants in step (b) can regenerate.
• transgenic plants long countered an economically useful use of a marker gene-free method for producing a transgenic plant.
• the production of a transgenic plant with selection based on a marker gene and the subsequent removal of the selection marker even if this was associated with immense work, cost and time, was still the method of choice for transgenic, selection marker to create free plants.
• transgenic maize and rice plants still using, in particular
• Marker gene-based selection takes place. To a not inconsiderable extent, this is also due to the continuing problem of the increased generation of chimeric plants in the absence of a selection marker and their subsequent necessary identification and sorting. Usually, the proportion of chimeric plants is significantly higher in the absence of the marker gene-based selection compared to the proportion which is achieved when using a marker gene.
• the method according to the invention describes for the first time the production of a transgenic plant of the genus Triticum using a Rhizobiaceae-mediated
• Transformation wherein no selection of a transformed cell based on a property mediated by the introduced during the transformation of the genetic component or a part thereof, takes place. Contrary to expectations, the process of the present invention exhibited a surprisingly high transformation efficiency, which was significantly higher than known in the art
• the method preferably has one
• Transformation efficiency of at least 5%, 6%, 7%, 8%, 9% or 10% more preferably at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19 %, 20% or even more preferably at least 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34 %, 35%, 36%, 37%, 38%, 39%, 40% or more than 40%.
• a method described above is characterized in that the transformation efficiency is increased by a treatment for increasing the transformation efficiency.
• the treatment for increasing the transformation efficiency may have a transformation efficiency of at least 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more preferably at least 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32% , 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or more than 40%.
• Various treatments to increase the transformation efficiency may have a transformation efficiency of at least 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more preferably at least 21%, 22%, 23%, 24%, 25%, 26%, 27%,
• the treatment for enhancing the transformation efficiency may include at least one treatment selected from
• Transformation efficiency also represent a combination of known treatments to increase the transformation efficiency.
• a method described above is either characterized in that the regeneration of a transgenic plant of the genus Triticum in step (b) comprises non-chimeric, transgenic plants with a frequency of at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 22% at least 24%, at least 26% at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38% or at least 40%, preferably of at least 45%, at least 50% at least 55%, at least 60%, at least 65% or at least 70%, particularly preferably at least 75% at least 80%, at least 85% or at least 90 produces, or characterized the regeneration of a transgenic plant of the genus Triticum in step (b) preferably less than 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 28%, 26%, 24%, 22%, 20%, 18% 16%, 15%
• the proportion of non-chimeric, transgenic plants of the genus Triticum from step (b) is comparable to the proportion of non-chimeric, transgenic plants of the genus Triticum, which are regenerated in a corresponding comparison method, the differs in that selecting a transformed cell based on a
• chimeric transgenic plants can arise when a regenerating shoot has formed from multiple cells of origin, with some of these cells transgenic and another non-transgenic. It can for example
• Sectoral chimera or periclinic chimera arise. Due to the proportion of non-transgenic tissue in the chimeric plants, these may e.g. by the quantitative PCR
• Another detection method for chimeric transgenic plants is the analysis of the first progeny of a starting transformant
• the initial transformant introduced genetic component or part thereof may be passed on to the next generation according to Mendel's rules.
• Mendel's rules When integrating a copy of the genetic component or a part thereof into the genome of the plant cell, it is integrated on only one chromosome of the diploid genome.
• the genetic component or part thereof When integrating a copy of the genetic component or a part thereof into the genome of the plant cell, it is integrated on only one chromosome of the diploid genome.
• meiosis the genetic
• transgenic plants in chimeras are also formed from the non-transgenic parts of the plant gametes. In these tissues, only gametes are formed that do not contain the genetic component or part of it. Thus, in chimera transgenic plants, the proportion of non-transgenic gametes increased to> 50%. In the selfing offspring of the chimeras
• Mendel's rules would be expected.
• Method is the proportion of chimeras, transgenic plants of the genus Triticum from step (b) comparable to the proportion of chimeras, transgenic plants of the genus Triticum, which are regenerated in a corresponding comparison method, which differs in that a selection of a transformed cell based on a
• the method according to the invention is characterized in that after step (b) it comprises a further step (c) selecting the regenerated transgenic plant from step (b). Preferably, this is done
• Step (c) is to demonstrate the successful transformation of the genetic component or a part thereof into the cell of a plant of the genus Triticum, i. also the transfer of the genetic component or a part thereof into the genome of the plants.
• nucleic acid complementary to the introduced genetic component with the genomic DNA of the transgenic plant e.g. in the so-called Southern Blot (Southern, 1975) or via sequencing of the genome of the transgenic plant (Kovalic et al., 2012).
• the molecular structure of the genetic component or a part thereof can also be the molecular structure of a derived component which can be obtained, for example, by transcription, processing and / or translation from the
• RNA formed from the genetic component or a part thereof into cDNA and subsequent polymerase chain reaction RT-PCR, Sambrook et al., 2001
• Hybridization of a detectable single-stranded nucleic acid complementary to the introduced genetic component with the RNA of the transgenic plant (Northern Blot, Sambrook et al., 2001) or the transcription of an RNA formed from the genetic component or a portion thereof into cDNA followed by sequencing the entire pool of obtained cDNA.
• the encoded peptide / polypeptide / protein can be identified, for example, by immunodetection using different methods such as Western blot or ELISA. Furthermore, to select a phenotypic
• Such phenotypic detection may also include detection of altered chemical composition of the plant cell. This altered chemical composition can then be detected by known methods of chemical analysis.
• the at least one cell of a plant of the genus Triticum is transformed with the complete genetic component in step (a), in particular stably transformed.
• Completely means preferably that the at least one cell of a plant of the genus Triticum is transformed with the genetic component, whereby the genetic component does not produce truncations (for example from the 5 'or 3' end) which affects the intended functionality of the genetic component in the cell of a plant of the genus Triticum, and particularly preferred that the at least one cell of a plant of the genus Triticum has been transformed with all nucleotides of the genetic component.
• the genetic component after transformation in step (a), the genetic component exhibits an expression level in the cell of a plant of the genus Triticum, which enables the intended functionality of the genetic component.
• the genetic component Preferably that is
• a method according to the invention characterized in that 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the transformed cells of step ( a) have a detectable level of expression, preferably an expression level, which the intended functionality of the genetic component
• Step (b) comprises cells which have a detectable level of expression, preferably one
• FIG. 1 shows an estimate of the costs for the generation of 100 TO transgenic lines in a co-transformation approach as well as in the selection marker-free transformation.
• the generated initial transformants are further analyzed in the next generations, with the aim of homozygous, selection marker-free
• the present invention further encompasses a transgenic plant of the genus Triticum, which has been produced by one of the methods described above, as well as a descendant, a part or a seed thereof, the offspring, the part or the seed containing the genetic component which is present in step ( a) of the invention
• a part can mean a cell, a tissue or an organ.
• a “gene of interest” may be any type of DNA or RNA molecule encoding, for example, a protein, or a nucleic acid molecule.
• a "plant of the genus Triticum” means a plant of the species Triticum aestivum, a plant of the species Triticum durum or a plant of the species Triticum spelta.
• a "regulatory sequence” in the context of the present invention is a nucleic acid sequence which controls the expression of a gene of interest, examples being promoters, operators, enhancer elements, attenuators, cis elements etc.
• selection marker is used in the context of the present invention to mean selection marker genes or marker genes Examples of useful selection markers are described above.
• Transformation efficiency may mean the ratio of the number of explants with positive transgenic sprouts to the number of initial explants
• Transformation efficiency is preferably given as a percentage.
• FIG. 1 Cost comparison for the generation of 100 T0 plants by means of cotransformation (left) and by means of a method of the present invention and further identification of homozygous, selection marker-free seed pools.
• Figure 2 View of the scutellum of a tDT-transformed wheat embryo 5 days after infection with A. tumefaciens (left: in fluorescent light, right: in daylight); Arrows show fluorescent regions in the starting explant, which give an example of the presence of the agrobacteria.
• FIG. 3 Binary vector pLH70SubiintrontDT (tDT is tandem dimer Tomato, a red
• FIG. 4 Southern blot of selected, selection-marker-free transgenic lines of the transformation experiment WA1. 20 ⁇ g of the genomic DNA of the respective line were completely digested with the enzyme HindIII, separated in a 0.8% agarose gel, blotted onto a nylon membrane and then hybridized with a DIG-labeled PCR product (tDT-rev / tDT-for).
• FIG. 5 Expression analysis of the introduced tDT gene by qRT-PCR in
• FIG. 6 Determination of the zygotic status by means of qPCR on the introduced transgene tDT and on the introduced nos terminator (see FIG. 3).
• Cultivation conditions were 18 ° C during the day and 16 ° C during the night, the daylength being 16 hours.
• Sodium lamps were used as the illumination source (Master SON-T Agro 400W).
• the embryo size in the developing ears was regularly checked and ears containing embryo-sized grains about 1.5-1.5mm in size were harvested and stored in the dark at 4 ° C until further use in water .
• the grains were isolated from the wheat spikes and then surface sterilized. For this purpose, the grains were first incubated for 45 seconds in 70% ethanol and then incubated for 10 minutes in 1% sodium hypochlorite solution. After sterilization, the grains were freed from adhering sodium hypochlorite by repeated washing in sterile water. The sterilized granules were then stored in the dark at 4 ° C until further use.
• Agrobacterium tumefaciens for transformation was carried out starting from a glycerol culture of the A. tumefaciens strain AGL1, which carries the gene construct to be transformed in the binary vector pLH70SubiintrontDT (FIG. 3). After smear on selective LB medium (with 100 mg / L rifampicin, 100 mg / L carbenicillin, 50 mg / L
• Liquid culture in MG / L medium (Wu et al., 2009) with 100 mg / L rifampicin, 100 mg / L
• the immature embryos were isolated and collected in the Inf liquid medium (Table 1). Subsequently, the embryos were washed once with fresh inf liquid medium and then pretreated by centrifugation at 15,000 rpm for 10 minutes. For infection with the agrobacteria was prepared
• FIG. 2 shows the scutellum of a transformed wheat embryo after several days after infection with A. tumefaciens.
• the embryo axis was removed from each embryo using a sharp scalpel and the remaining scutella were placed on a resting medium (Table 1). The plates with the scutella were then incubated for 5 days at 25 ° C in the dark. Subsequently, the developing callus was again subcultured at 25 ° C in the dark on the resting medium (Table 1) for 21 days.
• the induced callus was reacted to LSZ medium as a whole (Table 1) and exposed to light for 14 days. Forming green shoots were separated from callus and transplanted to LSF medium (Table 1) for rooting. The shoots were, as far as this was possible, separated from each other to obtain single shoots. Sprouts from an original explant (scutellum) were kept together. After sufficient growth of the shoots, leaf samples were taken for DNA extraction and subsequent PCR analysis.
• Embryos 89 embryos are stimulated to regenerate sprouts.
• the regenerated shoots were initially totaled 341 for PCR analysis
• Sprout pools summarized. For this purpose, depending on the number of regenerated shoots per initial explant, in each case 2 to 3 shoots of an explant were combined in a sample vessel for the purpose of DNA extraction. Should more than three shoots be regenerated per initial explant, then several sprout pools of one
• the primers tDT-1 (SEQ ID NO: 1) and tDT-2 (SEQ ID NO: 2) were used. DNA's in which a 287 bp fragment is amplified was able to demonstrate the presence of the introduced recombinant DNA and were considered transgenic.
• a quantitative PCR was carried out with the primers nosTxxxfO1 (SEQ ID NO: 3) and nosTxxxr03 (SEQ ID NO: 4) and the probe nosTxxxMGB (SEQ ID NO: 5). The quantitative PCR confirmed the previously obtained results with the classical PCR.
• the transgene could be detected in a total of 82 sprouts.
• the 82 shoots were from 37 explants / embryos that were initially infected with A. tumefaciens.
• the WA1 experiment achieved a transformation efficiency of ⁇ 25%. This efficiency is calculated from the 37 explants with positive sprouts, from originally 151 explants.
• transgenic single shoots could be identified in 56% (WA2) and 75% (WA3) of the regenerable explants.
• control experiments WA1 K, WA2K and WA3K were carried out, in which the selection marker hygromycin phosphotransferase (hpt) was integrated together with the gene of interest into the genome of the wheat variety typhoon.
• the transformations were carried out as described in EP 2 460 402, i. while the callus and
• Regeneration phase was added to the medium hygromycin in concentrations of 15 mg / L or 30 mg / L.
• the found transformation efficiency without application of selection corresponds to the efficiency that is usually achieved in wheat transformation experiments with marker gene-based selection, in some cases the efficiencies even seem to be higher.
• Table 2 Results of three transformation trials without the use of a marker gene-based selection in Triticum aestivum (variety Taifun); WAKx refers to the control experiments with marker gene-based selection, WAx refers to experiments without marker gene-based selection
• experiment WA2 identified twelve independent single copy lines using the qPCR approach. Since a total of 27 independent transgenic events were generated, this equals a rate of 44% single copy events. The experiment WA3 also generated 12 independent single copy events, resulting in a total of 42 generated independent events at a rate of 29%.
• T-DNAs are often designed so that the selection marker used for selection is positioned on the LB side of the T-DNA. Thus, only events with complete T-DNA, including completely transferred marker gene, can then be selected. Since only gene of interest as T-DNA is present in marker gene-free transformation, the gene of interest could thus be shortened involuntarily during transfer, which usually leads to erroneous expression of the transmitted gene of interest in the plant genome.
• Hybridization probe used. The genomic DNA was digested with HindIII so that a fully integrated T-DNA yielded a hybridization fragment greater than 3.0 kb. As can be seen in Figure 4, in all tested, PCR positive lines could be used.
• Hybridization fragment can be found. Genomic DNA from the negative control (Typhoon) did not hybridize to the probe. Since all hybridization fragments obtained have a size of> 3.0 kb, this demonstrates that the T-DNA is completely integrated in all the lines shown. This shows that the quality of the transgene after transfer is comparable to that using transformation with LB-side marker gene. This was not to be expected for a professional.
• Transformation method with respect to the level of expression of the integrated transgene studied more closely.
• T-DNA with selection marker it is for the successful selection of transgenic lines necessary that the gene of
• Selection marker is expressed, and thus it comes to the formation of the functional protein. T-DNA integrations in genomic regions that do not require reading of the
• transgenic line can not be identified as a transgenic line.
• events integrated into regions of the genome that do not allow the transgene to be read are also identified as a transgenic line by molecular biology techniques such as PCR.
• molecular biology techniques such as PCR.
• the expression level of the introduced transgene was determined from randomly selected lines of the transformation experiment WA1 by means of qRT-PCR (FIG. 5). In only 3 of the analyzed 13 transgenic lines no expression of the transgene could be detected. All other lines show a clear expression of the transgene, although the level of expression between the individual lines is clearly different. However, this is also the case in transgenic lines using a
• the introduced transgene is passed on to the next generation according to the Mendelian rules.
• the seeds of 6 transgenic lines were designed (each 30 grains / line) and the presence of the transgene and its zygote status were determined by qPCR on the introduced transgene tDT and on the introduced nos terminator.
• An example is the result of an analysis of a
• transgenic line azygot hemizygot homozygous total cleavage ratio chi 2
• PCTOC Plant Cell, Tissue and Organ Culture
• Mußmann, V., Serek, M., & Winkelmann, T. (201 1). Selection of transgenic petunia plants using green fluorescent protein (GFP). Plant Cell, Tissue and Organ Culture (PCTOC), 107 (3), 483-492.
• GFP green fluorescent protein
• PCTOC Plant Cell, Tissue and Organ Culture
• Phosphomannose isomerase an efficient selectable marker for plant
• Vasil V V Srivastava, AM Castillo, ME Fromm, IK Vasil (1993) Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos.
• WO 2002/012520 A1 Japanese Tobacco Inc.
• EP 2 274 973 A1 (Japan Tobacco Inc.) "Agrobacterium mediated method for producing transformed plant”
• EP 2 460 402 A1 (Japan Tobacco Inc.) "Method for gene transfer into Triticum plants using Agrobacterium bacterium, and Method for production of transgenic plant of triticum plant”

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