EP3080278A1

EP3080278A1 - Selection marker-free rhizobiaceae-mediated method for producing a transgenic plant of the genus triticum

Info

Publication number: EP3080278A1
Application number: EP14835695.9A
Authority: EP
Inventors: Klaus Schmidt
Original assignee: KWS SAAT SE and Co KGaA
Current assignee: KWS SAAT SE and Co KGaA
Priority date: 2013-12-15
Filing date: 2014-12-13
Publication date: 2016-10-19
Also published as: US20160312235A1; WO2015085990A1; AU2014361303A1; AR098760A1; AU2014361303B2; DE102013020605A1; CA2933922A1; CN106164277A

Abstract

The present invention relates to an improved method for producing a transgenic plant of the genus Triticum, with steps of a) Rhizobiaceae-mediated transforming of at least one cell of a plant of the genus Triticum with a genetic component, and b) regenerating of a transgenic plant of the genus Triticum from a transformed cell, where from step (a) to step (b) there is no selecting of a transformed cell based on a trait mediated by the genetic component, or of a part thereof.

Description

SELECTION MARKERS-FREE RHIZOBIA CEAE COMPOUND TE S METHOD FOR PRODUCING A TRANSGENIC PLANT OF THE GENUS TRITICUM

Field of the invention

The present invention relates to the field of biotechnology and comprises

improved process for the production of a transgenic plant of the genus Triticum by means of bacterial cells from the family Rhizobiaceae, in particular the genus

Agrobacterium, as well as transgenic plants or parts thereof, which were produced by the improved method.

Background of the invention

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

With regard to various features, such as Yield significantly lower than in other crops such as corn, sugar beet or rapeseed. The development of transgenic plants of the genus Triticum is a way to make up for this failed progress at least in part. However, the production of transgenic plants of the genus Triticum via Rhizobiacea (e.g., Agrobacterium tumefaciens) -mediated transformation has always been extremely difficult. In general, this is achieved, for example, by for wheat only efficiencies of 1% - 3% transgenic lines per isolated

Starting explants. In individual cases, transformational protocols with efficiencies of up to 10% are described in the literature (Hensel et al., 2009; Shrawat and Lörz, 2006), although these are often not feasible in practice. Known protocols include almost exclusively the use of marker genes for selection (selection marker) in a cotransformation. The selection marker is usually coupled with the gene of interest (goi) to be transformed. As a marker gene is usually used either an antibiotic resistance gene or a herbicide resistance gene, which transformed cells under certain in vitro conditions a survival advantage during the

Regeneration phase. Thus, marker genes provide a way to distinguish the transgenic from the non-transgenic plants. Ultimately, the application allows the selection with marker genes a more efficient transformation, or makes a

Transformation possible in the first place.

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.

Furthermore, the use of particular antibiotic resistance genes as

Selection markers in transgenic plants in the public in the criticism, so that in principle in the regulatory approval and in commercialization only transgenic plants without selection markers are acceptable. The removal of the

However, selection marker is associated with a considerable work, cost and time.

Technically, the skilled person today various methods and tools for the removal of a selection marker from the genome of a transgenic line

Available. First, one can use highly specific nucleases (e.g., zinc finger nucleases). Such nucleases must do so by crossing with a nuclease

expressing line into the genome of the transgenic plant containing the selection marker to be removed. After successful elimination of the selection marker further removal of the nuclease from the genome of the transgenic plant is required, which is done by meiotic segregation. As a result, at least two further generations are necessary for the identification of selection marker-free plants. As a variant of this method, the use of specific recombinases (e.g., cre recombinase) may be considered, but always leading to the fate of the recombination sites in the transgenic plant. From a regulatory point of view, this is problematic, as it is also unnecessary, ie superfluous sequence motifs within the transgenic plant.

On the other hand, 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. 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. However, identification of selection marker-free plants is only in the first

Branch generation of the initial transformants possible. However, the segregation of selection marker and goi by segregation is very inefficient due to the frequent co-integration of the two transformed T-DNAs in closely spaced genomic regions, so that a large number of starting transformants must be made to generate a sufficient number of transgenic, selection marker-free Being able to identify lines.

The production of transgenic plants without the use of a selection step during the transformation process has long been considered impossible (Potrykus et al., 1998, Erikson et al., 2005, Joersbo et al., 2001). In their review article in 2006, Shrawat and Lörz describe various ways to produce selection marker-free crop plants, but all methods are based on the application of one of the above

described strategies, ie either the implementation of co-transformations (gene of interest and selection markers are located on two separate T-DNAs) with

subsequent segregation of the selection marker and the goi via meiosis, or the subsequent removal of the selection marker via specific recombinases. An application of a selection marker-free transformation is not described.

In a recent review article by Tuteja et al. (2012) also lists numerous methods for the generation of marker gene-free plants. However, in this article only once again the possibilities of co-transformation and the subsequent ones already mentioned in Shrawat and Lörz (2006)

Selection marker removal shown. A transformation without selection marker in plants of the genus Triticum by means of Rhizobiaceae bacteria such as Agrobacterium

tumefaciens is not mentioned. A transformation of plants by means of Agrobacterium without the presence and application of a selection marker is for some others

Plant species described, inter alia. Potato (De Vetten et al., 2003, Ahmad et al., 2008), Tobacco (Li et al., 2009), Orange (Ballester et al., 2010), and Lucerne (Ferradini et al., 2011).

Today, the following undesirable phenomena are known which can occur when a selection with a marker gene is omitted:

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. Thus, 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. Thus, if 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

Have a vitality disadvantage compared to untransformed cells. Thus, in a selection marker-free transformation increases the probability that predominantly non-transgenic shoots regenerate. Consequently, the transformation efficiency decreases in the

Compared to a transformation with selection clearly off. This has been studied very well in the selection marker-free potato transformation, which has efficiencies of 1-4% (De Vetten et al., 2003), while selectable markers can achieve efficiencies of ~ 30% (Chang et al Chan, 1991).

Furthermore, it is regularly observed that, in the absence of marker gene-based selection, also shoots which consist of both transgenic and non-transgenic tissue (chimeric shoots) regenerate. There may be different forms of the chimeric character. If a periclinic chimera is present, it may happen that the L2 cell layer necessary for the formation of the gametes is not transgenic in the meristems of the plants. Thus, only non-transgenic gametes are formed in this plant and introduced into the plant

Transgen is not passed on to the next generation. When generative too

propagating plants, such chimeric transgenic plants are then lost. In sectoral chimeric plants, some areas of the plants are transgenic, other areas are not transgenic. In the non-transgenic areas / parts of the plant only non-transgenic gametes are formed. The proportion of non-transgenic gametes is thus significantly increased, so that an increased proportion of non-transgenic offspring can be detected in the subsequent generation. The splitting conditions in the branch generation do not correspond to the Mendelian rules. By the application By marker gene-based selection, the formation of chimeric shoots is usually suppressed or the proportion of transgenic tissue in a Sektorialchimäre is so high by the applied selection pressure that no or very little negative effects of the chimeric character of the regenerated transgenic plant, especially one of the Mendelschen Rules corresponding inheritance, occurs.

For monocotyledonous crops, only a few are known from the prior art

Applicable methods for the transformation and production of marker gene-free plants known. Especially for wheat, only Liu et al. 2011 a successful one

Selection marker-free production of transgenic wheat plants described. However, the yield obtained is extremely low at only 0.28%, which is why the described

Procedure is inappropriate for routine use. In addition, the authors use microprojectile bombardment for transformation and not Rhizobiaceae bacteria such as Agrobacterium tumefaciens.

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. As expected, 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

Generation of transgenic maize plants using the selection marker bar a proportion of only about 5% of the plants produced is chimeric, or 95% of the plants produced are not chimeric, and thus the transgene according to the Mendelian rules pass on the next generation. Furthermore, the authors in EP 2 274 973 describe the transformation of rice without the use of a selection marker, but no analyzes are carried out which show how high the proportion of chimeric plants in the population of the generated, selection marker-free plants. In this

In connection with this, it is interesting to note that chimeric plants already occur in the transformation of rice using a selection pressure (Hiei et al., 1994). Thus, it can be expected that the proportion of chimeric plants in rice in the

Selection marker-free transformation is significantly increased. The authors of EP 2 274 973 also propose the disclosed production method for the production of transgenic wheat, but there are no experimental data on which efficiencies and chimeric tendencies to expect for wheat. Although wheat as well as corn and rice belong to the monocotyledonous plants, it is known to the person skilled in the art that cells of this crop species can exhibit significantly different behavior in the process of transformation and regeneration, which is why it must be doubted that results from the transformation of other monocotyledonous plants can be easily transferred to wheat plants. For example, Hensel et al. Also in a comparison of the transformation of barley, corn, triticale and wheat in 2009 such differences.

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. However, the transformation protocol used here always involves the use of the selection markers hygromycin phosphotransferase (hpt) or phosphinotricin acetyltransferase (PAT / bar). Although the authors state that selection for the generation of transgenic wheat plants is not absolutely necessary, appropriate experimental evidence is lacking.

Summary of the invention

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.

The objects are achieved according to the invention by 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

Component by co-culturing cells of an explant of the plant of the genus Triticum with at least one bacterial cell of the family Rhizobiaceae, which comprises the genetic component, and (b) regenerating a transgenic plant of the genus Triticum from at least one transformed cell from (a), wherein 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.

Preferably, a bacterial cell of the family Rhizobiaceae is a bacterial cell of the genus Agrobacterium and more preferably a bacterial cell of the species

Agrobacterium tumefaciens (Broothaerts et al., 2005). Preferably, the

Bacterial cell the genetic component on a vector, in particular on a binary vector, a super binary vector or a vector of a cointegrative

Vector system.

Preferably, 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. Furthermore, 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).

In a method of the present invention, 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. Such transformation conditions are known to those skilled in the art (Cheng et al., 1997). Preferably, 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). However, 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)). Furthermore, 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). In addition, wheat plants have already been regenerated from flower tissue (Amoah et al., 2001) and from callus of mature embryos (Wang et al , 2009)

In the method according to the invention, from step (a) to step (b) no selection of a transformed cell from (a) based on a property mediated by the genetic component or a part thereof. Here, 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. Preferably, 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).

In addition, resistance to the herbicidal classes of the sulfonylureas, sulfonylaminocarbonyltriazolinones, imidazolinones, triazolopyrimidines and

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

Confer resistance to aminoglycoside class antibiotics (e.g., kanamycin, paromomycin, geneticin). As more commonly used antibiotic resistance is

For example, hygromycin phosphotransferase is used, which mediates resistance to the antibiotic hygromycin B. An overview of

Antibiotic resistance used in plant transformation can be found in Tuteja et al (2012), Kraus (2010) or Shrawat and Lörz (2006).

In addition to antibiotics and herbicide resistance but also other selection markers can be used, which allow a differentiation between transgenic and non-transgenic cells. Examples of this are e.g. the stimulation of the production of

Anthocyanins or other plant dyes by the expression of certain

Transcription factors (Kortstee et al., 2011), the expression of fluorescent proteins (Mußmann et al., 2011) or the expression of auxotrophic markers, such as

Phosphomannose isomerase (PMI), expression of which allows transgenic cells to grow on mannose as the only carbohydrate source, whereas non-transgenic cells can not utilize this carbon source (Reed et al., 2001).

The skilled worker is aware that due to the lack of selection pressure on the transformed as well as the untransformed cells from step (a) to step (b) of the method according to the invention not only transgenic plants but also transgenic or chimeric plants in step (b) can regenerate. The low yield

utilizable transgenic plants (non-chimeric) long countered an economically useful use of a marker gene-free method for producing a transgenic plant. In general, 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. To increase the efficiency of generating transgenic monocotyledonous plants, the experts agree that this can only be done by significantly increasing the infection rate already at the time of culturing the cells of the explant with the Agrobacterium. This should then lead to an increased transformation rate, ie the presence of more transformed cells in the explant, from which then more transgenic plants should regenerate. Various approaches for a deratige increased transformation efficiency are known from the prior art (US 201 1/0030101 A1). They have also been used successfully in methods for the marker gene-free production of corn and rice. Nevertheless, the marker gene-free methods for the production of transgenic maize and rice plants for transformational efficiency remain behind the methods with marker gene-based selection, so that the production of

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

Transformation efficiencies of known marker gene-free production methods of transgenic plants of the genus Triticum without the use of bacteria of the family Rhizobiaceae such as Agrobacterium tumefaciens. 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%.

In a preferred embodiment of the method according to the invention is

Transformation efficiency comparable to the transformation efficiency of a

corresponding comparison method, which is that selecting a transformed cell based on a property, mediated by the genetic component or a part thereof, that is based on at least one Selection marker takes place. Furthermore, the transformation efficiency of the

at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30% or at least 25% of the transformation efficiency of an equivalent method, in which a selection of a transformed cell based on a property mediated by the genetic component or a part thereof, that is based on at least one selection marker. Due to the high workload, which is associated with the subsequent removal of the selection marker from stable, transgenic plants, a specialist will then

View method according to the invention still advantageous and superior to the prior art, if such a transformation efficiency can be achieved in the method according to the invention. Furthermore, such high transformation efficiencies should surprise the skilled person, as he from the experience with methods for marker gene-free production of, for example, transgenic maize and rice plants a much lower

Transformation efficiency expected.

In a further preferred embodiment of the method according to the invention, 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 in processes for producing a transgenic plant,

in particular a transgenic monocotyledonous plant, are described in the prior art. The treatment for enhancing the transformation efficiency may include at least one treatment selected from

i. physical and / or chemical damage to the tissue or a part

of which during co-cultivation or after co-cultivation (EP 2 460 402), ii. Centrifugation before co-cultivation, during co-cultivation or after co-cultivation (Hiei et al., 2006, WO 2002/012520),

iii. Add silver nitrate and / or copper sulfate to the coculture medium

(Zhao et al., 2002, Ishida et al., 2003, WO 2005/017152), iv. thermal treatment of the explant before co-cultivation or during co-cultivation (WO 1998/054961),

v. Pressure treatment before co-cultivation or during co-cultivation or after co-cultivation (WO 2005/017169),

vi. Inoculation with Agrobacterium in the presence of a powder (WO 2007/069643) and vii. Add cysteine to the coculture medium (Frame et al., 2002).

In addition, other prior art enhancement transformation efficiencies are known which can be used in the method of the present invention. In addition, a treatment to increase the

Transformation efficiency also represent a combination of known treatments to increase the transformation efficiency.

In a further preferred embodiment of the method according to the invention, 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%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6% or 5% chimeric, transgenic plants produces.

In a particularly preferred embodiment of the method according to the invention, 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

Property mediated by the genetic component or a part thereof, that is based on at least one selection marker. This is just as surprising since a person skilled in the art expects a significantly lower proportion of non-chimeric, transgenic plants of the genus Triticum from experience with processes for the marker gene-free production of, for example, transgenic maize plants. Due to the high Workload for the preparation of selection marker-free plants of the genus Triticum with the above-described subsequent removal of the selection marker gene will be considered by a person skilled in the invention even then the method according to the invention as advantageous and superior to the prior art, if the proportion of non-chimeric, transgenic plants of the genus Triticum from step (b) is lower than in the comparison method. In this case, the proportion by a maximum of a factor of 10, by a maximum of a factor of 9, by a maximum of a factor of 8, by a maximum of a factor of 7, by a maximum of a factor of 6, by a maximum of a factor of 5, by a maximum of a factor of 4.5, to a maximum a factor of 4, by a maximum of a factor of 3.5, by a maximum of a factor of 3, by a maximum of a factor of 2.5 or by a maximum of a factor of 2 lower.

As described above, 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

be identified (Faize et al., 2010).

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. 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. In a non-chimeric plant then in meiosis the genetic

Component or part of it can then be found in 50% of the formed gametes. However, 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

Initial transformants thus increase the proportion of non-transgenic

Offspring to a value> 25%, which is larger than this according to the

Mendel's rules would be expected. An example of non-Mendelian segregation in the first branch generation of a chimera transgenic

Plants is in Coussens et al. (2012). In a further particularly preferred embodiment of the invention

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

Property mediated by the genetic component or a part thereof, that is based on at least one selection marker. This is just as surprising since a person skilled in the art expects a significantly higher proportion of chimeras, transgenic plants of the genus Triticum, from experience with processes for marker gene-free production of, for example, transgenic maize plants. Due to the high workload for the preparation of selection marker-free plants of the genus Triticum with the above-described subsequent removal of the selection marker gene, a person skilled in the art will consider the method according to the invention even more advantageous and superior to the prior art, if the proportion of chimeras, transgenic plants of the Genus Triticum from step (b) is higher than in the comparison method. In this case, the proportion by a maximum of a factor of 10, by a maximum of a factor of 8, a maximum of a factor of 6, by a maximum of a factor of 5, by a maximum of a factor of 4, by a maximum of a factor of 3.5, by a maximum of a factor of 3, by a maximum a factor of 2.5, by a maximum of a factor of 2, by a maximum of a factor of 1, 8, by a maximum of a factor of 1, 6, by a maximum of a factor of 1, 4, by a maximum of a factor of 1, 2, or by a maximum of a factor of 1, 1 higher.

In a particularly preferred embodiment, 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

Selecting based on the molecular structure of the genetic component or a portion thereof or based on the property, in particular a phenotypic property, which is directly or indirectly mediated by the genetic component (e.g., herbicide resistance, pathogen resistance, plant height, yield, leaf structure). In particular, the molecular structure of the genetic component or a part thereof means the sequential order of the nucleotides of the genetic component or a part thereof. 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. For the expert are numerous different methods of

Molecular biology available from the prior art. So is the proof of in the Cell-incorporated genetic component, for example, via polymerase chain reaction (Mullis, 1988), via hybridization of a detectable, single-stranded

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). Furthermore, 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

genetic component results mean. Thus, the detection of the transcript or the encoded peptide / polypeptide / protein of the introduced genetic component or a part thereof in the transgenic plant is also evidence of the successful

Transformation of the genetic component or part of it, ie for the

Select suitable. Examples of methods known to the person skilled in the art which can be used for the purpose of detecting the transcript are: the rewriting of an RNA formed from the genetic component or a part thereof into cDNA and subsequent polymerase chain reaction (RT-PCR, Sambrook et al., 2001 ), the

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

Property which is mediated directly or indirectly by the genetic component. 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.

In a further particularly preferred embodiment of the invention

Method, 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.

In a further particularly preferred embodiment of the invention

Method, 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. 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

allows, or that 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the regenerated transgenic plant of the genus Triticum out Step (b) comprise cells which have a detectable level of expression, preferably one

Have expression level, which allows the intended functionality of the genetic component.

Methods for the production of a transgenic plant of the genus Triticum described above can be used to advantage in that high-quality selection-marker-free transgenic lines can be developed from the transgenic plants. In order to create transgenic lines of comparable quality, there is currently only the possibility of co-transformation and subsequent segregation, selection marker-free

Create plants. Comparing the effort required to generate a selection marker-free transgenic line via the co-transformation approach with the effort of a method of the present invention, the cost of developing a homozygous, selection marker-free transgenic line is approx 50 times higher. 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

To obtain seed pools. While the yield of single copy, selection marker-free lines in the co-transformation approach is due to the only 30-50% co-transformation rate and the requirement that both the gene of interest and the selection marker be present as a single copy event need to be sufficiently high To get probability of segregation of the two transgenes, with only 2 homozygous seed pools, can be expected starting from 100 initial transformants in the selection marker-free transformation according to the invention with 30 homozygous seed pools.

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

Transmitted as transgene has. A part can mean a cell, a tissue or an organ.

Some of the terms used in this application are explained in more detail below:

A "gene of interest" may be any type of DNA or RNA molecule encoding, for example, a protein, or a nucleic acid molecule.

For example, 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.

The term "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.

The term "comparable" means in connection with two or more

numerical data that the data differ by no more than +/- 5%. Embodiments and embodiments of the present invention will become more apparent by reference to the attached figures and sequences

described:

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.

Figure 3: Binary vector pLH70SubiintrontDT (tDT is tandem dimer Tomato, a red

fluorescent protein)

FIG. 4: Southern blot of selected, selection-marker-free transgenic lines of the transformation experiment WA1. 20 μg 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).

Figure 5: Expression analysis of the introduced tDT gene by qRT-PCR in

selected, transgenic wheat plants.

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).

Selection marker-free transformation of wheat plants of the typhoon variety:

Wheat plants of the typhoon variety were grown in the greenhouse. The

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 , In preparation for the isolation of the immature wheat embryos, 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.

The cultivation of 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

Spectinomycin, 25 mg / L streptinomycin) was compared with a single colony 2 ml

Liquid culture in MG / L medium (Wu et al., 2009) with 100 mg / L rifampicin, 100 mg / L

Carbenicillin, 50 mg / L spectinomycin, 25 mg / L streptinomycin inoculated and grown overnight at 28 ° C and 200 rpm. 250μl of liquid culture was added the next day for inoculation of 50ml of fresh MG / L medium (100mg / L rifampicin, 100mg / L

Carbenicillin, 50 mg / L spectinomycin, 25 mg / L streptinomycin) and the culture grown overnight at 28 ° C and 200 rpm. An aliquot of the overnight culture was then spun down (5 minutes at 4 ° C and 3500 x g), the supernatant discarded and the bacterial pellet resuspended in the same volume of Inf liquid medium (Table 1) with 100 μM acetosyringone. The agrobacteria suspension thus prepared could be used for the infection of immature embryos.

From the sterilized wheat grains, 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

Agrobacterium suspension was added to the embryos and the embryos were panned for 30 seconds in the Agrobacterium suspension. Subsequently, the embryos were incubated for a further 5 minutes at room temperature in the Agrobacterium suspension. The immature embryos were then placed on Co-cul medium (Table 1) with the scutellum side up. The treated explants were incubated for two days at 23 ° C in the dark. FIG. 2 shows the scutellum of a transformed wheat embryo after several days after infection with A. tumefaciens. The

Wheat embryos were transformed with a reporter gene construct which was added to the Formation of a red-fluorescent protein in the transformed cells leads. The left picture shows the scutellum in daylight, the right one the scutellum with fluorescent light. It can be clearly seen that a large part of the cells of the scutellum express the transgene and were thus successfully infected by A. tumefaciens.

After two days of co-culture of the immature wheat embryos with the agrobacteria, 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.

Table V. Composition of applicable media

Inf liquid medium Co-cul medium resting medium

1/10 x MS inorganic 1/10 x MS inorganic 1x MS inorganic

Salts salts

1X MS Vitamins 1X MS Vitamins 1X MS Vitamins

10 g / L glucose 10 g / L glucose 40 g / L maltose

0.5 g / L MES 0.5 g / L MES 0.5 g / L glutamine

100 μΜ acetosyringone 0.1 g / L casein hydrolyzate

5 μΜ silver nitrate 0.75 g / L MgCl2 x 7H20

5 μΜ copper sulfate 1, 95 g / L MES

8 g / L agarose 100 mg / L ascorbic acid

150 mg / L Timentin

2.2 mg / L Picloram

0.5 mg / L 2,4-D

2 g / L gelrites

8 g / L agar 3 g / L gelrites

Results:

Three independent transformation experiments in Triticum aestivum were performed as described above without the use of a selection marker. In all three experiments, transgenic plants could be obtained without the use of selection markers (see Table 2). Surprising was the high number of explants that led to transgenic sprouts. In experiment WA1 could be infected by 151

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

Initial explant created. However, leaf samples from sprouts of several initial explants were never pooled. Of the sprout pools analyzed, a surprisingly high number were positive (78 or ~ 23%). Out of the 341 sprout pools of the 89 explants, 78 transgenic sprout pools could be identified from 42 explants. The 1 1 1 shoots underlying the 78 sprout pools were then individually sampled and reassessed for the presence of the transgene.

For the detection of the transgene in the regenerated sprouts was from the

Sprout pools or isolated from the individual shoots DNA by PCR for the presence of recombinant DNA examined. For this purpose, 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. To determine the number of copies of the transgene introduced into the wheat genome, 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.

In experiment WA1, 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. Thus, despite the omission of the marker gene-based selection, the WA1 experiment achieved a transformation efficiency of ~ 25%. This efficiency is calculated from the 37 explants with positive sprouts, from originally 151 explants.

In the experiments WA2 and WA3, all single shoots were taken directly from the

Explanants regenerated by PCR were investigated since in experiment WA1 a

surprisingly high yield of transgenic sprouts was obtained and thus the application of the pool PCR strategy was superfluous. By direct analysis of the regenerated shoots transgenic single shoots could be identified in 56% (WA2) and 75% (WA3) of the regenerable explants.

Calculating the efficiency of the transformation on the basis of the number of initial explants used results in transformation efficiencies of 27% for the

Experiment WA2 and 40% for the experiment WA3.

Averaging across all three transformation trials in Triticum aestivum without the use of marker gene-based selection indicates that on average 55% of regenerable explants produced transgenic solitary sprouts and achieved a mean transformation efficiency of approximately 30%.

In parallel, the 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.

At WAK1, a transformation efficiency of 37% could be achieved (75

Explants with positive sprouts from 204 initial explants). In trial WAK2 transformation efficiency was 24% (37 explants with positive sprouts from 153 initial explants) and 27% in WAK3 (47 explants with positive sprouts from 175 initial explants). On average, an efficiency of 30% (0 CTE) could be achieved in these transformation experiments.

Thus, 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

Proof of the transgenicity of the generated, selection marker-free transgenic lines was provided via qPCR as described above. At the same time, this analysis allowed an estimate of the proportion of single copy lines that are of particular interest for further commercial use. Again, it turned out that the Do not distinguish results between transformations with and without the use of a selection marker.

For example, 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%.

To further verify the transgenicity of the created lines, were

selected T. plants of experiment WA1 a Southern blot performed. It is known to the person skilled in the art that transfer of the T-DNA from the Agrobacterium into the plant genome frequently only results in the transfer of truncated T-DNA fragments. These are deleted on the LB (left border) page. Therefore, for use in a transformation with marker genes, 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.

To check the completeness of the transferred T-DNA were

Hybridization experiments performed. The introduced tDT gene was described as

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

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.

Further, the transgenic lines prepared with the marker gene-free

Transformation method, with respect to the level of expression of the integrated transgene studied more closely. When using 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

thus, can not be identified as a transgenic line. Using selection marker-free transformation, 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. Thus, there is a risk that an increased proportion of transgenic lines will be produced that have no expression of the incorporated transgene.

Therefore, 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

Selection marker were transformed. Thus, there are no differences in the quality of the transgene between transgenic lines made using a selection marker and those without a selection marker.

To demonstrate the formation of chimeric transgenic plants, it was investigated whether the introduced transgene is passed on to the next generation according to the Mendelian rules. For this purpose, 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

Progeny shown in Figure 6. It is clear to observe a classical 1: 2: 1 pattern of inheritance for a monogenic inheritance. A summary of the results of progeny analysis is presented in Table 3.

Five of the six analyzed progeny show inheritance according to Mendel's rules (equivalent to 83%). Thus, it can be assumed that most of the generated transgenic starting transformants were homogeneous with respect to the transgene. The non-Mendelian inheritance in the transgenic line WA1-T-014 may be due to a non-homogeneous, so chimar transgenic plant, on the other hand, the integration of the transgene into an important gene of the plant may be. This leads to partially lethal plants / embryos, which also would explain the poor germination of this offspring (only 20 out of 30 seeds germinated).

Table 3: Results of a progeny analysis for the detection of chimeric transgenic wheat plants

transgenic line azygot hemizygot homozygous total cleavage ratio chi ²

WA1-T-006 8 16 3 27 1: 2: 1 0.25

WA1-T-008 8 14 7 29 1: 2: 1 0.95

WA1-T-009 4 16 9 29 1: 2: 1 0.36

WA1-T-014 11 6 3 20? 0.01

WA1-T-024 8 13 9 30 1: 2: 1 0.74

WA1-T-028 9 17 4 30 1: 2: 1 0.33

references

Ahmad, R., Kim, Y.H., Kim, M.D., Phung, M.N., Chung, W.I., Lee, H.S., ... & Kwon, S.Y. (2008). Development of selection marker-free transgenic potato plants with enhanced tolerance to oxidative stress. Journal of Plant Biology, 51 (6), 401-407.

Amoah, B.K., Wu, H., Sparks, C, & Jones, H.D. (2001). Factors influencing

Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. Journal of Experimental Botany, 52 (358), 1135-1142.

Ballester, A., Cervera, M., & Pena, L. (2010). Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all

regenerants. Plant Cell, Tissue and Organ Culture (PCTOC), 102 (3), 329-336.

Broothaerts, W., Mitchell, H.J., Weir, B., Kaines, S., Smith, L.M., Yang, W., ... & Jefferson, R.A. (2005). Gene transfer to plants by diverse species of bacteria. Nature, 433 (7026), 629-633.

Chang, H.H., & Chan, M.T. (1991). Improvement of potato (Solanum tuberosum L.) Transformation efficiency by Agrobacterium in the presence of silver thiosulfate. Bot. Bull. Acad. Sin, 32, 63-70.

Cheng, M., Fry, J.E., Pang, S., Zhou, H., Hironaka, C.M., Duncan, D.R., ... & Wan, Y. (1997). Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiology, 11 (3), 971-980.

Comai, L, Sen, L.C., & Stalker, D.M. (1983). An altered aroA gene product resistance to the herbicidal glyphosate. Science, 221 (4608), 370-371.

Coussens, G., Aesaert, S., Vereist, W., Demeulenaere, M., De Buck, S., Njuguna, E., ... & Van Lijsebettens, M. (2012). Brachypodium distachyon promoters as efficient building blocks for transgenic research in maize. Journal of experimental botany, 63 (1 1), 4263-4273.

De Block, M., Botterman, J., Vandewiele, M., Dockx, J., Thoen, C, Gossele, V &

Leemans, J. (1987). Engineering herbicide resistance in plants by expression of a detoxifying enzyme. The EMBO journal, 6 (9), 2513.

De Veiten, N., Wolters, A.M., Raemakers, K., van der Meer, I., terstege, R., Heeres, E

& Visser, R. (2003). A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nature biotechnology, 21 (4), 439-442. Erikson, O., Hertzberg, M., & Nasholm, T. (2005). The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformation. Plant molecular biology, 57 (3), 425-433.

Faize, M., Faize, L, & Burgos, L. (2010). Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation. BMC biotechnology, 70 (1), 53.

Ferradini, N., Nicolia, A., Capomaccio, S., Veronesi, F., & Rosellini, D. (2011). Assessment of simple marker-free genetic transformation techniques in alfalfa. Plant cell reports, 30 (11), 1991-2000.

Frame, B.R., Shou, H., Chikwamba, R.K., Zhang, Z., Xiang, C, Fonger, T.M., ... & Wang, K. (2002). Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiology, 729 (1), 13-22.

Hiei, Y., Ohta, S., Komari, T., & Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6 (2), 271-282.

Hiei, Y., Ishida, Y., Kasaoka, K., & Komari, T. (2006). Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens. Plant cell, tissue and organ culture, 87 (3), 233-243.

Hensel, G., Kastner, C, Oleszczuk, S., Riechen, J., & Kumlehn, J. (2009). Agrobacterium mediated gene transfer to crop crop plants: current protocols for barley, wheat, triticale, and maize. International Journal of Plant Genomics, 2009.

Ishida, Y., Saito, H., Hiei, Y., & Komari, T. (2003). Improved protocol for transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Plant Biotechnology, 20 (1), 57-66.

Ishida, Y., Hiei, Y., & Komari, T. (2007). Agrobacterium-mediated transformation of maize. Nature Protocols, 2 (7), 1614-1621.

Joersbo, M. (2001). Advances in the selection of transgenic plants using non-antibiotic marker genes. Physiology plantarum, 111 (3), 269-272.

Kortstee, AJ, Khan, SA, Helderman, C, Trindade, LM, Wu, Y., Visser, RGF, ... & Jacobsen, E. (2011). Anthocyanin production as a potential Visual selection marker during plant transformation. Transgenic research, 20 (6), 1253-1264. Kovalic, D., Garnaat, C, Guo, L., Yan, Y., Groat, J., Silvanovich, A & Bannon, G.

(2012). The Use of Next Generation Sequencing and Junction Sequence Analysis

Bioinformatics to Achieve Molecular Characterization of Crops Improved Through Modern Biotechnology. The Plant Genome, 5 (3), 149-163.

Kraus, J. (2010). Concepts of marker genes for plants. In Genetic Modification of Plants (pp. 39-60). Springer Berlin Heidelberg.

Li, B., Xie, C, & Qiu, H. (2009). Production of selectable marker-free transgenic tobacco plants using a non-selection approach: chimerism or escape, transgenic inheritance, and efficiency. Plant cell reports, 28 (3), 373-386.

Liu, X., Jin, W., Liu, J., Zhao, H., & Guo, A. (201 1). Transformation of wheat with the HMW-GS 1 Bx14 gene without markers. Russian Journal of Genetics, 47 (2), 182-188.

Maluszynski, M. (Ed.). (2003). Doubled haploid production in crop plants: a manual.

Springer.

Mullis, K.B., & Erlich, H.A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239 (4839), 487-491.

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.

Potrykus, I., Bilang, R., Futterer, J., Sautter, C, Schrott, M., & Spangenberg, G. (1998). Genetic engineering of crop plants. Agricultural Biotechnology, 1 19-159.

Reed, J., Privalle, L, Powell, M.L., Meghji, M., Dawson, J., Dunder, E., & Wright, M. (2001). Phosphomannose isomerase: an efficient selectable marker for plant

transformation. In Vitro Cellular & Developmental Biology-Plant, 37 (2), 127-132.

Sambrook, J., Russell, D.W., & Russell, D.W. (2001). Molecular cloning: a laboratory manual (3-volume set).

Shrawat, A. K., & Lörz, H. (2006). Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers. Plant Biotechnology Journal, 4 (6), 575-603.

Southern, EM (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology, 98 (3), 503-517. Tuteja, N., Verma, S., Sahoo, RK, Raveendar, S., & Reddy, IBL (2012). Recent advances in the development of marker free transgenic plants: regulation and biosafety concern. Journal of Bioscience, 37 (1), 162-197.

Vasil V, V Srivastava, AM Castillo, ME Fromm, IK Vasil (1993) Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos.

Bio / Technology 11: 1553-1558.

Wang, Y. L, Xu, M.X., Yin, G.X., Tao, L.L., Wang, D.W., & Ye, X.G. (2009). Transgenic wheat plants derived from Agrobacterium -mediated transformation of mature embryo tissues. Cereal Research Communications, 37 (1), 1-12.

Wu, H., Doherty, A., & Jones, H.D. (2009). Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos. In Transgenic wheat, barley and oats (pp. 93-103). Humana Press.

Zhao, Z.Y., Gu, W., Cai, T., Tagliani, L, Hondred, D., Bond, D & Pierce, D. (2002).

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Molecular Breeding, 8 (4), 323-333.

WO 1998/054961 A2 (Novartis AG et al.) "Plant transformation methods"

WO 2002/012520 A1 (Japan Tobacco Inc.) "Method of improving gene transfer efficiency into plant cells"

WO 2005/017152 A1 (Japan Tobacco Inc. et al.) "Method for improving plant transformation efficiency by adding copper ion"

WO 2005/017169 A1 (Japan Tobacco Inc. et al.) "Method of transducing gene into plant material"

WO 2007/069643 A1 (Japan Tobacco Inc. et al.) "Method for Improving Transformation Efficiency Using Powder"

WO 2008/028121 A1 (Monsanto Technology LLC et al.) "Methods for producing transgenic plants"

EP 0 672 752 B1 (Japan Tobacco Inc.) "Method of transforming monocotyledons using scutella of immature embryos"

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"

US 201 1/0030101 A1 (Japan Tobacco Inc. et al.) "Agrobacterium mediated method for producing transformed plant"

Claims

claims

A process for producing a transgenic plant of the genus Triticum comprising the steps

(a) transforming at least one cell of a plant of the genus Triticum with a genetic component by co-culturing cells of an explant of the plant of the genus Triticum with at least one bacterial cell of the family Rhizobiaceae comprising the genetic component, and

(b) regenerating a transgenic plant of the genus Triticum from at least one transformed cell from (a),

wherein from step (a) to step (b) there is no selection of a transformed cell from (a) based on a property mediated by the genetic component or a part thereof.

2. The method according to claim 1, characterized in that the plant of the genus Triticum is a plant of the species Triticum aestivum, Triticum durum or Triticum spelta.

3. The method according to any one of claims 1 or 2, characterized in that the explant is an embryonic tissue, in particular radicle, embryo axis, scutellum or germ, or a part thereof.

A method according to claim 3, characterized in that the embryonic tissue is part of an immature embryo or mature seed.

5. The method according to any one of claims 1 to 4, characterized in that no selection based on a property mediated by the genetic component or a part thereof, no selection based on a herbicide or

Antibiotic resistance is.

6. The method according to any one of claims 1 to 5, characterized in that the method has a transformation efficiency of at least 5%.

7. The method according to any one of claims 1 to 5, characterized in that the method has a transformation efficiency which is comparable to the

Transformation efficiency of a corresponding comparison method, which is in it distinguishes that selecting a transformed cell based on a

Property mediated by the genetic component or part of it occurs.

8. The method according to any one of claims 1 to 7, characterized in that the transformation efficiency by a treatment to increase the

Transformation efficiency is increased.

9. The method according to claim 8, characterized in that the treatment for

Increasing the transformation efficiency causes a transformation efficiency of at least 5%.

10. The method according to any one of claims 8 and 9, characterized in that the treatment for increasing the transformation efficiency comprises at least one treatment which is selected from

i. physical and / or chemical damage to the tissue or a part thereof during co-cultivation or after co-cultivation,

ii. Centrifugation before co-cultivation, during co-cultivation or after co-cultivation,

iii. Adding silver nitrate and / or copper sulfate to the coculture medium, iv. thermal treatment of the explant prior to co-cultivation or during the

co-culturing,

v. Pressure treatment before co-cultivation or during co-cultivation or after co-cultivation,

vi. Inoculation with Agrobacterium in the presence of a powder and

vii. Add cysteine to the coculture medium.

11. The method according to claim 1 to 10, characterized in that step (b) produces non-chimeric transgenic plants with a frequency of at least 15%.

12. The method according to any one of claims 1 to 1 1, characterized in that the method comprises a further step

(c) selecting the regenerated transgenic plant from step (b).

13. The method according to claim 12, characterized in that the selection in step (c) based on the molecular structure of the genetic component or a part thereof or based on the property, in particular a phenotypic Property which is mediated directly or indirectly by the genetic component takes place.

14. Transgenic plant of the genus Triticum, which were prepared by a method according to any one of claims 1-13, and a descendant, a part or a seed thereof.