Patent Application
20190159417
The present invention relates to hybrid plants, in particular polyploid plants obtainable via a novel method crossing of more than two parent plants as well as to organs, tissue, parts, cells, seeds or offspring of such plants. Furthermore the present invention relates to a marker-assisted method for generating such plants.
These days, the agricultural sector faces tremendous challenges in providing sufficient food for the constantly growing world population which comes along with a reduced availability of arable land. Simultaneously, the need of agricultural crops has increased due to their use for the generation of energy and as substitutes for chemical compounds. Thus, due to the growing demand for agricultural goods, the key task of plant breeding is the increase of the crop yield as well as the improvement of certain plant characteristics such as heat or pest resistance. Nowadays, also the supply with convenient food, such as seed reduced eggplants or melons becomes more and more important. The improvement of plants requires the development of fast and efficient breeding procedures to render it economic.
Hitherto, the commonly used tool in plant breeding is hybridization of two parental lines which are preferably genetically distinct as it allows the combination of different germ plasms conferring agriculturally valuable traits in a single plant by intercrossing. However, hybridization of two different parental lines does not always result in a positive outcome but might lead to hybrid incompatibility resulting in offspring that is impaired in its growth and productivity, which is sterile or even inviable. In many cases, this incompatibility is mounted in the endosperm, which is the nurturing tissue for the developing embryo in the seed and forms after fertilization of the central cell.
Furthermore, the ability of plants to tolerate the presence of supernumerary genomic copies is used to improve the crop yield of plants, to establish novel variations and morphologies or to improve the genetic diversity of plants to allow them to efficiently adapt to changing environments. These supernumerary genomic copies can arise spontaneously in nature by several mechanisms, including meiotic or mitotic failures, fusion of unreduced (2n) gametes or somatic doublings and can be used for breeding processes. For example, the domestic common wheat was bred to contain six sets of chromosomes, i.e. it is hexaploid which results in a higher crop yield in comparison to the wild wheat which is diploid (2n). The selective generation and use of plants which produce unreduced microspores and/or pollen grains is also a well-known mechanisms in plant breeding; see, e.g., international application WO 2010/149322 A1.
In many plants, the introgression of supernumerary paternal copies by diploid sperm is associated with a postzygotic hybrid incompatibility reaction. Notably, this so called triploid block is mounted in the endosperm (Kohler et al., Tends Genetic. 26 (2010), 142-148) and can lead to mortality, reduced viability or reduced fertility of the hybrid. This latter phenomena is used, e.g., for the generation of seed reduced triploid plants, e.g. eggplants via crossing of a tetraploid with a diploid plant; see, e.g., international application WO 2009/095266. However, the offspring is still derived from two parent plants, i.e. it is biparental and to establish and combine favorable genes of different plant sources in the offspring, still multiple breeding cycles are necessary and there is no possibility that at the end the desired genetic information is equally distributed. On the other hand, the approach of tailor-made plants by genetically engineering, i.e. transgenic plants may not always be possible since many traits in crops are polygenic and dependent on the genomic background of the parent plant.
Thus, there is a need for high-performance plants, in particular hybrid and polyploid plants, respectively, which are suitable in breeding programs as well as for a method of obtaining such plants that can be performed in a time and cost effective manner. The above mentioned technical problems are solved by the embodiments characterized in the claims, which are described further below and illustrated in the Examples and Figures.
The present invention generally relates to polyploid plants obtainable by crossing of more than two parent plants (polypaternal crossing) within one generation as well as to a novel marker-assisted system allowing such polypaternal crossing. More specifically, the present invention relates to polyparental, preferably triparental plants and their use in agriculture and agronomics. The polyparental plants of the present invention which arise from fusion of at least three germ cells and hence contain the full genome of at least three genetically distinct parents are to be distinguished from plants simply derived from generic triparentage, i.e. from plants obtained by successive hybridization leading to the segregation of genetic material from more than two parents. The origin of plants from more than two ancestral plants is known as hybridization of multiple parents over an evolutionary timescale. In this case, the segregation of genetic material from more than two parents is, however, easily explained by successive crossings: A×B=C; C×D=E. This successive hybridization mode neither involves the fusion of three germ cells, nor does any of the offspring have three biological parents. The poly-, e.g. triparental origin of the polyploid plants of the present invention is to be distinguished from that generic triparentage, since in accordance with the present invention the triparental plant arises from fusion of three germ cells and hence contains the full genome of three genetically distinct parents: A+B+C=F.
In order to enable the provision of such triparental plants, in accordance with the present invention, a novel marker-assisted system has been established coined “High-throughput polypaternal breeding design (HIPOD) method” allowing the generation of polyploid plants via crossing of more than two parent plants within one generation and the generation of marker-free polyploid plants in the next generation. Thus, the present invention provides polyploid plants that have been generated via crossing of one female parent plant with at least two male parent plants as well as to organs, tissue, parts, cells, seeds or offspring of the plants, wherein the male parent plants can be genetically identical or distinct.
Although it is commonly known that several plants tolerate the presence of supernumerary genomic copies, plants nevertheless evolved polyspermy barriers that hinder the fusion of more than two gametes since in most eukaryotes polyspermy is lethal or results in reduced fertility or even sterility due to the genomic imbalance; see supra. Polyspermy barriers are implemented at different levels in the reproductive process and a common mechanism for polyspermy barriers found in animals and plants are egg cell blocks, which are mounted after gamete fusion and impose chemical or physical barriers to reduce the risk of further sperm entry (Iwao, Reproduction 144 (2012), 11-22; Scott et al., Mol. Plant. 1 (2008), 611-619; Jaffe, Proc. Natl. Acad. Sci. USA 88 (1991), 9883-9887 (1991); Tsaadon et al., Mol. Cell. Endocrinol. 252 (2006), 107-114; Spielman and Scott, Sexual Plant Reproduction 21 (2008), 53-65). So far, in vitro studies on rice have been performed in which two sperm cells of one rice plant have been electrically fused with the egg cell of another rice plant in order to investigate pathways which may contribute to the formation of autopolyploids (Toda et al., Plant Physiol. 171 (2016), 206-214). However, this approach was not considered for the generation of triploid plants with different male parents or implantation in plant breeding in general, especially since this approach uses sperm cells derived from one single plant still leading to biparental offspring and not to polyparental plants as described in the present invention. This method does not integrate naturally occurring in-planta mechanisms for polyspermy, but is based on a purely synthetic in vitro approach. Therefore, in one embodiment plants are specifically excluded from the present invention, which have been generated in vitro, e.g. by electrofusion such as the rice plants described in Toda et al. (2016).
In contrast, as demonstrated in the appended Examples, the method of the present invention enables crossing of three parental lines including at least two different male parental lines and gives rise to viable offspring which can be used in further breeding programs. In particular, the method of the present invention encompasses labeling the male parent plants each with a component of a selectable marker, crossing the male parent plants with a female parent plant and selecting the offspring for the presence of a selectable marker, which preferably confers herbicide resistance and/or fluorescence when combined in one single plant. Alternatively, the method of the present invention encompasses labeling the male parent plants each with a different selectable marker, crossing the male parent plants with a female parent plant and selecting the offspring for the presence of the two different selectable markers, which preferably confer herbicide and antibiotic resistance, respectively, potentially combined with fluorescence when combined in one single plant. Advantageously, since the F1 offspring of the method of the present invention is heterozygous for the selectable marker gene(s) but “homozygous” as regards its polyploidy, i.e. triparental selfing or crossing of the F1 generation inter alia results in marker gene-free F2 plants devoid of any DNA which is foreign to the plant. Accordingly, also the provision of marker-free polyploid plants in the following generation is encompassed by the present invention. Furthermore, the method of the present invention can be used to successfully cross at least one male parent plant which is incompatible with the female parent plant. Thus, the method of the present invention is suitable to overcome hybrid incompatibility or even species barriers in angiosperms via providing sperm from a compatible and an incompatible crossing partner.
The embodiments of the present invention are described with reference to the attached Figures and sequences.
Initially, some of the terms used throughout the specification are defined in the following.
The term “crossed” or “cross” means the fusion of gametes via pollination to produce a progeny and encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant individual). The term “crossing” refers to the act of fusing gametes via pollination to produce a progeny. Thus, the expression “crossing of one female parent plant with more than one male parent plants” is used interchangeably with the expression “fertilization of one egg cell with more than two sperm cells”. Of course, in angiosperms “crossing” also includes the fertilization of the endosperm with the sperm cell from male parent plant, preferably from the first male parent plant in case of polypaternal crossings.
The term “cultivar” as used throughout the present description refers to a plant or group of plants selected and bred by man for desirable characteristics such as size, color, yield, disease resistance, taste, etc. that can be maintained by propagation. Thus, cultivars are distinct, uniform and stable. Cultivars are subgroups within the same species. For uncultivated plants (wild flora) the term “variety” is accordingly used.
“Flowering plants” and “angiosperms” as used in the present description are seed-producing plants (spermatophytes) and are distinguished from other seed-producing plants (gymnosperms) by several characteristics including the presence of flowering organs, stamens with two pairs of pollen sacs, reduced male and female gametophytes, closed carpel enclosing the ovules and endosperm formation, wherein the endosperm is a highly nutritive tissue inside the seeds of the flowering plant and provides nutrition to the embryo.
In the context of encoding sequences, promoters, and other genetic elements, the term “heterologous” indicates that the element is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
The term “heterozygous” means a genetic condition of a given individual wherein different alleles reside at corresponding loci on homologous chromosomes. The term “homozygous” means a genetic condition wherein identical alleles reside at corresponding loci on homologous chromosomes.
The term “hybridization” in the context of plant breeding can be used interchangeable with “hybrid cross” and means a cross between two genetically distinct parent plants produced by crossing plants of different lines or breeds or species, including but not limited to the cross between two inbred lines (e.g., a genetically heterozygous or mostly heterozygous individual) to produce a hybrid plant (offspring).
The term “hybrid incompatibility” is commonly used as the collective for hybrid inviability and sterility. This incompatibility can be mounted at different levels of the reproduction process. One can distinguish between hybrid incompatibility which occurs due to differences in the ploidy status of the crossing partners and hybrid incompatibility which is “intrinsic” meaning that it occurs in the absence of ploidy differences or apparent ecological factors isolating lineages. The latter one is thought to be due to negative epistasis between newly derived alleles that have arisen in isolation but are dysfunctional against the genetic background of the other diverged lineage. For example, hybrid seed inviability (abortion) is a common early interspecific barrier among many plant groups and has frequently been attributed to species-specific changes in the endosperm.
In context of the present application, the term “offspring” plant refers to any plant resulting as progeny from a sexual reproduction from one or more parent plants or descendants thereof. For instance, an offspring plant may be obtained by crossing two or more parent plants and include selfings as well as the F1 or F2 or still further generations.
Genetic elements are said to be “operatively linked” if they are in a structural relationship permitting them to operate in a manner according to their expected function. For instance, if a promotor helps to initiate transcription of the coding sequence, the coding sequence can be referred to as operatively linked to (or under control of) the promoter. There may be intervening sequences between the promoter and coding region so long as this functional relationship is maintained.
Unless otherwise stated, a “plant” of the present invention is any plant at any stage of development, preferably a seed plant including dicotyledons and monocotyledons, preferably a flowering plant. “Plant organs” mean for example leaves, stem, roots, root tips, buds, meristems, embryos, anthers, ovules, seeds, flowers or fruits.
The term “plant part” refers to a part of a plant including but not limited to single cells and cell tissues such as plant cells that are intact in plants, cell clumps and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems shoots, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, inflorescence, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, calli, and the like. The terms “plant cells” or “cells of a plant” are used to describe for example isolated cells containing a cell wall or aggregates thereof or protoplasts.
“Polyploidy” defines a status in which more than two sets of chromosomes are present in a cell, whereas “triploid” plant cells possess three sets of chromosomes, “tetraploid” plant cells four sets, “pentaploids” five sets, “hexaploids” six sets, “heptaploids” seven sets, “octaploids” eight sets, “nonaploids” nine sets, “decaploids” ten sets, “undecaploids” eleven sets and “dodecaploids” twelve sets of chromosomes.
Particular gene sequences referred to as “promoters”, like the NOS promoter, the RPS5a promoter or the UAS promoter, are polynucleotide sequences derived from the gene referred to that promotes transcription of an operatively linked gene expression product. It is recognized that various portions of the upstream and intron untranslated gene sequence may in some instance contribute to promoter activity via proving the binding domain for the RNA-polymerase and via initiating transcription of DNA. The promoter contains further elements functioning as regulator of the gene expression (e.g. cis-regulatory elements). The promoter may be based on the gene sequence of any species having the gene, unless explicitly restricted, and may incorporate any additions, substitutions or deletions desirable, as long as the ability to promote transcription in the target tissue is maintained.
For the purpose of this description the term “polyspermy” refers to the fertilization of one egg cell with more than one sperm cell that may be derived from different pollen tubes and is not to be confused with “double fertilization” which is a commonly known mechanism in angiosperms comprising the fertilization of not only the egg cell but also the egg-adjoining central cell, which comprises the two polar nuclei, to generate the embryo nourishing tissue, i.e. the endosperm which is usually triploid. The two sperm cells necessary for this double fertilization are delivered by a single pollen tube. Furthermore, in the sense of the present invention, polyspermy it is not to be confused with the “physiological polyspermy”, in which the entry of several sperm cells into the egg cell is permitted but only one sperm nucleus participates in the formation of a zygote nucleus, while the other sperm nuclei undergo degeneration.
A “transgenic plant” refers to a plant stably transfected with at least one polynucleotide, preferably a heterologous polynucleotide. Preferably, the polynucleotide is stably integrated meaning that the integrated polynucleotide is stably maintained in the plant, is expressed and also stably passed to the offspring. The stably integration of a polynucleotide into the genome of a plant includes the integration into the genome of the previous parent generation as well, wherein the polynucleotide is stably inherited.
The present invention relates to the embodiments characterized in items [1] to [19] and illustrated in the Examples.
Since flowering plants are commonly known to reproduce sexually and thus carry the genetic material from a male and a female parent plant, and since it is known that plants exhibit polyspermy barriers preventing the egg cell to be fertilized with more than one sperm cell due to incompatibility reactions that might occur otherwise, the generation of a polyploid plant having more than two parents has not been thought about before. As described in the present invention, the inventors developed a high-throughput polypaternal breeding design (HIPOD) method for the generation of polyploid plants having more than two parents i.e. polyparental plants that have been generated via fertilization of one egg cell with more than two sperm cells with is referred to as polyspermy. Thus, the present invention provides a polyploid plant characterized in that it is obtained via crossing of one female parent plant with more than one male parent plant as well as the polyploid seed which is obtained by this crossing and from which the plant develops. The male parent plants can be either genetically distinct or genetically identical. In the following, when referred to the plant of the present invention, it encompasses, unless otherwise stated, the seed from which the plant develops as well.
In one embodiment, the polyploid plant of the present invention is obtained via crossing of one female plant with two, three, four, five, six, seven, eight, nine or ten male parents plants. Preferably, said polyploid plant is obtained via crossing of three parent plants, i.e. via crossing of one female parent plant with two male parent plants, also referred to as triparental.
In one embodiment, the polyploid plant of the present invention comprises three, four, five, six, seven, eight, nine, ten, eleven or twelve sets of chromosomes, i.e. it is triploid, tetraploid, pentaploid, hexaploid, heptaploid, octaploid, nonaploid, decaploid, undecaploid or dodecaploid and has been obtained by crossing of one female plant with two, three, four, five, six, seven, eight, nine or ten male parents plants, wherein the crossing can occur between parent plants with different ploidy, i.e. they can be haploid, diploid, triploid, tetraploid, pentaploid, hexaploid, heptaploid, octaploid, nonaploid, decaploid, undecaploid or dodecaploid. Preferably, the polyploid plant of the present invention is triploid or hexaploid and has been obtained via crossing of three diploid or tetraploid parent plants, i.e. by one diploid or tetraploid female parent plant and two diploid or tetraploid male parent plants.
The polyploid plant of the present invention is furthermore viable. In experiments regarding phenotypic characterization of triploid Arabidopsis plants that have been generated via crossing of one diploid female parent plant with two diploid male parent plants it has been shown that the overall plant size of triparental plants was increased 1.5 fold and the plants gave rise to bigger inflorescences and flowers when compared to diploid biparental plants; see
An inherent consequence of meiosis in triploid plants is aneuploidy, i.e. an imbalance in chromosome number. In fact, it has been observed that fertility in triparental triploid plants, similarly to biparental triploids is substantially reduced accompanied by the segregation of shriveled and malformed ovules and seeds;
In a further embodiment, a polyploid plant of the present invention is provided, which is obtained by crossing of one female parent plant with at least two male parent plants, wherein the two male parent plants are genetically distinct, preferably wherein they belong to different cultivars or varieties. Thus, in case of wild flora, although the different varieties belong to the same species and therefore can be bred with each other, in general this would not occur naturally since usually they either originate from different areas; i.e. they are geographically isolated and/or they are reproductively isolated. For plant breeding, this opens new possibilities to increase the genetic variety in cultured plants within one generation and due to the fact that the chromosomes of all three partners are equally represented in the polyparental polyploid plant, which would not be possible in a traditional two parent cross as this requires two generations, the traits and protein products are balanced resulting in a better predictability of the outcome of this breeding process.
The method of the present invention even allows the crossing of incompatible cultivars or varieties or even different species. Since the recent doctrine teaches that hybrid incompatibility is often mounted in the endosperm, either due to the presence of two paternal genomic copies which could occur e.g. during fertilization with diploid sperm cells or due to genetic incompatibilities between specific genes of the crossing partners, without wishing to be bound to theory it is well conceivable that polyspermy induced triploid embryos develop in seeds where the endosperm did not receive an extra paternal copy, which could bypass the triploid block. This principle is illustrated in
Thus, in one embodiment of the present invention, the polyploid plant of the present invention is obtained by crossing of one female parent plant with at least two male parent plants, wherein at least one of the male parent plants is incompatible with the female parent plant.
Experiments for enabling the triparental crosses have been performed with Arabidopsis thaliana. Since Arabidopsis thaliana is well known in the art to be a model plant for higher plants, especially for angiosperms, i.e. flowering plants, the ability to generate polyparental polyploid Arabidopsis plants can be transferred to other angiosperms. Thus, according to one embodiment, the present invention comprises, next to Arabidopsis, polyploid angiosperms in general.
In a preferred embodiment, the present invention relates to in polyploid dicotyledons and monocotyledons that have been obtained via crossing of more than two parent plants. More particularly, the present invention encompasses crop plants and cultured plants, wherein the first ones include cultivated plants as well as uncultivated plants (wild flora) and encompass all plants that are directly or indirectly used by man, e.g. as food, medicine, luxury food, as feed for livestock or wood supplier and wherein the latter ones, i.e. the cultivated plants refer to a plant which is grown and bred by man and used as crop plant or ornamental plant. These cultured plants include amongst others food crops, industrial crops (e.g. fiber crops), feed crops and ornamental plants. Important characteristics of these cultured plants are amongst others the increase in size of the plant, in particular of the used plant organs, the loss of bitter compounds, pest resistance and/or a high content of nutrients. More preferably, in one embodiment, the present invention relates to plants which are selected from the plants mentioned below.
Furthermore, the present invention provides populations of polyploid plants as characterized above, wherein a population comprises 7, 10, 20, 50, 100, 500, 1000, 2000, 5000, 10000 or even more of said polyploid plants and wherein the proportion of non-polyparental plants is 75% at the most, but preferably less than 50%, more preferably less than 25%, even more preferably less than 10%, and most preferably less than 1%.
In a further embodiment, the present invention also relates to organs, parts, tissues, cells, seed or offspring of the polyploid plant of the present invention.
The method of the present invention, i.e. HIPOD method is illustrated in
Thus, the method of the present invention may encompass the following steps:
As mentioned, in principle the means and methods described in European patent application EP 0 589 841 A2, for example the expression cassettes, may be used in the method of the present invention except that instead of an anther-specific 5′-regulatory region a constitutive promoter such as the 35S or NOS promoter employed in the Examples may be used and instead of a nucleotide sequence which encodes anti-sense RNA or a polypeptide which disrupts the formation of viable pollen when expressed, a nucleotide sequence is used encoding a product that provides for a selectable phenotype such as the BAR gene or a fluorescent protein. Thus, the components necessary and sufficient to arrive at the marker system for use in accordance with the present invention are well known in the art. Furthermore, as mentioned, more than two male parent plants may be used for generating multi-parental plants, for example using a three-hybrid component system which may comprise the following steps:
Thus, in principle steps (a1) and (a2) may be extended to (a2+n) to obtain polyploid, (2+n)+1-parental offspring by designing the marker system such that (2+n) components must act in an concerted manner in order to give rise to the selectable marker when combined in one single plant.
In an alternative embodiment, the method of the present invention comprises as a first step the generation of at least two transgenic male parent plants or plant lines each of which comprises a different selectable marker which together during sperm-egg fusion gives rise to plants resistant to the at least two selectable markers.
Thus, the method of the present invention may encompass the following steps:
Furthermore, as mentioned, more than two male parent plants may be used for generating multi-parental plants being resistant to multiple selectable markers.
Advantageously, the F1 offspring of the method of the present invention is heterozygous for the selectable marker gene but “homozygous” as regards its polyploidy, e.g. triparental. Typically, triploid hybrids with 3 sets of chromosomes (ABD), and plants with an uneven set of chromosomes in general have a reduced fertility and may be sterile. However, as described in Example 4 and illustrated in
The selfing of the hexaploid version of the triparental plant would then result in a typical F2 segregation of the offspring including plants which are triparental but free of the components of the marker gene system, i.e. free of the (different) marker(s) or parts thereof and thus non-transgenic. Accordingly, in an additional step the method of the present invention comprises the selfing of the F1 generation inter alia resulting in marker gene-free F2 plants devoid of any DNA which is foreign to the plant. Alternatively, meanwhile techniques exist to generate marker-free transformed/transgenic plants, for example the CRISPR-Cas system (see, e.g., Lowder et al., Plant Physiology 169 (2015), 971-985) and the cre/lox site-specific recombination (see, e.g., Wang et al., Transgenic Res. 14 (2005), 605-614) which though less preferred may be used for generating marker-free plants.
Thus, the present invention generally relates to a method for the generation of polyparental polyploid plants comprising (a) providing more than two parental plants, wherein (i) each of the male parent plants comprises a part of a components of a selectable marker system, or (ii) each of the male parent plants comprises a different selectable marker; (b) crossing the more than two parental plants; (c) selecting the offspring obtained by step (b) for the presence of the marker(s); and optionally (d) generating marker-free plants via (i) crossing or selfing at least two of the offspring of step (c); and (ii) selecting the offspring of step (d)(i) for the absence of the marker(s).
Means and methods for generating transgenic plants including the transformation of dicotyledonous and monocotyledonous plants are known in the art, see, e.g., Plant Cell and Tissue Culture—A Tool in Biotechnology: Basics and Application, Springer 2009 ISBN 978-3-540-93883-5. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants are well known in the art; see, e.g., Mitsuhara et al., Plant Cell Physiol. 37 (1996), 49-59 and Potenza et al., In Vitro Cellular & Developmental Biology—Plant 40 (2004), 1-22, the latter for review. Likewise, engineering herbicide resistance in plants by expression of a detoxifying enzyme is well known since 1980ths; see, e.g., De Block et al., EMBO J. 6 (1987), 2513-2518 and Miki et al., J. Biotechnol. 107 (2004), 193-232, the latter for review. Specific examples for the construction of the plant vectors and the generation of the transgenic plants of the present invention are described in Examples 1, 2 and 3 and can be adapted according to the selectable markers of interest.
Thus, in one embodiment, the method of the present invention generally relates to a method comprising the crossing of one female parent plant with more than one male parent plant, wherein the male parent plants each comprise a part of a selectable marker system which when present in one single plant allows for selection of said plant.
After crossing of the transgenic male parent plants comprising the individual part of the selectable marker system with a female parent plant, selection of the seedlings is performed. Seedlings resulting from monospermy, i.e. from crossing of the female parent plant with only one male parent plant are rendered sensitive for the selectable marker. In contrast, combinations of both constructs, which can only result from polyspermy will give rise to plants resistant to the selectable marker or which show a selectable phenotype.
In an alternative embodiment, the method of the present invention comprises the crossing of one female parent plant with more than one male parent plant, wherein the male parent plants each comprise a different selectable marker which when present in one single plant allow for selection of said plant for the presence of these markers.
After crossing of the transgenic male parent plants comprising the different selectable markers with a female parent plant, selection of the seedlings is performed. Seedlings resulting from monospermy, i.e. from crossing of the female parent plant with only one male parent plant are are only resistant to the one selectable marker comprised in one male parent plant. In contrast, combinations of both constructs, which can only result from polyspermy will give rise to plants resistant to the different selectable markers or which show a selectable phenotype. Preferably, a first selectable marker is used conferring herbicide resistance, preferably resistance to an herbicide that inhibits the glutamine synthase, such as the glufosinate-based herbicide BASTA (Biolophos) and a second selectable marker is used conferring antibiotic resistance, preferably resistance to hygromycin.
Preferably, said methods allow the crossing of one female parent plant with two, three, four, five, six, seven, eight, nine or ten male parents plants, wherein the crossing can occur between parent plants with different ploidy, i.e. they can be haploid, diploid, triploid, tetraploid, pentaploid, hexaploid, heptaploid, octaploid, nonaploid, decaploid, undecaploid or dodecaploid. Thus, the selectable marker system consists of two, three, four, five, six, seven, eight, nine or ten parts or of two, three, four, five, six, seven, eight, nine or ten different selectable markers, which when combined in one single plant allows for selection of said plant.
In a preferred embodiment, the method of the present invention comprises the crossing of one diploid or tetraploid female parent plant with two diploid or tetraploid male parent plants, wherein the two male parent plants comprise each a part of the selectable marker system; thus said maker system consists of two parts either of one selectable marker or alternatively of two different selectable marker.
In one embodiment, the selectable marker system of the present invention comprises a first part comprising a driver expressing a heterologous transcription factor under the control of a ubiquitous promoter and a second part comprising the selectable marker gene(s) under the control of a corresponding responsive promoter. More particular, when combined in one plant, preferably in close proximity, i.e. in the same part of the plant, the individual parts of the selection system resemble to a functional selectable marker and/or to a functional expression unit expressing the gene product which serves as selectable marker comprising the selectable marker gene operably linked to a regulatory sequence, preferably to a promotor and/or a heterologous transcription factor.
Since transgenic plants are obtained by the method of the present invention (steps (a) to (c)), which are still under debate in the public and have poor acceptance in particular with respect to food, said method provides the possibility to generate marker-free, non-transgenic plants (steps (d) and (e)) via crossing or selfing of the F1 generation resulting in marker gene-free plants that are devoid of any DNA which is foreign to the plant.
In one embodiment, the selectable marker of the present invention confers herbicide resistance. Preferably, the herbicide can be selected from but is not limited to a herbicide that inhibits acetyl coenzyme A carboxylase, such as aryloxyphenoxypropionate (FOPs)-, cyclohexanedione (DIMs)-, and phenylpyrazolin (DENs)-based herbicides, a herbicide that inhibits acetolactate synthase including sulfonylureas (such as flazasulfuron and metsulfuron-methyl), imidazolinones, triazolopyrimidines, pyrimidinyl oxybenzoates, and sulfonylamino carbonyl triazolinones, a herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase EPSPS, such as glyophysate-based herbicides (Roundup), a herbicide that inhibits the glutamine synthase, such as the glufosinate-based herbicide BASTA (Biolophos), a synthetic auxin herbicide, such as 2,4-D and Dicamba, a herbicide that inhibits the photosystems, including the triazine herbicides, such as atrazine, the urea derivatives, such as diuron, the bipyridinium herbicides, such as diquat and paraquat, the diphenyl ether herbicides, such as nitrofen, nitrofluorfen, acifluorfen, and oxyfluorfen, and the nitrile-herbicides, such as bromoxynil-based herbicides, and a herbicide that inhibits the phytoen-desaturase, including norflurazon-based, fluridon-based and difunon-based herbicides. Preferably, the herbicide is a glufosinate-based herbicide such as the non-selective herbicide (“total weedkiller”) BASTA. The antibiotic can be selected, but is not limited to kanamycin, neomycin, geneticin, paromomycin, hygromycin, gentamicin, spectinomycin, streptomycin or tobramycin.
In a further embodiment, the selectable marker of the present invention confers a detectable, preferably visible output such as fluorescence when the parts of the selectable marker system are brought into close proximity, i.e. are present in one plant, preferably in one part of the plant.
In a further embodiment, the selectable marker system of the present invention comprises the selectable marker conferring herbicide, antibiotic and/or antimetabolite resistance as well as a selectable marker conferring the visible detectable output, wherein the first one is preferably used for selection of the plants and the second one is preferably used as reporter for visualization. In a preferred embodiment, the selectable marker confers herbicide resistance and a reporter gene is used for visualization.
Thus, after crossing of the transgenic male parent plants comprising the individual part of the selectable marker system, i.e. either of one selectable marker or alternatively of two different selectable marker with a female parent plant, selection of the seedlings is performed with an herbicide. Seedlings resulting from monospermy, i.e. from crossing of the female parent plant with only one male parent plant are rendered sensitive for the herbicide and die upon herbicide treatment and/or remain colorless. In contrast, combinations of both constructs, which can only result from polyspermy will give rise to herbicide resistant and/or fluorescent progeny.
Of course, next to the selectable markers conferring herbicide resistance, further selectable markers for plants commonly known in the art can be used as well, e.g. selectable markers conferring resistance to antibiotics or antimetabolites and further markers conferring a visible output can be used, for example the lacZ system.
Since plants are eukaryotic organisms, they possess two or more copies of its genetic information per cell. Usually, every gene is represented by two alleles, which are identical in the homozygous state and are different in the heterozygous state, respectively. In a further embodiment, the male parent plants are homozygous for the individual parts of the selectable marker system. In particular, the first male parent plant (first pollen donor) is homozygous for a construct comprising a driver expressing a heterologous transcription factor under the control of an ubiquitous promoter and wherein the second pollen donor is homozygous for a construct comprising a herbicide resistance conferring gene, preferably the BAR gene or the pat gene under the control of a corresponding responsive promoter, optionally wherein the herbicide resistance conferring gene is linked to a fluorescence conferring gene, preferably the YFP gene. Alternatively, the male parent plants are homozygous for the different selectable marker.
The selectable marker system of the present invention is adapted to the mGAL4-VP16/UAS-based system well known in the art; see e.g. European patent application EP 0 589 841 A2. Consequently, the heterologous transcription factor of the construct of the first male parent plant (first pollen donor) is mGAL4-VP16 and the promoter is the NOS promoter or the RPS5a promoter and wherein the responsive promoter of the construct of the second male parent plant (second pollen donor) is the UAS promoter. Thus, both selectable markers, the one conferring herbicide resistance and the other conferring fluorescence are under control of the same regulatory elements. Of course, equivalent marker systems may be used as well for example those based on the first and second expression cassettes described in European patent application EP 0 589 841 A2. As mentioned, preferably the selectable marker gene confers herbicide resistance, most preferably the selectable marker system of the present invention comprises the BAR gene conferring resistance to the glufosinate-based herbicide BASTA.
The fluorescence conferring selectable marker, also referred to as reporter, can be of any kind as long as it confers an observable or measurable phenotype. According to the present invention, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria (described in international applications WO95/07463, WO96/27675 and WO95/121191) and its derivates “Blue GFP” (Heim et al, Curr. Biol. 6 (1996), 178-182), Redshift GFP” (Muldoon et al., Biotechniques (1997), 162-167) as well as enhanced “EGFP” variants can be used. Further embodiments are the yellow, cyan and blue fluorescent proteins (YFP, CFP and BFP, respectively) as well as its enhanced variants EYFP, ECFP and EBFP, the red fluorescent proteins (DsRed, HcRed, mCherry, dtTOMATO) and further fluorescent proteins such as the Venus protein, mPlum, mOrange, zoanFP and Cerulan. Further fluorescent proteins are known to the person skilled in the art and can be used according to the invention. The detection of fluorescent proteins takes place through per se known fluorescence detection methods. A summary of fluorescent proteins used in transgenic plants as well as methods for detection is for example provided in “Fluorescent Proteins in Transgenic Plants by Reginald J. Millwood, Hong S. Moon, and C. Neal Stewart Jr.”. In a preferred embodiment, the YFP gene is used as selectable marker/reporter.
Naturally, the present invention also extends to the plants obtained and obtainable, respectively, by the method of the present invention or any of the individual steps (a1+n) as well to parts, tissue, cells, seed and offspring as well as progeny of those plants, including the following:
These and other embodiments are disclosed and encompassed by the description and Examples of the present invention. Further literature concerning any one of the materials, methods, uses, and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database “Medline” may be utilized, which is hosted by the National Center for Biotechnology Information (NCBI) and/or the National Library of Medicine at the National Institutes of Health (NLM.NIH). Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.
The above disclosure generally describes the present invention. Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2. Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application including the background section and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.
In the following, specific materials and methods used in context with the present invention are listed.
Wild-type Arabidopsis strains used are Ler, Col-0, and C24. Plants were germinated on soil in a Conviron MTPS growth chamber under long-day conditions (16 hr light/8 hr dark) at 23° C. Upon bolting, plants were transferred to 18° C. both at otherwise constant growth conditions. Plant selection was either performed on soil by spraying the herbicide BASTA, or seeds were surface sterilized with ethanol and plated on MS medium containing hygromycin.
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded during experiments and analysis. Statistics were performed using two tailed t-test for unequal variances: * p<0.05; ** p<0.01; ***p<0.001.
The HIPOD constructs have been generated via adapting the mGAL4-VP16/UAS-based system, comprising a driver expressing the heterologous transcription factor mGAL4-VP6 under the control of the ubiquitous RPS5a promoter described by Haselhoff, Methods Cell Biol. 58 (1999), 139-151; see
First, the system was tested in a transient expression assay using Arabidopsis protoplasts. Transformation with only one of the constructs yielded YFP-negative protoplasts. By contrast, protoplasts that have been co-transfected with both HIPOD constructs exhibit YFP fluorescence; see
For the generation of triparental plants, PD1 (homozygous for the pRPS5a::mGAL4-VP6 construct) and PD2 (homozygous for the responder construct UAS::BAR_YFP) were provided as distinct pollen donors onto wild-type flowers. Therefor pollen grains from PD1 and PD2 were independently collected using a vacuum cleaner based collection device adopted from Jose Feijo lab (Johnson-Brousseau and McCormick, Plant. J. 39 (2004), 761-775) and the 3-8 oldest closed flower buds were emasculated and pollinated after two to three days using a brush. The procedure was repeated 4 times and in total 2575 double pollinations were performed yielding 120644 seeds. The seeds can be counted using open source imaging processing software, such as ImageJ (http://imagej.net/; e.g. Schneider et al., Nature methods 9 (2012), 671-675. Following herbicide treatment of the F1, 7 herbicide (BASTA) resistant seedlings have been detected; see
To determine whether these plants were indeed of triparental origin, the seven seedlings were subjected to genotyping by multiplex PCR using primers 5′-TATAGGGCGAATTGGGTACC-3′ (SEQ ID NO: 14) and 5′-GGAACTGGCATGACGTGGGTTT-3′ (SEQ ID NO: 15) that target the PD2 construct (UAS::BAR_YFP) as well as primers 5′-TCGTTTTCTCTGCCGTCTCTCT-3′ (SEQ ID NO: 16) and 5′-CCCTTGTTGCTGCTCTCCTC-3′ (SEQ ID NO: 17) that target the PD1 construct (pRPS5a:: mGAL4-VP16). Importantly, all plants segregated both HIPOD constructs (
The introgression of the two paternal copies implies that the resulting plants are triploid. In order to determine the ploidy of the seedlings, flow-cytometry has been performed. For this, one or two leaves were harvested and chopped using a razor blade in a petri dish with nuclei extraction buffer (Partec CyStain®). Afterwards, staining reagent (Partec CyStain UV Precise-Kit®) was added and incubated at RT for 1 min. The liquid was passed through a 50 μM nylon mesh and analyzed using Partec CyFlow® ploidy analyzer. Notably, all seven plants exhibited a profile characteristic to triploid plants; see
Growth height, sepal length, petal length and cell size were measured from digital images. Dissected sepals and petals were mounted in 70% alcohol to measure proximodistal dimensions. For cell size measurements petals were cleared in chloral hydrate:glycerine:ddH2O (8:2:1) and average cell sizes were calculated at the adaxial side, from the number of cells per unit area of digital micrographs as described in Disch et al., Curr. Biol. 16 (2006), 272-279. All size measurements were calculated using ImageJ software. Fertility assay was carried out as described in Volz et al., Dev. Cell 25 (2013), 310-316. The total number of seeds produced per wild-type plant was assessed by collecting all mature siliques during the plants life-time. Digital images of the respective seeds were processed the above mentioned software tool.
In order to discriminate attributes caused by the introgression of additional paternal copies and potential negative effects specifically associated with the unique polyspermic origin of these plants, biparental triploid plants have been included, which have been recovered from an interploidy cross (2n×4n). In comparison to diploid plants, overall plant size of triparental plants was increased 1.5 fold and the plants gave rise to bigger inflorescences and flowers. The size increase was comparable to that of biparental triploid plants;
As an approximation, the polyspermy rate was calculated on the basis of total seed counts (120644) and the number of recovered triparental seedlings (7), as evidenced by BASTA resistance and positive identification of both paternal HIPOD constructs. In addition, it was taken into account that via using the HIPOD method, only ⅓ of all polyspermy events can be selected as supernumerary sperm contributions involving sperm from single father escape detection: Frequency=(3×# triparental plants/# seeds)×100. Thereby, a polyspermy frequency of 0.017% has been yielded. When extrapolating this polyspermy frequency to the approximately 45000 seeds generated on average by Arabidopsis under the growth conditions used in this study, the data imply that every plant can give rise to about 7 polyspermy-induced polyploid plants.
Three genetically distinct Arabidopsis accessions have been combined in a three parent cross. For this three accession HIPOD cross more than 200 double pollinations were performed in two independent experiments, yielding 9493 seeds. In order to facilitate the detection of triparental offspring, the UAS HIPOD responder construct (see Example 1) has been introduced into the Arabidopsis Ler accession and the GAL4 HIPOD driver construct (see Example 1) into the C24 accession. Following pollination of a third accession, Col-0, with pollen from the distinct fathers resulted in the recovery of two viable herbicide resistant plants, TP3 1 and TP3 2; see
| Number | Date | Country | Kind |
|---|---|---|---|
| 16179967.1 | Jul 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/068133 | 7/18/2017 | WO | 00 |
