WO2007015238A2 - Methods of preventing gene flow in plants - Google Patents

Abstract

Methods of reducing stable transgene introgression from cultivated transgenic plants to non-cultivated plants are provided. Specifically, the present invention provides methods of producing transgenic plants having an exogenous genomically integrated sequence that is incapable of stably introgressing into related crops or wild or weedy species.

Description

METHODS OF PREVENTING GENE FLOW IN PLANTS

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to methods of reducing gene flow from cultivated transgenic plants to non-cultivated plants and more particularly, to methods of producing transgenic plants having an exogenous genomically integrated sequence which is incapable of introgressing into related crops or wild or weedy species.

One of the major concerns regarding the cultivation of transgenic crops is the uncontrolled spread of transgenes among fields to related wild species or to other varieties of the transgenic species. This is of particular importance when transgenic herbicide resistant genes are introduced. In 2004, approximately 58.6 million hectares of a total of >80 million hectares planted of transgenic crops were herbicide-resistant crops. Transgenes can flow from herbicide resistant crops to other varieties of the same crop or to fully or partially compatible wild or weedy species through cross pollination. Thus, herbicide resistance in oilseed rape has moved from one variety to others in oilseed rape, and from cultivated rice (Oryza sativa) to weedy red rice {Oryza sativa). Other transgenes that may confer a selective advantage on a related weed or wild species can move at a similar rate in a similar manner. Recently, a commercial release of new transgenic herbicide resistant wheat was abandoned. The reasons for this were not fully stated, but it was implied, in part, that the transgene would easily escape into populations of weedy relatives, notably Aegilops species. Ae. cylindrica is a pernicious weed in the US western grain belt (Jasieniuk, M. et al, 2001; Stone, A. E. & Peeper, T. F., 2004) although not in its center of origin in northern Asia Minor. Ae. longissima, previously a species that grows on dry plains on non-agricultural rocky outcroppings near fields, is increasingly becoming a pesky weed in southern Israel. It is not clear whether that is due to introgression of wheat genes, evolution by itself, and/or the availability of an ecological niche due to the eradication of other grass weeds. Many other Aegilops and wheat-related species reside in ruderal ecosystems in close proximity to wheat. Of these several Aegilops species, namely, Ae. cylindrica (2n = 4X = 28, genomes CCDD), Ae. ventricosa (2n = 4x = 28; genome DDUⁿUⁿ), Ae. crassa tetraploid (2n = 4x = 28; genome DDM^crM^cr) and hexaploid (2n = 6x = 42; genome DDD²D²M^crM^cr), Ae. vavilovii (2n = 6x = 42; genome DDM⁰TVfS¹S¹), Ae. juvenalis (2n = 6x = 42; genome DDM^crM^crUU) and Ae. tauschii (2n = 2x = 14: genome DD) (Kihara, H. 1954; Kimber, G. & Tsunewaki, K., 1988), has one of their genomes homologous to the D genome of bread wheat Triticum aestivum (2n = 6X = 42, genomes BBAADD). Thus, it was suggested that only wheat with transgenes on the A or B genome be released, to prevent gene flow to these species (Gressel, J., 2002; Wang, Z. N., et al., 2001). This is specious, as breeders have commonly incorporated alien genes from homoeologous chromosomes of Aegilops species into wheat (Dhaliwal, H. S., et al., 2002; Ganeva, G et al., 2000; Martin-Sanchez, J. A. et al. 2003; Spetsov, P., et al., 1997; Sharma, H. et al. 1995).

Studies have demonstrated gene flow from wheat to related genera. Groups in Oregon and Switzerland have demonstrated that hybrids easily form between wheat and Ae. cylindrica, with wheat genes remaining in the first two backcrosses, but have not yet demonstrated that wheat gene introgression is stabilized in the C genome of Ae. cylindrica, which is homoeologous to the genomes of bread wheat (Wang, Z. N., Z., et al., 2001; Guadagnuolo, R., et al., 2001a; Hegde, S. G. & Waines, J. G., 2004; Morrison, L. A., et al., 2002a, b; Wang, Z. N. et al., 2000; Wang, Z. N. et al., 2002; Zemetra, R. S., et al., 1998). In addition, the present inventors have shown that a specific wheat DNA sequence appears sporadically in Ae. peregrina (Ae. variabilis) (2n = 4X = 28, genomes S^VS^VUU) in nature (Weissmann, S., et al. 2005), and have also demonstrated that wheat genes can introgress and are stable in Ae. peregrina BC₃F₂ plants with 28 chromosomes after manual hybrid formation between Ae. peregrina and bread wheat and backcrossing to Ae. peregrina (Weissmann, Feldman and Gressel, unpublished data). Similarly, wheat-specific DNA sequences have been found in an accession of sea barley (Hordeum marinum), which had the appearance of the weed (Guadagnuolo, R., et al., 2001b). It is not clear that the DNA has introgressed into a Hordeum chromosome as no chromosome counts nor progeny segregation analyses were reported.

Farmers need transgenic wheat varieties to deal with problems where breeding cannot help, or where breeding is slow. Weed control is one problem where breeding has been of little assistance (Lyon, D. J., et al., 2002). Graminicides (herbicides that control grass weeds) are continually falling by the wayside due to grass weeds evolving resistance, either by mutating the herbicide target sites to non binding forms, or up-regulating cytochrome P450s that degrade the herbicides, the same mode used by wheat to be resistant to graminicides (Gressel, J., 2002; Gressel, J., 1988). Much of Australian wheat lands as well as smaller areas scattered throughout the world are plagued by the weed Lolium rigidum (annual or rigid ryegrass) (De Prado, J. et al., 2005; Neve, P., et al., 2004; Yu, Q., et al., 2004), which has evolved resistance to all selective graminicides commonly used in wheat. Alopecurus myosuroides (blackgrass) has evolved the same way in Britain, but has been spread to a less extent (Price, L., et al., 2003; Cocker, K. M., et al., 1999). In North America there are large areas without a graminicide to control Avena spp. (wild oats) (Beckie, H. J. et al., 1999; Mengistu, L. W., et al., 2003; Kern, A. J. et al., 2002), in other areas there are limited graminicide choices to deal with Setaria (foxtail) species (Delye, C, et al., 2004; De Prado, R., et al, 2004; Volenberg, D. S., et al., 2002; De Prado, R., et al., 2000). The extent of these resistance problems is continuously documented on the web site http://www.weedscience.org.

It is clear that resistant grass weeds will rear their ugly heads throughout the wheat lands of the world. The chemical industry has been of little help; it is decades since a graminicide for wheat with a new mode of action has been released, or a pre- release described at scientific meetings. If selectivity of new herbicides for wheat is based on the same P450s used by wheat at present, then some grass weeds will be resistant to new graminicides before they are tested (Gressel, J. 2002; Gressel, J. 1988; Preston, C, 2004). Thus, transgenes, where genes encode novel mechanisms of selectivity not found in any plants, offer a solution although no solution is forever in agriculture (Yenish, J. R. & Young, F. L., 2004). This is especially true with the weedy relatives of wheat, such as Ae. cylindrica, which cannot be controlled by wheat selective herbicides, but could be controlled by transgenic herbicide resistance, or wheat with specific mutations conferring herbicide resistance (Ball, D. A., et al., 1999). Unfortunately, the herbicide resistant wheat marketed has the mutant gene localized on the D genome of wheat (Anderson, J. A., et al., 2004), which homologously recombines with D genome of Ae. cylindrica. Indeed, a high rate of wheat D genome retention was found in manually produced BC₂ plants of wheat X Ae. cylindrica hybrids backcrossed to Ae. cylindrica (Kroiss, L. J. et al., 2004).

Similarly, a new violent race of stem rust has appeared in Africa on wheat lines having the commonly used rye Sr3l gene, for which no resistance had previously been reported anywhere in the world

(http://www.cdl.umn.edu/crb/1999crb/99crb2.html). Some sort of transgenic rust resistance may be a quicker fix than trying to find resistance genes within the diversity of wheat. One does not want transgenic disease resistance stably introgressing into wild relatives, which would render them healthier and more competitive with crops. If wheat were transformed to greater mineral-nutrient use efficiency, the transgenes would be of great utility to its wild/weedy relatives, to the detriment of agriculture. Other allopolyploid crops such as cotton and oilseed rape also have needs that transgenic technologies have fulfilled, as is apparent from farmer acceptance. As discussed below, oilseed rape can easily transfer genes to one of its weedy/wild diploid progenitors.

As transgenics are imperative to the future of wheat (Gressel, J., 2002; Gressel, J., 1988) and other allopolyploid crops, the issue of gene flow to related weeds and wild species must be dealt with (Ellstrand, N. C. 2003; Stewart, C. N., et al., 2003; Snyder, J. R., 2000). Thus, herbicide or disease resistant, nutrient efficient weedy or ruderal species of the Triticeae can spread in wheat fields, when all other grass weeds are eliminated, or diseased, creating a niche for these species that had been less competitive than the other grass weeds.

As mentioned before, inserting transgenes to allopolyploid crop chromosomes that are homoeologous to related weed/wild species does not preclude transfer. Other solutions have been suggested such as GURT (Genetic Use Restriction Technologies) (terminator) but they have not yet been demonstrated to work in the field (Oliver, M. J., et al., 2004; U.S. Pat. 5,723,765, 1998 to Oliver, M. J., et al.). This system allows crosses between the wild/weedy relatives or other varieties in the areas used for seed production, so it is also "leaky".

It has also been proposed that transforming transgenes into the chloroplast genome (plastome) would prevent gene flow due to the maternal mode of plastid inheritance (Daniell, H., 2002; Maliga, P., 2004). This is also a highly leaky technology with 0.4 % pollen transmission of plastid genes in the field between the crop Setaria italica (Italian millet) and its biologically con-specific (i.e., same species), weedy progenitor S. viridis (Wang, T. et al., 2004) as well as in other cases (Darmency, H. 1994). Moreover, in many natural wheat x wild relative hybrids, wheat was the predominant male parent (Morrison, L. A., 2002b), but the wild species can pollinate wheat producing the same F₁ plants albeit bearing the wheat plastome.

As gene containment systems seem leaky, it was suggested to augment them with transgenic mitigation, i.e. coupling the gene of choice in tandem with a gene that is neutral or useful to the crop, but would render weedy offspring unfit to compete (Gressel, J. 1999, US Pat. Appl. No. 20040172678 to Gressel and Al-Ahmad). Such mitigating genes, which would be inherited as a genetically linked group, include dwarfing (increases crop yield) and anti-shattering (preventing the premature seed drop that keeps weed seeds from being harvested and removed from the field). The utility of mitigation has been demonstrated through the stage of transgenic screen house in two species (Al-Ahmad, H., et al., 2004; Al-Ahmad, H., et al., 2005a and b). It is a technology that should be coupled with other technologies to further lower the risk of transgene flow.

Polyploid crops can be divided into two groups, those that arose from a single progenitor by genome duplication, such as potato, alfalfa, strawberry, and yam, and those 'allopolyploids' such as durum and bread wheat, oilseed rape, cotton, sugarcane, and tobacco, that resulted from the hybridization of more than one progenitor of different, but closely related species having homoeologous chromosomes. Despite the synteny and similarity between the constituent genomes, pairing of chromosomes during meiosis of allopolyploid crops appears similar to that of diploid crops, namely, only bivalent pairing.

There is thus a widely recognized need for, and it would be highly advantageous to have, a method of preventing genetic introgression of plant transgenes into wild type plants devoid of the above limitations.

SUMMARY QF THE INVENTION

According to one aspect of the present invention there is provided a method of generating a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the method comprising inserting an exogenous polynucleotide into a genetic locus of the transgenic cultivated plant, the genetic locus being in linkage disequilibrium with a PhX locus of the transgenic cultivated plant thereby preventing the stable transgene introgression into the related, non-cultivated plant. According to another aspect of the present invention there is provided a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the transgenic cultivated plant comprising an exogenous polynucleotide positioned in a genetic locus of the transgenic cultivated plant, the genetic locus being in linkage disequilibrium with a PhI locus of the transgenic cultivated plant.

According to yet another aspect of the present invention there is provided a method of reducing introgression of an exogenous polynucleotide of a transgenic cultivated plant into a related, non-cultivated plant, the method comprising inserting the exogenous polynucleotide into a genetic locus of the transgenic cultivated plant, the genetic locus being in linkage disequilibrium with a PhI locus of the transgenic cultivated plant thereby preventing the transgene flow into the related, non-cultivated plant. According to still another aspect of the present invention there is provided a method of generating a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the method comprising inserting an exogenous polynucleotide into a centromeric region of a chromosome of the transgenic cultivated plant thereby preventing the transgene flow into the related, non-cultivated plant.

According to an additional aspect of the present invention there is provided a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the transgenic cultivated plant comprising an exogenous polynucleotide positioned in a centromeric region of a chromosome of the transgenic cultivated plant.

According to yet an additional aspect of the present invention there is provided a method of reducing introgression of an exogenous polynucleotide of a transgenic cultivated plant into a related, non-cultivated plant, the method comprising inserting the exogenous polynucleotide into a centromeric region of a chromosome of the transgenic cultivated plant.

According to still an additional aspect of the present invention there is provided a method of generating a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the method comprising inserting an exogenous polynucleotide into a genetic locus of the transgenic cultivated plant, the genetic locus being in linkage disequilibrium with a locus including a gene capable of preventing homoeologous recombination, thereby preventing the stable introgression into the related, non-cultivated plant.

According to a further aspect of the present invention there is provided a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the transgenic cultivated plant comprising an exogenous polynucleotide positioned in a genetic locus of the transgenic cultivated plant, the genetic locus being in linkage disequilibrium with a locus including a gene capable of preventing homoeologous recombination. According to yet a further aspect of the present invention there is provided a method of reducing introgression of an exogenous polynucleotide of a transgenic cultivated plant into a related, non-cultivated plant, the method comprising inserting the exogenous polynucleotide into a genetic locus of the transgenic cultivated plant, the genetic locus being in linkage disequilibrium with a locus including a gene capable of preventing homoeologous recombination thereby reducing introgression of the exogenous polynucleotide into the related, non-cultivated plant.

According to further features in preferred embodiments of the invention described below, the exogenous polynucleotide comprises a nucleic acid sequence encoding a polynucleotide or a polypeptide product. According to still further features in the described preferred embodiments the genetic locus being in linkage disequilibrium with the PhI locus is set forth by SEQ ID NO:13, 14, 15, 16, 17 and 39 .

According to still further features in the described preferred embodiments the transgenic cultivated plant is wheat. According to still further features in the described preferred embodiments the wheat is Triticum aestivum ssp. aestivum or Triticum turgidum ssp. durum.

According to still further features in the described preferred embodiments the related, non-cultivated plant is selected from the group consisting of Aegilops spp., Hordeum spp., Elymus spp., Agropyron spp., Eremopyrum ssp., Secale spp., Dasypyrum spp., Heteranthelium ssp., Amblyopyrum ssp., Henrardia ssp., Thinopyrum spp., Leymus spp., Psathyrostachys spp., Hystrix ssp.,Hordelymus ssp., Taeniatherum ssp., and Crithopsis ssp.

According to still further features in the described preferred embodiments the exogenous polynucleotide comprises a nucleic acid sequence encoding a suppressor of a gene or gene product lethal to the related, non-cultivated plant.

According to still further features in the described preferred embodiments the method further comprising inserting a second exogenous polynucleotide into a second genetic locus of the transgenic cultivated plant, the second genetic locus being in random association with the PhX locus of the transgenic cultivated plant. According to still further features in the described preferred embodiments the transgenic cultivated plant further comprising a second exogenous polynucleotide positioned in a second genetic locus of the transgenic cultivated plant, the second genetic locus being in random association with the PhX locus of the transgenic cultivated plant.

According to still further features in the described preferred embodiments the second exogenous polynucleotide comprises a nucleic acid sequence encoding a product lethal to the related, non-cultivated plant.

According to still further features in the described preferred embodiments the second exogenous polynucleotide further comprises a second nucleic acid sequence encoding a transgene.

According to still further features in the described preferred embodiments the transgene is a polynucleotide or a polypeptide.

According to still further features in the described preferred embodiments the polynucleotide or polypeptide product endows the transgenic cultivated plant with a commercially desirable trait selected from the group consisting of herbicide resistance, disease resistance, insect resistance and nematode resistance, environmental stress resistance, high productivity, modified agronomic quality, enhanced yield, modified ripening, and bioremediation. According to still further features in the described preferred embodiments the exogenous polynucleotide further comprises a promoter for directing an expression of the nucleic acid sequence in the transgenic cultivated crop.

According to still further features in the described preferred embodiments the promoter is selected from the group consisting of FMV, 35S, E8, E4, E17, J49, 2Al 1, and Tapl.

According to still further features in the described preferred embodiments the product lethal to the related, non-cultivated plant is barnase.

According to still further features in the described preferred embodiments the suppressor of the product lethal to the related, non-cultivated plant is bar star. According to still further features in the described preferred embodiments the centromeric region of the transgenic cultivated plant is a sub-centromeric region of the transgenic cultivated plant. The present invention successfully addresses the shortcomings of the presently known configurations by providing a method of preventing stable gene introgression into non-cultivated plants.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings: FIG. 1 is an AFLP analysis depicting introgression of DNA from the B genome of T. aestivum (line TAAOl) into Ae. peregrina (line TKE02). The introgressed band (marked by an arrow) is present in T. aestivum [TAAOl, genome BBAADD], T. turgidum ssp. durum (TTR (lines TTR298 and TTRl 6), genome BBAA), F₁ of the cross between T. aestivum and Ae. peregrina (F₁, 2n =35; genome S^VUBAD), BC₃F₂ [having 2n =28 chromosomes (14 bivalents at meiotic metaphase)], and BC₃ [having 29 chromosomes (14 bivalents + 1 univalent , presumably of T. aestivum)], but missing from Ae. peregrina (TKE02 genome S^VS^VUU), T. urartu [TMU (lines TMU38 and TMU05), genome AA), and Ae. tauschii [TQ (lines TQ96, TQ81 and TQ71), genome DD]. FIG. 2 is an AFLP analysis depicting introgression from the D (band No. 1) and A (band No. 2) genomes of T. aestivum (TAAOl) into Ae. peregrina (TKE02). Band No. 1 is present in T. aestivum [TAAOl (genome BBAADD)], Ae. tauschii (TQ71, TQ81 and TQ96); genome DD), F₁ of the cross between T. aestivum wxά Ae. peregrina [having 2n = 28 chromosomes (14 bivalents at meiotic metaphase)], and BC₃ [having 29 chromosomes (14 bivalents + 1 univalent, presumably of T. aestivum), but missing from Ae. peregrina [TKE02 genome S^VS^VUU], T. turgidum ssp. durum [(TTR16 and TTR298); genome BBAA], and T. urartu [(TMU05 and TMU38); genome AA). Band No. 2 is present in T. aestivum [TAAOl (genome BBAADD)], T. turgidum ssp. durum [(TTR16 and TTR298); genome BBAA], T. urartu [(TMU05 and TMU38); genome AA], F₁ of the cross between T. aestivum and Ae. peregrina (Fi, 2n 35; genome S^VU BAD), BC₃F₂ [having 2n = 28 chromosomes (14 bivalents at meiotic metaphase)], and BC₃ [having 29 chromosomes (14 bivalents + 1 univalent , presumably of T. aestivum),, but missing from Ae. tauschii [(TQ96, TQ81 and TQ71), genome DD] and^e. peregrina [TKE02 genome S^VS^VUU].

FIG. 3 is an AFLP analysis depicting introgression of a band from an undetermined genome of T. aestivum (TAAOl) into Ae. peregrina (TKE02). The introgressed band (marked by an arrow) is present in T. aestivum (TAAOl, genome BBAADD), T. turgidum ssp. durum (TTR298 and TTRl 6; genome BBAA), F₁ of the cross between T. aestivum and Ae. peregrina [F₁, 2n = 35; genome S^VUBAD), BC₃F₂ [having 2n=28 chromosomes (14 bivalents at meiotic metaphase)], and BC3 [having 29 chromosomes (14 bivalents + 1 univalent , presumably of T. aestivum), , T. urartu (TMU05 and TMU38; genome AA), and Ae. tauschii (TQ96, TQ81 and TQ71; genome DD), but missing from Ae. peregrina [TKE02, genome S^VS^VUU]. Note that this band segregates; it is missing from one of the BC₃ plants (right BC3 lane) and from four BC₃F₂ plants.

FIG. 4 is a schematic illustration depicting random segregation of chromosomes in the F₁ hybrids (due to the presence of PhI gene that suppresses homoeologous pairing) may lead to the formation of PhI lacking plants in backcross generations. The absence of PhI causes an increase of homoeologous pairing and thus promotes recombination and introgression in backcross plants.

FIG. 5 is a schematic illustration depicting two failsafe mechanisms (b and c) to suppress the movement of transgene from bread wheat into a wild relative and one failsafe mechanism (a) which is used by other studies, a, possible transgene movement, by homoeologous recombination, from wheat chromosomes into wild relatives without linkage to PhX; b, transgene movement by homoeologous recombination is suppressed by linkage of the transgene to PhX on the long arm of wheat chromosome 5B; c, a double failsafe mechanism that includes the placement of a transgene in a tandem construct with barnase, a gene encoding a lethal RNase, on any chromosome arm other than 5BL, and by inserting barstar on chromosome arm 5BL in proximity to PhX. Barstar encodes a protein that suppresses the specific RNase produced by barnase. Any backcross progeny having the transgene without chromosome arm 5BL will die. The presence of PhI, in backcross plants containing 5BL, will prevent the establishment of barstar and the transgene (if present) in the wild population by preventing homoeologous pairing.

FIG. 6 is a photomicrograph depicting chromosomal staining of an F₁ hybrid plant. The chromosomes were stained with acetocarmine. Note the expected number of 35 chromosomes (21 bread wheat chromosomes + 14 Ae. peregrina chromosomes) in the F₁ hybrid plant of Ae. peregrina x T. aestivum.

FIGs. 7a-d are photomicrographs depicting chromosomal staining of BC₃F₂ plants derived from backcrossing the F₁ hybrid Ae. peregrina x T. aestivum three times to Ae. peregrina. Figure 7a - a stabilized BC₃F₂ plant containing 28 chromosomes. This plant is among the 15 BC₃F₂ plants that were used to analyze introgression from T. aestivum into Ae. peregrina. Figure 7b - BC₃ plants contained trivalents (marked by an arrow) at meiosis. This may indicate a possible translocation from T. aestivum into Ae. peregrina. One (or two) of the homologous chromosomes of Ae. peregrina may contain a wheat segment homologous to that of the third chromosome (possibly a wheat chromosome) attached to the bivalent. Figure 7c - In meiosis, all BC₃F₂ plants with 28 chromosomes had 14 bivalents. Figure 7d - A meiotic cell of wild type parental species Ae. Peregrina. Note that the BC₃F₂ plants shown in Figure 7c exhibit a significantly (P = 1.07 x e^"46) higher number of rod bivalents (range 3-7, average 4.03) as compared to their wild type parental species Ae. peregrina (range 1-4, average 2.01) shown in Figure 7d. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of transgenic plants and methods of preventing stable introgression of transgenes into non-target plants. The present method can be utilized to generate a transgenic cultivated plant which is failsafe, i.e., is unable to introgress genes into a genome of related, non-cultivated plants such as weeds.

The principles and operation of the method of preventing stable genome introgression according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Domesticated wheat (durum and bread wheat) are in dire need of transgenic traits such as herbicide and disease resistance, especially in light of the recent evolution of herbicide resistant grass weeds and an intractable new strain of rust. However, such transgenes can be transferred to wild/weedy relatives of the wheat through cross-pollination. Such naturally occurring gene flow has been documented in closely related Aegilops spp. (Weissmann et al., 2005) as well as in a more distantly related barley (Hordeum) spp (Guadagnuolo, R., et al., 2001a). Other allopolyploid crops, such as oilseed rape and cotton have similar biotic and abiotic constraints that genetic engineering can rapidly solve. This is evident from the large number of experimental as well as commercial releases of transgenic oilseed rape and cotton from the Animal and Plant Health Inspection Services website (APHIS; http://www.aphis.usda.gov/).

Studies have focused on the formation of crop-wild inter-generic hybrids, and assumed that introgression between homoeologous genomes is mainly the result of homoeologous pairing in the hybrid (Guadagnuolo et al. 2001a; Hegde and Waines 2004; Seefeldt et al. 1998; Snyder et al. 2000; Wang et al. 2001; Zemetra et al. 1998). However, due to the PhI gene, which suppresses pairing between homoeologous chromosomes (Wall et al. 1971, Genetical Research 18: 329-339), F₁ hybrid plants between wild relatives and durum or bread wheat are characterized by low homoeologous pairing [Jauhar, P.P., Peterson, T.S. (2001). Hybrids between durum wheat and Thinopyrum junceiforme: Prospects for breeding for scab resistance. Euphytica 118 (2): 127-136; Anamthawat-Jonsson and Bodvarsdottir, 1998, Chromosome Research 6: 339-343].

In sharp contrast to these studies, the present inventors have uncovered that the recombination events occurring between homoeologous chromosomes of cultivated wheat (7". aestivum cv. Chinese Spring; TAAOl) and Ae. peregrina is significantly higher than the number of bivalents per cell observed in Fl hybrids between T. aestivum cv. Chinese Spring and Ae. peregrina TKE02 (Ozkan and Feldman 2001, Genome 44: 1000-1006.; Sears 1977, Canadian Journal of Genetics and Cytology 19: 585-593). As is shown in Figures 1-3 and is described in Example 1 of the Examples section that follows, the present inventors have uncovered 17 DNA fragments which introgressed from cultivated wheat (TAAOl) to BC3 and BC₃F₂ backcrosses. These results demonstrate that stable genomic introgression occurs in a frequency higher than expected during backcrosses of the transgenic plant to the wild type.

These results have led the present inventors to postulate that cultivated transgenic plants can be generated such that gene flow of the transgene to related wild/weedy relatives of the cultivated plant is substantially prevented. As is shown in Examples 2 and 3 of the Examples section that follows, the present inventors have uncovered methods of generating transgenic plants characterized by reduced stable transgene introgression.

Thus, according to one aspect of the present invention there is provided a method of generating a transgenic cultivated plant characterized by a reduced stable transgene introgression to a related, non-cultivated species. As used herein the phrase "reduced stable transgene introgression" refers to a plant in which any transgene (e.g., an exogenous polynucleotide) being inserted into a genome thereof is characterized by reduced gene introgression. The phrase "gene introgression" or "transgene introgression", which is interchangeably used herein, refers to a stable recombination (transfer of nucleic acid sequences) between plant species having homoeologous chromosomes. Homoeologous chromosomes are non- identical yet related chromosomes of two related species from the same genus or related genera that have a common ancestor, but have diverged from each other during evolution. Such chromosomes are partial homologous, similar in synteny and in many sequences and thus can pair at meiosis. Thus, homoeologous chromosomes share some homologous DNA sequences along with some significantly nonhomologous DNA sequences.

The method according to this aspect of the present invention is effected by inserting an exogenous polynucleotide into a genetic locus that is in linkage disequilibrium with the PhX locus of the transgenic cultivated plant.

The term "PM locus" as used herein, refers to the genomic location of a polynucleotide or a gene encoding the PhI gene product on a chromosome region. In durum and bread wheat PhX is located in the central region of the long arm of chromosome 5B (5BL) (Jampates, R. & Dvorak, J. 1986), about 1 centi-Morgan (cM) from the centromere (Sears, E. R., 1984). PhI (or a PhX homolog) exists only in polyploid species of Triticum having the B genome.

The phrase "linkage disequilibrium (LD)" refers to the non-random association between DNA markers or genes at two adjacent loci. LD is quantified with either Lewontin's parameter of association (D') or with Pearson correlation coefficient (r) [Devlin B, Risch N. (1995). A comparison of linkage disequilibrium measures for fine-scale mapping. Genomics. 29: 311-322.]. Two loci with a LD value of 1 are considered to be in complete LD. At the other extreme, two loci with a LD value of 0 are termed to be in linkage equilibrium. LD values according to the present invention for neighboring loci are selected above 0.1, preferably, above 0.2, more preferable above 0.5, more preferably, above 0.6, still more preferably, above 0.7, preferably, above 0.8, more preferably above 0.9, ideally about 1.0 to 1.0.

It will be appreciated that the genetic distance of two loci which are in linkage disequilibrium is smaller than the genetic distance (measured in centiMorgans) of loci which are in linkage equilibrium. Preferably, the genetic distance of the two loci of the present invention {i.e., the locus in which the exogenous polynucleotide is inserted and the PhX gene locus) is smaller than 50 centiMorgan, preferably smaller than 40 centiMorgan, more preferably smaller than 30 centiMorgan, more preferably smaller than 20 centiMorgan, more preferably smaller than 10 centiMorgan, more preferably smaller than 5 centiMorgan, more preferably smaller than 1 centiMorgan, most preferably in the range of 0 to 1 centiMorgan, wherein 0 centiMorgan refers to juxtaposed sequences.

Although the gene coding to PhX has not yet been isolated, various sequences are genetically mapped to the PhX locus using genetic studies (e.g., recombination analyses between markers and the like). Recently, Griffiths S., et al., have localized the PhI locus to a 2.5-megabase interstitial region of wheat chromosome 5B (Nature Letters, 2006, 439: 749-752, incorporated herein by reference). This 2.5-megabase region, was sequenced and a group of three cdc2-related genes was identified. Although the cdc2 genes are good candidates for being involved in PhI function, as cdc2-related genes affect chromosome condensation (http://www.ihop- net.org/UniPub/iHOP/), none of the genes that were identified in the region showed differences in expression between the presence and absence of PhI.

Non-limiting examples of sequences (genes, markers or loci) which are in linkage disequilibrium with the PhX locus in wheat, and which can be used according to this aspect of the present invention, are provided in Table 1, hereinbelow.

Table 1

Genes, markers and loci in linkage disequilibrium with the PhI locus on the long arm of wheat chromosome 5B.

Table 1: Data from: http://wheat.pw.usda.gov/GG2/index.shtml, http://ncbi.nlm.nih.gov/. The PhI locus is mapped to chromosomal interval 5L-0.53-O.53 (http://wheat.pw.usda.gov/, Wheat, Physical - Ta-Physical-Gill-5ABD).

According to one preferred embodiment of this aspect of the present invention, the genetic locus being in linkage disequilibrium with the PhX locus is set forth by SEQ ID NO: 13, 16 or 17.

It will be appreciated that other loci of genes that are capable of preventing recombination between homoeologous chromosomes can be also used along with the present invention.

As used herein the phrase "transgenic cultivated plant" refers to a cultivated plant genetically modified to include an exogenous polynucleotide (i.e., a transgene) in a genome thereof. The transgenic cultivated plant of the present invention can include the PhX locus (e.g., wheat) or another locus of another gene capable of preventing homoeologous recombination. Preferably, the transgenic cultivated plant of the present invention is wheat. It will be appreciated that since the wheat genome comprises three sets of complete genomes, i.e., genomes A, B and D, the exogenous polynucleotide can be integrated into any one of the chromosomes of each genome (i.e., chromosomes 1-7 of genome A, B or D).

Preferably, the wheat used by the present invention is Triticum aestivum ssp. aestivum and/or Triticum turgidum ssp. durum.

As used herein in the specification and in the claims section that follows, the phrase "related, non-cultivated plant" refers to any wild type species or related species or related genera of the cultivated plant including weeds. Non-limiting examples of related, non-cultivated plants that can introgress genes into wheat or vice versa include Aegilops spp., Hordeum spp., Elymus spp., Agropyron spp., Eremopyrum ssp., Secale spp., Dasypyrum spp., Heteranthelium ssp., Amblyopyrum ssp., Henrardia ssp., Thinopyrum spp., Leymus spp., Psathyrostachys spp., Hystrix ssp.,Hordelymus ssp., Taeniatherum ssp., Crithopsis ssp and other species of the tribe Triticeae.

The term "weed" includes undesirable plants growing wild, especially those growing on cultivated ground to the disadvantage of a cultivated crop, lawn, or flower bed. In the broadest sense, the term weed is defined as all plants that grow in locations where they are undesired, including, for example: dicotyledonous weeds of the genera: Abutilon, Amaranthus, Ambrosia, Anoda, Anthemis, Aphanes, Atriplex, Bellis, Bidens, Capsella, Carduus, Cassia, Centaurea, Chenopodium, Cirsium, Convolwlus, Datura, Desmodium, Emex, Erysimum, Euphorbia, Galeopsis, Galinsoga, Galium, Hibiscus, Ipomoea, Kochia, Lamium, Lepidium, Lindernia, Matricaria, Mentha, Mercurialis, Mullugo, Myosotis, Papaver, Pharbitis, Plantago, Polygonum, Portulaca, Ranunculus, Raphanus, Rorippa, Rotala, Rumex, Salsola, Senecio, Sesbania, Sida, Sinapis, Solanum, Sonchus, Sphenoclea, Stellaria, Taraxacum, Thlaspi, Trifolium, Urtica, Veronica, Viola, Xanthium, weedy varieties of dicotyledonous crops of the genera: Arachis, Beta, Brassica, Cucumis, Cucurbita, Helianthus, Daucus, Glycine, Gossypium, Ipomoea, Lactuca, Linum, Lycopersicon, Nicotiana, Phaseolus, Pisum, Solanum, Vicia; monocotyledonous weeds of the genera: Aegilops, Agropyron, Agrostis, Alopecurus, Apera, Avena, Brachiaria, Bromus, Cenchrus, Commelina, Cynodon, Cyperus, Dactyloctenium, Digitaria, Echinochloa, Eleocharis, Eleusine, Eragrostis, Eriochloa, Festuca, Fimbristylis, Heteranthera, Imperata, Ischaemum, Leptochloa, Lolium, Monochoria, Panicum, Paspalum, Phalaris, Phleum, Poa, Rottboellia, Sagittaria, Scirpus, Setaria, Sorghum, Thinopyrum spp., Leymus spp., and Psathyrostachys and weedy varieties of monocotyledonous crops of the genera: Allium, Ananas, Asparagus, Avena, Hordeum, Oryza, Panicum, Saccharum, Secale, Sorghum, Triticale, Triticum, Zea. The term further includes various forms of the crop species that are undesirable to agriculture: feral forms that have escaped cultivation and have evolved weedy characters, wild (uncultivated) forms that have escaped cultivation and evolved weedy characters undesirable, uncultivated interbreeding species related to the cultivated crop, other varieties of the crop that do not possess the same transgenes, and the transgenic crop when it is a volunteer weed in following crops.

According to one preferred embodiment of this aspect of the present invention, the transgenic cultivated plants are generated such that they harbor a gene which is beneficial to plant such as a commercially desirable trait. Non-limiting examples of commercially desirable traits include herbicide resistance, disease resistance, insect resistance, nematode resistance, environmental stress resistance (e.g., cold resistance and drought resistance), high productivity, modified agronomic quality, enhanced yield, modified ripening, and bioremediation. Table 2, herein below, summarizes the various genetic elements endowing desired traits into transgenic plants.

Table 2 Commercially desirable traits that have been engineered into crop plants

Table 2: Data is derived from the Animal and Plant Health Inspection Services website (APHIS; http://www.aphis.usda.gov/).

Some specific crops have been broadly engineered to include the commercially advantageous traits listed above. For example, some of the more well- known transgenic traits, and the genes responsible for them, that have been introduced into use in oilseed rape production, are listed in Table 3, hereinbelow. Table 3

Examples of primary advantageous genes inserted into oilseed rape for potentially commercial purposes in USA

Tvpe ofsene/phenotype Gene APHIS #

Herbicide Resistance

CBI CBI 03-254-03

Glyphosate EPSP synthase 01-080-04 glyphosate oxidoreductase 96-045-02

Glufosinate CBI 00-023-05

Phosphinothricin acetyl transferase 98-274-10

Brotnoxynil nitrilase 98-243-02

Sulfonylurea acetolactate synthase 96-102-01

Insect Resistance

Lepidopteran CrylA(c) 02-312-02

Trypsin inhibitor 99-098-05 proteinase inhibitor II + Cryl A(c) 94-326-01

Disease resistance (fungal)

Cylindrosporium +Phoma chitinase + glucanase 01-074-11 post harvest coat protein 97-078-02

Asronomic traits

Nitrogen metabolism altered alanine amino transferase 03-276-04

Cold tolerance cold regulated gene binding factor 01-066-07

Male sterility/fertility CBI 03-254-03

Barnase/Barstar 98-119-01

Yield increase sucrose phosphate synthase 98-064-20

Product Oualitv

Oil profile altered Acyl CoA reductases, Acyl-ACP 97-022-02 thioesterases, elongase, ketoacyl-ACP synthases

Acyl-ACP-thioesterase + Delta 12saturase, 01-068-01

Delta 9 + 15 desaturases

Acetyl CoA carboxylase 99-067-02

34 different genes 96-071-07

Lysine increased Dihydropicolinate synthase 98-099-03

Pharmaceutical protein CBI 96-215-01

Polymer CBI 96-061-02

Industrial enzymes CBI 93-048-02

The term "exogenous polynucleotide" as used herein, refers to any DNA or RNA molecule that is transformed {i.e., integrates) into the genome of a target plant.

According to one preferred embodiments of this aspect of the present invention, such a polynucleotide comprises a nucleic acid sequence encoding a polypeptide (a gene product). The polypeptide encoded by the polynucleotide can be involved in the synthesis of a target polypeptide or nutrient (e.g., dihydropicolinate synthase), can be a regulatory polypeptide which is involved in the inhibition, degradation or inactivation of a target polypeptide or nutrient [e.g., barstar (GenBank Accession No. X15545; SEQ ID NO:40)] can regulate its production, stabilization or activation (e.g. herbicide resistance such as glyphosate resistance) or can be a lethal product (e.g., ribosome inhibitor protein).

According to another preferred embodiments of this aspect of the present invention, such a polynucleotide is a DNA or an RNA that comprises a nucleic acid sequence encoding a regulatory agent (i.e., an upregulation agent or a downregulation agent) which affects the level of gene expression. Non-limiting examples of such downregulation polynucleotides are antisense nucleic acid sequences, siRNA and the like. Non-limiting examples of upregulating polynucleotides are promoter sequences, enhancers and the like. Methods of inserting the exogenous polynucleotide into a cultivated plant are further described hereinbelow.

Thus, the exogenous polynucleotide of the present invention is inserted into the genome of the cultivated plant in a locus that is in high linkage disequilibrium with the PhX locus. Once integrated in close proximity to the PhX locus of the cultivated plant, the stable transgene introgression of the transgene is prevented, thus leaving the transgene in the target cultivated plant and avoiding intogression to homoeologous chromosomes of related, non-cultivated plants.

Stable transgene introgression can be also prevented using alternative approaches. For example, the exogenous polynucleotide of the present invention that is inserted into a locus being in linkage disequilibrium of the PhI locus can be a suppressor of a product that is lethal to related, non cultivated plants. Such a suppressor can be for example, the barstar, which inhibits the action of barnase (a suicide gene encoding RNase which is lethal to plants over-expressing it).

Thus, according to another preferred embodiments of this aspect of the present invention, the exogenous polynucleotide comprises a nucleic acid sequence encoding a suppressor of a product lethal to the related, non-cultivated plant.

A product lethal to the related, non-cultivated plant can be any polynucleotide (DNA or RNA) or polypeptide that causes death of a plant harboring such a product. Preferably, the product lethal to the related, non-cultivated plant can be barnase (e.g., GenBank Accession No. Ml 4442; SEQ ID NO:41). Accordingly, a suppressor of such a product can be any polynucleotide or polypeptide which is capable of suppressing, inhibiting, downregulating or degrading the lethal product. Non-limiting examples of such suppressors include the gene encoding barstar (e.g., which is isolated from the Bacillus amyloliquefaciens species, GenBank Accession No. X15545; SEQ ID NO: 40) whose product suppresses the action of the gene barnase (e.g., barnase isolated from B. amyloliquefaciens species, GenBank Accession No. M14442; SEQ ID NO: 41).

Once this configuration is utilized, the transgene of interest, which encodes the commercially desired trait, is preferably inserted into a second locus which segregates independently of the PhX locus (i.e., which is not linked to the PhX locus or that is in linkage equilibrium with the PhX locus).

Thus according to additional preferred embodiments of the present invention, the method according to this aspect of the present invention further comprising inserting a second exogenous polynucleotide into the second genetic locus of the transgenic cultivated plant, when the second genetic locus being in random association with the PhX locus of the transgenic cultivated plant.

Preferably, the second exogenous polynucleotide comprises a nucleic acid sequence encoding a product lethal to the related, non-cultivated plant.

To prevent stable introgression of the transgene into the related, non-cultivated plant, the second exogenous polynucleotide preferably includes a second nucleic acid sequence encoding a transgene-of-interest.

It will be appreciated that to ensure the co-localization of the transgene with the lethal product, the nucleic acid sequences encoding the transgene and the lethal product are preferably conjugated in tandem a single nucleic acid construct. Thus, under such configurations (i.e., when the transgene forms a part of the same polynucleotide having the nucleic acid sequence encoding the lethal product), in case a stable genomic introgression occurs between the chromosomes of the transgenic cultivated plant and the chromosomes of the related, non-cultivated plant (i.e., stable introgression of the transgene), the plants to which the transgene has introgressed is expected to die (due to the effect of the lethal product). On the other hand, the progeny of the transgenic cultivated plant, which undergoes homologous recombination, are protected from the effect of the lethal product due to the over- expression of the suppressor of the lethal product that is encoded by the exogenous polynucleotide inserted in the locus being in linkage disequilibrium with the PAl locus.

The exogenous polynucleotide of the present invention (i.e., the polynucleotide comprising the nucleic acid sequence of the transgene, the lethal product or the suppressor of the lethal product) can be engineered for plant expression using a suitable chimeric gene and transformation vector. A typical chimeric gene for transformation into a plant includes a promoter region, a heterologous structural DNA coding sequences (e.g., the nucleic acid sequence of the transgene, the lethal product and the suppressor of the lethal product) and a 3' non-translated polyadenylation site. A heterologous structural DNA coding sequence means a structural coding sequence that is not native to the plant being transformed. Heterologous with respect to the promoter means that the coding sequence does not exist in nature in the same gene with the promoter to which it is now attached. Chimeric means a novel non-naturally occurring gene which is comprised of parts of different genes. It will be appreciated that the exogenous polynucleotide of the present invention can also utilize a natural promoter of a specific desired gene. In preparing the transformation vector, the various DNA fragments may be manipulated as necessary to create the desired vector. This includes using linkers or adaptors as necessary to form suitable restriction sites or to eliminate unwanted restriction sites or other similar manipulations that are known to those of ordinary skill in the art. Promoters that are known or found to cause transcription of selected gene or genes in plant cells can be used to implement the present invention. Such promoters may be obtained from plants, plant pathogenic bacteria or plant viruses, or any other organism, and include, but are not necessarily limited to, strong constitutive promoter such as a 35S promoter [Odell et al (1985) Nature 313, 810-812], a 35S'3 promoter [Hull and Howell (1987) Virology 86, 482-493] and the 19S promoter of cauliflower mosaic virus (CaMV35S and CaMV 19S), the full-length transcript promoter from the fϊgwort mosaic virus (FMV35S) and promoters isolated from plant genes such as EPSP synthase, ssRUBISCO genes and promoters obtained from T-DNA genes of Agrobacterium tumefaciens such as the promoter of the nopaline synthetase gene ("PNOS") of the Ti-plasmid [Herrera-Estrella (1983) Nature 303, 209-213], the mannopine synthase promoter, or the promoter of the octopine synthase gene ("POCS" [De Greve et al (1982) J. MoI. Appl. Genet. 1 (6), 499-511]). Also useful can be expression in wound tissue, for example, using a second promoter which is a TR promoter such as the TRi' or TR2¹ promoter of the Ti-plasmid [Velten et al (1984) EMBO J. 3, 2723-2730]. Selective expression in green tissue can be achieved by using, for example, the promoter of the gene encoding the small subunit of Rubisco (European patent application 87400544.0 published Oct. 21, 1987, as EP 0 242 246). Promoters can be selected so that the transgenes are expressed in specific cells, such as petal cells, leaf cells or seed cells. AU of these promoters have been used to create various types of DNA constructs that have been expressed in plants. See, for example PCT publication WO 84/02913 (Rogers et al., Monsanto, herein incorporated by reference in its entirety). The particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of the respective proteins to confer the traits.

Particularly useful promoters for use in some embodiments of the present invention are fruit specific promoters and the full-length transcript promoter from the figwort mosaic virus (FMV35S). The FMV35S promoter is particularly useful because of its ability to cause uniform and high levels of expression in plant tissues. The DNA sequence of a FMV35S promoter is presented in U.S. Pat. No. 5,512,466 and is identified as SEQ ID NO: 17 therein. Other examples of fruit specific promoters include the E8, E4, El 7 and J49 promoters from tomato (Lincoln et al., 1988), as well as the 2Al 1 promoter as described in U.S. Pat. No. 4,943,674. Tapetum and anther specific promoters can be used to isolate gene expression to reproductive organs, to achieve, for example, male sterility. Promoters that show this specificity are well known. Especially useful are the tapetum-specific promoter Tapl as described in Nacken et al. Sol. Gen. Genet. 229, 129-136, 1991), the tapetum specific promoters A9 (WO 92/11379), T29, PTA 29, PTA 26 and PTAl 3 as well as any promoter of a gene encoding a tapetum-specific mRNA hybridizable to the genes TA29, TA26 or TA13 from which genes the PTA29, PTA26 and PTA13 promoters have been isolated (U.S. Patent Nos. 5,652,354 and 6,046,382 to Mariani et al), the anther specific promoters described in WO 92/18625, WO 90/08826 and European patent application EP 93810455.1, and the tapetum specific promoter MFS 14 (WO 97/04116). Examples of female organ-specific promoters and sterility promoters are: the style and/or stigma-specific promoters, such as PSTMG07, PSTMG08, PSTMG4B12 and PSTMG3C9, and the ovule-specific promoter corresponding to the cDNA clone pMON9608 as described in U.S. Pat. No. 5,633,441; as well as a promoter of a gene encoding i) a style-stigma specific or ii) an ovule-specific mRNA hybridizable respectively to i) a STMG-type style-stigma specific gene or ii) CDNA clone pMON9608 of U.S. Pat. No. 5,633,441. Several other promoters are known in the art (see, e.g., McCormick et al. "Anther-Specific Genes: Molecular Characterization and Promoter Analysis in Transgenic Plants" in Plant Reproduction: From Floral Induction to Pollination, Lord et al. (ed.), 128-135, 1989; and Scott et al., 1992, The Plant Cell 4, 253, and U.S. Patent No. 6,603,064 to Van Dun) and as long as they give specific expression in the reproductive system, choice of the promoter is not critical to the invention.

The 3' non-translated region contains a polyadenylation signal that functions in plants to cause the addition of polyadenylated nucleotides to the 3' end of an RNA sequence. Examples of suitable 3' regions are the 3' transcribed, non-translated regions containing the polyadenylation signal of the tumor-inducing (Ti) plasmid genes of Agrobacterium, such as the nopaline synthase (NOS) gene, and plant genes like the 7S soybean storage protein genes and the pea E9 small subunit of the RuBP carboxylase gene (ssRUBISCO). The RNAs produced by a DNA construct of the present invention also preferably contain a 5' non-translated leader sequence. This sequence can be derived from the promoters selected to express the genes, and can be specifically modified so as to increase translation of the mRNAs. The 5' non-translated regions can also be obtained from viral RNA's, from suitable eukaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non- translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. Rather, the non-translated leader sequences can be part of the 5' end of the non-translated region of the native coding sequence for the heterologous coding sequence, or part of the promoter sequence, or can be derived from an unrelated promoter or coding sequence as discussed above.

In a preferred embodiment according to the present invention, the vector that is used to introduce the encoded proteins into the host cells of the plant comprises an appropriate selectable marker. In a more preferred embodiment according to the present invention the vector is a plant expression vector comprising both a selectable marker and an origin of replication. In another most preferred embodiment according to the present invention the vector is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation or integration in the genome of the plant organism of choice. According to some embodiments of the present invention, secretion of the protein or proteins out of the cell is preferred. In this embodiment the construct comprises a signal sequence to effect secretion as is known in the art. For some applications, a signal sequence that is recognized in the active growth phase will be most preferred. As will be recognized by the skilled artisan, the appropriate signal sequence should be placed immediately downstream of the translational start site

(ATG), and in frame with the coding sequence of the gene to be expressed.

The exogenous polynucleotide of the present invention is utilized to stably transform plant cells. In stable transformation, the exogenous polynucleotide of the present invention is integrated into the plant genome and as such it represents a stable and inherited trait.

There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants [Potrykus, L, Annu. Rev. Plant.

Physiol., Plant. MoI. Biol. (1991) 42:205-225; Shimamoto et al., Nature (1989) 338:274-276].

The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:

(i) Agrobacterium-m.Qdia.ted gene transfer: Klee et al. (1987) Annu. Rev.

Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell,

J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25;

Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth

Publishers, Boston, Mass. (1989) p. 93-112.

(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988)

Bio/Technology 6:1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment,

Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988)

6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and

Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No.

5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S.

H. and Daniels, W. Longman, London, (1985) p. 197-209; and OhIa, Proc. Natl.

Acad. Sci. USA (1986) 83:715-719. The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledenous plants, but with the addition of suitable chemicals also works with monocotyledonous plants. Other bacteria related to Agrobacterium can perform the same function (Broothaerts, W. et al. (2005) Gene transfer to plants by diverse species of bacteria. Nature 433, 632 -633).

There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as gold or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues. Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. It is preferred that the seed be produced on a transformed plant such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by a process that provides a rapid, consistent reproduction of the transformed plants.

Regeneration is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the regeneration process involves: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established contaminant-free. During stage two, the tissue samples grown in stage one are divided and grown into individual plantlets. At stage three, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment. It will be appreciated that the nucleic acid construct of the present invention (which includes the exogenous polynucleotide of the present invention) can be randomly inserted or directly targeted into the genome of the cultivated plant.

To directly target the integration of the nucleic acid construct of the present invention (which includes the exogenous polynucleotide of the present invention) into a specific desired locus (e.g., a locus that is in linkage disequilibrium with the PhI locus, or a locus from the centromeric region of a chromosome), gene knock-in methods (also called site specific recombination) are employed. Thus, sequences homologous to regions upstream or downstream of the desired locus (e.g., the PhI locus) are generated to enable recombination events between the vector and the target genomic region. Non-limiting examples of such sequences are provided in Table 1, hereinabove. It will be appreciated that in case the length of such sequences is insufficient to enable recombination, additional sequences can be uncovered using PCR with primers derived from the known sequence (e.g., from SEQ ID NO: 13) and a random primer using methods well known in the art.

Such DNA constructs preferably include positive and negative selection markers and may therefore be employed for selecting homologous recombination events. One ordinarily skilled in the art can readily design a knock-in construct including both positive and negative selection genes for efficiently selecting transformed plant cells that underwent a homologous recombination event with the construct. Such cells can then be grown into full plants. Standard methods known in the art can be used for implementing knock-in procedures. Such methods are set forth in, for example, U.S. Pat. Nos. 5,487,992, 5,464,764. It will be appreciated that to increase the efficiency of the targeted insertion, a gene that promotes homologous pairing such as the yeast RAD54 gene can also be engineered as part of the construct inserted into the plant, essentially as described elsewhere (Shaked, H., Melamed- Bessudo, C, Levi, A. A. 2005. High-frequency gene targeting in Arabidopsis plants expressing the RAD54 gene. Proceedings of the National Academy of Sciences USA 102: 12265-12269) and references therein. For example, the first exogenous polynucleotide of the present invention can include a constitutive promoter (e.g., 35S), the barstar gene (GenBank Accession No. Xl 5545), a marker gene [e.g., hygromycin resistance (GenBank Accession No. E00287; SEQ ID NO: 42)], and two flanking regions that contain non coding regions specific to the long ami of wheat chromosome 5B, such as WPG90 (SEQ ID NO:46) (Segal, G. et al., 1997, Theoretical and Applied Genetics 94, 968-970; for more information see: http://wheat.pw.usda.gov/GG2/index.shtml). This construct will target the long arm of 5BL in the vicinity of Phλ .

The second exogenous polynucleotide of the present invention can contain a constitutive promoter, such as 35 S, the transgene of choice, such as glufosinate resistance (GenBank Accession No. M37389; SEQ ID NO: 43), a marker gene, such as GUS (GenBank Accession No. Y82477; SEQ ID NO: 44), and two flanking regions that contain non coding sequences specific to any wheat chromosome other than 5BL, such as WPGl 18 (; SEQ ID NO: 45) (Liu, B., et al., 2003, A chromosome- specific sequence common to the B genome of polyploid wheat and Aegilops searsii. Plant Systematics and Evolution 241, 959-965; Weissmann, S., et al., 2005. Sequence evidence for sporadic inter-generic DNA introgression from wheat into a wild Aegilops species. Molecular Biology and Evolution 22:2055-2062; for more information see http://wheat.pw.usda.gov/GG2/index.shtml). This construct can be targeted to any wheat chromosome except to the long arm of chromosome 5B. The two constructs can be simultaneously inserted into wheat callus by Agrobacterium transformation as described by Terada et al [Terada, R., et al., 2004, A large-scale Agrobacterium-medisted transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.). Plant Cell Reports 22, 653-659]. For random insertion of the exogenous polynucleotide, a DNA construct comprising the gene-of-interest, for example the glyphosate resistance gene (SEQ ID NO:43) and a marker gene, such as GUS (SEQ ID NO:44) are inserted into immature wheat embryos using particle bombardment (Stoger, E.,Williams, S., Keen, D.,Christou, P. 1999. Molecular characteristics of transgenic wheat and the effect on transgene expression. Transgenic Research 7: 463-471). A screen for the desired location of the transgene in the vicinity of the PhI locus on the long arm of wheat chromosome 5B is further performed using FISH analysis with a gene-specific probe (e.g., SEQ ID NO:43), essential as described in Stoger, E., et al., 1999 (Molecular characteristics of transgenic wheat and the effect on transgene expression. Transgenic Research 7: 463-471) which is fully incorporated herein by reference. Following FISH analysis, the stained chromosomes are photographed (e.g., using a camera connected to a microscope or an image analysis system) and the position of the labeled chromosome is recorded. Prior to C-banding, the fluorescent probe is removed by incubating for 4 minutes the chromosomes specimen in a warm solution (at 60 ⁰C) of 70 % formamide in 300 niM NaCl₃ 30 mM NaCitrate (2XSSC), followed by dehydration in a series of cold (at -20 ⁰C) ethanol (Le., 70, 95 and 100 %). Following dehydration the chromosome specimen is subjected to a standard C banding essentially as described in Rodriguez, S., et al. 2000 (Chromosome structure of Triticum timopheevn relative to T. turgidum. Genome 43: 923-930). Embryos in which the gene-of-interest has been inserted into 5BL near the PhI locus are further selected.

Once a transgenic plant tissue that contains an expression vector has been obtained, transgenic plants are regenerated from this transgenic plant tissue. The term "regeneration" as used herein, means growing a whole plant from a plant cell, a group of plant cells, a plant part or a plant piece. Transformed plant cells that are derived by any of the above transformation techniques are cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the nucleic sequence of interest. Regeneration techniques for regenerating plants from plant callus, explants, organs, or parts thereof are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987). The culture media will generally contain various organic components including vitamins, sugars, and plant hormones, such as auxin and cytokinins, as well as inorganic salts. The regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner.

Confirmation of the transgenic nature of the cells, tissues, and plants may be performed by PCR analysis, antibiotic or herbicide resistance, enzymatic analysis and/or Southern blots to verify transformation. Progeny of the regenerated plants may be obtained and analyzed to verify whether the transgenes are heritable. Heritability of the transgene is further confirmation of the stable transformation of the transgene in the plant. Progeny may be sexually or asexually derived progeny.

After the desired genes or construct is stably incorporated into regenerated transgenic plants, they can be transferred to other plants by sexual crossing. The plants are then grown and harvested using conventional procedures.

Thus, the teachings of the present invention can be used to prevent DNA introgression between homoeologous chromosomes. Studies have shown that recombination in wheat occurs mainly in the distal one-third of the chromosomes (Akhunov et at 2003; Lukaszewski and Curtis 1993).

While further reducing the present invention to practice, the present inventors have uncovered that insertion of the transgene near the centromere will reduce the probability of stable transgene introgression.

Thus, according to yet an additional aspect of the present invention there is provided a method of generating a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant. The method is effected by inserting an exogenous polynucleotide near a centromeric region of a chromosome of the transgenic cultivated plant thereby preventing the stable transgene introgression into the related, non-cultivated plant.

As used herein, the phrase "centromeric region of a chromosome" refers to the nucleic acid sequence encompassed by the centromere of the chromosome of the transgenic cultivated plant. Preferably, the method is effected by inserting the exogenous polypeptide near a sub-centromeric region of the chromosome of the transgenic cultivated plant. The phrase "sub-centromeric" refers to a portion of the centromere of the chromosome, preferably, a portion that includes 0.5 Morgan unit on each side of the centromere.

Tables 4 and 5 hereinbelow, list genes, markers and loci which can be used for targeting the exogenous polynucleotide to the sub-centromeric region of wheat chromosomes.

Table 4 Non-coding sequences in sub-centromeric regions

Table 4: Non-coding sequences from a sub-centromeric region of the chromosome. The distance from the centromere is provided using deletion lines; the distance from the centromere is represented by the remaining fraction of the specific chromosomal arm.

Table s Markers tightly linked to the centromere

Table 5: Data from http://wheat.pw.usda.gov/GG2/index.shtral

According to one preferred embodiment of this aspect of the present invention, the exogenous polynucleotide of the present invention can be any of the polynucleotides set forth by SEQ ID NOs: 17-39.

It will be appreciated that such a method can be used in most species (including those of the tribe Triticeae), except for in those species (e.g., family Liliaceae others) within which there is a high incidence of recombination of genes near the centromere.

Following is a list of species in which recombination occurs near the centromeric region (i.e., species in which the transgene should not be inserted into the centromeric region):

Several species of Fritilaria (Darlington, C. D. 1965. Cytology. Part I: recent advances in cytology, 1937: Part II: Recent advances in cytology, 1937-1964. J. & A. Churchill ltd, London).

Several species of Bellevalia [Gopal-Ayengar, A. R. 1941. The origin and behaviour of chiasmata in Bellevalia species, (abstr.) Amer. J. Bot. 28: 3s]

Several species of Allium [Levan, A. 1933. Cytological studies in Allium, IV. Allium fistulosum. Svensk Bot. Tidskr. 27: 211-232; Levan, A. 1935. Cytological studies in Allium, VI. The chromosome morphology of some diploid species of

Allium. Hereditas 20: 289-330; Levan, A. 1940. Meiosis of Allium porrum, a tetraploid species with chiasma localisation. Hereditas 26: 454-462; Ved Brat, s.

1965. Genetic system in Allium. III. Meiosis and breeding systems. Heredity 20: 325- 339; Koul, A. K. and R. N. Gohil, 1970. Cytology of the tetraploid Allium ampeloprasum with chiasma localisation. Chromosoma 29: 12-19].

According to one preferred embodiments of the this aspect of the present invention, the exogenous polynucleotide is inserted into the genome of the target cultivated plant in a locus which is in linkage disequilibrium with the PhI (as described hereinabove) and which is also included in the centromeric region (preferably the sub-centromeric region) of the targeted chromosome (e.g., chromosome 5 of subgenome B of T. aestivum or Triticwn turgidum). For example, the exogenous polynucleotide encoding a transgene of interest (e.g., glyphosate resistance, GenBank Accession No. M37389) is inserted using homologous recombination to the centromeric sequences (e.g., SEQ ID NO: 17 and/or 39), that are near the PhI locus and also near the centromere.

Similarly, the exogenous polynucleotide having the nucleic acid sequence encoding the suppressor of the lethal product (e.g., Barstar, SEQ ID NO: 40) can be inserted within the sub-centromeric region of the target chromosome. Preferably, it can be inserted in a locus which is in linkage disequilibrium with the PhI locus and yet within the sub-centromeric region of the chromosome (e.g., SEQ ID NOs: 17 and/or 39). As is mentioned before, under such a configuration, the second exogenous polynucleotide which includes the transgene and the lethal gene is inserted in a locus which independently segregates from the first locus (which is near the centromere and PhI), such a second locus can be on another chromosome.

As used herein the term "about" refers to ± 10 %.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions; illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include cytogenetic, molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Chromosome Engineering in Plants" Parts A and B, Gupta PK and Tsuchiya T. Eds., (1991); "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., Ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Eds.) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, L E., Ed. (1994); "Oligonucleotide Synthesis" Gait, M. J., Ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course . Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE l

HOMOEOLOGOUSINTROGRESSIONOFDNA SEQUENCES FROM THE GENOME OF T. AESTIVUM {CULTIVATED WHEAT) INTO THE GENOME

OFAE. PEREGRINA

Materials and Experimental Methods

Amplified fragment length polymorphism (AFLP) analysis - AFLP analysis was performed essentially according to Vos et al. 1995 [Vos, P.H.R., Bleeker, M., Reijans, M., Van der Lee, T., Homes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M. AFLP: a new technique for DNA fingerprinting. (1995). Nucleic Acids Research 23 (21): 4407-4414] except that the primers were labeled with one of the fluorescent dyes FAM (6-carboxyfluorescein), HEX (4, 7, 2', 4', 5', 7'-hexachloro-6- carboxyfluorescein), or TET (4, 7, 2', 7'-tetrachloro-6-carboxyfluorescein) (Eisenberg Bros. Ltd.) and following electrophoresis, the gel was scanned using the appropriate laser with a Typhoon™ 9400 scanner (Amersham Bioscience) at medium sensitivity and the intensity of the gel digital image was adjusted using the ImageQuant™ software. Ten primer combinations were randomly chosen for the analysis. The selective nucleotides and the primer dye are listed in Table 6, hereinbelow. Table 6 Primers used for AFLP analysis

Msel selective nucleotides EcoRI selective nucleotides

GATGAGTCCTGAGTAACTG(SEQIDNO:1) GACTGCGTACCAATTCACT(FAM)(SEQ ID

NO:8) GATGAGTCCTGAGTAACTT(SEQ IDNO:2) GACTGCGTACCAATTCACA(TET) (SEQ ID

NO:9) GATGAGTCCTGAGTAACTT(SEQ IDNO:2) GACTGCGTACCAATTCAAC (HEX) (SEQ ID

NO:10) GATGAGTCCTGAGTAACTT(SEQ IDNO:2) GACTGCGTACCAATTCACC (FAM) (SEQ ID

NO:11) GATGAGTCCTGAGTAACAG(SEQ IDNO:3) GACTGCGTACCAATTCACA(TET) (SEQ ID

NO:9) GATGAGTCCTGAGTAACAT(SEQ IDNO:4) GACTGCGTACCAATTCACA(TET) (SEQ ID

NO:9) GATGAGTCCTGAGTAACTA(SEQ IDNO:5) GACTGCGTACCAATTCACA(TET)(SEQID

NO:9) GATGAGTCCTGAGTAACTC (SEQIDNO:6) GACTGCGTACCAATTCACA(TET)(SEQID

NO:9) GATGAGTCCTGAGTAACAG (SEQID NO:7) GACTGCGTACCAATTCACC(FAM) (SEQ ID

NO:11) GATGAGTCCTGAGTAACAG (SEQ ID NO:7) GACTGCGTACCAATTCACG (HEX) (SEQ ID NO: 12)

Table 6: The selective nucleotides combination of primers that were randomly chosen to detect, by AFLP, introgression from T. aestivutn into Ae. peregrina. The dye of the EcoBI primers is in brackets

Cytogenetic analysis of hybrids and successive backer oss generations - Root tips from F₁ hybrids and backcross seedlings were collected and placed in vials containing cold water, placed in crushed ice, refrigerated for 30 hours and then transferred into a fixative solution of 3 ethanol: 1 acetic acid. The fixative solution was changed several times during the first week. The material was kept at 4 ⁰C until analyzed. Anthers from mature plants were placed immediately after collection in a fixative solution. Fixed root tips or anthers were placed on a glass slide with a drop of 2 % acetocarmine, heated over a moderate flame for several seconds and then squashed. The number of chromosomes (at mitosis) and bivalents (at meiosis) was determined by examination of the slides with an Olympus inverted system microscope IX, and photographed by an Imago CCD camera with TiLLvisION 3.3 software (T. I. I. L Photonics). Experimental results

Fifteen BC₃F₂ plants were analyzed. All of them had 2n = 28 chromosomes (14 bivalents at first meiotic metaphase). A total of 17 amplified fragment length polymorphism (AFLP) fragments introgressed from T. aestivum into the BC₃F₂ plants: 2 bands from the B genome of T. aestivum (Table 7 herein below, and Figure 1), 2 bands from the A genome (Table 7 and Figure 2), and 7 from the D genome (Table 7 and Figure 2) of T. aestivum. The origin of 6 additional introgressed bands was not determined (Table 7, Figure 3). Although 17 bands introgressed from T. aestivum into Ae. peregrina, it is not necessarily certain that these bands result from 17 separate recombination events. It is possible that the introgressed bands that originated from a certain genome of T. aestivum are all localized on the same large fragment that introgressed into Ae. peregrina. If the relative size of the different chromosome arms of BC₃F₂ plants are ignored, each chromosome arm in these plants constitutes about 1.78 % of the plant's genome i.e., 1/(28 chromosomes x two arms each). If the three putative events of recombination result in the exchange of three chromosome arms between the chromosomes of the parental species, then the introgressed bands are expected to constitute about 5.36 % of the bands scored for BC₃F₂ plants. Interestingly, the 17 introgressed bands constitute 5.39 % of all the bands scored for BC₃F₂ plants (Table 8, herein below). Since the 17 bands originated from three different genomes of T. aestivum, it is more than likely that these bands introgressed by at least three events of recombination.

Table 7 Introgressed bands from T. aestivum into Ae. peregrina

The genomic origin No. of introgressed No. of segregating Minimal no. of of the band in wheat bands bands recombination events

B B 2 2 1 2

A A 2 2 0 1

D D 7 7 4 2

N Noott ddeetteerrmmiinneedd 6 6 2 -

Total 17 7 5 Table 7: Seventeen bands introgressed into Ae. peregrina from all three sub genome of T. aestivum. Segregation of introgressed bands from the same wheat genome origin indicates more than one events of recombination. The data indicate at least two recombination events between chromosomes of Ae. peregrina and those of the wheat genome D, at least two with those of wheat genome B, and at least one with those of wheat genome A. Table 8 Introgression off. aestivum DNA in stabilized 28 chromosomes BC₃F₂ plants

F₁ BC₃F₂ Ae. peregrina T. aestivum

% of Band % of parental Band Band Band parental sharing unique bands sharing sharing sharing unique bands

Ae. peregrina 17.2 0.8 24.8 0.9 - 0.7

T. aestivum 19.0 0.8 5.4 0.7 0.7 -

Table 8: Introgression of T. aestivum DNA in stabilized 28 chromosomes BC₃F₂ plants. Similar values of band sharing were found between hybrids of T. aestivum and Ae. peregrina with the two parental species. The percentage of Ae. peregrina unique bands was five time higher than the percentage of T. aestivum unique bands in BC₃F₂ plants.

Three of the bands segregated among the 15 BC₃F₂ plants (that are all offspring of the same BC₃ plant). They were missing from 1/3 to 1/4 of the 15 plants and were present in 2/3 - 3/4 of them (see an example in Figure 4). One of the bands originated from the D genome of wheat; the origin of the other two was not determined. Since the other 6 bands that introgressed from the D genome of wheat did not segregate, it is possible that two separate segments introgressed from the D genome of wheat. In this case it is more than likely that at least four recombination events occurred in the process of these crosses.

Seven of the 17 introgressed bands showed segregation. They were missing from one or both tested BC₃ plants, but were present in all or some of the BC₃F₂ plants. Five of these bands originated from the D genome of wheat, one from the B genome and the origin of the seventh band was not determined (Table 7, hereinabove). These results further support the assumption of two separate introgressions from the D genome of wheat, since all BC₃ plants are offspring of the same BC₂ plant. It indicates that one of the fragments introgressed from the D genome has reached homozygosity and thus does not segregate but the other segments have not reached homozygosity and are still segregating. In addition, since one band of the B genome segregated among BC₃ plants and the other did not it is possible that two separate segments introgressed from the B genome as well. That brings the total number of possible recombination events in the process of these crosses to at least five. Determination of chromosome number in the Fj hybrids - To ascertain that the seeds collected from the parental Ae. peregrina plants are indeed F₁ hybrids, root tips were collected from both plants, fixed, stained with acetocarmine, and then squashed on a glass slide to separate the chromosomes. Both plants had the expected number of 35 chromosomes (14 of chromosomes of Ae. peregrina + 21 chromosomes of T. aestivum) (Figure 6). These results demonstrate that these plants are hybrids resulting from the cross between Ae. peregrina and wheat.

Stabilization of chromosome number in backcross generations of crosses between Ae. peregrina and T. aestivum - Hybrid plants exhibited the expected number of 35 chromosomes (14 of chromosomes of Ae. peregrina + 21 chromosomes of T. aestivum) (Figure 6). BC₃ plants exhibited between 29 to 32 chromosomes. This indicates a tendency of the chromosome number to stabilize at 28 chromosomes through backcrossing to Ae. peregrina. Three BC₃ plants contained trivalents at meiosis (Figure 7b). This may indicate a possible translocation from T. aestivum into Ae. peregrina. One (or two) of the homologous chromosomes of Ae. peregrina may contain a wheat segment homologous to that of the third chromosome (possibly a wheat chromosome) attached to the bivalent. One of the BC₃ plants (No. 49) was chosen for further analysis. This plant exhibited 29 chromosomes with 14 bivalents and one univalent. Thirty four of this plant's seeds were planted. Root tips from these BC₃F₂ plants were collected, and their chromosome numbers were determined. Five BC₃F₂ plants had 29 chromosomes and 29 plants had 28 chromosomes (Figure 7a). In meiosis, all the BC₃F₂ plants with 28 chromosomes had 14 bivalents. Interestingly, these BC₃F₂ plants had a significantly (P = 1.07 ^χ e^"46) higher number of rod bivalents (range 3-7, average 4.03, Figure 7c) compared to their wild parental species Ae. peregrina (range 1-4, average 2.01, Figure 7d). As recombination tends to occur at higher rates towards the terminal ends of chromosome arms (Akhunov, E.D. et al., 2003, The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms. Genome Res. 13: 753-763), this may indicate introgression of wheat segments into the chromosomes of Ae. peregrina and thus the loss of homology between the edges of the homologous chromosomes.

Analysis and Discussion

Many publications in recent years focused on the formation of crop x wild inter-generic hybrids, and assumed that introgression between homoeologous genomes is mainly the result of homoeologous pairing in the F₁ hybrid (Guadagnuolo et al 2001a; Hegde and Waines 2004; Seefeldt et al. 1998; Snyder et al 2000; Wang et al. 2001; Zemetra et al. 1998). Although formation of hybrids is crucial for the occurrence of introgression, inter-specific and inter-generic wheat F₁ hybrids are characterized by low homoeologous pairing. For example Jauhar and Peterson (2001; Hybrids between durum wheat and Thinopyrum junceiforme: Prospects for breeding for scab resistance. Euphytica. 118: 127-136.) Reported an average of 2 bivalents per cell between the chromosomes of durum wheat and Thinopyrum junceiforme. Anamthawat-Jonsson and Bodvarsdottir (1998; Chromosome Research 6: 339-343) reported 1 bivalent per cell between the chromosome of T. aestivum and Leymus arenarius and L. mollis). This is due to the presence of the PhX gene that suppresses pairing between homoeologous chromosomes (Wall et al. 1971, Genetical Research 18: 329-339). The results described hereinabove, indicate the transfer of at least 5 different fragments from wheat into Ae. peregrina i.e. five recombination events (Table 4, hereinabove). This is ten times higher than the average number of bivalents per cell observed in hybrids between T. aestivum cv. Chinese Spring and Ae. peregrina TKE02 (Ozkan and Feldman 2001, Genome 44: 1000-1006.; Sears 1977, Canadian Journal of Genetics and Cytology 19: 585-593). Farooq et al. reported an increase of such magnitude in hybrids between T. aestivum and Ae. peregrina in the absence of PhI (Farooq et al. 1990, Genome 33: 825-828). However, all the F₁ hybrids of the present study contained 21 wheat chromosomes (Figure 6) including chromosome 5B with PhX. Hence, it is very unlikely that all the five putative recombination events that transferred wheat bands into Ae. peregrina occurred in the F₁ hybrids. Indeed, not all backcross plants contain PhX (Figure 5). The low level of pairing in the F₁ hybrid causes chromosomes to segregate at random. An unpaired chromosome (univalent) has a probability of only 0.25 to be present in gametes (due to random segregation in the two meiotic divisions) (Morris and Sears 1967 ). F₁ hybrids of wide crosses with wheat are highly male sterile (Wang et al. 2001), meaning that most (75 %) BC₁ plants produced may not carry PhX. As a result, all progeny of these PhX -lacking plants also will not contain PhX. Homoeologous pairing may thus sharply increase in these backcross progeny compared to the Fl hybrids (Figures 4 and 5). Considering the facts that at least five introgressions in backcross plants all originated from a single F₁ plant; the probability for homoeologous pairing in the parental F₁ hybrid is extremely low, and the probability for homoeologous pairing in backcross plants lacking PhX is high, it can be concluded that most introgression events from T. aestivum to Ae. peregrina discovered in the present study occurred in backcross rather than in F₁ hybrid plants (Figures 4 and 5).

Since there is no information available on a homolog PhX gene for species other than Triticum, the transition of PhX to backcross plants is crucial to the prevention of homoeologous pairing and thus to the transfer of the transgene in backcross plants.

EXAMPLE 2

CHROMOSOMAL PLACEMENT AS A FAILSAFE TO PREVENT TRANSGENE MOVEMENT TO THE WILD POPULATION

Placement on chromosomes belonging to one of the A, B, or D genomes has been suggested as a possible failsafe mechanism for transgenic wheat. It has been suggested that placing the transgene on a wheat genome not shared by the neighboring wild or weedy relatives will reduce the risk of transgene movement (compared to genes on homologous chromosomes) because homoeologous chromosomes rarely pair in the F₁ hybrid (Gressel 2000; Wang et al. 2001). However, low homoeologous pairing occurs only in hybrid and backcross plants that contain PhI but may increase sharply in backcross plants not containing PhL PhI specifically prevents the promiscuous pairing of homoeologous chromosomes, preventing recombination within the three genomes of wheat (in the absence of homologous chromosomes) and with related species (Okamoto, M., 1957; Riley, R. & Chapman, V. 1958; Sears, E. R. & Okamoto, M. 1958; Sears, E. R. 1976). PhI is located in the middle of the long arm of chromosome 5B (5BL) (Jampates, R. & Dvorak, J. 1986), about 1 centi Morgan (cM) from the centromere (Sears, E. R., 1984). PhI (or a PhX homolog) has not been found to occur in other Graminae where it had been actively sought except in Triticum, hence if Triticum PhI is not present in the backcross plant homoeologous pairing is not suppressed. Consequently, a transgene placed on a genome homoeologous to that of the wild relative may not introgress in the hybrid plant but has a higher probability of introgressing in backcross generations (Figure 4).

Oilseed rape (Brassica napus, 2N = 38, AACC) another polyploid crop, has the PrBn gene that stringently controls the extent of meiotic pairing in haploids (Jenczewski, E. et al., 2003). PrBn is somewhat different from PhX as PhX prevents homoeologous pairing at both haploid and diploid stages while PrBn controls pairing only in haploids. A system suppressing homoeologous pairing in the diploid stage of oilseed rape probably evolved long after the formation of the allopolyploid, as recently synthesized allopolyploids that mimic the original one have high rates of homoeologous recombination (Udall, J. A., et al., 2005).

When wheat with PhI gene hybridizes with a wild polyploid species there is little or no homoeologous pairing in the F₁, as long as PhI is watchfully present as a chaperon. As an unpaired chromosome has a probability of only 0.25 of being present in the gametes (due to random segregation in the two meiotic divisions) (Morris, R. and Sears, E. R.,1967), about 75 % of the progeny resulting from the backcross Of F₁ to the wild parent will lack wheat chromosome 5B, and consequently, Phi [Figure 5, fail mechanism (a)]. In these plants single wild and wheat chromosomes will promiscuously homoeologous pairing and recombine with abandon, transferring genes with impunity. The present inventors uncovered methods that significantly reduce the probability of homoeologous transgene introgression and stabilization in backcross generations of the transgene in the wild or weedy population.

/. A transgene is located in proximity to the centromere (in a region which is not subject to recombination) - As discussed hereinabove, recombination events in wheat occur mainly in the distal one-third of the chromosomes (Akhunov et al. 2003; Lukaszewski and Curtis 1993). Therefore, release of transgenic wheat cultivars with the transgene located in close proximity to the centromere would significantly reduce the chance of homoeologous transgene movement It will be appreciated that such a method can be used in most species (including Triticeae), except than in those species (e.g., family Liliaceae and others) within which there is a high incidence of recombination of genes near the centromere.

Following is a list of species in which recombination occurs within the sub- centromeric region (i.e., species in which there would be no advantage if the transgene is inserted into the sub-centromeric region): Several species of Fritilaria (Darlington, C. D. 1965. Cytology. Part I: recent advances in cytology, 1937: Part II: Recent advances in cytology, 1937-1964. J. & A. Churchill ltd, London).

Several species of Bellevalia [Gopal-Ayengar, A. R. 1941. The origin and behaviour of chiasmata in Bellevalia species, (abstr.) Amer. J. Bot. 28: 3 s] Several species of Allium [Levan, A. 1933. Cytological studies in Allium, IV. Allium fistulosum. Svensk Bot. Tidskr. 27: 211-232; Levan, A. 1935. Cytological studies in Allium, VI. The chromosome morphology of some diploid species of Allium. Hereditas 20: 289-330; Levan, A. 1940. Meiosis of Allium porrum, a tetraploid species with chiasma localisation. Hereditas 26: 454-462; Ved Brat, s. 1965. Genetic system in Allium. III. Meiosis and breeding systems. Heredity 20: 325- 339; Koul, A. K. and R. N. Gohil, 1970. Cytology of the tetraploid Allium ampeloprasum with chiasma localisation. Chromosoma 29: 12-19].

//. A transgene is located in tight linkage to the PhI locus (gene) which inhibits recombination of homoeologous chromosomes - PhX is located on the long arm of wheat chromosome 5B (5BL) (Okamoto 1957) and strongly suppresses homoeologous pairing when present. If the transgene is located on this chromosome arm the probability of the transgene to transfer into a homoeologous genome is vastly reduced even in backcross plants. PhI will be present in the backcross plant with the closely linked transgene and thus prevent homoeologous pairing and the transfer of the transgene into the homoeologous chromosomes of the other species.

Using the novel systems of targeted introgression (also called, confusingly homologous recombination by molecular biologists) (Puchta, H., 2002; Reiss, B., 2003; Li, H. Q. & Li, M. R., 2004), it is possible to insert the transgenes of choice in close proximity to PhX on chromosome arm 5BL. Thus, the transgene of choice will remain genetically linked with watchful PhI, and will segregate with it in the F₁ hybrid and backcrosses, and thus not introgress into the chromosomes of wild/weedy relatives [Figure 5, failsafe mechanism (b)]. During backcrosses with the wild/weedy species, the excess wheat chromosomes are selectively eliminated due to lagging during meiotic anaphases (Wang, Z. N., et al., 2001). Chromosome arm 5BL with the PhX gene and the linked transgene will be retained in a small proportion of offspring only as long as the transgene confers a selective advantage, e.g. when the transgene is for a herbicide resistance, and the particular herbicide is used. In seasons where other herbicides are used, the selective disadvantage will eliminate 5BL. Such a solution, especially if coupled with other solutions, such as mitigating genes, could considerably lower the risk of stable transgene introgression between wheat and its relatives. Indeed, when PhI is finally isolated, it too could be used as a mitigator gene, no longer necessitating targeted insertion (US Pat. Appl. No. 20040172678). It will be appreciated that release of cultivars with the transgene located in the sub-centromeric region of the long arm of chromosome 5B is preferred. In this way even if the chromosome containing the transgene segregates into a backcross plant, and persists in 2-3 more generations until stabilization of the chromosome number, the transgene will not be able to transfer into the homoeologous genome and stabilize in the wild population. Methods such as site-specific gene integration (knock-in) (Srivastav and Ow, 2004) assist in creating wheat lines with specific insertion sites in the vicinity of PhI that would allow the insertion of any desired transgene as well as its safe release. Following are sequences that can be used for targeted insertion of the transgene into the sub- centromeric region of a chromosome and for targeting the insertion in tight linkage to the PhI gene: BARC58 (non-coding; at break point interval C-5BL6-0.29; GenBank Accession No. BV211574; SEQ ID NO:39), and BARC89 (non-coding; at break point interval C-5BL6-0.29; GenBank Accession No. BV211400; SEQ ID NO: 17).

EXAMPLE 3

ANADDITIONAL SOLUTION TO PREVENT ALLOPOLYPLOID CROP GENE INTEGRATIONINTO WILD/WEEDY RELATIVES

An additional way to curtail unwanted stable introgression of a wheat transgenes into wild or weedy relatives also utilizes insertion on chromosome 5BL, while providing another layer of protection. The transgene of choice (e.g., the transgene of commercial significance of which introgression is undesired) is inserted together with a suicide gene on any chromosome arm other than 5BL, and inserting a gene encoding a suppressor of the suicide gene product on chromosome arm 5BL [Figure 5, failsafe mechanism (c)]. The linkage between the suicide-suppressor gene and PhI on 5BL will prevent the transfer of the suppressor to a wild chromosome and consequently, the establishment of this gene in the wild population. The suicide gene can encode any heterologous protein that is toxic to plants possessing it. Such a gene is barnase, isolated from Bacillus amyloliquefaciens (GenBank Accession No. M14442), which encodes a RNase that destroys RNA (Mariani, C. et al., 1992). The suicide-suppressor gene can be any gene that encodes a heterologous protein that inactivates either the suicide gene or the toxic protein encoded by the suicide gene. Barstar is an intracellular suppressor of barnase that binds the barnase encoded protein and blocks its active site (Korchuganov, D. S. et al., 2004). The gene encoding barstar protein also was isolated from B. amyloliquefaciens (Hartley, R. W. 1989) (GenBank Accession No. X15545). Any backcross progeny having the tandem transgene of choice - suicide gene, but are without the suppressor of the suicide gene will die. Due to the presence of PhI, 5BL will not homoeologously pair with chromosomes in the wild or weedy relative, and like extraneous chromosomes that are unpaired, 5BL will be eliminated during the continuous backcrossing to the wild or weedy parents. It is appreciated that PhX will prevent homoeologous recombination only in the absence of a PhX suppressor gene such as that present in several diploid species, e.g., Ae. speltoides, Amplyopyrum muticum, (Riley, R., et al., 1966) and several diploid and tetraploid Agropyron species have such a suppressor (Chen, Q., et al., 1993). Thus, this might necessitate ascertaining that transgenic wheat will not transfer genes to the indigenous wild/weedy relatives in each locality where transgenic wheat is to be cultivated. Clearly these two interrelated mechanisms can be effective in preventing stable transgene introgression to Ae. cylindrica in the parts of the world where it is the sole problem- weed known to stably introgress genes from wheat.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

References (Additional references are cited in text)

1. Jasieniuk, M. et al. Evaluation of models predicting winter wheat yield as a function of winter wheat and jointed goatgrass densities. Weed Science 49, 48-60 (2001).

2. Stone, A. E. & Peeper, T. F. Characterizing jointed goatgrass (Aegilops cylindricd) x winter wheat hybrids in Oklahoma. Weed Science 52, 742-745 (2004).

3. Kihara, H. Considerations on the evolution and distribution of Aegilops species based on the analyser-method. Cytologia 19, 336-342 (1954). 4. Kimber, G. & Tsunewaki, K. Genome symbols and plasma types in the wheat group, in 7th International Wheat Genetics Symposium 1209-1211 (Cambridge,

1988).

5. Gressel, J. Molecular biology of weed control (Taylor and Francis, London,

2002). 6. Wang, Z. N., Zemetra, R. S., Hansen, J. & Mallory-Smith, C. A. The fertility of wheat x jointed goatgrass hybrid and its backcross progenies. Weed Science 49,

340-345 (2001).

7. Dhaliwal, H. S., Harjit, S. & William, M. Transfer of rust resistance from Aegilops ovata into bread wheat (Triticum aestivum L.) and molecular characterization of resistant derivatives. Euphytica 126, 153-159 (2002).

8. Ganeva, G., Georgieva, V., Panayotova, M., Stoilova, Z. & Balevska, P. The transfer of genes for brown rust resistance from Aegilops umbellulata Eig, to Triticum aestivum L. genome. Russian Journal of Genetics 36, 60-65 (2000).

9. Martin-Sanchez, J. A. et al. A new Hessian fly resistance gene (H30) transferred from the wild grass Aegilops triuncialis to hexaploid wheat. Theoretical and Applied Genetics 106, 1248-1255 (2003).

10. Spetsov, P., Mingeot, D., Jacquemin, J. M., Samardjieva, K. & Marinova, E. Transfer of powdery mildew resistance from Aegilops variabilis into bread wheat. Euphytica 93, 49-54 (1997). 11. Sharma, H. et al. Introgression and characterization of barley yellow dwarf virus-resistance from Thinopyrum intermedium into wheat. Genome 38, 406-413 (1995).

12. Guadagnuolo, R., Savova-BiancM, D. & Felber, F. Gene flow from wheat (Triticum aestivum L.) to jointed goatgrass (Aegilops cylindrica Host), as revealed by RAPD and microsatellite markers. Theoretical and Applied Genetics 103, 1-8 (2001a).

13. Hegde, S. G. & Waines, J. G. Hybridization and introgression between bread wheat and wild and weedy relatives in north America. Crop Science 44, 1145—1155 (2004).

14. Morrison, L. A., Cremieux, L. C. & Mallory-Smith, C. A. Infestations of jointed goatgrass (Aegilops cylindricά) and its hybrids with wheat in Oregon wheat fields. Weed Science 50, 737-747 (2002).

15. Morrison, L. A., Riera-Lizarazu, O., Cremieux, L. & Mallory-Smith, C. A. Jointed goatgrass {Aegilops cylindrica Host) x wheat (Triticum aestivum L.) hybrids:

Hybridization dynamics in Oregon wheat fields. Crop Science 42, 1863-1872 (2002).

16. Wang, Z. N. et al. Visualization of A and B genome chromosomes in wheat {Triticum aestivum L.) x jointed goatgrass {Aegilops cylindrica Host) backcross progenies. Genome 43, 1038-1044 (2000). 17. Wang, Z. N. et al. Determination of the paternity of wheat {Triticum aestivum L) x jointed goatgrass {Aegilops cylindrica Host) BCl plants by using genomic in situ hybridization (GISH) technique. Crop Science 42, 939-943 (2002).

18. Zemetra, R. S., Hansen, J. & Mallory-Smith, C. A. Potential gene transfer between wheat {Triticum aestivum) and jointed goatgrass {Aegilops cylindrica). Weed Science 46, 313-317 (1998).

19. Weissmann, S., Feldman, M. & Gressel, J. Sequence evidence for sporadic inter-generic DNA introgression from wheat into a wild Aegilops species. Molecular Biology and Evolution (2005) (in press at time of patent submission).

20. Guadagnuolo, R., Savova-Bianchi, D., Keller-Senften, J. & Felber, F. Search for evidence of introgression of wheat {Triticum aestivum L.) traits into sea barley

{Hordeum marinum s.str. Huds.) and bearded wheatgrass {Elymus caninus L.) in central and northern Europe, using isozymes, RAPD and microsatellite markers. Theoretical and Applied Genetics 103, 191-196 (2001b).

21. Lyon, D. J., Bussan, A. J., Evans, J. O., Mallory-Smith, C. A. & Peeper, T. F. Pest management implications of glyphosate-resistant wheat (Triticum aestivum) in the western United States. Weed Technology, 680-690 (2002).

22. Gressel, J. Multiple resistances to wheat selective herbicides: New challenges to molecular biology. Oxford Surveys of Plant Molecular and Cellular Biology 5, 195-203 (1988). 23. De Prado, J. L., Osuna, M. D., Heredia, A. & De Prado, R. Lolium rigidum, a pool of resistance mechanisms to ACCase inhibitor herbicides. Journal of Agriculture Food and Chemistry 53, 2185-2191 (2005).

24. Neve, P., Sadler, J. & Powles, S. B. Multiple herbicide resistance in a glyphosate-resistant rigid ryegrass (Lolium rigidum) population. Weed Science 52,

920-928 (2004).

25. Yu, Q., Cairns, A. & Powles, S. B. Paraquat resistance in a population of Lolium rigidum. Functional Plant Biology 31, 247-254 (2004).

26. Price, L. J., Herbert, D., Moss, S. R., Cole, D. J. & Harwood, J. L. Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-

CoA carboxylase isoforms. Biochemistry Journal 375, 415-423 (2003).

27. Cocker, K. M., Moss, S. R. & Coleman, J. O. D. Multiple mechanisms of resistance to fenoxaprop-P-ethyl in United Kingdom and other European populations of herbicide-resistant Alopecurus myosuroides (black-grass). Pesticide Biochemistry and Physiology 65, 169-180 (1999).

28. Beckie, H. J. et al. Nature, occurrence, and cost of herbicide-resistant wild oat (Avenafatud) in small-grain production areas. Weed Technology 13, 612-625 (1999).

29. Mengistu, L. W., Messersmith, C. G. & Christoffers, M. J. Diversity of herbicide resistance among wild oat sampled 36 yr apart. Weed Science 51, 764-773 (2003).

30. Kern, A. J. et al. Two recessive gene inheritance for triallate resistance in Avenafatua L. Journal of Heredity 93, 48-50 (2002).

31. Delye, C, Menchari, Y., Michel, S. & Darmency, H. Molecular bases for sensitivity to tubulin-binding herbicides in green foxtail. Plant Physiology 136, 3920- 3932 (2004).

32. De Prado, R., Osuna, M. D. & Fischer, A. J. Resistance to ACCase inhibitor herbicides in a green foxtail (Setaria viridis) biotype in Europe. Weed Science 53, 506-512 (2004).

33. Volenberg, D. S., Stoltenberg, D. E. & Boerboom, C. M. Green foxtail (Setaria viridis) resistance to acetolactate synthase inhibitors. Phytoprotection 83, 99-

109 (2002).

34. De Prado, R., Lopez-Martinez, N. & Gonzalez-Gutierrez, J. Identification of two mechanisms of atrazine resistance in Setaria faberi and Setaria viridis biotypes. Pestecide Biochemistry and Physiology 67, 114-124 (2000). 35. Preston, C. Herbicide resistance in weeds endowed by enhanced detoxification: complications for management. Weed Science 52, 448-453 (2004).

36. Yenish, J. R. & Young, F. L. Winter wheat competition against jointed goatgrass {Aegilops cylindήcd) as influenced by wheat plant height, seeding rate, and seed size. Weed Science 52, 996-1001 (2004).

37. Ball, D. A., Young, F. L. & Ogg, A. G. Selective control of jointed goatgrass {Aegilops cylindricd) with imazamox in herbicide-resistant wheat. Weed Technology 13, 77-82 (1999).

38. Anderson, J. A., Matthiesen, L. & Hegstad, J. Resistance to an imidazolinone herbicide is conferred by a gene on chromosome 6DL in the wheat line cv. 9804.

Weed Science 52, 83-90 (2004).

39. Kroiss, L. J. et al. Marker-assessed retention of wheat chromatin in wheat {Tήticum aestivum) by jointed goatgrass {Aegilops cylindricd) backcross derivatives. Crop Science 44, 1429-1433 (2004). 40. Ellstrand, N. C. Dangerous Liaisons? When Cultivated Plants Mate with Their Wild Relatives (John Hopkins University Press, Balitmore, 2003). 41. Stewart, C. N., Halfhill, M. D. & Warwick, S. I. Transgene introgression from genetically modified crops to their wild relatives. Nature Reviews: Genetics 4, 806- 817 (2003). 42. Snyder, J. R., Mallory-Smith, C. A., Baiter, S., Hansen, J. L. & Zemetra, R. Seed production on Tήticum aestivum by Aegilops cylindrica hybrids in the field. Weed Science, 588-593 (2000).

43. Oliver, M. J., Luo, H., Kausch, A. & Collins, H. in Proceedings of the International Symposium on the Biosafety of Genetically Modified Organisms 154- 161 (INRA, Montpellier, 2004).

44. Oliver, M. J., Quisenberry, J. E., Trolinder, N. L. G. & Keim, D. L. (US patent 5,723,765, 1998).

45. Daniell, H. Molecular strategies for gene containment in transgenic crops. Nature Biotechnology 20, 581-586 (2002). 46. Maliga, P. Plastid transformation in higher plants. Annual Review of Plant Biology 55, 289-313 (2004).

47. Wang, T. et al. Low frequency transmission of a plastid-encoded trait in Setaria italica. Theoretical and Applied Genetics 108, 315-320 (2004). 48. Darmency, H. in Herbicide Resistance in Plants: Biology and Biochemistry (eds. Powles, S. B. & Holtum, J. A. M.) 263-298 (Lewis, Boca-Raton, 1994).

49. Gressel, J. Tandem constructs: preventing the rise of superweeds. Trends in Biotechnology 17, 361-366 (1999). 50. Al-Ahmad, H., Dwyer, J., Moloney, M. & Gressel, J. Mitigation of establishment of Brassica napus transgenes in volunteers using a tandem construct containing a selectively unfit gene. Plant Biotech. Journal (2005a). In Press. 51. Al- Ahmad, H., Galili, S. & Gressel, J. Tandem constructs to mitigate transgene persistence: tobacco as a model. Molecular Ecology 13, 697-710 (2004). 52. Al- Ahmad, H., Galili, S. & Gressel, J. Poor competitive fitness of transgenically mitigated tobacco in competition with the wild type in a replacement series. Planta (2005b). In Press.

53. Al- Ahmad, H. & Gressel, J. Mitigation using a tandem construct containing a selectively unfit gene precludes establishment of Brassica napus transgenes in hybrids and backcrosses with weedy Brassica rapa. Plant Biotech. Journal (2005b). In Press .

54. Okamoto, M. Asynaptic effect of chromosome V. Wheat Information Service 5, 6-8 (1957).

55. Riley, R. & Chapman, V. Genetic control of the cytologically diploid behavior of hexaploid wheat. Nature 182, 713-715 (1958). 56. Sears, E. R. & Okamoto, M. Intergenomic chromosome relationships in hexaploid wheat, in Proceedings of the 10th International Congress of Genetics 258-

259 (University of Toronto, Toronto, 1958).

57. Sears, E. R. Genetic control of chromosome pairing in wheat. Annual Review of Genetics 10, 31-51 (1976). 58.

60. Jenczewski, E. et al. PrBn, a major gene controlling homoeologous pairing hi oilseed rape (Brassica napus) haploids. Genetics 164, 645-653 (2003).

61. Udall, J. A., Quijada, P. A. & Osborn, T. C. Detection of chromosomal rearrangements derived from homoeologous recombination in four mapping populations of Brassica napus L. Genetics 169, 967-979 (2005).

62. Morris, R. & Sears, E. R. in Wheat and wheat improvement (ed. Quisenberry, K. S.) 19-88 (American Society of Agronomy Inc., Madison, Wisconsin, 1967). 63. Jatnpates, R. & Dvorak, J. Location of the PhI locus in the metaphase chromosome map and the linkage map of the 5Bq arm of wheat. Canadian Journal of Genetics and Cytology 28, 511-519 (1986).

64. Sears, E. R. Mutations in wheat that raise the level of meiotic chromosome pairing, in 16th Stadler Genetics Symposium pp. 295-300 (Plenum Press, New York,

1984).

65. Puchta, H. Gene replacement by homologous recombination in plants. Plant Molecular Biology 48, 173-182 (2002).

66. Reiss, B. Homologous recombination and gene targeting in plant cells. International Review of Cytology 228, 85-139 (2003).

67. Li, H. Q. & Li, M. R. RecQ helicase enhances homologous recombination in plants. FEBS letters 574, 151-155 (2004).

68. Mariani, C. et al. A chimeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 357, 384-387 (1992). 69. Logemann, J., Jach, G., Tommerup, H., Mundy, J. & Schell, J. Expression of a barley ribosome-inactivating protein leads to increased fungal protection in transgenic tobacco plants. Bio-Technology 10, 305-308 (1992).

70. Jach, G. et al. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant Journal 8, 97-109 (1995).

71. Korchuganov, D. S. et al. 187E mutation prevents barstar dimerization. Russian Journal of Bϊoorganic Chemistry 30, 577-581 (2004).

72. Hartley, R. W. Barnase and barstar - two small proteins to fold and fit together. Trends in Biochemical Sciences 14, 450-454 (1989). 73. Riley, R., Campbell, C. S., Young, R. M. & Belfield, A. M. Control of meiotic chromosome pairing by the chromosomes of homoeologous group 5 of Triticum aestivum. Nature 212, 1475-1477 (1966).

74. Chen, Q., Jahier, J. & Cauderon, Y. The B-chromosome system of inner Mongolian Agropyron Gaertn .3. Cytogenetical evidence for B-A pairing at metaphase-I. Hereditas 119, 53-58 (1993).

Claims

WHAT IS CLAIMED IS:

1. A method of generating a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the method comprising inserting an exogenous polynucleotide into a genetic locus of the transgenic cultivated plant, said genetic locus being in linkage disequilibrium with a PhI locus of the transgenic cultivated plant thereby preventing the stable transgene introgression into the related, non-cultivated plant.

2. A transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the transgenic cultivated plant comprising an exogenous polynucleotide positioned in a genetic locus of the transgenic cultivated plant, said genetic locus being in linkage disequilibrium with a PhX locus of the transgenic cultivated plant.

3. A method of reducing introgression of an exogenous polynucleotide of a transgenic cultivated plant into a related, non-cultivated plant, the method comprising inserting the exogenous polynucleotide into a genetic locus of the transgenic cultivated plant, said genetic locus being in linkage disequilibrium with a PhI locus of the transgenic cultivated plant thereby preventing the transgene flow into the related, non-cultivated plant.

4. The method or the transgenic cultivated plant of any of claims 1, 2 and 3, wherein said exogenous polynucleotide comprises a nucleic acid sequence encoding a polynucleotide or a polypeptide product.

5. The method or the transgenic cultivated plant of any of claims 1, 2 and 3, wherein said genetic locus being in linkage disequilibrium with said PhX locus is set forth by SEQ ID NO:13, 14, 15, 16, 17 and 39 .

6. The method or the transgenic cultivated plant of any of claims 1, 2 and 3, wherein the transgenic cultivated plant is wheat.

7. The method or the transgenic cultivated plant of claim 6, wherein said wheat is Triticum aestivum ssp. aestivum and Triticum turgidum ssp. durum.

8. The method or the transgenic cultivated plant of any of claims 1, 2 and 3, wherein said related, non-cultivated plant is selected from the group consisting of Aegilops spp., Hordeum spp., Elymus spp., Agropyron spp., Eremopyrum ssp., Secale spp., Dasypyrum spp., Heteranthelium ssp., Amblyopyrum ssp., Henrardia ssp., Thinopyrum spp., Leymus spp., Psathyrostachys spp., Hystrix ssp., Hordelymus ssp., Taeniatherum ssp. and Crithopsis ssp.

9. The method or the transgenic cultivated plant of any of claims 1, 2 and 3, wherein said exogenous polynucleotide comprises a nucleic acid sequence encoding a suppressor of a gene or gene product lethal to the related, non-cultivated plant.

10. The method of claim 9, further comprising inserting a second exogenous polynucleotide into a second genetic locus of the transgenic cultivated plant, said second genetic locus being in random association with said PhX locus of the transgenic cultivated plant.

11. The transgenic cultivated plant of claim 9, further comprising a second exogenous polynucleotide positioned in a second genetic locus of the transgenic cultivated plant, said second genetic locus being in random association with said PhI locus of the transgenic cultivated plant.

12. The method or the transgenic cultivated plant of any of claims 10 and 11, wherein said second exogenous polynucleotide comprises a nucleic acid sequence encoding a product lethal to the related, non-cultivated plant.

13. The method or the transgenic cultivated plant claim 12, wherein said second exogenous polynucleotide further comprises a second nucleic acid sequence encoding a transgene.

14. The method or the transgenic cultivated plant of any of claims 4 and 13, wherein said transgene is a polynucleotide or a polypeptide.

15. The method or the transgenic cultivated plant of any of claims 4 and 13, wherein said polynucleotide or polypeptide product endows the transgenic cultivated plant with a commercially desirable trait selected from the group consisting of herbicide resistance, disease resistance, insect resistance and nematode resistance, environmental stress resistance, high productivity, modified agronomic quality, enhanced yield, modified ripening, and bioremediation.

16. The method or the transgenic cultivated plant of any of claims 4 and 13, wherein said exogenous polynucleotide further comprises a promoter for directing an expression of said nucleic acid sequence in the transgenic cultivated crop.

17. The method or the transgenic cultivated plant of claim 16, wherein said promoter is selected from the group consisting of FMV, 35S, E8, E4, El 7, J49, 2Al 1, and Tap 1.

18. The method or the transgenic cultivated plant of claim 12, wherein said product lethal to the related, non-cultivated plant is barnase.

19. The method or the transgenic cultivated plant of claim 9, wherein said suppressor of said product lethal to the related, non-cultivated plant is barstar.

20. A method of generating a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the method comprising inserting an exogenous polynucleotide into a centromeric region of a chromosome of the transgenic cultivated plant thereby preventing the transgene flow into the related, non-cultivated plant.

21. A transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the transgenic cultivated plant comprising an exogenous polynucleotide positioned in a centromeric region of a chromosome of the transgenic cultivated plant.

22. A method of reducing introgression of an exogenous polynucleotide of a transgenic cultivated plant into a related, non-cultivated plant, the method comprising inserting the exogenous polynucleotide into a centromeric region of a chromosome of the transgenic cultivated plant.

23. The method or the transgenic cultivated plant of any of claims 20, 21 and 22, wherein said exogenous polynucleotide comprises a nucleic acid sequence encoding a polynucleotide or a polypeptide product.

24. The method or the transgenic cultivated plant of any of claims 20, 21 and 22, wherein the transgenic cultivated plant is wheat.

25. The method or the transgenic cultivated plant of claim 24, wherein said wheat is Triticum aestivum ssp. aestivum and/or Triticum turgidum ssp. durum.

26. The method or the transgenic cultivated plant of any of claims 20, 21 and 22, wherein said related, non-cultivated plant is selected from the group consisting of Aegilops spp., Hordeum spp., Elymus spp., Agropyron spp., Eremopyrum ssp., Secale spp., Dasypyrum spp., Heteranthelium ssp., Amblyopyrum ssp., Henrardia ssp., Thinopyrum spp., Leymus spp., Psathyrostachys spp., Hystrix ssp.,Hordelymus ssp., Taeniatherum ssp., and Crithopsis ssp.

27. The method or the transgenic cultivated plant of claim 23, wherein said polynucleotide or polypeptide product endows the transgenic cultivated plant with a commercially desirable trait selected from the group consisting of herbicide resistance, disease resistance, insect resistance and nematode resistance, environmental stress resistance, high productivity, modified agronomic quality, enhanced yield, modified ripening, and bioremediation.

28. The method or the transgenic cultivated plant of claim 23, wherein said exogenous polynucleotide further comprises a promoter for directing an expression of said nucleic acid sequence in the transgenic cultivated crop.

29. The method or the transgenic cultivated plant of claim 28, wherein said promoter is selected from the group consisting of FMV, 35S₅ ES₅ E4₅ El 7, J49, 2Al 1, and Tap 1.

30. The method or the transgenic cultivated plant of any of claims 20, 21 and 22, wherein said centromeric region of the transgenic cultivated plant is on chromosome 5 of sub genome B of Triticum aestivum ssp. aestivum and/or Triticum turgidum ssp. durum.

31. The method or the transgenic cultivated plant of any of claims 20, 21 and 22, wherein said centromeric region of the transgenic cultivated plant is a sub- centromeric region of the transgenic cultivated plant.

32. A method of generating a transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the method comprising inserting an exogenous polynucleotide into a genetic locus of the transgenic cultivated plant, said genetic locus being in linkage disequilibrium with a locus including a gene capable of preventing homoeologous recombination, thereby preventing the stable introgression into the related, non-cultivated plant.

33. A transgenic cultivated plant characterized by reduced stable transgene introgression to a related, non-cultivated plant, the transgenic cultivated plant comprising an exogenous polynucleotide positioned in a genetic locus of the transgenic cultivated plant, said genetic locus being in linkage disequilibrium with a locus including a gene capable of preventing homoeologous recombination.

34. A method of reducing introgression of an exogenous polynucleotide of a transgenic cultivated plant into a related, non-cultivated plant, the method comprising inserting the exogenous polynucleotide into a genetic locus of the transgenic cultivated plant, said genetic locus being in linkage disequilibrium with a locus including a gene capable of preventing homoeologous recombination thereby reducing introgression of the exogenous polynucleotide into the related, non- cultivated plant.

35. The method or the transgenic cultivated plant of any of claims 32, 33 and 34, wherein said exogenous polynucleotide comprises a nucleic acid sequence encoding a polynucleotide or a polypeptide product.

36. The method or the transgenic cultivated plant of any of claims 32, 33 and 34, wherein said related, non-cultivated plant is selected from the group consisting of Aegilops spp., Hordeum spp., Elymiis spp., Agropyron spp., Eremopyrum ssp., Secale spp., Dasypyrum spp., Heteranthelium ssp., Amblyopyrum ssp., Henrardia ssp., Thinopyrum spp., Leymus spp., Psathyrostachys spp., Hystrix ssp.,Hordelymus ssp., Taeniatherum ssp., and Crithopsis ssp.

37. The method or the transgenic cultivated plant of any of claims 32, 33 and 34, wherein said exogenous polynucleotide endows the transgenic cultivated plant with a commercially desirable trait selected from the group consisting of herbicide resistance, disease resistance, insect resistance and nematode resistance, environmental stress resistance, high productivity, modified agronomic quality, enhanced yield, modified ripening, and bioremediation.