WO2002052924A2 - Procede permettant de maintenir une ligne parentale genique male-femelle sterile pour la production de ble hybride - Google Patents

Procede permettant de maintenir une ligne parentale genique male-femelle sterile pour la production de ble hybride Download PDF

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WO2002052924A2
WO2002052924A2 PCT/US2002/000549 US0200549W WO02052924A2 WO 2002052924 A2 WO2002052924 A2 WO 2002052924A2 US 0200549 W US0200549 W US 0200549W WO 02052924 A2 WO02052924 A2 WO 02052924A2
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male
plants
gene
heterologous
allele
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PCT/US2002/000549
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WO2002052924A3 (fr
Inventor
Moshe Feldman
Eitan Millet
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Yeda Research And Development Co. Ltd.
Fleit, Lois
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Priority to AU2002248325A priority Critical patent/AU2002248325A1/en
Priority to EP02717309A priority patent/EP1347678A4/fr
Publication of WO2002052924A2 publication Critical patent/WO2002052924A2/fr
Publication of WO2002052924A3 publication Critical patent/WO2002052924A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • the present invention relates to the production of hybrid lines of common and durum wheat, and more specifically, relates to a new method for maintaining a male-sterile female parental line for use in the production of hybrid wheat seeds, which female line is homozygous for a recessive male-sterility mutant allele, and relates to a new maintainer line for maintaining the female parental line, which is isogenic to the female line, but has an alien chromosomal arm, as a monotelosomic addition, carrying a dominant male-fertility allele homoeoallelic to the recessive male-sterility allele, a heterologous microspore-suicide gene capable of killing the microspores or pollen grains carrying it, and a heterologous selectable marker.
  • the selectable marker is seed color such as blue grains
  • the maintainer seeds the blue seeds
  • the invention further provides the maintainer line.
  • the resulting hybrid plants do not contain the heterologous genes and are all heterozygous for the dominant male-fertility allele and for the recessive male-sterility allele and are, therefore, male-fertile.
  • Hybrid wheat lines yield higher than pure, true breeding lines, and exhibit improved quality and greater tolerance to environmental and biotic stresses (Wilson and Driscoll, 1983; Pickett, 1993; Bruns and Peterson, 1998; Jordaan et al., 1999).
  • Common (bread) wheat, Triticum aestivum L. ssp. aestivum MacKey, and durum (macaroni) wheat, T. turgidum L. ssp. durum (Desf.) Husn. are predominantly self-pollinating species and every flower contains both female and male organs. To produce hybrid seeds, it is therefore necessary to male-sterilize the female parent. Since hand emasculation is impractical in wheat, male-sterility may be brought about by application of chemical hybridizing agents (CHAs) or by genetic means. Utilization of a CHA to male-sterilize wheat plants is expensive, inefficient and pollutant.
  • CHAs chemical hybridizing agents
  • CMS cytoplasmic male-sterility
  • GMS genie male-sterility
  • Genie male sterility is expressed in a normal common or durum wheat cytoplasm and does not involve deleterious effects on plant performance. Further, using a female parent homozygous for a recessive male-sterility allele, any wheat cultivar which is by its nature homozygous for the dominant allele conferring male-fertility, can be used as a male parent that will restore complete fertility to the Fi hybrids. There is no need to breed for male lines and no limitation exists for the number of males which can be crossed with the male-sterile females and evaluated for their combining ability. Several chromosome arms have been described in common wheat which carry genes affecting male-fertility, e.g.
  • chromosome arms of group 4 the long arm of chromosome 4A (4AL), the short arm of chromosome 4B (4BS) and the short arm of chromosome 4D (4DS), carrying the normal male-fertility Ms-A1, Ms-B1 and Ms-D1 genes, respectively, and the long arms of the group 5 chromosomes: 5A, 5B and 5D (5AL, 5BL and 5DL, respectively), carrying the Ms-A2, Ms-B2 and Ms-D2 genes, respectively.
  • group 5 chromosomes 5A, 5B and 5D (5AL, 5BL and 5DL, respectively
  • Ms-A2, Ms-B2 and Ms-D2 genes respectively.
  • the maintainer (Y) Since the maintainer (Y) is not a true-breeding line, it is produced by pollinating the male-sterile female (Z) by the disomic alien addition line (X).
  • This system was characterized by two major drawbacks: some transmission of the alien chromosome occurred through the pollen of the maintainer line which introduced male fertility to the new generation of the male-sterile female line; and addition decay occurred in the X line impairing its purity. These are possibly the reasons why this system has never come into practical (commercial) use.
  • Driscoll (1985) proposed a modification of the above XYZ system of producing hybrid wheat.
  • a selfed Y-line replaces the Y-line to maintain and propagate the male-sterile Z-line.
  • This modification eliminates the need for the X-line that was originally needed to generate a large quantity of Y-line plants.
  • the newly proposed Y-line carries an alien isochromosome so that the compensating male-fertility homoeoallele is in two doses.
  • Such genetically .altered common wheat plants contain a modified chromosome with a dominant normal male-fertility allele from the diploid wheat Triticum monococcum as an addition or substitution for one of the wheat 4B chromosomes.
  • the modified chromosome carries the short arm of chromosome 4A m of T. monococcum (4A m S) carrying the Ms-A m 1 allele and a second arm with a proximal segment from the long arm of either chromosome 4A m of T.
  • the above hybrid-production system has too a number of drawbacks as regards the efficient maintenance of the parental lines.
  • pollination of female plants by the maintainer will yield a larger number of seeds with the recombinant alien/4BL chromosome, which will develop into male-fertile plants.
  • the maintenance of the male-sterile female parent involves a complex procedure of progeny selection based on marker genes.
  • the maintainer line for the female (male-sterile) parental line is also a genetically unstable line in that it carries 20 pairs of normal common wheat chromosomes, one 4B chromosome carrying the male-sterility (ms-B1-b) mutant allele (known as 'Probus') (Wilson and Driscoll, 1983) and one recombinant alien group 4/4BL chromosome having the normal, male-fertility Ms-A m 1 allele and the seed coloration allele.
  • the maintainer line is male-fertile, and upon selfing will yield fertile plants homozygous or heterozygous for the modified chromosome.
  • the male-sterile female line is homozygous for both ms and Gc1, which are tightly linked, while a male-fertile line (the maintainer) isogenic to the female line but having Ms and gc1 alleles, is used to pollinate the female line to yield a double heterozygote msMsGdgd. Due to abortion of gametes carrying gd, all the progeny of such selfed line will be homozygote msmsGclGd and identical to the female line.
  • the male line (R line) in the hybrid production system should also be bred to contain the Gel allele otherwise the fertility of the Fi hybrid will be reduced.
  • Gc1 causes the abortion of female as well as male gametes and therefore, a cross (between the female and the maintainer) and a self (of the double heterozygote) are required each year to renew the female seed stock. This is a drawback in time and cost.
  • Another disadvantage of Endo's proposal stems from the fact that the male-sterile female parent contains an alien chromosome segment carrying the Gc1 allele that was derived from Ae. speltoides. This segment may carry also alleles with negative effect on the performance of the female, increasing the cost of hybrid seed production, or even affecting the yield of the hybrid.
  • the maintainer line is isogenic to the female line but has an alien engineered chromosome carrying a dominant male-fertility allele that restores fertility to the maintainer line, a recessive pollen-killing allele that is susceptible to the killing effect of the native pollen killer thus preventing the transmission of this chromosome to the female line, and one or more selectable markers that facilitate the maintenance of the maintainer itself.
  • the engineered chromosome due to some frequency of transversal division of the centromere, was found to be unstable resulting in male gametes that contain the chromosome arm with the male-fertility allele. These male gametes were transmitted upon pollination of the female plants resulting in a certain degree of male-fertile female plants, which led to reduced hybrid purity. Consequently, this method was not used in commercial practice.
  • the male-sterility gene is a heterologous gene comprising foreign DNA that codes for a cytotoxic protein such as RNase (European Patent Application No. EP 0,412,911) or for a protein that binds an essential cellular factor such as biotin (International PCT Patent Application No. WO 96/40949), and a plant promoter that enables the expression of the foreign DNA only in stamen cells.
  • a cytotoxic protein such as RNase (European Patent Application No. EP 0,412,911)
  • biotin International PCT Patent Application No. WO 96/40949
  • This method requires the transformation of the male parent too with another heterologous gene comprising antisense DNA that is capable to inhibit the cytotoxic effect by suppressing the transcription activity of the male-sterility DNA or coding for a protein that inhibits the cytotoxic effect, with a plant constitutive promoter.
  • a plant constitutive promoter To maintain the male-sterile female line there is a need for additional heterologous gene with an inducible promoter or to treat the male-sterile female plants with an exogenous compound such as biotin, to supply the essential factor.
  • the method requires breeding of the male parent too rendering the hybrid seed production expensive.
  • the hybrid plants contain at least two heterologous genes rendering their grains unsuitable for human consumption in the present time.
  • 4BS the short arm of chromosome 4B of common and durum wheat.
  • 4S S S the short arm of chromosome 4S S of Aegilops searsii.
  • AA an alien chromosomal arm 4S S S of Aegilops searsii, added to the complement of common wheat as a monotelosomic addition.
  • Ms a dominant allele responsible for male-fertility in wheat.
  • Ms-B1 a dominant allele for male-fertility in durum and common wheat located on
  • ms a recessive mutant allele of Ms that confers male-sterility.
  • ms-B1 a recessive mutant allele of Ms-B1, that confers male-sterility in durum and common wheat, when present in homozygous state.
  • ms-B1-a msla which is the 'Pugsley' mutant ms-B1 allele.
  • ms-B1-b mslb which is the 'Probus' mutant ms-B1 allele.
  • ms-B1-c mslc which is the 'Cornerstone' mutant ms-B1 allele.
  • Ms-S s 1 a dominant allele for male-fertility, homoeoallelic to Ms-B1, on 4S S S.
  • Msu a heterologous gene capable of killing microspores, microgametophytes or pollen grains carrying it, controlled by a microspore-or microgametophytic-specific promoter.
  • Ski a heterologous gene capable of killing seedlings carrying it, controlled by a seedling-specific promoter.
  • Ski inhibitor a heterologous gene capable of inhibiting the expression of Ski, controlled by an inducible promoter.
  • BaRNase a gene from Bacillus amyloliquefaciens coding for RNase.
  • Barstar a gene from Bacillus amyloliquefaciens coding for a protein that inhibits
  • Hr a heterologous specific herbicide-resistance gene by which plants having this gene can be selected, controlled by a constitutive promoter.
  • Ba a dominant glufosinate (Basta)-resistance gene.
  • Female line a male-sterile line of common and durum wheat homozygous for one of the recessive male-sterility alleles of ms-B1.
  • Male line a male-fertile line of common and durum wheat homozygous for the dominant male-fertility allele Ms-B1.
  • Maintainer line a male-fertile line isogenic to the male-sterile female line but contains the alien chromosomal arm AA, carrying a dominant male-fertility allele (Ms) linked to a heterologous microspore-suicide gene (Msu) capable of killing microspores or pollen grains carrying it, controlled by a microspore-specific promoter, and a heterologous selectable marker gene, by which plants having this chromosomal arm can be selected, controlled either by a constitutive promoter or a tissue- specific promoter.
  • Ms dominant male-fertility allele
  • Msu microspore-suicide gene
  • Yet another object of the present invention is to provide a maintainer line for use in the above method, which maintainer line is easily, rapidly and stably propagated.
  • These three genes on the alien chromosomal arm are permanently linked due to lack of pairing and recombination between the alien chromosomal arm and the wheat chromosomes.
  • the presence of the microspore-suicide gene on the alien chromosomal arm ensures that all the maintainer viable male gametes (about 80% of the gametes) lack this chromosomal arm, and consequently, the dominant male-fertility allele and the selectable marker allele. Hence, pollination of male-sterile female plants with such a maintainer will yield only male-sterile female plants.
  • the selectable marker is a gene conferring resistance to a specific herbicide
  • the selectable marker is seed color such as blue grains
  • the invention further provides the maintainer line.
  • the resulting hybrid plants do not contain the heterologous genes and are all heterozygous for the dominant male-fertility allele and for the recessive male-sterility allele and are, therefore, male-fertile.
  • the present invention makes possible the commercial production of hybrids of common and durum wheat.
  • the invention provides a novel method for the maintenance of a male-sterile female parental line that is homozygous for a recessive male-sterility mutant (ms) allele.
  • the maintainer Figs.
  • AA a monotelosomic addition
  • Ms dominant male-fertility allele
  • Msu a heterologous microspore-suicide gene
  • Hr specific herbicide-resistance gene
  • Sc seed-color gene
  • the male-sterile female parental line is maintained by pollinating it with the male-fertile maintainer line, and all of the resulting progeny are male-sterile female plants (Figs. 2 and 3).
  • the maintainer line is itself easily maintained by self-pollination, resulting in a mixture of progeny seeds of which about 20% carry the AA alien chromosomal arm, when grown, developing into male-fertile plants, and about 80% lack this alien chromosomal arm, when grown, developing into male-sterile plants.
  • Spraying said progeny plants with the specific herbicide kills the male-sterile plants thereby allowing only the male-fertile maintainer plants to grow (Fig. 2).
  • the selectable marker is seed color
  • the red or white seeds when grown, developing into male-sterile female plants, can be separated by a seed sorter from the blue seeds, when grown, developing into male-fertile maintainer plants (Fig. 3).
  • the AA alien chromosomal arm of the maintainer line can carry one (Fig. 1b) or two (Fig. 1c) additional heterologous genes facilitating the use of the progeny of the selfed maintainer as a source for male-sterile female plants (Figs. 4a and 5a) or of male-fertile maintainer plants (Figs. 4b and 5b).
  • the alien chromosomal arm was derived from Ae. searsii. It is the short arm of chromosome 4S S (4S S S). Being an alien single telocentric chromosome that was added to the complement of common wheat, it does not pair during meiosis and only enters to about 20% of the gametes, either male or female. Hence, -while it is not transmitted through the male gametes because of the presence of the microspore-suicide gene, the rate of its transmission through the female gametes is about 20%.
  • the Ms- ⁇ l gene is homoeoallelic to the Ms-B1 gene and dominant over the ms- B1 alleles of common and durum wheat and is expressed in all tested genetic backgrounds.
  • the heterologous microspore-suicide gene is any gene that codes for heterologous protein that is toxic to the microspores, microgametophytes or pollen grains possessing it or disturbs their metabolism. It consists of a foreign DNA sequence coding for a toxic protein, which is toxic to the microspores, microgametophytes or pollen grains, or codes for a protein that significantly disturbs the metabolism and thereby the viability of the microspores, microgametophytes or pollen grains, inserted into an expression cassette under control of a microspore-specific promoter.
  • the heterologous specific herbicide-resistance gene (Hr) codes for a heterologous protein that confers resistance to a specific herbicide under control of a constitutive promoter.
  • the heterologous seed-color gene codes for an anthocyanin and thus conferring coloration of the grains containing it, under control of an endosperm-specific promoter.
  • heterologous genes are introduced to the AA alien chromosomal arm by standard genetic-engineering manipulations.
  • the invention relates to a method for producing a hybrid plant line of common or durum wheat, wherein the male-sterile female parental line is crossed with any cultivar of the same species, which by its nature is male-fertile homozygous for the Ms-B1 allele, to yield Fi hybrid progeny that are all heterozygous
  • the invention further provides a plant homozygous for a recessive male-sterility allele and having in the nuclear genome of its cells an alien chromosomal arm carrying a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene (Msu) comprising a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable, and a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; and a heterologous selectable marker comprising a selectable-marker DNA encoding for a heterologous protein that facilitates the selection of plants carrying it, and a plant promoter, either constitutive or tissue specific, directing the expression of said selectable marker DNA in all plant tissues or in a specific tissue, respectively.
  • Ms male-fertility dominant
  • the invention provides a plant homozygous for a recessive male-sterility allele and having in the nuclear genome of its cells an alien chromosomal arm carrying a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene comprising a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable; and a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; a heterologous seedling-killer gene comprising a seedling-killer DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those seedlings that possess it, thus killing the seedlings, and a seedling-specific promoter directing the expression of said seedling-killer DNA selectively in the seedlings that possess it; and
  • the invention provides a plant homozygous for a recessive male-sterility allele, and having in the nuclear genome of its cells an alien chromosomal arm carrying a male-fertility dominant allele (Ms); a heterologous microspore-suicide gene comprising a microspore-suicide DNA encoding a heterologous protein that is either cytotoxic to, or disturbs, the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable, and a microspore-specific promoter directing the expression of said microspore-suicide DNA selectively in the microspores that possess it; a heterologous seedling-killer gene comprising a seedling-killer DNA encoding a heterologous protein that is either cytotoxic to or disturbs the metabolism of those seedlings that possess it, and a seedling-specific promoter directing the expression of said seedling-killer DNA selectively in the seedlings that possess it; a heterologous gene
  • the male-sterility allele can be a heterologous gene.
  • the male-sterility allele can be one of the male-sterility alleles of common wheat, durum wheat or another species of the Triticeae.
  • the male-sterility allele may be one of the male-sterility alleles of the Ms-B1 locus either ms- B1-a, ms-B1-b, ms-B1-c or any other allele of MS-B1.
  • the dominant male-fertility allele (Ms) can be a heterologous gene.
  • the dominant male-fertility allele (Ms) may be one of the male-fertility alleles of the Triticeae.
  • the dominant male-fertility allele (Ms) may be Ms- ⁇ l of chromosome arm 4S S S of Aegilops searsii.
  • the microspore-suicide gene may be any of the genes affecting the viability of microspores, microgametophytes, pollen grains or male gametes that carry it.
  • the microspore-suicide gene encodes avidin or sterptavidin of variant thereof.
  • the microspore-suicide gene may encode RNase of variant thereof.
  • the microspore-suicide DNA may encode ribosomal-inhibitor protein (RIP) of variant thereof.
  • the microspore-specific promoter may be any promoter that is expressed in plants directing gene expression in the microspore, male- gametophyte or pollen grain.
  • the heterologous selectable marker may be any gene encoding for a heterologous protein that facilitates the selection of plants carrying it.
  • the heterologous selectable marker can be a specific herbicide-resistance gene.
  • the specific herbicide-resistance gene may be Bar conferring resistance to glufosinate (Basta).
  • the constitutive promoter may be of the specific herbicide-resistance gene expressed in plants.
  • the heterologous selectable marker may be a seed-color gene coloring the aleurone layer or the endosperm.
  • the promoter of the seed-color gene may be an endosperm-specific promoter or an aleurone-specific promoter.
  • the heterologous selectable marker may be a seed-size, weight or morphology gene.
  • the promoter of this selectable marker gene may be a seed-specific promoter.
  • the seedling-killer gene may encode a protein that renders the cells containing it inviable.
  • the seedling-killer DNA may encode avidin or sterptavidin of variant thereof.
  • the seedling-killer gene may encode Rnase of variant thereof.
  • the seedling-killer gene may encode ribosomal-inhibitor protein (RIP) of variant thereof.
  • the seedling-specific promoter may be any promoter that is expressed in plants directing gene expression in the seedling.
  • the DNA counteracting the effect of the seedling-killer gene may encode a protein that neutralizes the effect of the seedling-killer gene of variant thereof. More particularly, the DNA counteracting the effect of the seedling-killer gene may encode biotin of variant thereof. The DNA counteracting the effect of the seedling-killer gene may encode RNase antisense of variant thereof or RIP antisense of variant thereof.
  • the constitutive promoter may be any promoter that is expressed in plants directing gene expression in the plants.
  • the inducible promoter is any promoter that is expressed in plants directing gene expression in the plants.
  • the inducible promoter can be induced by temperature, light, chemical or any other environmental treatment.
  • the inducible promoter can be induced by tetracyclin.
  • the alien chromosomal arm may be any arm of a Triticeae species carrying a dominant male-fertility allele.
  • the alien chromosome arm may be chromosomal arm 4S S S of Aegilops searsii.
  • the plants noted above may be selected from any of the Gramineae species.
  • the plants noted above may be selected from any of wheat, triticale, barely, rye, oat, rice, and maize.
  • the invention provides a method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat comprising:
  • the invention further provides a method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat, said method comprising:
  • the invention further provides a method for the maintenance of a male-sterile female parental line of common or durum wheat for use in the production of hybrid wheat comprising:
  • the invention further provides a method for the maintenance of a male-fertile maintainer line of common or durum wheat for use in the maintenance of male-sterile female line comprising:
  • the invention providing a method for a maintenance of a male-fertile maintainer line of common or durum wheat for use in the maintenance of male-sterile female line of wheat comprising:
  • the progeny seeds of (a) will be separated by a seed sorter, the blue seeds will be the maintainer seeds and the red or white seeds will be added to the male-sterile female line.
  • the blue seeds will be treated with biotin.
  • the selectable marker gene is a specific herbicide- resistance gene (Hr), controlled by a constitutive promoter.
  • the selectable marker gene may be a seed-color gene (Sc), controlled by an endosperm-specific promoter or a seed-size, weight or morphology gene, controlled by a seed-specific promoter.
  • the invention further provides a method for producing a hybrid plant line of common or durum wheat comprising :
  • male-sterile female parent of the same species, wherein the male parent is selected from any desired common or durum wheat cultivar which, by its nature, is homozygous for the dominant wild-type male-fertility (Ms-B1) allele, and said male-sterile female parent is a line of the wheat species being homozygous for both any one of the recessive mutant male-sterility (ms-B1) allele, the male-sterile female parent being maintained by a maintainer line as described above ; and
  • Fig. 1 depicts schematic drawings of the alien chromosomal arm 4S S S of Aegilops searsii (AA) carrying the dominant male-fertility allele Ms-S s 1, a heterologous microspore-suicide gene (Msu) with a microspore-specific promoter, and a heterologous selectable marker gene (Sm), either specific herbicide resistance (Hr) or seed color (Sc), with a constitutive promoter or an endosperm-specific promoter, respectively (1a); same arm as in (a) with one additional heterologous seedling-killer gene (Ski) with a seedling-specific promoter (1b); and same arm as in (b) with one additional heterologous seedling-killer inhibitor gene (Ski-inhibitor) with an inducible promoter (1c).
  • Ms-S s 1 a heterologous microspore-suicide gene
  • Sm heterologous selectable marker gene
  • Hr specific herbicide resistance
  • Sc seed color
  • Fig. 2 depicts a general scheme for maintaining a male-sterile female parental line by pollinating it by a maintainer line and maintaining the maintainer line by self- pollination.
  • the maintainer line is isogenic to the female line and has further the AA alien chromosomal arm added to the wheat complement, carrying the dominant male- fertility allele Ms-S s 1, a heterologous microspore-suicide gene (Msu) with a microspore- specific promoter, and a heterologous specific herbicide-resistance gene (Hr) with a constitutive promoter.
  • Fig. 3 depicts a general scheme for maintaining a male-sterile female parental line by pollinating it by a maintainer line as well as by separating the red or white seeds (the female line seeds) by a seed sorter from the progeny of the selfed maintainer, and maintaining the maintainer line by separating the blue seeds (the maintainer seeds) from the progeny of the selfed maintainer.
  • the maintainer line is isogenic to the female line and has further the AA alien chromosomal arm added to the wheat complement, carrying the dominant male-fertility allele Ms ⁇ l, a heterologous microspore-suicide gene (Msu) with a microspore-specific promoter, and a heterologous seed-color gene (Sc) with a endosperm-specific promoter.
  • Msu microspore-suicide gene
  • Sc seed-color gene
  • Fig. 4 depicts a general scheme for maintaining a male-sterile female parental line by selfing the maintainer line and obtaining progeny seeds of which 80%, when grown, developing into the female line, and 20%, when grown, developing into the maintainer line, the latter die in an early seedling stage due to the production of sterptavidin that specifically binds the essential factor biotin (a), and maintaining the maintainer line by treating the mixture of the said progeny seeds with exogenous biotin and spraying the grown seedlings with the specific herbicide, thereby killing all the plants lacking the Hr gene, i.e., the male-sterile female plants, leaving to grow only the male-fertile maintainer plants (b).
  • Fig. 5 depicts a general scheme for maintaining a male-sterile female parental line by selfing the maintainer line and obtaining progeny seeds of which 80%, when grown, developing into the male-sterile female line, and 20%, when grown, developing into the male-fertile maintainer line, the latter die in an early seedling stage due to the production of sterptavidin that specifically binds the essential factor biotin (a), and maintaining the maintainer line by separating the blue seeds (the maintainer seeds) from the red or white seeds (the female seeds) by a seed sorter and treating the blue seeds with biotin (b).
  • Fig. 6 depicts a general scheme for production of suitable material for transformation.
  • a simple system has been developed for common and durum wheat as depicted in Figs. 2 and 3 by which the male-sterile female parental line is maintained by pollinating it with the maintainer line, and all of the resulting progeny are male-sterile female plants.
  • the maintainer line is itself easily maintained by self-pollination, resulting in a mixture of seeds of which about 20%, when grown, developing into male-fertile plants identical to the maintainer line and carrying the AA alien chromosomal arm, and about 80%, when grown, developing into male-sterile plants due to the absence of the alien chromosomal arm and consequently, the Ms-S s 1 allele (Figs. 2 - 5).
  • Spraying the progeny plants of the selfed maintainer line with the specific herbicide kills the male-sterile plants while the male- fertile plants carrying the AA with the specific herbicide-resistance gene survive.
  • separating the seeds on the basis of their different colors with a seed sorter yields an additional amount of female seeds.
  • the male-sterile female parental line is crossed with any common or durum wheat cultivar, which by its nature is male-fertile homozygous for the Ms-B1 allele, to yield Fi hybrid offspring that are all heterozygous Ms-B1 ms-B1 and therefore, male-fertile.
  • the present invention provides a method for the maintenance of a male-sterile female parental line of common or durum wheat (Figs. 2 and 3) for use in the production of hybrid seed, said method comprising:
  • searsii carrying the dominant male-fertility allele Ms-S s 7, linked to a heterologous microspore-suicide gene (Msu) that disturbs the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable, controlled by a microspore-specific promoter, and a heterologous selectable marker gene by which plants having this chromosomal arm can be selected, controlled by a constitutive or a tissue-specific promoter. Pollen grains of the maintainer containing this alien chromosome arm are not functional, and
  • the present invention further provides an alternative method for the maintenance of a male-sterile female parental line of common or durum wheat (Figs. 4 and 5) for use in the production of hybrid wheat, said method comprising:
  • AA a monotelosomic addition
  • searsii carrying the dominant male-fertility allele Ms- S s 1, linked to three heterologous genes, a microspore-suicide gene (Msu) that disturbs the metabolism of those microspores, microgametophytes or pollen grains carrying it thus rendering them inviable, controlled by a microspore-specific promoter, a seedling-killer gene (Ski) coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, such as streptavidin gene coding for a protein that binds biotin, or a gene that codes for RNase or ribosome-inhibitor protein (RIP) that cause cell death, controlled by a seedling-specific promoter, and a selectable marker gene by which plants having this chromosomal arm can be selected, such as a specific herbicide-resistance gene (Hr) controlled by a constitutive promoter or a seed-color gene (Sc) controlled by
  • Any male-sterility ms-B1 allele may be used according to the invention such as, for example, the ms-B1-a, ms-B1-b and ms-B1-c alleles or any other allele of this locus or another locus of common or durum wheat inducing male sterility.
  • the heterologous microspore-suicide gene is any gene that codes for heterologous protein which is toxic to the microspores, microgametophytes or pollen grains possessing it, or codes for a protein that disturbs the metabolism of the microspores, microgametophytes or pollen grains possessing it.
  • RNA Ribonucleic acid
  • RIP ribosome-inhibitor protein
  • the heterologous selectable marker by which maintainer plants or seeds can be selected among the progeny of the selfed maintainer can be a specific herbicide- resistance gene (Hr) or a gene conferring seed color (Sc).
  • the heterologous specific herbicide-resistance gene (Hr) codes for a heterologous protein that confers resistance to a specific herbicide, such as, glufosinate (De Block et al., 1987; Weeks et al., 1993), under control of a constitutive promoter.
  • the present invention provides a male-fertile maintainer line of common or durum wheat which in itself is maintained by selfing (Figs. 2 - 5), said maintainer line being isogenic to the female parent and homozygous for any one of the ms-B1 male-sterility alleles of the female parent, and having an alien chromosomal arm (4S S S) as a monotelosomic addition, herein referred to as AA, carrying the Ms-S s 1 male-fertility allele, a heterologous microspore-suicide gene (Msu), under control of a microspore- specific promoter.
  • AA alien chromosomal arm
  • the selectable marker by which plants having this chromosome can be selected is a specific herbicide-resistance gene conferring resistance to specific herbicide such as glufosinate (basta), controlled by a constitutive promoter (Figs. 2 and 4).
  • specific herbicide such as glufosinate (basta)
  • Figs. 2 and 4 a constitutive promoter
  • the alien chromosomal arm does not pair during meiosis and consequently, enters only to about 20% of the gametes, either male or female.
  • the amount of seeds containing the AA chromosomal arm among the progeny of the selfed maintainer is about 20%.
  • the selectable marker is a heterologous seed-color gene (Sc) coding for an anthocyanin in the aleurone layer of the endosperm resulting in blue- grain coloring (Figs. 3 and 5).
  • the blue seeds containing the AA chromosomal arm can be separated from the red or white seeds lacking the AA chromosomal arm by a seed sorter.
  • the blue seeds when grown, developing into male-fertile maintainer plants, and the red or white seeds, when grown, developing into male-sterile female plants.
  • the present invention further provides an alternative male-fertile maintainer line of common or durum wheat (Figs.
  • Ms-S s 1 male-fertility allele and three heterologous genes a microspore-suicide gene (Msu), controlled by a microspore- specific promoter, a seedling-killer gene (Ski) coding for a protein that binds an essential factor thus causing a depletion in this essential factor and eventually death of the cell containing it, such as streptavidin gene coding for a protein that binds biotin, and a selectable marker gene by which plants having this chromosomal arm can be selected, such as a specific herbicide-resistance gene (Hr) controlled by a constitutive promoter or a seed-color gene (Sc) controlled by an
  • Treating the mixed progeny seeds of the selfed maintainer with biotin will allow also the seedlings carrying the AA chromosomal arm to develop, resulting in a mixture of plants of which 20% developing into male-fertile maintainer and 80% developing into male- sterile female plants.
  • the selectable marker by which plants having this chromosome arm can be selected, is a heterologous herbicide-resistance gene (Hr)
  • spraying the mixture with the specific herbicide will kill all the female plants and leave only the maintainer plants to grow (Fig. 4b).
  • the selectable marker by which plants having this chromosome arm can be selected is a heterologous seed-color gene (Sc) such as gene coding for an anthocyanin that is accumulated in the aleurone layer resulting in blue coloring of the grain
  • the mixture of the maintainer progeny seeds will consist of blue and red or white seeds. Separating the seeds by a seed sorter yields 80% red or white seeds, when grown, developing into male-sterile female plants, and 20% blue seeds, when grown, developing into male-fertile maintainer plants (Fig. 5b).
  • the present invention further provides an additional alternative male-fertile maintainer line of common or durum wheat which in itself is maintained by selfing, said maintainer line being isogenic to the female parent and homozygous for any one of the ms-B1 male-sterility alleles of the female parent, and having the AA alien chromosomal arm, as a monotelosomic addition, carrying the Ms-S s 1 male-fertility allele and four heterologous genes, a microspore-suicide gene (Msu), controlled by a microspore- specific promoter, a seedling-killer gene (Ski) coding for a toxic protein that kills the cells such as a gene that codes for RNase or RIP, controlled by a seedling-specific promoter, an anti seedling-killer gene (Ski inhibitor) coding for an antisense RNA or other types of RNA, protein or peptides that inhibit the Ski gene, or coding for biotin, controlled by an inducible promoter, and a select
  • Treating the mixed progeny of the selfed maintainer with an inducing chemical activates the promoter of the Ski-inhibitor gene resulting in the activity of the Ski-inhibitor gene thus inhibiting the seedling-killer gene or its effect, and consequently, also the seedlings carrying the AA chromosomal arm germinate and grow, resulting in a mixture of plants of which 20% developing into male- fertile maintainer and 80% developing into male-sterile female plants.
  • the selectable marker by which plants having this chromosome arm can be selected, is a heterologous herbicide-resistance gene (Hr)
  • spraying the mixture with the specific herbicide will kill all the female plants and leave only the maintainer plants to grow.
  • the selectable marker by which plants having this chromosome arm can be selected is a heterologous seed-color gene (Sc) such as gene coding for an anthocyanin that is accumulated in the aleurone layer resulting in blue coloring of the grain
  • the mixture of the maintainer progeny seeds will consist of blue and red or white seeds. Separating the seeds by a seed sorter yields 80% red or white seeds, when grown, developing into male-sterile female plants, and 20% blue seeds, when grown, developing into male-fertile maintainer plants.
  • Yet another aspect of the present invention is a method for producing hybrid plants of common or durum wheat, comprising:
  • the Fi hybrid seeds produced in this way are all male-fertile.
  • the female line is propagated either by pollination with the maintainer line or by planting the progeny of the selfed maintainer line, and the maintainer line is itself maintained by selfing, and the desired male-fertile plants among the progeny of the selfed maintainer are selected each generation by the use of a selectable marker characterizing the maintainer.
  • male-sterility recessive mutant alleles ms-B1ms-B1
  • Three such male-sterility alleles have been described (review in Wilson and Driscoll, 1983).
  • the EMS-treated mutants can be distinguished from the various deletions of the Ms-B1 locus by the presence in these mutants of a terminal C-band on 4BS.
  • the DNA marker Xpsr921 which is located on the distal region of 4BS, is absent in ms-B1-c and in several of our gamma-irradiated mutants (and possibly also in ms-B1-b) (our unpublished data), i.e., it is located in the deleted segment and its absence can mark homozygosity for the deletion.
  • the maintainer line is of the same cultivar as the female line, i.e., cv. 'One', is homozygous for the same ms-B1 allele present in the female line but has the AA alien chromosomal arm as a monotelosomic addition (Figs. 2 and 3), consisting of 4S S S of Aegilops searsii, that carries the dominant male-fertility allele Ms-S s 1, and the heterologous streptavidin gene coding for streptavidin which binds specifically biotin causing biotin depletion and consequently, cell death, controlled by a microspore- specific promoter.
  • the male-fertile offspring of the maintainer are either resistant to glufosinate while the male-sterile offspring are susceptible (Fig. 2), or the color of the seeds which, when grown, developing into male-fertile plants, is blue while that of the other seeds, when grown, developing into male-sterile plants, is red or white.
  • This differential reaction to glufosinate or the difference in seed color facilitates the selection of the male-fertile offspring, thus growing only the maintainer line.
  • the male parent (cv. 'Two') is any normal common or durum wheat cultivar, which by its nature is homozygous for the male-fertility Ms-B1 allele.
  • hybrid seeds of common or durum wheat are readily and efficiently produced as the Fi progeny, all of which are heterozygous for the male-sterility alleles (Ms-B1ms-B1) and therefore, are male-fertile. So far, all cultivars that were used as male parents were able to fully restore the male fertility of the Fi hybrid.
  • Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S S S carrying the dominant male-fertility allele Ms-S s 1, are transfected with the heterologous Streptavidin and Bar genes, the first is linked to a microspore-specific promoter and the second to a plant constitutive promoter.
  • the transgenic plants thus obtained are screened and those carrying the two heterologous genes on the alien chromosomal arm are selected and analyzed for their expression. Those plants with the desirable heterologous gene expression are selected.
  • Example 4 Example 4:
  • Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S S S carrying the dominant male-fertility allele Ms-S s 1, are transfected with the heterologous Streptavidin and seed-color (Sc) genes, the first is linked to a microspore-specific promoter and the second to an endosperm- specific promoter.
  • the transgenic plants thus obtained are screened and those carrying the two heterologous genes on the alien chromosomal arm are selected and analyzed for heterologous gene expression. Those plants with the desirable heterologous gene expression are selected.
  • Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S S S carrying the dominant male-fertility allele Ms-S s 1, are transfected with the three heterologous genes, BaRNase, controlled by a microspore-specific promoter, Streptavidin, controlled by a seedling-specific promoter, and Bar, controlled by a plant constitutive promoter.
  • the transgenic plants thus obtained are screened and those carrying the three heterologous genes on the alien chromosomal arm are selected and analyzed for gene expression. Those plants with the desirable heterologous gene expression are selected.
  • Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S S S carrying the dominant male-fertility allele Ms-S s 1, are transfected with the three heterologous genes, BaRNase, controlled by a microspore-specific promoter, Streptavidin, controlled by a seedling-specific promoter, and seed-color (Sc) gene controlled by an endosperm-specific promoter.
  • the transgenic plants thus obtained are screened and those carrying the three heterologous genes on the alien chromosomal arm are selected and analyzed for gene expression. Those plants with the desirable heterologous gene expression are selected.
  • Plants homozygous for the male-sterility recessive allele ms-B1-c and ditelosomic addition for the alien chromosomal arm 4S S S carrying the dominant male-fertility allele Ms-S s 1, are transfected with the four heterologous genes, Streptavidin, controlled by a microspore-specific promoter, BaRNase, controlled by a seedling-specific promoter, Barstar, controlled by an inducible promoter, and Bar, controlled by a plant constitutive promoter.
  • the transgenic plants thus obtained are screened and those carrying the four heterologous genes on the alien chromosomal arm are selected and analyzed for gene expression. Those plants with the desirable heterologous gene expression are selected.
  • the progeny seed from the cross were all heterozygous ms-B1-c Ms-B1 and had the alien chromosome arm as monotelosomic addition.
  • the seeds were germinated and the plants developed from them were male fertile. Selfing of these plants yielded, among other combinations, about 1% of plants homozygous for the male-sterility ms-B1-c allele and the alien chromosome arm as ditelosomic addition line. This is the suitable material for transformation.
  • the transmission of the male-sterility and male-fertility alleles was studied in several genetic combinations during several years and under greenhouse and field conditions. While the transmission of the ms-B1-c allele through the female gametes is as expected, i.e., 50%, the transmission through the male gametes is somewhat lower, i.e., 40-45%. This difference stems from the fact that the ms-B1-c allele is located within a small deletion.
  • the transmission of the alien chromosome arm 4S S S when present in two doses, as in the ditelosomic line, is almost normal, i.e., 95-98%.
  • the transmission of this arm when present in a single dose, as in monotelosomic line is only around 20%.

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Abstract

L'invention concerne un procédé permettant de maintenir stable une ligne parentale génique mâle-femelle stérile de blé à pain et de blé dur pour la production de blé hybride. Elle concerne également une ligne mâle-femelle stérile, homozygote pour un allèle à stérilité mâle récessif, et une ligne de conservation capable de se propager facilement et de manière stable. La ligne de conservation est isogénique vis-à-vis de la ligne femelle, et comprend un bras chromosomique étranger, ajouté au complément blé, porteur d'un allèle à fertilité mâle dominante qui restaure la fertilité à la ligne de conservation, un gène microspore suicide hétérologue capable de tuer des microspores ou des graines de pollen qui le portent, empêchant ainsi la transmission de ce bras chromosome à la ligne femelle, ainsi qu'un marqueur sélectionnable hétérologue qui facilite la sélection des plants de conservation parmi la progénie de la ligne autoentretenue et, par conséquent, le maintien de la ligne de conservation elle-même.
PCT/US2002/000549 2001-01-04 2002-01-02 Procede permettant de maintenir une ligne parentale genique male-femelle sterile pour la production de ble hybride WO2002052924A2 (fr)

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US7910802B2 (en) 2007-08-03 2011-03-22 Pioneer Hi-Bred International, Inc. MSCA1 nucleotide sequences impacting plant male fertility and method of using same
US7915478B2 (en) 2007-08-03 2011-03-29 Pioneer Hi-Bred International, Inc. Msca1 nucleotide sequences impacting plant male fertility and method of using same
US7919676B2 (en) 2007-08-03 2011-04-05 Pioneer Hi-Bred International, Inc. Msca1 nucleotide sequences impacting plant male fertility and method of using same
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CN108782226A (zh) * 2018-07-02 2018-11-13 周口师范学院 一种小麦不育系的创制方法及应用
WO2019043082A1 (fr) * 2017-08-29 2019-03-07 Kws Saat Se Aleurone bleue améliorée et autres systèmes de ségrégation
CN112899303A (zh) * 2021-02-08 2021-06-04 南京农业大学 一种利用胚乳特异性自杀获得非转基因的定向基因突变植株的方法及其应用
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WO2002052924A3 (fr) 2003-02-20
EP1347678A2 (fr) 2003-10-01
EP1347678A4 (fr) 2004-08-18

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