Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-01-04
2002-11-12
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S274000, C800S278000, C800S286000, C800S287000, C800S288000, C800S306000, C800S309000, C800S312000, C800S313000, C800S314000, C800S316000, C800S317100, C800S317200, C800S317400, C800S320000, C800S320100, C800S320200, C800S320300, C800S322000, C435S069700, C435S069800, C435S069900, C435S320100, C536S023400
Reexamination Certificate
active
06479735
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to hybrid nucleic acid sequences, comprising at least the coding region of an unedited mitochondrial gene from higher plants and allowing the control of male fertility in plants containing the said sequences, to the transgenic plants having such sequences, as well as to a method for producing transgenic male-sterile plants and to a method for restoring male-fertile plants.
BACKGROUND OF THE INVENTION
The control of male fertility in plants is one of the key problems for obtaining hybrids, and more particularly male-sterile lines which are of agronomic interest especially for controlling and improving seeds. Indeed, the large scale production of hybrid seeds with controlled characteristics is a real challenge since many crops have both male and female reproductive organs (stamens and pistils). This causes a high rate of self-pollination and makes difficult the control of crossings between lines for obtaining the desired hybrids.
In order to allow non-inbred crossings to be obtained which make it possible to produce hybrid seeds having advantageous properties, the inventors have developed new transgenic male-sterile plants capable of being restored and which facilitate the development of hybrid crops.
Cytoplasmic male sterility (MCS) is characterized by non-formation of the pollen after meiosis.
In alloplasmic systems, MCS is due to a nucleus-cytoplasm incompatibility which may occur at several levels: replication of DNA, transcription of genes, maturation of transcripts, translation or assembly of multiprotein complexes.
From the observations made on maize and petunia (Dewey R. E. et al., Cell, 1986, 44, 439; Young E. G. et al., Cell. 1987, 50, 41), comes the hypothesis that MCS is due to a deficiency in the mitochondrial bioenergetic machinery. Indeed, MCS manifests itself by a reduction in the ATP and NADP levels. At the cellular level, this deficiency is correlated with degeneration of the cells of the anther lawn, while having no effect on the development of the plant.
A number of methods have been proposed in the prior art for obtaining male-sterile plants.
There may be mentioned especially the backcrossings which lead to the substitution of the nuclear genome of a species by another genome and this, in the cytoplasmic environment of the first species (alloplasmy); this substitution may also appear spontaneously in field crops. MCS can also be obtained by protoplast fusion (Lonsdale D. M., Genetic Engineering, 1987, 6, 47).
In all these situations, the results are not reliable or reproducible; furthermore, in all cases, the manipulations are long, tedious and often difficult to control.
Male-sterile plants have also been obtained by transgenosis, with the aid of a gene encoding an RNAse, under the control of an anther-specific promoter (Mariani C. et al., Nature, 1990, 347, 737). This transgene, when expressed, has a toxic effect on the cell insofar as the endogenos RNAs are degraded, thereby causing cell death.
Another system, which also introduces a new artificial and destructive function, has been described by Worrall D. et al., (The Plant Cell, 1992, 4, 759-771) (callase system) and has the same disadvantages as the RNAse system.
Other methodologies have also been proposed forobtaining male-sterile plants; there may be mentioned especially the techniques which take advantage of the disruption of certain metabolic pathways (Van de Meer I. M. et al., The Plant Cell, 1992, 4, 253-262) (expression of a chalcone synthase antisense gene) or the techniques involving asymmetric somatic hybridization (Melchers C. et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 6832-6836) to bring into contact, as in alloplasmic male-sterile lines, the cytoplasm of a donor individual and the nucleus of a recipient partner. The latter two processes have the major disadvantage of being highly unpredictable as regards the desired objective, namely the obtaining of male-sterile plants which makes it possible to control reproduction in these plants.
SUMMARY OF THE INVENTION
The Applicant consequently set itself the objective of obtaining transgenic male-sterile plants in a controlled, reliable and reproducible manner which are capable of being used in agronomic programmes of seed improvement.
The subject of the present invention is transgenic plants having in their nuclei an expressible hybrid sequence (transgene) comprising at least one coding region of an unedited mitochondrial gene from higher plants and a sequence capable of transferring the protein expressed by the said coding region to the mitochondrion, which plants are characterized in that:
the coding regions of the unedited mitochondrial genes are chosen from among the genes encoding a protein of the ATP synthase complex which are chosen from among the wheat ATP9 gene fragment, of the following formula I:
(Seq ID No: 7)
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT
GCT ACA ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT
GGA AAC GTC CTC AGT TCT TTG ATT CAT TCC GTG GCG
CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT
GCC ATT TTG GGC TTT GCT CTC ACC GAA GCT ATT GCA
TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC
GTT TTC CGA TCG CAT AAA AAG TCA TGA
or the ATP6 gene, or from among the genes encoding a protein of the respiratory chain which are chosen from among the genes for subunits 1 to 7 of NAD dehydrogenase, the gene for apocytochrome b and the genes for subunits I, II or III of cytochrome oxidase and
the sequence capable of transferring the said expressed protein to the mitochondrion is selected from the group consisting of the fragments encoding yeast tryptophanyl tRNA synthetase (SCHMITZ, U. K. et al., 1989, The Plant Cell, 1, 783-791), and the .beta. subunit of Nicotiana plumbaginifolia ATPase (BOUTRY et al., 1987, Nature, 328:340-342), and the maize ATP/ADP translocator (BATHGATE et al., 1989, Eur. J. Biochem., 183:303-310) or a 303 base pair EcoRI/KpnI fragment including codons 1 to 62 of subunit IV of yeast cytochrome oxidase (MAARSE et al., 1984, EMBO J., 3, 2831-2837),
which hybrid sequence is capable of modifying male fertility in plants having incorporated the said transgene while not modifying the other phenotypic characteristics of the said plants.
REFERENCES:
patent: 5914447 (1999-06-01), Araya et al.
Handa et al. FEBS Letters 310(2): 111-114, Sep. 1992.*
“Novel Recombination in the Maize Mitochondrial Genome Produce a Unique Transcriptional Unit in the Texas Male-Sterile Cytoplasm,” by R.E. Dewey et al.,Cell, vol. 44, Feb. 14, 1986, p. 439-449.
“A Fused Mitochondrial Gene Associated with Cytoplasmic Male Sterility is Developmentally Regulated,” by Ellora G. Young et al.,Cell, vol. 50, Jul. 3, 1987, p. 41-49.
“The Molecular Biology and Genetic Manipulation of the Cytoplasm of Higher Plants,” by David M. Lonsdale, Molecular Genetics Department, Cambridge, United Kingdom, p. 47-102.
“Induction of Male Sterility in Plants by a Chimaeric Ribonuclease Gene,” by Celestina Mariani et al.,Nature, vol. 347, Oct. 25, 1990, p. 737-741.
“Premature Dissolution of the Microsporocyte Callose Wall Causes Male Sterility in Transgenic Tobacco,” by Dawn Worrall et al.,The Plant Cell, vol. 4, Jul. 1992, p. 759-771.
“Antisense Inhibition of Flavonoid Biosynthesis in Petunia Anthers Results in Male Sterility,” by Ingrid M. Van Der Meer et al.,The Plant Cell, vol. 4, Mar. 1992, p. 253-262.
“One Step Generation of Cytoplasmic Male Sterility by Fusion of Mitochondrial-Inactivated Tomato Protoplasts with Nuclear-Inactivated Solanum Protoplasts,” by Georg Melchers et al.,Proc. Natl. Acad. Sci. USA, Aug. 1992, p. 6832-6836.
“A Yeast Mitochondrial Presequence Functions as a Signal for Targeting to Plant Mitochondria in Vivo,” by Udo K. Schmitz et al.,The Plant Cell, vol. 1, Aug. 1989, p. 783-791.
“Targeting of Bacterial Chloramphenicol Acetyltransferase to Mitochondria in Transgenic Plants,” by Marc Boutry et al.,Nature, vol. 328, Jul. 23, 1987, p. 340-342.
“Two Genes Encode the Adenine Nucleotide Translocator of Maize Mitochondria,” by Brian Bathgate et al.,Eur. J. Biochem, vol. 183, 1989, p. 303-
Araya Alejandro
Mouras Armand
Centre National de la Recherche Scientifique "CNRS"
Fox David T.
Lowe Hauptman & Gilman & Berner LLP
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