Method to obtain male sterile plants

Details Method to obtain male sterile plants Method to obtain male sterile plants

1999-12-10

2002-02-05

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

C800S278000, C800S285000, C800S286000, C800S287000, C800S290000, C800S306000, C800S320100, C800S320200, C435S419000, C435S468000, C435S412000

Reexamination Certificate

active

06344602

ABSTRACT:

BACKGROUND OF THE INVENTION
(i) Field of the Invention
The present invention relates to an improved method to obtain male-sterile plants using foreign male-sterility genes that comprise plant promoters that direct expression of a male-sterility DNA in stamen cells, and to plants obtained by the method.
(ii) Description of Related Art
In many, if not most plant species, the development of hybrid cultivars is highly desired because of their generally increased productivity due to heterosis: the superiority of performance of hybrid individuals compared with their parents (see e.g. Fehr, 1987, Principles of cultivar development, Volume 1: Theory and Techniques MacMillan Publishing Company, New York; Allard, 1960, Principles of Plant Breeding, John Wiley and Sons, Inc.).
The development of hybrid cultivars of various plant species depends upon the capability to achieve almost complete cross-pollination between parents. This is most simply achieved by rendering one of the parent lines male sterile (i.e., bringing them in a condition so that pollen is absent or nonfunctional) either manually, by removing the anthers, or genetically by using, in the one parent, cytoplasmic or nuclear genes that prevent anther and/or pollen development (for a review of the genetics of male sterility in plants see Kaul, 1988, ‘Male Sterility in Higher Plants’, Springer Verlag).
For hybrid plants where the seed is the harvested product (e.g., corn, oilseed rape) it is in most cases also necessary to ensure that fertility of the hybrid plants is fully restored. In systems in which the male sterility is under genetic control this requires the existence and use of genes that can restore male fertility. The development of hybrid cultivars is mainly dependent on the availability of suitable and effective sterility and restorer genes.
Endogenous nuclear loci are known for most plant species that may contain genotypes which effect male sterility, and generally, such loci need to be homozygous for particular recessive alleles in order to result in a male-sterile phenotype. The presence of a dominant ‘male fertile’ allele at such loci results in male fertility.
Recently it has been shown that male sterility can be induced in a plant by providing the genome of the plant with a chimeric male-sterility gene comprising a DNA sequence (or male-sterility DNA) coding, for example, for a cytotoxic product (such as an RNase) and under the control of a promoter which is predominantly active in selected tissue of the male reproductive organs. In this regard stamen-specific promoters, such as the promoter of the TA29 gene of Nicotiana tabacum, have been shown to be particularly useful for this purpose (Mariani et al., 1990, Nature 347:737, European patent publication (“EP”) 0,344,029). By providing the nuclear genome of the plant with such a male-sterility gene, an artificial male-sterility is locus is created containing the artificial male-sterility genotype that results in a male-sterile plant Various stamen-specific promoters have been described (see e.g. WO 92/13956, WO 92/13957).
In addition it has been shown that male fertility can be restored to the plant with a chimeric fertility-restorer gene comprising another DNA sequence (or fertility-restorer DNA) that codes, for example, for a protein that inhibits the activity of the cytotoxic product or otherwise prevents the cytotoxic product to be active in the plant cells (EP 0,412,911). For example the barnase gene of
Bacillus amyloliguefaciens
codes for an RNase, the barnase, which can be inhibited by a protein, the barstar, that is encoded by the barstar gene of
B. amyloliguefaciens.
The barnase gene can be used for the construction of a sterility gene while the barstar gene can be used for the construction of a fertility-restorer gene. Experiments in different plant species, e.g., oilseed rape, have shown that a chimeric barstar gene can fully restore the male fertility of male sterile lines in which the male sterility was due to the presence of a chimeric barnase gene (EP 0,412,911, Mariani et al., 1991, Proceedings of the CCIRC Rapeseed Congress, Jul. 9-11, 1991, Saskatoon, Saskatchewan, Canada; Mariani et al., 1992, Nature 357:384). By coupling a marker gene, such as a dominant herbicide resistance gene (for example the bar gene coding for phosphinothricin acetyl transferase (PAT) that converts the herbicidal phosphinothricin to a nontoxic compound [De Block et al., 1987, EMBO J. 6:2513]), to the chimeric male-sterility and/or ferility-restorer gene, breeding systems can be implemented e.g., to select for uniform populations of male sterile plants (EP 0,344,029; EP 0,412,911).
Barnase is an etracellular ribonuclease produced by
Bacillus amyloliguefaciens.
Barstar is an inhibitor of barnase that is produced intracellularly by the same bacterium to protect it from the toxic effects of the intracellular barnase activity (Hartley, 1989, TIBS, 14:450-454). Initial attempts to clone the barnase gene in
E.coli
and
B.subtilis
under control of its own or another bacterial promoter were unsuccessful as the produced barnase proved to be toxic to the host cells. When the barnase gene was reconstructed from previously cloned parts on the same plasmid as the barstar gene, the lethal effects of barnase expression were suppressed (Hartley, 1988, J.Mol. Biol. 202:913-915).
Whenever bamase is cloned in a bacterial host cell, such as
E.coli,
it may be useful to have the barstar gene, under control of its native or another bacterial promoter, present in the host cell to prevent possible harmful effects of undesired bamase expression. Paul et al, 1992, Plant Mol. Biol. 19:611-622 for instance, constructed a chimeric bamase gene under control of a tapetum specific promoter of the A9 gene of Arabidopsis. Plasmids pWP127 and pWP128 contain a DNA fragment encoding barstar and the mature bamase cloned between the 1437 bp A9 promoter fragment and a CaMV polyadenylation sequence. The promoter and coding sequence of barstar were included on these plasmids since mature bamase could not be cloned in its absence in
E.coli.
As indicated above bamase DNA has been used to induce male-sterility in plants. However, other uses of bamase have also been described. WO 92/21757 describes inter alia a plant transfonrmed with a nematode-induced chimaeric gene comprising the following operably linked DNA sequences:
a nematode-induced promoter that is suitable to direct transcription of a foreign DNA substantially selectively in specific root cells, preferably in the cells of fixed-feeding sites of the plant and,
a first foreign DNA that encodes bamase; and which also contains a restorer chimaeric gene, preferably in the same genetic locus as the nematode-induced chimaeric gene, comprising the following operably linked DNA sequences:
a second promoter, such as a nematode-repressed promoter, which can direct transcription of a second foreign DNA in cells of the plant where the first foreign DNA is expressed, preferably substantially selectively in cells other than the specific root cells, preferably in cells other than the fixed feeding site cells, of the plant, and,
a second foreign DNA that encodes barstar. WO 93/19188 describes inter alia a plant transformed with a fungus-responsive chimaeric gene comprising the following operably linked DNA sequences:
a fungus-responsive promoter that is suitable to direct transcription of a foreign DNA substantially selectively in cells of a plant surrounding, preferably immediately surrounding, a site of infection of the plant by a fungus; and,
a first foreign DNA that encodes bamase; and which also contains a restorer chimaeric gene, preferably in the same genetic locus as the fungus-responsive chimaeric gene, comprising the following operably linked DNA sequences:
a second promoter, such as a constitutive promoter (e.g., 35S), which can direct transcription of a second foreign DNA in cells of the plant other than those surrounding, preferably in at least cells of the plant other than those immediately surrounding, said fungus infection site; and,

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