Callase-related DNAs and their use in artificial male sterility

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

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800287, 800298, 800300, 536 236, 536 241, 435200, 435209, 4353201, 435418, 435419, 435468, A01H 500, C12N 1529, C12N 1556, C12N 1582

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059556536

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BRIEF SUMMARY
BACKGROUND OF THE INVENTION

This invention relates to recombinant, isolated and other synthetic DNA useful in male-sterility systems for plants. In particular, the invention relates to restorable male-sterility systems. Male-sterile plants are useful for the production of hybrid plants by sexual hybridisation.
Hybrid plants have the advantages of higher yield and better disease resistance than their parents, because of heterosis or hybrid vigour. Crop uniformity is another advantage of hybrid plants when the parents are extensively homozygous; this leads to improved crop management. Hybrid seed is therefore commercially important and sells at a premium price.
Producing a hybrid plant entails ensuring that the female parent does not self-fertilise. There have been many prior proposals, mechanical, chemical and genetic, for preventing self-pollination. Among the genetic methods is the use of anther-specific genes or their promoters to disrupt the normal production of pollen grains. An anther-specific promoter, for example, can be used to drive a "male-sterility DNA" at the appropriate time and in the right place. Male sterility DNAs include those coding for lytic enzymes, including those that lyse proteins, nucleic acids and carbohydrates. Glucanases are enzymes which break down carbohydrates.
In EP-A-0344029 (Plant Genetic Systems (PGS)) and WO-A-9211379 (Nickerson International Seed Company Limited) glucanase-coding DNA features among possible malesterility DNAs. Although many plant glucanases have been characterised and the genes cloned in some cases (eg defence-related "PR" glucanases), to date no glucanase with properties consistent with a role in microspore release has been reported. Microspore release is the process by which the immature microspores are liberated from a protective coat of .beta.(1,3) poly-glucan (callose) laid down by the microsporogenous cells before meiosis (Rowley, Grana Palynol., 2, 3-31 (1959); and Heslop-Harrison, Can. J. Bot. 46, 1185-1191(1968) and New Phytol., 67, 779-786 (1968)). The anther-expressed glucanase responsible for the dissolution of this callose coat is known as callase. Callase is synthesised by the cells of the tapetum and secreted into the locule. The appearance of the enzyme activity is developmentally regulated to coincide precisely with a specific stage of microspore development.
The basis of the use of a glucanase as a sterility DNA lies in the fact that mis-timing of the appearance of callase activity is associated with certain types of male-sterility (Warmke and Overman, J. Hered. 63 103-108 (1972)). Two types are recognised depending on whether the appearance of glucanase activity is premature or late. Since both types are found in nature, one important attraction of glucanase as a potential sterility DNA is that it already occurs in a natural system. Although plants that fail to produce active callase have not been described in nature, mutants of this type almost certainly occur. Failure to produce callase would prevent microspore-release, thereby causing pollen abortion and male-sterility. So, preventing callase expression would form the basis of a male-sterility system.
Several studies suggest that callase is probably different from other types of glucanases, such as the "PR" glucanases. For example, callase activity may be subject to both transcriptional and post-transcriptional control. This is suggested by the fact that there is a strong relationship between locule pH, callase activity, and the timing of microspore release (Izhar and Frankel, Theor. and Appl. Genet. 41, 104-108 (1971)). Locule pH and callase activity change coordinately in a developmentally regulated manner. In fertile Petunia hybrida anthers, the pH during meiosis is 6.8-7.0 and callase activity is undetectable. Following meiosis, at the tetrad stage, the locule pH drops in a precipitous fashion to 5.9-6.2 and callase activity increases sharply resulting in microspore release.
In certain male-sterile Petunia strains, the drop in pH and the appearance of callase activity are prec

REFERENCES:
Paul, W., et al., "Aspects of the molecular biology of anther development," J. Exp. Bot. . Annual Meeting of the Society for Experimental Biology, Birmingham, AL, Apr. 7-12, 1991, vol. 42, 1991, 238 Suppl. p. 40.
Scott, R., et al. "Identification of genes exhibiting cell-specific and temporal regulation in developing anthers ofBrassica-napus," J. Exp. Botany, 1990 Annual Meeting of the Society for Experimental Biology, vol. 41, 1990 suppl., p. P5-3.
Barghchi, M., et al., "Genetic engineering of Arabidopsis," Abstracts VIIth International Congress on Plant Tissues and Cell Culture, 1990, Jun. 24-29, Amsterdam, p. 46, Abstract No. A2-10.
Scott, R. et al., "Patterns of gene expression in developing anthers of Brassica napus," Plant Molecular Biology, vol. 17, No. 2, 1991, pp. 195-207.
Del Campillo, E. et al., "Cell wall hydrolases in anther and abscission zones," Plant Phisiology Supplement, Annual Meeting American Society of Plant Physiologists, Jul. 29-Aug. 2, 1990, vol. 93, No. 1, May 1990, Abstract No. 771.
Hodge, R. P. et al., "A9--a tapetum specific gene," J. Expermental Botany, Annual Meeting of the Society for Experimental Biology, Birmingham, AL, Apr. 7-12, 1991, vol. 238, 1991, Suppl. p. 40.
Mascavenhas, J. 1989. Mol. Basis Plant Dev., Goldberg, R.,ed., Alan R. Liss, Inc.: New York, pp. 99-105.
Worrall et al 1992 (Jul.) The Plant Cell 4:759-771.
Krabel et al 1993 Plant Science 93:19-23.

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