Multicellular living organisms and unmodified parts thereof and – Method of using a plant or plant part in a breeding process... – Method of breeding using gametophyte control
Reexamination Certificate
1997-01-09
2001-09-18
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of using a plant or plant part in a breeding process...
Method of breeding using gametophyte control
C800S286000, C800S287000, C800S288000, C800S303000, C435S069100, C435S199000, C435S468000, C536S024500
Reexamination Certificate
active
06291741
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the production of plants having one or more desired phenotypic traits. In particular embodiments, the phenotypic trait is male sterility, and so the invention relates in those embodiments to male sterile plants, which are useful in hybrid seed production, and to the production of such plants.
Various routes for high level expression of foreign genes in plant cells have been explored over the years. Increasing levels of transcription using strong promoters have been obtained; promoters available include DNA virus promoters (such as p35S from CaMV; Fang et al,
The Plant Cell
1 141-150 (1989)), plant gene promoters (such as EFE1&agr;; Curie et al,
Plant Molecular Biology
18 183-1089 (1992)) and bacterial promoters (such as pMAS; Fox et al,
Plant Molecular Biology
20 219-233 (1992)). These promoters give high level of transcription and expression in many tissues (including leaves, stems, roots, flowers and seeds) of the plant. They are sometimes called constitutive promoters. Other promoters confer better expression in particular tissues (for example, the MAS promoter transcribes genes more efficiently in wounded tissues).
For certain applications, the expression of a gene is desired only in particular tissues or at a particular period of the plant development; for example to increase the level of expression of a heterologous protein, it is useful to express the gene specifically in seeds, as seeds are the principal organ for protein storage in plants. Expression of genes in certain tissues or organs of the plant or at a precise stage of plant development generally requires tissue-specific promoters. Seed-specific promoters have been characterised (for example, the promoter of the &bgr;-conglycine gene: Chen et al,
The EMBO Journal
7 297-302 (1988) and Calgene's EP-A-0255378), as have root-specific promoters (such as the
Agrobacterium tumefaciens
roll promoter: Leach and Aoyagi
Plant Science
79 69-76 (1991)).
Seed-specific promoters have been used to express pharmaceutical proteins in plant seeds (Vandekerckhove et al,
Bio/Technology
7 929-932 (1989)). Stable expression of a gene under the control of seed-specific promoters has been obtained, and heterologous proteins were expressed, only in seeds, at a high expression level. Plants recovered from those seeds expressed the gene in their seeds, and the phenotype was transmitted from progeny to progeny.
In the production of industrially or pharmaceutically useful proteins, or other heterologous proteins, under the control of constitutive or seed-specific promoters in plants, F1 seeds will be sold to farmers to grow the plants and to produce seeds which will contain the protein of interest. In this system of production, the F1 line which expresses the gene of interest is grown by farmers and can be multiplied or reproduced by growers themselves or by competitors. Moreover, if the plant breeding industry should become required by law to control the distribution of all seeds sold, the fact that the above production process does not prevent the dissemination of genetically modified plants becomes a very significant issue.
From the above, it is apparent that there is a need for hybrid plants which
(i) have a desired phenotypic trait as a result of the expression of a certain gene (or suite of genes), and
(ii) are the result of a cross between two parents, neither of which have, either at all or to a substantial degree, the phenotypic trait in question.
However, the provision of such plants is not the whole extent of the problem, as there are some phenotypic traits which result from the non-expression of a gene (or suite of genes. One trait which may be (but is not necessarily) the result of non-expression of a gene is male sterility.
Hybrid seed production involves the cross of two different plants. Because most crops are able to self-pollinate, the female parent in the cross must be prevented from self-pollinating (“selfing”) so that it will yield 100% of hybrid seeds. This has been achieved in several different ways:
(a) by mechanically removing or chemically inactivating the pollen-producing organs of the female parent before they reach maturity; this method has been used for example in maize (corn) and tomato;
(b) by using cytoplasmic male sterile (CMS) mutant plants; this method has been used for example in oilseed rape and sunflower;
(c) by using a recessive nuclear male sterile mutant plant; and
(d) by using a dominant nuclear male sterile genetically engineered plant (artificial male sterility or AMS) as described for example in Mariani et al,
Nature
347 737-741 (1990) or in Worrall et al,
The Plant Cell
4 759-771 (1992).
There are practical difficulties with each of the above. Mechanical male sterilisation ((a) above) is easy and flexible. But it is labour intensive, costly and also prone to human error, giving a problem of quality of hybrid seeds batches. It is practical only for species where the flower is big enough to be emasculated manually; it is not practical, for example, for most cereals. An attempt to overcome this difficulty and reduce costs is by using chemical instead of mechanical emasculation. The efficiency of this technique is very dependent on environment conditions at the time of spraying the gametocide, and leads the seed producer to take a considerable risk each season. The cost of the gametocide and the spraying is also significant.
Cytoplasmic male sterility ((b) above) is very convenient, and allow easy maintenance of the female line. But its use is limited by availability of the appropriate mutant plant in each species of interest. The loss of cytoplasmic genetic diversity when all breeders use the same cytoplasm in their breeding program can be a serious problem as seen in the U.S. on maize in the 1970s. And the maintenance of sterility relies on the existence of a maintaining cytoplasm.
Recessive nuclear male sterility ((c) above) is not practical. Because the male sterility gene is recessive, maintenance of the male sterile line involves screening the ¼ of male sterile plants out of ¾ fertile in the selfed progeny of an heterozygous plants. In the absence of a tightly linked selectable or easily screenable marker this is practically impossible. This is the problem that artificial male sterility has tried to solve.
Artificial male sterility ((d) above) has solved the problems of the other systems but only to some extent. The AMS gene system is potentially universal, being limited only to genetically transformable species. It does not rely on the existence of a mutant as in CMS. The maintenance of the male sterile line is obtained by engineering a dominant male sterility gene linked to a marker gene that allows selection of AMS plants in a population segregating ½ AMS plants. To be practical, this marker is often a herbicide resistance gene. But this process is undesirable for several reasons:
Agronomy: the seed production can be affected by the need to eliminate half the female plants (the fertile segregants) from the field. The result is a heterogenous plant density, and potentially lower yield and lower quality of the seeds. The cost of spraying the herbicide is significant.
Use of the herbicide: on some crops, such as vegetables, the use of a herbicide resistance gene, and the subsequent use by farmers of the herbicide is not desirable. Generally speaking, there is a tendency to restrict the use of herbicide as much as possible.
The female line can be stolen easily. Once sold on distant and not easily controllable markets, the basic seeds can be multiplied by the local seed producer without the breeder's knowledge, control or share of the profit.
Disadvantages with a conventional AMS system can be appreciated by looking at a particular example. The AMS system which is the subject of EP-A-0344029 (Plant Genetic Systems (PGS)) is based on the tissue-specific expression of a toxin gene (Ribonuclease, “Barnase”) to generate the male-sterile parent for hybrid seed production. The genetic linkage of the toxi
Betzner Andreas
Huttner Eric
Lenee Phillipe
Paul Wyatt
Perez Pascual
Cooper & Dunham LLP
Fox David T.
Gene Shears Pty. Limited
White John P.
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