Hybrid seed production

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

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C800S274000, C800S278000, C800S287000, C800S303000, C800S306000, C800S312000, C800S314000, C800S317400, C800S320000, C800S320100, C800S320200, C800S320300, C800S322000, C435S468000

Reexamination Certificate

active

06683230

ABSTRACT:

This application is a 371 of PCT/GB99/00238 filed Jan. 22, 1998.
FIELD OF THE INVENTION
The present invention relates to methods of preparing hybrid seed.
In particular, the present invention relates to the molecular control of sterility in crop plants. Such male and female sterility in plants can be used in the preparation of hybrid seed from crops which are naturally self-pollinators.
The present invention also provides for a method of restoring fertility in the parent plants to allow self-pollination, thereby allowing the maintenance of the parental lines.
The present invention further relates to expression cassettes for incorporation into plants and to the use of such expression cassettes in a male/female sterility restorer system.
Hybrid plants grown from hybrid seed benefit from the heterotic effects of crossing two distinct genetic backgrounds. The production of hybrid seed depends on the ability to control self-pollination and ensure cross-pollination of male and female parent plants.
A number of methods are available to control pollen fertility. For example, in the case of maize, which has separate male and female flowers, control of pollen fertility is achieved by physically removing the male inflorescence or tassel, prior to pollen shed, thus preventing self-pollination.
Most major crops, however, have both functional male and female reproductive organs within the same flower. In this instance, removal of the pollen producing organs is very labour intensive and expensive. The use of chemicals (gametocides), particularly in wheat, maize (corn) and rice, to kill or block pollen production produces transitory male sterility but the use of such chemicals is expensive. The reliability of the chemicals and their length of action are also issues.
There is considerable interest in developing systems of pollen control based on genetic mechanisms producing male sterility. There are two general types: a) nuclear male sterility caused by the failure of pollen production due to one or more nuclear genes and b) cytoplasmic male sterility (CMS) in which pollen production is blocked because of a defect in a gene in the mitochondria.
Currently available nuclear systems are based on the introduction of a male sterility trait to one parent plant followed by the introduction of a fertility restoration gene as a result of cross-pollination with another plant to produce fertile hybrid plants. The Paladin system, which is described in WO96/01799, is different and is based on the separation during hybrid seed production of genes which, when expressed together in one plant, have a cytotoxic effect leading to male sterility.
Rice and wheat are self-pollinating plants and have small hermaphrodite flowers and so the detasseling approach taken for hybrid seed production in maize is not applicable. Manual removal of anthers is difficult and time consuming. Moreover, wheat pollen is relatively heavy and is viable only for a short time, rarely remaining viable for longer than 30 minutes. The technique of planting used in hybrid corn production i.e. planting the male parent in a block physically separated from the female parent (the male sterile) and allowing wind pollination does not, therefore, work well in wheat or rice. The male and female parents for these crops have to be interplanted to ensure cross pollination. As hybrid seed needs to comprise more than 95% hybrids, it is necessary to remove seed arising from self-pollination of the male parent or to make the male parent incapable of self-fertilisation and therefore incapable of producing non-hybrid seed. Clearly, the interplanting of the parent plants means that the first option is difficult unless the male plants are susceptible to some chemical treatment to which the female parent is tolerant e.g. herbicide treatment.
Our International Patent Application No. PCT/GB90/00110 describes a cascade of gene sequences which expresses a protein which disrupts the biosynthesis of viable pollen in a female parent plant. In this case, however, only one of the parent plants i.e. the female parent is sterile to minimise self-pollination of the female plant and this female plant is crossed with a fertile male parent plant to yield fertile hybrid seed. There is no description in the literature, however, of a method of producing hybrid seed wherein both parent plants are unable to self-pollinate.
SUMMARY OF THE INVENTION
The present invention relates to two methods by which hybrid seed may be produced which seeks to overcome the problems presently associated with the production of hybrid seed, particularly with the production of hybrid wheat and rice seed.
According to a first aspect of the present invention, there is provided a method of preparing hybrid seed comprising interplanting a male parent plant which is male fertile and homozygous recessive female sterile and a female parent plant which is homozygous recessive male sterile and female fertile, allowing cross-pollination and obtaining seed produced therefrom.
According to a second aspect of the present invention, there is provided the use of the above method to produce hybrid seed.
According to a third aspect of the present invention, there is provided fertile plants produced by the aforementioned method.
According to a fourth aspect of the present invention there is provided the progeny of the aforementioned plants, the seeds of such plants and such progeny.
According to a fifth aspect of the present invention there is provided an expression cassette comprising:
(a) a first gene promoter sequence which is a male flower specific promoter sequence;
(b) a disrupter gene encoding a product capable of disrupting male fertility operably linked to the first gene promoter sequence;
(c) a second gene promoter sequence which is a female flower specific promoter sequence optionally operably linked to one or more translational enhancer or intron sequences;
(d) a restorer gene encoding a product capable of restoring female fertility operably linked to the second gene promoter sequence;
(e) a third gene promoter sequence responsive to the presence of an exogenous chemical inducer optionally operably linked to one or more translational enhancer or intron sequences; and
(f) a restorer gene encoding a product capable of restoring male fertility operably linked to the third gene promoter sequence;
whereby the presence of the exogenous chemical inducer controls male fertility.
According to a sixth aspect of the present invention there is provided an expression cassette comprising:
(a) a first gene promoter sequence which is a female flower specific promoter sequence;
(b) a disrupter gene encoding a product capable of disrupting female fertility;
(c) a second gene promoter sequence which is a male flower specific promoter sequence optionally operably linked to one or more translational enhancer or intron sequences;
(d) a restorer gene encoding a product capable of restoring male fertility operably linked to the second gene promoter sequence;
(e) a third gene promoter sequence responsive to the presence or absence of an exogenous chemical inducer optionally operably linked to one or more translational enhancer or intron sequences; and
(f) a restorer gene encoding a product capable of restoring female sterility operably linked to the third gene promoter sequence;
whereby the presence of the exogenous chemical inducer controls female fertility.
According to a seventh aspect of the present invention there is provided a further method of producing hybrid seed comprising incorporating a first expression system according to the fifth aspect of the present invention into a first plant to generate a hemizygous female parent plant and incorporating a second expression system according to the sixth aspect of the present invention into a second plant to generate a hemizygous male parent plant;
applying an exogenous chemical inducer to the transformants thereby allowing the plants to self-pollinate;
growing up plants from the resulting seed;
selecting for male and female homozygous plants;
crossing the selected male and female

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