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
2000-05-17
2003-10-21
McElwain, Elezabeth F. (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, C800S290000, C800S298000, C800S320000, C435S069100, C435S320100, C435S468000, C536S023600
Reexamination Certificate
active
06635811
ABSTRACT:
TECHNICAL FIELD
The present invention relates to materials and methods which may be used in the detection and manipulation of Pre-Harvest Sprouting (PHS) and other dormancy-related phenotypes in plants. The present invention also relates to materials and methods for use in plant breeding—in particular to molecular-biology based methods for generating, identifying, characterising or manipulating genetic variation which affects the PHS and other dormancy related traits.
PRIOR ART
Pre-Harvest Sprouting (PHS)
Pre-Harvest Sprouting (PHS) of non-dormant grains is a major limiting factor in achieving consistent bread making quality of UK wheat. Average annual losses due to PHS in the UK wheat crop have been estimated at some £17 million, but the problem is erratic and is much more severe in cool, damp seasons. Variation in the degree of sprouting damage from year to year makes this problem difficult to select against in conventional breeding programmes.
VP1 in Maize and Other Species
Previous work in other plant species has shown that the VIVIPAROUS 1 gene is a major regulator of embryo maturation in maize. Thus McCarty et al (1989) in The Plant Cell 1, 523-532 disclosed that vp1 mutants in maize were abscisic acid (ABA) insensitive, and demonstrated its role in controlling the developmental responses associated with the maturation phase of seed formation. VP1 mutants were shown to germinate precociously. Similarly McCarty et al (1991) in Cell 55, 895-905 disclosed that VP1 encoded 73 kDa transcription factor.
Giraudat et al (1992) in The Plant Cell 4, 1251-1261 showed that Arabidopsis ABI3 mutants had altered seed development & germination. The predicted gene product was similar to VP1 protein. These results and others show that ABI 3/VP1 function as developmental regulators during the maturation stage of embryogenesis by regulating transcription of sets of genes that determine the embryonic phenotype in preparation for desiccation of the seed prior to shedding.
A VP1 homolog in rice has also been isolated (Hattori et al (1994) Plant Molecular Biology 24, 805-810). Similarly in Abstract, Poster No P184 “Poaceae sequence analysis: cloning of a VP-1 homolog from genomic barley DNA via PCR”, at the Plant and Animal Genome V Conference in San Diego, USA, January 1997, Wilson & Sorrells disclosed the use of conserved primers to pick out vp1 homologs in Barley.
Wilson speculated that a wheat VP1 homolog may be obtainable by comparison with maize, rice and barley sequences, and (on the basis of comparison with these species) that the R locus may contain a wheat VP1 homolog.
Interestingly, earlier work by Cadle et al (1994) in Genome 37, 129-132 had already shown that the maize vp1 from McCarty didn't hybridise strongly to wheat DNA and could not therefore be used as a probe to map the wheat gene, although various ABA-inducible genes were mapped successfully.
Dormancy
Other recent studies of the genetics of the transition from embryogenesis to germination in maize and Arabidopsis show that mutation of GA (gibberellic acid) and ABA synthesis and sensitivity can alter dormancy levels (Koornneef and Karssen, 1994). For example, whereas the Arabidopsis mutation ga 1 causes a loss of germination due to GA deficiency, aba/abi mutations (that affect ABA synthesis and perception respectively) cause a loss of dormancy (and in strong alleles, loss of viability) because embryos fail to develop desiccation tolerance during maturation (for example alleles of abi 3, Ooms et al., 1993).
It has been suggested that PHS in wheat is the result of the lack of induction of dormancy during embryo development (Gale and Lenton 1987). As is known to those skilled in the art, dormancy is one of two possible developmental states which mature seeds may show following desiccation and shedding (the other being germination). Embryo dormancy develops during late embryogenesis, and results in a lack of germination. Following imbibition of the mature shed seed it results in an inactive phase of plant growth during which development is deferred, although the embryo still maintains a high metabolic activity. Dormancy of mature imbibed seeds occurs even under environmental conditions that would favour germination, indicating that the process is not simply a lack of correct conditions. During dormancy, cells within the mature embryo are maintained in an arrested state, and nuclear DNA values obtained from A. fatua embryos indicate that the cell-cycle is held in G1 and DNA replication does not occur during imbibed dormancy (Elder and Osborne 1993). Dormancy is probably an evolutionary strategy that allows survival of seeds through adverse conditions, and dispersal of seed germination through time. Dormancy is therefore a very important phase of plant development required both for the inhibition of germination prior to completion of embryogenesis, and for the pre-germinative survival of mature seeds. It is also an important agronomic trait, with the market value of wheat being determined, inter alia, by its Hagberg Falling Number, which measures the degree to which some germination-related processes have progressed (discussed in relation to plant breeding hereinafter).
Seeds of the persistent weed A. fatua can show very high levels of embryo dormancy (Simpson 1978).
Embryos with primary dormancy go through a time and environment sensitive process of after-ripening in the dry seed, that is manifested by loss of dormancy in the imbibed seed (Mayer and Poljakoff-Mayber 1989). Dormancy can subsequently be reimposed on after-ripened dry embryos under specific environmental conditions (‘induced’ or ‘secondary’ dormancy). These features indicate that signals perceived by the dry seed influence developmental choices following imbibition, resulting in either dormancy or germination (Hilhorst and Karssen 1992). Recent work on the water status of embryos of A. fatua has demonstrated that individual enzymatic and non-enzymatic reactions, rather than metabolic processes control this dormancy
on-dormancy switch in the dry seed (Foley 1994). Others have proposed that the process may involve kinase-phosphatase interactions (Trewavas 1987).
Many studies have analysed the genetic control of embryo dormancy (Hilhorst and Karssen 1992). Results obtained from experiments with inbred lines of A. fatua have suggested that in this species dormancy may be controlled by three loci, two that promote dormancy (L1 and L2), and one that promotes after-ripening (E) (Jana et al., 1979,
Jana et al. 1988). These dormancy genes have not been cloned or characterised, their existence was inferred from statistical analysis of segregation for dormancy phenotypes among the progeny from a cross between two different strains.
There is currently a requirement for materials and methods which have utility in the identification or molecular tagging of the genes responsible for PHS in wheat, or which could be used in the manipulation of the PHS trait in wheat or other plants.
Thus it can be seen that the provision of such materials or methods would provide a contribution to the art.
DISCLOSURE OF THE INVENTION
The present inventors have for the first time identified a gene from the oat Avena fatua which encodes a protein which has a high degree of similarity to known VP1/ABI 3 related transcription factors. The expression product of this gene has been termed afVP1.
By studying imbibed mature seeds, the expression of afVP1 has been correlated with the dormant phenotype (primary dormancy, secondary dormancy and after-ripening) in oat. In particular, the present inventors have demonstrated that wild oat has the potential for extremely high levels of dormancy in the mature dry seed, and that expression of afVP 1 is absolutely correlated to the dormant phenotype in imbibed mature seeds. This is the first demonstration of expression of a VP 1-homologue in a developmental situation other than embryogenesis. It indicates that afVP1 activity keeps mature seeds dormant, and inhibits germination—it can thus be used to maintain or impose sufficient intensity and duration of
Flintham John Ellis
Gale Michael Denis
Holdsworth Michael John
Lenton John R.
Baum Stuart F.
Dann Dorfman Herrell and Skillman
McElwain Elezabeth F.
Plant Bioscience Limited
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