Multicellular living organisms and unmodified parts thereof and – Method of using a plant or plant part in a breeding process... – Method of breeding involving a genotypic or phenotypic marker
Reexamination Certificate
1998-06-19
2004-06-15
Nelson, Amy J. (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 involving a genotypic or phenotypic marker
C800S271000, C800S272000, C800S275000
Reexamination Certificate
active
06750377
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of producing herbicide resistant plants. This method is useful in the production of hybrids as well as in the production of transgenic plants generally.
BACKGROUND OF THE INVENTION
Incorporation of a gene which confers resistance to the herbicides such as glyphosate into plants is well known (see. for example Gasser et al., Recent Adv—Phytochem, New York, Plenum Press 1988 22, p45-59). Such plants, in particular corn plants, are available on the market, for example from DeKalb Genetics. In the case of glyphosate resistance, the plants include a gene which brings about a glyphosate-resistant, GA21, which is based on the gene encoding an altered an altered active site enzyme.
For example diploid plants may be homozygous (termed RR where R is glyphosate resistance) or heterozygous Rr for glyphosate resistance, depending upon their method of production and lineage.
When used in plant breeding programmes, heterozygous plants will result in the production of a significant proportion of plants which do not possess the herbicide resistance trait. For example, when dealing with glyphosate resistant plants, this may be represented diagrammatically by:
Rr*Rr>RR+
2
Rr+rr
R=the transgene and r=azygous wildtype
Where one of the parents does not carry the resistance trait, even more of the progeny will lack the trait i.e.
rr*Rr>rR+rr
This means that heterozygous plants cannot normally be used as gene donors in the production of commercial seed for farmers togrow, because much of the seed would lack the trait, and desired levels of purity would not be achieved.
However, the use of heterozygous plants could actually be preferred to the use of homozygotes in seed production. This is because the phenomenon of gene silencing, in which transformation events become unstable or stop functioning after several generations, occurs more frequently in homozygous plants.
There is a need for a process whereby the yield of plants demonstrating a desired trait, such as herbicide resistance, is increased from breeding programmes which include heterozygous plants.
SUMMARY OF THE INVENTION
The applicants have found that by applying a herbicide at a particular stage of plant development, maintenance of the herbicide resistance in the pollen and therefore in the progeny can be enhanced.
The invention provides a method of producing a plant which shows resistance to a herbicide, said method comprising
(i) applying said herbicide to a population of herbicide resistant plants at an advanced vegetative stage before flowering,
(ii) using pollen from said plants to fertilise female plants; and
(iii) obtaining progeny therefrom.
The method of the invention may be applied to plants which are resistant to various herbicides. In particular it may be applied to non-selective herbicides in which the method of plant resistance to the herbicide is achieved through a modified endogenous gene which produces a plant that is unaffected by the presence of the herbicide.
A particular herbicide for use in the method of the invention is glyphosate and salts thereof such as those sold as Roundup™ and Touchdown™. Other herbicides which may be used in this way are Acetolactate Synthase (ALS) and (AHAS)inhibitors (sold as Pursuit™ and Sceptre™), where the herbicide resistant mechanism of the plant is alteration of the active site as described above.
The invention is particularly applicable where the plants are crop plants such as grasses particularly corn.
What constitutes a suitable “advanced vegetative stage” may vary from plant to plant, depending upon the pollen development. This may be determined in each case using routine test methods. However in general, it will comprise a stage equivalent to V5 or above, for example up to V16 in corn. A description of these growth stages in corn is given by S. W. Ritchie et al., “How a Corn Plant Develops. Rev. Feb. 1982” Special Report (Iowa State University of Science and Technology, Cooperative Extension Service, No. 48) 1982, 21 p: col.ill;, the content of which is incorporated herein by reference.
The invention is based upon the statistically significant deviance of observed segregation from expected segregation that observed in series of experiments. The glyphosate resistance allele, designated as R, behaves in a dominant fashion. Therefore, plants either homozygous R (i.e. RR) or heterozygous R (i.e. Rr) will be resistant to foliar applications of glyphosate. Starting from a known allelic composition, the observed segregation can be statistically compared with the expected segregation by using a Chi-Square test, a readily accepted statistical tool for comparing the deviation of observed from expected values. It was found that, using this method of analysis, the segregation varied in a statistically significant manner.
Specifically, it has been observed that a corn plant which is heterozygous for the Glyphosate-resistance event GA21 behaves as if it were RR when used as a pollen parent in a cross to another corn plant Specifically, it has been observed that a corn plant which is heterozygous for the Glyphosate-resistance event GA21 behaves as if it were RR when used as a pollen parent in a cross to another corn plant and when it has first been sprayed with glyphosate at a relatively advanced vegetative stage, but before flowering. Conversely, if the Rr plant is not sprayed with glyphosate, or if the spray is delivered at an early vegetative growth stage, the plant behaves normally, and contributes both R and r pollen to the cross.
Diagrammatically:
With glyphosate
rr*Rr>Rr
only
Without glyphosate, or glyphosate early
rr*Rr>rR+rr
Similar effects are not seen with female gametes, or seed set.
Without being limited by any theory or mode of actions, it is thought that residual herbicide within the plant kills, or inactivates, r pollen during development. This is consistent with the known mode of action of the herbicide glyphosate, which is translocated readily in the plant and which will accumulate at sites of high metabolic activity (sinks) such as the developing tassel and anthers. It is noteworthy also that GA21 is an alternative-enzyme method of protection, not a herbicide degradation system, so GA21 itself should not affect the distribution and persistence of the herbicide in the plant.
The method of the invention can be used in several ways. In one way, it may be used to improve the purity of inbred and hybrid seed production (especially herbicides resistant crops, by preventing the production of pollen lacking the desired genes.
Normal standards for seed production allow 2-4% offtypes in hybrid seed. However, if the crop contains a herbicide resistance trait, and the trait is not present in the offtypes, these will be killed when the farmer uses the herbicide for weed control. Death of these plants can cause farmer concern and, theoretically at least, could somewhat reduce yield (to the extent that the offtypes, had they survived, would have set grain). This has become a significant issue in the USA during the last couple of years.
One possible source of offtypes occurs when the male parent is used as the trait donor in a production field, and (using glyphosate as an example) contains either rr or Rr genotypes as well as the desired homozygote, RR. Obviously, we can use glyphosate itself to eliminate rr from the male rows, so they are not a problem. But, because the trait is dominant, the herbicide will not eliminate the Rr plants. However, using a late vegetative spray of pollen as described above will eliminate the r pollen from these heterozygotes, so they will behave as RR. Hence, one source of susceptible offtypes will be prevented.
Thus in a further aspect, the invention provides, in the production of herbicide resistant hybrid seed, a method of reducing the numbers of herbicide susceptible offtypes in a population of herbicide resistant hybrids, said method comprising spraying male parent plants with said herbicide at late vegetative stage of growth such that any hetero
Hawkins Alan F.
Huang Tzao Fen
Kaster, Jr. Larry V.
Reysack James J.
Advanta Technology Ltd.
Kruse David H
Nelson Amy J.
Rewaldt Dana S.
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