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
1999-08-05
2003-09-09
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 involving a genotypic or phenotypic marker
C800S269000, C800S320000, C800S320100, C435S006120
Reexamination Certificate
active
06617492
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the fields of molecular genetics and plant breeding. More particularly, it relates to a means of moving novel, stably inherited, variant forms of DNA into maize (
Zea mays L
.), also referred to as corn in the United States. These novel DNA sequences, derived from intergeneric hybridization between Eastern gamagrass (
Tripsacum dactyloides L
.) and perennial teosinte (
Zea diploperennis Iltis
, Doebley and Guzmán), provide unique markers for assisting selection of desirable traits in plant breeding programs, for detection of target DNA sequences in genetic analyses, and for the identification of new genes for corn improvement that may enhance resistance to insect pests and diseases, drought stress tolerance, cold tolerance, perennialism, grain yield, totipotency, apomixis, improved root systems, tolerance of water-logged soils, tolerance of high-aluminum and acidic soils, improved grain quality, enhanced forage quality, and adaptability to a CO2 enriched atmosphere.
BACKGROUND OF THE INVENTION
Molecular Genetics. Genetics is the study of genes and heritable traits in biological organisms. In plant breeding, the goal of molecular genetics is to identify genes that confer desired traits to crop plants, and to use molecular markers (DNA signposts that are closely associated with specific genes) to identify individuals that carry the gene or genes of interest in plants (Morris 1998), to determine the DNA sequences and characterize gene expression and function. A genetic marker is a variant allele that is used to label a biological structure or process throughout the course of an experiment. Variants in DNA and proteins are used as markers in molecular genetics. Genetic analysis of molecular variants can identify a particular gene that is important for a biological process. Mutation is the process whereby nucleotide sequences and genes change from the reference form generally designated wild type to a different form, and mutants are the source of variant genotypes in genetic analysis that allow selection of new phenotypes (Griffiths et al. 1993). Mutations occur at the level of a specified nucleotide sequence, the gene (i.e. DNA sequence), or the chromosome (i.e. the hereditary package in which units of DNA containing specific nucleotide sequences and genes are supercoiled with proteins). In a genetic mutation, the nucleotides that comprise the wild type allele of a gene (i.e. reference form that exists at a particular locus) is altered. In chromosome mutations, segments of chromosomes, whole chromosomes, or entire sets of chromosomes change via inversion, translocation, fusions and deletions.
In general, mutations are very rare, and most newly formed mutations are deleterious. Data on mutation frequencies for seven genes in maize provides a baseline indicating the rarity of mutations in maize (Stadler 1951). Mutation frequency ranged from 0.000492% (i.e. 492 mutants out of a million gametes) in the red color(R) gene; 0.000106% (i.e. 106 out of a million) for the inhibitor of R (I) gene; 0.000011% (i.e. 11 out of a million) for the purple aleurone (Pr) gene; 0.0000024% (i.e. 2.4 out of a million) for the starchy (Su) gene; 0.0000022% (i.e. 2.2 out of a million) for the yellow color (Y) gene; 0.0000012% (i.e. 1.2 out of a million) for the normal kernel (Sh) gene, and 0% (i.e. 0 out of a million for the waxy gene (Wx).
Because spontaneous mutations are rare, geneticists and plant breeders typically use mutagens (i.e. agents such as chemicals and radiation to increase the frequency of mutation rates) to obtain variant forms that can be used in genetic analysis and selection of new varieties. Another method of inducing mutagenesis in maize is transposon tagging whereby a maize line is crossed with a line containing one of the three systems of transposable elements found in maize. When a transposable element inserts into a gene, it causes a mutation. The reported mutation frequencies for transposable element mutator lines varies from 1 in a thousand to 1 in a million (Chomet 1994). To find a mutation using one of these mutagenic lines, a breeder must screen a minimum of 100,000 progeny.
Plant Breeding. Conventional plant breeding is the science that utilizes crosses between individuals with different genetic constitutions. The resulting recombination of genes between different lines, families, species, or genera produces new hybrids from which desirable traits are selected. Plant breeding is achieved by controlling reproduction. Since maize is a sexually reproducing plant, techniques for controlled pollination are frequently employed to obtain new hybrids. Controlling reproduction in maize involves continually repeating two basic procedures: (1) evaluating a series of genotypes, and (2) self-pollinating or crossing among the most superior plants to obtain the next generation of genotypes or progeny. Controlled pollinations in maize utilize two procedures: (1) detasseling, and (2) hand pollination.
Maize is a monoecious grass that has separate male and female flowers on the same plant. The male or staminate flowers produce pollen in the tassel at the apex of the maize stalk, and the female or pistillate flowers that produce the grain when pollinated are borne laterally in leaf axils tangential to the stalk. Pollination is accomplished by transfer of pollen from the tassel to silks which emerge from the axillary pistillate ears. Since maize is wind-pollinated, controlled pollination in which pollen collected from the tassel of one plant and transferred by hand to the silks of the same or another plant, is a technique used in maize breeding. The steps involved in making controlled crosses and self-pollinations in maize are standard practice (Neuffer 1982) and are as follows: (1) the ear emerging from the leaf shoot is covered with an ear shoot bag one or two days before the silks emerge to prevent contamination by stray pollen; (2) prior to making a pollination, the ear shoot bag is quickly removed and the silks cut with a knife to form a short brush, then the bag is immediately placed back over the ear; (3) also prior to making a pollination, the tassel is covered with a tassel bag to collect pollen; (3) on the day crosses are made, the tassel bag with the desired pollen is carried to the plant for crossing, the ear shoot bag is removed and the pollen dusted on the silk brush, the tassel bag is then fastened in place over the pollinated shoot to protect the developing ear.
Zea diploperennis
(hereafter referred to as diploperennis), is a diploid perennial teosinte and a wild relative of maize endemic to the mountains of Jalisco, Mexico. Diploperennis is in the same genus as maize, has the same chromosome number (2n=20), and can hybridize naturally with it.
Tripsacum is a polyploid, rhizomatous perennial grass that is a more distant wild relative of maize and has a different chromosome number (x=18, 2n=36 or 2n=72). Tripsacum is not know to naturally form fertile hybrids with maize or the wild Zeas. The progeny of (maize X Tripsacum) obtained by artificial methods have ten maize chromosomes and either 18 or 36 Tripsacum chromosomes and are male sterile. Female fertility can be partially restored using special techniques that eliminate most of the Tripsacum chromosomes (Mangelsdorf 1974). Plants obtained by crossing Tripsacum and maize (
Zea mays L
.) employing Tripsacum as the pollen donor have unreduced gametes with a complete set of Zea chromosomes and a complete set of Tripsacum chromosomes. There is one report of a successful reciprocal cross in which Tripsacum was pollinated by maize that required embryo culture techniques to bring the embryo to maturity, and the plants were sterile (Farquharson 1957). Maize-Tripsacum hybrids have been crossed with teosinte to created a trigenomic hybrid that has a total of 38 chromosomes; 10 from maize, 18 from Tripsacum and 10 from teosinte. The resulting trigenomic plants were all male sterile and had a high degree of female infertility (Mangelsdorf 1974; Galina
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