Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
2000-05-22
2002-05-21
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C536S023600, C536S024100
Reexamination Certificate
active
06392123
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the production of hybrid seed in plants. In particular, the invention relates to a method of hybrid seed production using as one parent of the hybrid a plant transformed with a chimeric gene which when expressed in the female reproductive structures yields a protein that catalyzes the conversion of an exogenously-applied protoxin to a toxin, thereby rendering fertilization ineffective. Also included in the invention is the use of the conditional female-sterile plants in combination with conditional male-sterile plants to more efficiently produce hybrid seed, chimeric genes useful for the invention, transgenic plants comprising the chimeric genes, and novel promoters useful for expression in the female reproductive structures of a plant.
BACKGROUND OF THE INVENTION
Heterosis in crop plants has received considerable attention because of its marked effect on yield improvement. The increased productivity on crossing different strains of maize was first noted in the late 19th century and was then developed according to systematic genetic procedures. The usual method for raising hybrid maize is to establish many inbred lines, make intercrosses, and determine which hybrids are more productive in a given locality.
The success of hybrid maize motivated plant breeders to explore the existence and magnitude of hybrid vigor in many other species with economic importance. In general, hybrids exhibit increase yields in comparison to non-hybrid varieties. Hybrids are usually more efficient in use of growth factors and give a greater return per unit for the growth factors such as water and fertilizer. Under stress hybrids are generally superior to parental cultivars, with a more stable performance over a wide range of environments. With hybrids, there is uniformity in product and maturity that often facilitates harvest and increases the value of the product in the marketplace. The hybrid may also combine characters that are difficult or impossible to combine in other ways. This is particularly true of many interspecific and intergeneric hybrids. The general conclusion from research is that hybrid vigor, a common phenomenon in plants, is of sufficient magnitude to warrant commercial exploitation if appropriate techniques can be devised.
Hybrid vigor has been recognized as a wide-spread phenomenon in plants and animals for many years. Commercial hybrids are now used extensively in many crops, including maize, sorghum, sugar beet, and sunflower. Other large acreage crops such as wheat, barley and rice are still primarily grown as inbred varieties. Research is being conducted on these and other crops that may permit the wide-spread use of commercial hybrids in the future, but the primary limiting factor in new hybrid crop development is the lack of economical hybrid seed production methods in these crops.
Traditionally, large-scale hybrid seed production is accomplished by planting separate rows or blocks of female parent lines and the male parent lines to pollinate them. Only the seed produced on the female parent rows is harvested. To ensure that this seed is hybrid seed uncontaminated with selfed seed, pollination control methods must be implemented on the female parent plants to ensure that seeds formed on them result from cross-pollination and not self-pollination. Known pollination control mechanisms are generally mechanical, chemical, or genetic.
Elimination of fertile pollen from the female parent can be achieved by hand emasculation in some species such as maize, a monoecious species. Such elimination of fertile pollen is achieved by pulling or cutting the male inflorescence (tassel) from plants in the female parent population. This simple procedure prevents self-fertilization by mechanically detasseling female plants before pollen shed to prevent selfing. However, most major crop plants of interest have functional male and female organs within the same flower making emasculation impractical. Even where practical, this form of hybrid seed production is extremely labor intensive and hence expensive. To eliminate the laborious detasseling that is necessary to prevent self-fertilization in hybrid crosses, genetic factors which produce male-sterility have been used in some species.
Male-sterility in the female parent can be controlled by nuclear genes or by a cytoplasmic-genetic system. Genetic male-sterility is controlled by nuclear genes in which the alleles for sterility generally are recessive to the alleles for fertility. Genetic male-sterility occurs in many species. Usually, it is controlled by a single recessive gene that must be homozygous to cause male-sterility. Breeders who use genetic male-sterility for hybrid seed production usually develop a phenotypically uniform female line that segregates 50% male-sterile and 50% male-fertile individuals. Seed for these lines is increased in isolation by harvesting seed from plants homozygous for the male-sterility gene that are pollinated by plants heterozygous for the male-sterility gene, and hence male-fertile. To produce commercial hybrid seed with genetic male-sterility, the 50 percent of male-fertile female plants must be rogued from the field as soon as their fertility can be identified. The labor associated with roguing fertile plants from female plants has greatly restricted the use of genetic male-sterility in producing hybrid seed. There are several problems associated with this system for producing commercial hybrid seed. First, it is not possible to eliminate fertile plants from the desired male-sterile plants in the female population. Genetic male-sterile plants must be maintained by mating them with male-fertile individuals. Half of the F
1
plants from such a cross would be sterile, but the remaining plants would be fertile. Thus, the unwanted male-fertile plants in the female population may disseminate pollen and reduce the effectiveness of the desired male parent.
The successful use of cytoplasmic male-sterility for commercial hybrid seed production requires a stable male-sterile cytoplasm, an adequate pollen source, and an effective system of getting the pollen from the male parent to the male-sterile female. Also, the cytoplasmic-genetic system of male sterility requires three lines to produce a single crossed hybrid; the A line (male-sterile), B line (male-fertile maintainer), and R line (male-fertile with restorer genes). Three-way crosses produced with cytoplasmic-genetic male sterility involved maintenance and production of four lines, an A and B line of one inbred and male-fertile inbreds of the other two.
Furthermore, the southern maize blight caused by
Helminthosporium maydis,
Race T, which severely attacked all maize hybrids with cytoplasmic male-sterile T cytoplasm, demonstrated the vulnerability of a hybrid seed production industry based on a single source of male-sterile cytoplasm. For hybrid maize, most seed producers have returned to hand or mechanical emasculation and wind pollination.
Hybrid seed may also be produced by the use of chemicals that block or kill viable pollen formation. These chemicals, called gametocides, are used to impart a transitory male-sterility. However, the expense and availability of the chemicals and the reliability of the applications limits the production of hybrid seed through the use of gametocides.
Molecular methods for hybrid seed production have also been described. Such methods transform plants with constructs containing anti-sense DNA and other genes which are capable of controlling the production of fertile pollen into plants. Such regenerated plants are functionally male-sterile and are used for the production of hybrid seed by crossing with pollen from male-fertile plants. The primary deficiencies of these approaches stem from the fact that the genetically engineered male sterility gene, whether it is an anti-sense or RNAse, can only be maintained in a heterozygous state. They are fundamentally the same as natural genetic male steriles in that they must be maintained by crossing to isogenic male fertile lines. T
Crossland Lyle Dean
Harper Stacy Marie
Pascal Erica
Fox David T.
Lebel Edouard G.
Meigs J. Timothy
Pascal Erica
Syngenta Participations (AG)
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