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
1994-12-16
2002-05-28
Benzion, Gary (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
C800S288000, C800S265000, C800S267000, C800S320100
Reexamination Certificate
active
06395966
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the fields of plants, such as maize, and to improved methods of plant breeding. More particularly, it provides methods of increasing yield in plants by introducing a gene encoding phosphinothricin acetyltransferase. The increased yield phenotype may be transferred to other lines of plants by crossing.
DESCRIPTION OF THE RELATED ART
Ever since the human species emerged from the hunting-gathering phase of its existence, and entered an agricultural phase, a major goal of human ingenuity and invention has been to improve crop yield and to alter and improve the characteristics of plants. In particular, man has sought to alter the characteristics of plants to make them more tasty and/or nutritious, to produce increased crop yield or render plants more adaptable to specific environments.
Up until recent times, crop arid plant improvements depended on selective breeding of plants with desirable characteristics. Initial breeding success was probably accidental, resulting from observation of a plant with desirable characteristics, and use of that plant to propagate the next generation. However, because such plants had within them heterogenous genetic complements, it was unlikely that progeny identical to the parent(s) with the desirable traits would emerge. Nonetheless, advances in controlled breeding have resulted from both increasing knowledge of the mechanisms operative in hereditary transmission, and by empirical observations of results of making various parental plant crosses.
Attempts to improve commercially important traits in plants, for example, grain yield in corn and wheat, have consumed the energies of commercial plant breeders in the twentieth century. Clever and sophisticated breeding schemes have been devised, yet the rate of improvement of economically important characters has been only a few to several percent of the mean per year for the past several decades. For various crop plants, it has been established that roughly half of this improvement is due to improved husbandry practices, i.e., environmental effects rather than genetic changes effected by selection. (Lande & Thompson, 1990).
The record available from the crude crop breeding programs of the late nineteenth century through the present is littered with dead ends—failures, for one reason or another. For example, data on the ineffectiveness of mass selection for several corn ear characters as presented by Williams and Welton in 1915 are reproduced and discussed by Sprague & Eberhart (1977). Selection for long and short ears was not effective in separating the population into two distinct subpopulations defined by ear length. Yield, one of the most commercially valuable traits, has been the least responsive to selective breeding programs. Selection from 1907-1914 had no overall effect on yield. An examination of data on corn yield trials published by study stations in Illinois from 1860 to 1900 shows that many corn varieties were included for short test periods, then discarded because of poor yielding ability. (Sprague & Eberhart, 1977). This article refers to a report that visual selection practiced during inbreeding had little, if any, direct influence on yield in hybrid combinations. However, selection was effective for some other traits, e.g., maturity. Recurrent selection was somewhat more effective in improving breeding populations.
These failures to substantially alter plant characteristics are costly. Even the successes with recurrent selection may generally be described as incremental and long range improvements rather than mercurial saltatory jumps. Divergence of corn varieties for oil and protein content of grain was achieved if results over the 70 year history of a long-term study in Illinois are considered. However, improvement in yield has been less dramatic. Over the past 60 years, increases in yield due to genetic improvement have averaged only about one bushel/acre/year (Hallauer et al., 1988). Only a small population of hybrid plants produced commercially ever show enough improvement to be worth marketing. World-wide needs for plant derived food, both for animals and humans, warrant improved strategies. Plants are also finding uses in non-food products necessitating increased production. New methods are necessary for more efficient and successful plant breeding programs than are currently available.
Recent advances in molecular biology have expanded man's ability to manipulate the germplasm of animals and plants. Genes controlling specific phenotypes, for example specific polypeptides that lend antibiotic or herbicide resistance, have been located within certain germplasm and isolated from it. Even more important has been the ability to take the genes which have been isolated from one organism and to introduce them into another organism. This transformation may be accomplished even where the recipient organism is from a different phylum, genus or species from that which donated the gene (heterologous transformation).
Attempts have been made to genetically engineer desired traits into plant genomes by introduction of exogenous genes. These techniques have been successfully applied in some plant systems, principally in dicotyledonous species. The uptake of new DNA by recipient plant cells has been accomplished by various means, including Agrobacterium infection (Nester et al., 1984), polyethylene glycol (PEG)-mediated DNA uptake (Lorz et al., 1985), electroporation of protoplasts (Fromm et al., 1986) and microprojectile bombardment (Klein et al., 1987). Unfortunately, the introduction of exogenous DNA into monocotyledonous species and subsequent regeneration of transformed plants has proven much more difficult than transformation and regeneration in dicotyledonous plants. However, techniques are now available for transformation of barley (Wan & Lemaux, 1994), wheat (Weeks et al., 1993), corn (Gordon-Kamm et al., 1990), and sorghum (Casas et al., 1993). The availability of these transformation techniques suggests that it may now be possible to address improvement of agronomic performance of a crop plant through the techniques of genetic engineering.
With the advent of genetic engineering techniques that are new opportunities for increasing yield in crops. One of the goals of this technology is to introduce genes into a crop that will increase yield and/or stabilize yield across multiple environments. However, no genetic elements that contribute to important agronomic characteristics, such as yield, have been identified and introduced into crops using the techniques of genetic engineering.
The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicides bialaphos or phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., 1986; Twell et al., 1989) causing rapid accumulation of ammonia and cell death. Genes that encode the enzyme phosphinothricin acetyltransferase are obtainable from species of Streptomyces (e.g., ATCC No. 21,705) and include the bar gene from
Streptomyces hygroscopicus
or the pat gene from
Streptomyces viridochromogenes
. These organisms synthesize numerous unique compounds, secondary metabolites, that often possess antibacterial, antitumor, or antiparasitic activity (Demain et al., 1983).
Streptomyces viridochromogenes
produces a broad spectrum tripeptide antibiotic phosphinothricyl-alanyl-alanine (phosphinothricin) [2-amino-4-(methylphosphinyl)-butanoic acid] (Bayer et al., 1972). The gene that encodes for phosphinothricin resistance has been designated pat and was first isolated from
S. Viridochromogenes
and shares extensive nucleotide sequence homology with the bar gene of
S. hygroscopicus
(Murakami et al., 1986; Thompson et al., 1987). The bar gene has been well studied and serves as a model to explain the mode of action of the pat gene. The bar gene encodes a phosphinothricin acetyltransferase, which acetylates the free NH
2
group of phosphinothricin and thereby prevents autotoxicity in the producing organism (Murakami et al.,
Mumm Rita Hogan
Spencer T. Michael
Benzion Gary
DeKalb Genetics Corp.
Schwegman Lundberg Woessner & Kluth P.A.
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