Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters pigment production in the plant
Reexamination Certificate
1998-02-13
2003-11-25
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters pigment production in the plant
C800S286000, C800S287000, C800S288000, C800S298000, C800S306000, C800S310000, C800S312000, C800S313000, C800S314000, C800S320000, C800S320100, C800S320200, C800S320300, C800S322000, C800S281000
Reexamination Certificate
active
06653530
ABSTRACT:
INCORPORATION OF SEQUENCE LISTING
A paper copy of the Sequence Listing and a computer readable form of the sequence listing (“CRF”) on diskette, containing the file named “16516.122. txt”, which is 14,451 bytes in size (measured in MS-DOS), and which was created on Jan. 22, 2002, are herein incorporated by reference.
FIELD OF THE INVENTION
The invention relates to genetic modification of plants, plant cells and seeds, particularly altering carotenoid biosynthesis, tocopherol biosynthesis, and fatty acid composition.
BACKGROUND OF THE INVENTION
Carotenoids
Carotenoids are pigments with a variety of applications. They are yellow-orange-red lipids which are present in green plants, some molds, yeast and bacteria. Carotenoid hydrocarbons are referred to as carotenes, whereas oxygenated derivatives are referred to as xanthophylls. The carotenoids are part of the larger isoprenoid biosynthesis pathway, which, in addition to carotenoids, produces such compounds as chlorophyll and tocopherols, Vitamin E active agents. The carotenoid pathway in plants produces carotenes, such as &agr;- and &bgr;carotene, and lycopene, and xanthophylls, such as lutein.
The biosynthesis of carotenoids involves the condensation of two molecules of the C
20
precursor geranyl PP
i
to yield the first C
40
hydrocarbon phytoene. In a series of sequential desaturatioris, phytoene yields lycopene. Lycopene is the precursor of the cyclic carotenes, &bgr;-carotene and a &agr;-carotene. The xanthophylls, zeaxanthin and lutein are formed by hydroxylation of &bgr;-carotene and a &agr;-carotene, respectively.
&bgr;carotene, a carotene whose color is in the spectrum ranging from yellow to orange, is present in a large amount in the roots of carrots and in green leaves of plants. &bgr;carotene is useful as a coloring material and also as a precursor of vitamin A in mammals. Current methods for commercial production of &bgr;-carotene include isolation from carrots, chemical synthesis, and microbial production.
A number of crop plants and a single oilseed crop are known to have substantial levels of carotenoids, and consumption of such natural sources of carotenoids have been indicated as providing various beneficial health effects. The below table provides levels of carotenoids that have been reported for various plant species.
CAROTENOID CONTENTS OF VARIOUS CROPS
(&mgr;g/g)
Beta-
Alpha-
Crop
Carotene
Carotene
Lycopene
Lutein
Total
Carrots
30-110
10-40
0-0.5
0-2
65-120
Pepper (gr)
2
—
—
2
8
Pepper (red)
15
1
—
—
200
Pumpkin
16
0.3
tr
26
100
Tomato
3-6
—
85
—
98
Watermelon
1
tr
19
—
25
Marigold petals
5
4
—
1350
1500
Red palm oil
256
201
8
—
545
The pathway for biosynthesis of the carotenoids has been studied in a variety of organisms and the biosynthetic pathway has been elucidated in organisms ranging from bacteria to higher plants. See, for example, Britton, G. (1988)
Biosynthesis of carotenoids
, p. 133-182, In T. W. Goodwin (ed.),
Plant pigments
, 1988. Academic Press, Inc. (London), Ltd., London. Carotenoid biosynthesis genes have also been cloned from a variety of organisms including
Erwinia uredovora
(Misawa et al. (1990)
J. Bacteriol
. 172:6704-6712
; Erwinia herbicola
(Application WO 91/13078, Armstrong et al. (1990)
Proc. Nat. Acad. Sci., USA
87:9975-9979);
R. capsulatus
(Armstrong et al. (1989)
Mol. Gen. Genet
. 216:254-268, Romer et al. (1993)
Biochem. Biophys. Res. Commun
. 196:1414-1421);
Thermus thermophilus
(Hoshino et al. (1993)
Appl. Environ. Microbiol
. 59:3150-3153); the cyanobacterium Synechococcus sp. (Genbank accession number X63873). See also, application WO 96/13149 and the references cited therein.
While the genes have been elucidated, little is known about the use of the genes in plants. Investigations have shown that over expression or inhibition of expression of the plant phytoene synthase (Psy1) gene in transgenic plants can alter carotenoid levels in fruits. See, Bird et al. (1991)
Biotechnology
9:635-639; Bramley et al. (1992)
Plant J
. 2:343-349; and Fray and Grierson (1993)
Plant Mol. Biol
. 22:589-602. Further, as reported by Fray et al. (1995)
The Plant Journal
8:693-701, constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway.
Application WO 96/13149 reports on enhancing carotenoid accumulation in storage organs such as tubers and roots of genetically engineered plants. The application is directed towards enhancing colored native carotenoid production in specific, predetermined non-photosynthetic storage organs. The examples of the application are drawn to increasing colored carotenoids in transformed carrot roots and in orange flesh potato tubers. Both of these tissues are vegetative tissues, not seeds, and natively have a high level of carotenoids.
Carotenoids are useful in a variety of applications. Generally, carotenoids are useful as supplements, particularly vitamin supplements, as vegetable oil based food products and food ingredents, as feed additives in animal feeds and as colorants. Specifically, phytoene finds use in treating skin disorders. See, for example, U.S. Pat. No. 4,642,318. Lycopene, &agr;- and &bgr;-carotene are used as food coloring agents. Consumption of &bgr;-carotene and lycopene has also been implicated as having preventative effects against certain kinds of cancers. In addition, lutein consumption has been associated with prevention of macular degeneration of the eye.
Plant oils are useful in a variety of industrial and edible applications. Novel vegetable oils compositions and/or improved means to obtain oils compositions, from biosynthetic or natural plant sources are needed. Depending upon the intended oil use, various different fatty acid compositions are desired. The demand for modified oils with specific fatty acid compositions is great, particularly for oils high in oleic acid. See, Haumann, B. F. (1996)
INFORM
7:320-334. As reported by Haumann, the ideal frying oil would be a low-saturate, high oleic and low linolenic oil. Furthermore, studies in recent years have established the value of monounsaturated fatty acids as a dietary constituent.
Attempts have been made over the years to improve the fatty acid profiles of particular oils. For example, the oxidative stability of vegetable oil is related to the number of double bonds in its fatty acids. That is, molecules with several double bonds are recognized to be more unstable. Thus, scientists have attempted to reduce the content of &agr;-linolenic acid in order to improve shelf life and oxidative stability, particularly under heat.
It is apparent that there is needed a method for producing significant levels of carotenoid compounds in crop plants and particularly in plant seeds. It would additionally be beneficial to alter the fatty acid content of the plants and seeds. Such altered seed products would be useful nutritionally as well as provide a source for producing more stable oils. There is no report of methods to substantially altering the levels and composition of carotenoids produced in a plant seed, particularly with respect to increasing the level of production of carotenoids. There is therefore needed, a useful method for altering carotenoid levels in plants, particularly seeds, and for producing oils with modified carotenoid composition and/or content.
Tocopherols
A number of unique and interconnected biochemical pathways leading to secondary metabolites, including tocopherols, exist in chloroplasts of higher plants. Tocopherols not only perform vital functions in plants, but are also important from mammalian nutritional perspectives. In plastids, tocopherols account for up to 40% of the total quinone pool. As shown in
FIG. 15
, the biosynthesis of &agr;-tocopherol in higher plants involves condensation of homogentisic acid and phytylpyrophosphate to form 2-methyl-6 phytylbenzoquinol that can, by cyclization and subsequent methylations (Fiedler et al., 1982
, Planta
, 155: 511-515, Soll et al., 1
Bhat B. Ganesh
Boddupalli Sekhar S.
Kishore Ganesh M.
Rangwala Shaukat H.
Shewmaker Christine K.
Arnold & Porter
Bonner Grace L.
Calgene LLC
Collins Cynthia
Fox David T.
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