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
1999-01-15
2001-08-28
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...
C800S286000, C800S294000, C800S306000, C800S312000, C800S317400, C800S322000, C435S468000, C435S469000
Reexamination Certificate
active
06281410
ABSTRACT:
INTRODUCTION
1. Technical Field
This invention relates to regulated genetic modification of plant material, particularly for tissue and/or developmental specific trascription and expression. Heterologous constructs are provided whereby production of endogenous products can be modulated or new capabilities provided.
2. Background
While the ability to manipulate bacterial and mammalian cells by hybrid DNA technology has been available for almost a decade, only in 1983 was it first reported that successful expression of an exogenous gene was achieved in a plant cell. Plants have a highly complex genome and differ in numerous ways from both bacterial and mammalian genes. Therefore, while as a first approximation, one may extrapolate from the experience with other species, the relevance of such experience must be determined by experimentation. In general, genetic engineering techniques have been directed to modifying the phenotype of individual prokaryotic and eukaryotic cells, especially in culture. Plant cells have proven more intransigent than other eukaryotic cells due not only to the lack of suitable vector systems but also a result of the different goals involved. Plant genetic engineering has for the most part been directed to modifying the entire plant or a particular tissue rather than modifying a single cell in culture.
In order to be able to successfully modify plant cells, it will be necessary to develop a large number of different systems for introducing the exogenous DNA into the plant cell, for directing, as appropriate, the introduced DNA either randomly or to particular genomic sites, to provide for constitutive or regulated expression and, as appropriate, to provide for transport of the product to an appropriate site. Toward this end, it will be necessary to develop a wide variety of regulatory signals involved with replication, transcription, translation, integration, and the like. To varying degrees these regulatory signals will have general application across species or be species-specific, will be associated with specific stages of plant growth, or be subject to external control. To that extent, it will be necessary to develop a wide spectrum of regulatory sequences to allow for expression under predetermined conditions.
For many applications, it will be desirable to provide for transcription in a particular plant tissue and/or at a particular time in the growth cycle of the plant or maturation cycle of the tissue. Toward this end, there is substantial interest in identifying endogenous plant products transcription or expression of which is regulated in a manner of interest. In identifying such products, one must first look for a product which appears at a particular time in the cell growth cycle or in a particular plant tissue, demonstrate its absence at other times or in other tissue, identify nucleic acid sequences associated with the product and then identify the sequence in the genome of the plant in order to obtain the 5′-untranslated sequence associated with transcription. Identifying the particular sequence, followed by establishing that it is the correct sequence and isolating the desired transcriptional regulatory region requires an enormous outlay in time and effort. One must then prepare appropriate constructs, and demonstrate that the constructs are efficacious in the desired manner.
There has been substantial interest in modifying the seed with transcriptional initiation regions to afford transcription and expression of the gene introduced into the seed, rather than constitutive expression which would result in expression throughout the plant. Also of interest is the ability to change the phenotype of fruit, so as to provide fruit which will have improved aspects for storage, handling, cooking, organoleptic properties, freezing, nutritional value, and the like.
In addition, different systems may be required for the introduction of nucleic acid into plant cells to obtain reasonable efficiencies of transformation and functioning of the nucleic acid. In many instances, such as the tumor inducing plasmids and viruses, the vectors have found limited utilization in their range of hosts. Therefore, different transformation and replication systems may be required for different plant species.
RELEVANT LITERATURE
Lack of transformation by Agrobacterium of soybean is reported by DeCleene and DeLey,
The Botanical Review
(1976) 42:389-446. Encouraging results in the transformation (Pederson et al.,
Plant Cell Repts.
(1983) 2:201-204 and Hood et al.,
Bio/Technology
(1984) 2:702-708) and regeneration (Christianson et al.,
Science
(1983) 222: 632-634) of soybean have recently been reported. A light inducible soybean SSU gene (small subunit SSU) of ribulose-1,5-bisphosphate-carboxylase (RuBP-carboxylase) is reported by Berry-Lowe et al.,
J. Mol. Appln. Gen.
(1982) 1:483-498. Sequences 5′ to the pSSU gene were recently shown to direct foreign gene expression in a light-inducible manner when transferred into tobacco callus (Herrera-Estrella et al.,
Nature
(1984) 310:115-120).
Crouch et al., In:
Molecular Form and Function of the Plant Genome,
eds. van Vloten-Doting, Groot and Hall, Plenum Publishing Corp. 1985, pp 555-566; Crouch and Sussex,
Planta
(1981) 153:64-74; Crouch et al.,
J. Mol. Appl. Genet.
(1983) 2:273-283; Simon et al.,
Plant Molecular Biology
(1985) 5:191-201; and Scofield and Crouch,
J. Biol. Chem.
(1987) 262:12202-12208, describe various aspects of
Brassica napus
storage proteins. Rose et al.,
Nucl. Acids Res.
(1987) 15:7197 and Scherer and Knauf,
Plant Mol. Biol.
(1987) 9:127-134 describe ACP genes. Beachy et al.,
EMBO J.
(1985) 4:3047-3053; Sengupta-Gopalan et al.,
Proc. Natl. Acad. Sci. USA
(1985) 82:3320-3324; Greenwood and Chrispeels,
Plant Physiol.
(1985) 79:65-71 and Chen et al.,
Proc. Natl. Acad. Sci. USA
(1986) 83:8560-8564 describe studies concerned with seed storage proteins and genetic manipulation. Eckes et al.,
Mol. Gen. Genet.
(1986) 205:14-22 and Fluhr et al.,
Science
(1986) 232:1106-1112 describe the genetic manipulation of light inducible plant genes.
cDNA clones from tomato displaying differential expression during fruit development have been isolated and characterized (Mansson et al.,
Mol. Gen. Genet.
(1985) 200:356-361; Slater et al.,
Plant Mol. Biol.
(1985) 5:137-147). The studies have focused primarily on mRNAS which accumulate during fruit ripening. One of the proteins encoded by the ripening-specific cDNAs has been identified as polygalacturonase (Slater et al.,
Plant Mol. Biol.
(1985) 5:137-147). A cDNA clone which encodes tomato polygalacturonase has been sequenced. Grierson et al.,
Nucleic Acids Research
(1986) 14:8395-8603. The concentration of polygalacturonase mRNA increases 2000-fold between the immature-green and red-ripe stages of fruit development. This suggests that expression of the enzyme is regulated by the specific mRNA concentration which in turn is regulated by an increase in transcription. Della Penna et al.,
Proc. Natl. Acad. Sci. USA
(1986) 83:6420-6424. Mature plastid mRNA for psbA (one of the components of photosystem II) reaches its highest level late in fruit development, whereas after the onset of ripening, plastid mRNAS for other components of photosystem I and II decline to nondetectable levels in chromoplasts. Piechulla et al.,
Plant Mol. Biol.
(1986) 7:367-376.
SUMMARY OF THE INVENTION
Novel methods and DNA constructs are provided for transforming plants employing T-DNA and a Ti- or Ri-plasmid for heterologous DNA introduction and integration into the plant genome. Transformation without gall formation of plant cells which have historically not been Agrobacterium hosts is achieved with successful expression of the heterologous DNA. Additionally, DNA constructs are provided which are employed in manipulating plant cells to provide for regulated transcription, such as light inducible transcription, in a plant tissue or plant part of interest at particular stages of plant growth or in response to external control. Particularly, transcr
Knauf Vic C.
Kridl Jean C.
Calgene LLC
Fox David T.
Rae-Venter Barbara
Rae-Venter Law Group, PC
Wahlsten Jennifer
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