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
1999-06-25
2001-09-04
Fox, David T. (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
C800S278000, C800S287000, C800S293000, C435S069100, C435S194000, C435S204000, C435S209000, C435S320100, C435S418000, C435S419000, C435S468000, C435S470000
Reexamination Certificate
active
06284956
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an expression cassette containing a selectable marker gene, for cotransforming a monocot plant, to plant transformation methods using the selectable marker expression cassette, and to plants and plant cells transformed with the selectable marker expression cassette.
BACKGROUNG OF THE INVENTION
Transgenic plants carrying one or more expressible heterologous genes in a transgene expression cassette have a variety of potential advantages. The plants carrying such a transgene expression cassette may carry one or more genes which confer herbicide tolerance, pesticide tolerance insect resistance, tolerance to stress, enhanced flavor or stability of the fruit or seed, or the ability to synthesize useful, non-plant proteins, e.g., medically valuable proteins or the ability to generate altered concentrations of plant proteins, and related impacts on the plant, e.g., altered levels of plant proteins catalyzing production of plant metabolites including secondary plant metabolites.
Ideally, the expression of the heterologous protein from the transgene expression cassette is largely confined to a particular differentiated plant tissue, e.g., the fruit or seed, and/or induced under selected conditions, e.g., plant hormone induction. To this end, it is desirable to place the gene encoding the heterologous protein in a gene expression cassette under the control of a promoter that is induced or inducible in a selected plant tissue, such as roots or leaves or seeds, and/or during selected plant induction states, such as seed maturation or seed germination.
There are multiple technologies, methods and biological materials that are needed in order to successfully genetically engineer a plant cell so that it can express recombinant molecules from a transgene expression cassette, and to make the transgenic plant cell commercially potentially usable and acceptable, including the following.
First, it is important to the success of methods used to transform plant cells to be able to readily and quickly detect successful transformant events. In the usual case, this means being able to screen cells for successful transformation within a few days to a few weeks of the transformation procedure. For many plants, including monocots, positive screening for successful transformants is performed most rapidly by co-transforming one or more transgene expression cassettes with a selectable marker expression cassette and conveniently by screening callus cells taken through the transformation process for a selectable marker in culture or on media plates.
The selectable marker gene in the selectable marker expression cassette is operably linked to selectable marker regulatory elements including a promoter and terminator. The expression in the transgenic plant cell of the selectable marker gene generally encodes a protein which confers resistance to an antibiotic or herbicide. Common selectable marker genes include, for example, the nptll kanamycin resistance gene, for selection in kanamycin-containing media, or the phosphinothricin acetyltransferase gene, for selection in media containing phosphinothricin (PPT), or the hph hygromycin phosphotransferase gene, for selection in media containing hygromycin B.
These selectable marker genes in the selectable marker expression cassette are expressed by promoters which are active in the undifferentiated callus tissues into which the selectable marker and heterologous genes are inserted. Heretofore, promoters generally used to drive expression of the selectable markers genes have been constitutive gene promoters, such as the Cauliflower Mosaic virus (CaMV) 35s promoter, the ubiquitin ubil promoter, and the actin promoter, have been constitutive promoters which express in a wide range of tissues, including the tissues in which expression of the heterologous gene is desired.
Second, it is important to the success of methods used to transform plant cells to be able to perform the transformation process in an efficient matter, that is, the process of plant cell transformation, selection and regeneration ought to require limited post-transformation manipulation of the plant tissue such as callus subjected to transformation, so as to enable processing of relevant number of transformed plant tissues so as to take advantage of items such as position effects.
Third, it may be important to the successful use and commercial acceptance of transgenic plant organs or tissues such as transgenic plant seeds in a food or feed formulation incorporating as ingredients transgenic plant seeds or extracts thereof, that the selectable marker protein not be present, or present only in very low amounts. In other words, the presence of the selectable marker protein in a transgenic seed for use in food or feed is potentially a negative or block to the food or feed use of said transgenic seed.
Finally, in many cases, it is useful to be able to simultaneously transform plant cells with one or more transgene expression cassettes. Such a procedure would allow the introduction of multiple transgenic traits in a single plant cell, e.g., multiple proteins promoting human or animal health in a single transgenic plant cell or transgenic plant tissue or organ such as a plant seed. Such a procedure would also allow for the introduction of multiple genes in a single plant transgenic plant cell with the intended purpose of using said genes in combination as a way to metabolically engineer a plant cell pathway, e.g., a pathway coding for plant secondary metabolites such as the phenylpropanoid pathway. Transformation and selection methods employing such promoters have generally not been successful and efficient in transforming monocot plants with multiple, e.g., 5-10, heterologous genes found in multiple transgene expression cassettes.
Heretofore, there have been no convenient methods, technologies and biological materials enabling efficient transformation events and selection and regeneration with the resulting transgenic plant tissues not expressing the selectable marker protein from the selectable marker expression cassette.
For example, the use of the double cassette vector or multiple single cassette vectors in conjunction with Agrobacterium transformation requires that the resulting transformants go through a breeding program in order to determine if it is possible to segregate the selectable marker expression cassette from the transgene expression cassette so as to get a transgenic plant and resulting seed carrying the transgene expression cassette but not the selectable marker expression cassette. This breeding program takes substantial time to the development of a product from a transgenic plant.
A similar problem is encountered with the use of the ballistic transformation methodology in which there is co-transformation of a selectable marker expression cassette with a constitutive promoter directing expression of the selectable marker gene, along with the transgene expression cassette. Again, in order to generate a transgenic plant cell and resulting transgenic plant or plant seed without the selectable marker protein, the selectable marker expression cassette and the transgenic expression cassette need to be genetically segregated using a breeding program, a timely procedure that limits the commercial utility transgenic protein.
The current invention solves these current problems. First, this invention includes a method of selecting and regenerating transgenic plants that significantly reduces the time and labor processing transgenic plant material. Second, the present invention includes a regulated promoter for use in controlling expression of the selectable marker gene during selection, but not after regeneration so that the resulting transgenic seed does not contain the selectable marker protein.
SUMMARY OF THE INVENTION
In one aspect, the invention includes a set of expression cassettes, including vectors containing expression cassettes, for use in transforming monocot plants with one or more heterologous genes capable of producing heterologous
Huang Ning
Rodriguez Raymond L
Applied Phytologics, Inc.
Fox David T.
Judge Linda R.
Perkins Coie LLP
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