Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-02-24
2003-07-29
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S320100, C800S312000, C800S288000, C424S009100, C504S153000
Reexamination Certificate
active
06600088
ABSTRACT:
FIELD OF THE INVENTION
The invention relates in general to methods for detecting the presence of a selectable marker in a plant. In particular, a simple and efficient method for detecting the presence of the neomycin phosphotransferase II protein in plants is disclosed.
BACKGROUND OF THE INVENTION
In the production of transgenic plants, a gene encoding a selectable marker that confers resistance to a selective agent is often included in the transformation vector to provide a means for distinguishing plant tissue that has been transformed from that which has not. The selection is typically made by growing the transformed tissue in an environment containing the selective agent and only those tissues expressing the selectable marker gene product are able to survive.
Neomycin phosphotransferase II (NPTII) is a protein of bacterial origin that confers resistance to a number of selective agents, such as kanamycin, paromomycin and genenticin, and when genetically engineered to be expressed in plant tissues, has been used as a selectable marker. Although the use of NPTII as a selectable marker to select for transformed plant tissue in the early stages of producing a transgenic plant has become routine, no efficient method for identifying NPTII-containing plants in the field has been disclosed. Typical methods involve laboratory analysis of harvested tissue from potential transgenic plants through methods including Southern blotting, immunoassays, and enzyme activity assays. These laboratory analyses are labor-intensive and time-consuming endeavors, particularly when large numbers of plants must be tested.
One method for identifying NPTII-containing transgenic tomato plants in the field has been reported. Weide et al. (
Theor. Appl. Genet.
78:169-172, 1989) disclosed that young (three-leaf stage) transgenic tomato plants could be phenotypically distinguished from nontransgenic counterparts by spraying the plants with a solution containing kanamycin. In the Weide et al. process, trays of seedlings were sprayed with a kanamycin solution that did not include a surfactant over a three-day period, with a total application of 0.5-2.0 mg kanamycin per plant. Transgenic plants containing the NPTII protein were distinguished from non-transgenic plants in that transgenic plants did not develop bleached spots on the treated tissue within 7 days of treatment. NPTII activity in the putative transgenic plants was subsequently confirmed by a radiolabel transfer assay. This approach is disadvantageous in at least one respect in that it necessitated the use of large amounts of selective agent and a lengthy period of incubation before results were obtained and has not been shown to be an effective method for any plant other than tomato.
Thus, there exists a need for a rapid and more efficient method by which transgenic plants comprising a selectable marker may consistently be identified in the field.
SUMMARY OF THE INVENTION
This invention relates to an improved method for identifying plants expressing a selectable marker gene product, such as NPTII, and growing in the absence of a selective agent. More specifically, in one embodiment of the invention there is provided a method for detecting the presence of a selectable marker in a plant comprising contacting a composition comprising a selective agent and an effective amount of an organosilicone surfactant with putative transgenic plants, assessing the physical appearance of the plants for evidence of necrosis and/or bleaching of the treated plant tissue, and assigning the status of transgenic or non-transgenic to such plants based on the physical appearance of such plant tissue. A plant with little or no necrosis or bleaching evidences the presence of a selectable marker gene product in the plant and is determined to be a transgenic plant.
In a further embodiment of the present invention there is provided a method for detecting the presence of a selectable marker in a plant comprising contacting an effective amount of a selective agent with putative transgenic plant tissue and separately contacting an effective amount of an organosilicone surfactant with said putative transgenic plant tissue, assessing the physical appearance of the treated plant tissue for evidence of necrosis and/or bleaching of the treated plant tissue, and assigning the status of transgenic or non-transgenic to such plants based on the physical appearance of such plant tissue.
Among the many objects and advantages of the present invention include the provision of a method that utilizes a significantly reduced amount of the selective agent in the method as a result of the use of an organosilicone surfactant in cooperation with the selective agent; and the provision of a rapid, non-destructive method that may be utilized in field conditions on growing plants.
DETAILED DESCRIPTION OF THE INVENTION
It has been discovered that existing methods for detecting the presence of a selectable marker gene product in a transgenic plant growing in a non-selective environment may be improved by applying an organosilicone surfactant to the plant in addition to the selective agent or by utilizing a composition comprising a selective agent with an effective amount of an organosilicone surfactant. In accordance with the invention, significantly reduced amounts of the selective agent may be used as compared to other methods. In general, subsequent to treating the putative transgenic plant tissue with the selective agent and organosilicone surfactant (whether together or separately), the physical appearance of the treated tissue is assessed to determine is whether the treated tissue contains a selectable marker gene product or not. Treated plants that contain the selectable marker gene product will exhibit one of two phenotypes:
1) no bleaching or necrosis of the treated plant tissue; or
2) reduced bleaching or necrosis relative to that seen in a similarly treated nontransgenic plant of the same genetic background. Transgenic plants are thereby distinguished from plants that have undergone the same selective agent/organosilicone treatment and do not contain the selectable marker gene product, which display relatively more bleaching and/or necrosis of the treated plant tissue.
The nucleic acid sequence serving as the source of the selectable marker gene product functions to produce a phenotype in cells that facilitates their identification relative to cells not containing the marker. Characteristics of useful selectable markers for plants, both dicotyledonous and monocotyledonous, have been outlined in a report on the use of microorganisms (Advisory Committee on Novel Foods and Processes, July 1994). These characteristics include stringent selection with minimal contaminating nontransformed tissue, high numbers of independent transformation events without interference in subsequent regenerative steps, application to a large number of species, and availability of an assay to detect the marker. Several antibiotic and herbicide resistance markers satisfy these criteria (Dekeyser et al.,
Plant Physiol.,
90: 217-223, 1989; Della-Cioppa et al.,
Bio/Technology,
5:579-584, 1987). For example, NPTII confers resistance to kanamycin, paromomycin and GENENTICIN; aph IV confers resistance to hygromycin B; aac3 and aacC4 confer resistance to gentamycin; the pat and bar genes confer resistance to phosphinothricin; and the enolpyruvylshikimate-phosphate synthase (EPSPS) and glyphosate oxidoreductase (GOX) genes confer resistance to glyphosate. In a preferred embodiment of the present invention, the selectable marker confers resistance to an antibiotic and, more preferably, the selectable marker is NPTII.
The detection method of this invention may be utilized with any species compatible with transformation with a nucleic acid sequence of interest and subsequent regeneration to form a transgenic plant. The plant may be a monocotyledonous or dicotyledonous plant. More preferably, it will be monocotyledonous of the Gramineae (Poaceae) family or dicotyledonous of the Leguminosae family. Most preferably, it will be a co
Feng Paul C. C.
Howe Arlene R.
Fox David T.
Kubelik Anne
McBride Thomas P.
Monsanto Company
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