Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2001-12-18
2004-05-04
Mehta, Ashwin (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S314000
Reexamination Certificate
active
06730824
ABSTRACT:
FIELD OF INVENTION
This invention relates to a method of using elongated, needle-like microfibers or “whiskers” to transform plant cell aggregates and selected plant tissues.
BACKGROUND OF THE INVENTION
Until recently, genetically manipulated plants were limited almost exclusively to those events created by application of classical breeding methods. Creation of new plant varieties by breeding was reserved primarily for the most agronomically important crops, such as corn, due to the cost and time needed to identify, cross, and stably fix a gene in the genome, thus creating the desired trait. In comparison, the advent of genetic engineering has resulted in the introduction of many different heterologous genes and subsequent traits into diverse crops including corn, cotton, soybeans, wheat, rice, sunflowers and canola in a more rapid manner. However, the intergression of a new transgene into elite germplasm is still quite a laborious task due to the tissue culturing and back-crossing needed to produce a commercially viable, elite, line.
Several techniques exist which allow for the introduction, plant regeneration, stable integration, and expression of foreign recombinant vectors containing heterologous genes of interest in plant cells. One such technique involves acceleration of microparticles coated with genetic material directly into plant cells (U.S. Pat. Nos. 4,945,050 to Cornell; 5,141,131 to DowElanco; and 5,538,877 and 5,538,880, both to Dekalb). This technique is commonly referred to as “microparticle bombardment” or “biolistics”. Plants may also be transformed using Agrobacterium technology (U.S. Pat. No. 5,177,010 to University of Toledo, 5,104,310 to Texas A&M, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500 all to Max Planck, European Patent Applications 604662,627752 and U.S. Pat. No. 5,591,616 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba-Geigy, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135 both to Agracetus). Another transformation method involves the use of elongated needle-like microfibers or “whiskers” to transform cell suspension cultures (U.S. Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca). In addition, electroporation technology has been used to transform plant cells from which fertile plants have been obtained (WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and 5,384,253 both to Dekalb; U.S. Pat. Nos. 5,679,558, 5,641,664, WO9209696 and WO9321335 to Plant Genetic Systems).
Despite all of the technical achievements, genetic transformation and routine production of transgenic plants in a commercially viable, elite, germplasm is still a laborious task. For example, microparticle bombardment, while capable of being used either on individual cells, cell aggregates, or plant tissues, requires preparing DNA-attached gold particles and optimization of an expensive and not yet widely available, “gun” apparatus. Techniques involving Agrobacterium are extremely limited because not all plant species or varieties within a given species are susceptible to infection by the bacterium. Electroporation techniques are not preferred due to the extreme difficulties and cost typically encountered in routinely making protoplast from different plant species and tissues thereof and the concomitant low viability and low transformation rate associated therewith.
As disclosed herein, applicants have invented a method whereby plant cell aggregates and plant tissues from non-elite and elite germplasm can be directly and inexpensively transformed with a recombinant vector containing the gene of choice using whiskers. Applicants' invention is advantageous over currently used methods in that it is simple, quick and easy to use. Furthermore, applicants' invention is superior to that described in the art in that it eliminates the need to establish Type III callus cultures or establish and maintain cell suspension cultures, and can be used with either Type I or Type II callus, thus, is less germplasm limited. This means that applicants' invention, as described herein, can be used to transform elite genotypes directly thus eliminating the problems and time generally associated with gene intergression.
SUMMARY OF THE INVENTION
The present invention relates to the production of fertile, transgenic,
Zea mays
plants containing heterologous DNA preferably integrated into the chromosome of said plant and heritable by the progeny thereof.
One aspect of the present invention relates to
Zea mays
plants, plant parts, plant cells, plant cell aggregates, and seed derived from transgenic plants containing said heterologous DNA.
The present invention also relates to the production of fertile, transgenic,
Oryza sativa
L. plants containing heterologous DNA preferably integrated into the chromosome of said plant and heritable by the progeny thereof.
Another aspect of the present invention relates to
Oryza sativa
L. plants, plant parts, plant cells, plant cell aggregates, and seed derived from transgenic plants containing said heterologous DNA.
The present invention also relates to the production of fertile, transgenic,
Gossypium hirsutum
L. plants containing heterologous DNA preferably integrated into the chromosome of said plant and heritable by the progeny thereof.
Another aspect of the present invention relates to
Gossypium hirsutum
L. plants, plant parts, plant fibers, plant cells, plant cell aggregates, and seed derived from transgenic plants containing said heterologous DNA.
The invention further relates to a process for producing fertile transformed plants from Type I callus, Type II callus, hypocotyl-derived callus, or cotyledon-derived callus by whisker-mediated transformation.
The invention yet further relates to a process for producing fertile transformed plants from meristematic tissue by whisker-mediated transformation.
Another aspect of the invention relates to fertile, mature maize plants regenerated from Type I or Type II callus and transgenic seed produced therefrom.
Another aspect of the invention relates to fertile, mature rice plants regenerated from Type I callus and transgenic seed produced therefrom.
Yet, another aspect of the invention relates to fertile, mature cotton plants regenerated from hypocotyl-derived callus or cotyledon-derived callus and transgenic seed and fiber produced therefrom.
In a preferred embodiment, this invention produces the fertile transgenic plants described herein by means of whisker-mediated cell perforation and heterologous DNA uptake, said whisker-mediated cell perforation being performed on plant cell aggregates and plant tissues, followed by a controlled regimen for selection and production of transformed plant lines.
Other aspects, embodiments, advantages, and features of the present invention will become apparent from the following specification.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods for production of fertile transgenic plants and seeds of, for example, the species
Zea mays, Oryza sativa
L.,
Gossypium hirsutum
L., and
Brassica napus
by transforming plant cell aggregates and plant tissues of said species with the DNA construct of interest via whisker-mediated transformation. After transformation, transgenic plants are regenerated from said transformed plant cell aggregates and plant tissues and said regenerated plants express the chimeric DNA construct of interest. The transgenic plants produced herein by the methods described include: all species of corn including but not limited to field corn, popcorn, sweet corn, flint corn, dent corn and the like; all species of cotton; and all species of rice.
The following phrases and terms are defined below:
By “antisense” is meant an RNA transcript that comprises sequences complementary to a target RNA
Armstrong Katherine
Hopkins Nicole L.
Pareddy Dayakar R.
Petolino Joseph F.
Dow AgroSciences LLC
Mehta Ashwin
Stuart Donald R.
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