Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Via agrobacterium
Reexamination Certificate
1999-07-29
2003-08-05
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Via agrobacterium
C800S278000, C800S312000, C800S314000, C800S306000, C800S320100, C800S322000, C435S468000, C435S469000, C435S419000, C435S426000, C435S427000, C435S428000, C435S424000, C435S430000, C435S431000
Reexamination Certificate
active
06603061
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the field of plant biotechnology. More specifically, it concerns methods of incorporating genetic components into a plant comprising a T-DNA transfer process. In particular, provided herein are systems for genetically transforming monocotyledonous plants including corn, rice, and wheat.
The method comprises novel conditions during the inoculation, co-culture, or infiltration of Agrobacterium with a transformable plant cell or tissue. Exemplary methods include an improved method using a bacterial growth suppressing agent during the Agrobacterium-mediated transformation process. The improved method can be used for introducing nucleic acids into transformable cells or tissues using a variety of selectable and/or screenable marker systems, and with a number of different plant species. The present invention also provides transgenic plants, in particular, corn, rice, and wheat. In other aspects, the invention relates to the production of stably transformed plants, gametes, and offspring from these plants.
During the past decade, it has become possible to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology. This advance has provided enormous opportunities to improve plant resistance to pests, disease and herbicides, and to modify biosynthetic processes to change the quality of plant products (Knutson et al., 1992; Piorer et al., 1992). However, the availability of efficient Agrobacterium-mediated transformation methods suitable for high capacity production of economically important plants is limited. In particular, a novel culture system that generates reproducible transformants with a simple integration pattern of the introduced DNA into the host genome, more specifically, the integration of a low copy number (one to two copies) of the introduced DNA is needed.
There have been many methods attempted for plant transformation, but only a few methods are highly efficient. Moreover, few methods are both highly efficient and result in transformants with simple integration pattern and low copy number of the introduced DNA. Copy number refers to the number of complete or incomplete copies of T-DNA introduced in host cell. The technologies for the introduction of DNA into cells are well known to those of skill in the art and can be divided into categories including but not limited to: (1) chemical methods (Graham and van der Eb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation ( Fromm et al., 1985; U.S. Pat. No. 5,384,253) and the gene gun (Christou, 1992; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988);(4) receptor-mediated mechanisms (Curiel et al., 1992); and (5) Agrobacterium-mediated plant transformation methods.
Until recently, the methods employed for some monocot species included direct DNA transfer into isolated protoplasts and microprojectile-mediated DNA delivery (Fromm et al, 1990). The protoplast methods have been widely used in rice, where DNA is delivered to the protoplasts through liposomes, PEG, and electroporation. While a large number of transgenic plants have been recovered in several laboratories (Datta et al., 1990), the protoplast methods require the establishment of long-term embryogenic suspension cultures. Some regenerants from protoplasts are infertile and phenotypically abnormal due to the long-term suspension culture (Davey et al., 1991; Rhodes et.al.,1988). U.S. Pat. No. 5,631,152 describes a rapid and efficient microprojectile bombardment method for the transformation and regeneration of monocots and dicots.
To date, microparticle- and Agrobacterium-mediated gene delivery are the two most commonly used plant transformation methods. Microparticle-mediated transformation refers to the delivery of DNA coated onto microparticles that are propelled into target tissues by several methods. This method can result in transgenic events with a higher copy number, complex integration patterns, and fragmented inserts. Agrobactenum-mediated plant transformation can also result in transformed plants with multiple copies of inserts and complex integration patterns. A reduction in copy number can result from a decrease in the frequency of T-DNA transfer. Accordingly, novel culture conditions can be manipulated to impact the frequency of T-DNA transfer and can produce transformation events containing the optimum number of copies of the introduced DNA.
A reproducible Agrobacterium-mediated method that consistently results in low copy number inserts and is applicable to a broad number of plant species is desirable for a number of reasons. For example, the presence of multiple inserts can lead to a phenomenon known as gene silencing which can occur by several mechanisms including but not limited to recombination between the multiple copies which can lead to subsequent gene loss. Also, multiple copies can cause reduced levels of expression of the gene which in turn can result in the reduction of the characteristic(s) conferred by the gene product(s). Despite the number of transformation methods available for specific plant systems, it would be advantageous to have a method of introducing genes into plants that is applicable to various crops and a variety of transformable tissues.
Agrobacterium-mediated transformation is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrobacterium. Several Agrobacterium species mediate the transfer of a specific DNA known as “T-DNA”, that can be genetically engineered to carry any desired piece of DNA into many plant species. The major events marking the process of T-DNA mediated pathogenesis are: induction of virulence genes, processing and transfer of T-DNA, This process is the subject of many reviews (Ream, 1989; Howard and Citovsky, 1990; Kado, 1991; Hooykaas and Schilperoort, 1992; Winnans, 1992; Zambryski, 1992; Gelvin, 1993; Binns and Howitz, 1994; Hooykaas and Beijersbergen 1994; Lessl and Lanka, 1994; Zupan and Zambryski, 1995).
Agrobacterium-mediated genetic transformation of plants involves several steps. The first step, in which the Agrobacterium and plant cells are first brought into contact with each other, is generally called “inoculation”. Following the inoculation step, the Agrobacterium and plant cells/tissues are usually grown together for a period of several hours to several days or more under conditions suitable for growth and T-DNA transfer. This step is termed “co-culture”. Following co-culture and T-DNA delivery, the plant cells are often treated with bacteriocidal and-or bacteriostatic agents to kill the Agrobacterium. If this is done in the absence of any selective agents to promote preferential growth of transgenic versus non-transgenic plant cells, then this is typically referred to as the “delay” step. If done in the presence of selective pressure favoring tranasgenic plant cells, then it is referred to as a “selection” step. When a “delay” is used, it is followed by one or more “selection” steps. Both the “delay” and “selection”. steps typically include bacteriocidal and-or bacteriostatic agents to kill any remaining Agrobacterium cells because the growth of Agrobacterium cells is undesirable after the infection (inoculation and co-culture) process.
Although transgenic plants produced through Agrobacterium-mediated transformation generally contain a simple integration pattern as compared to microparticle-mediated genetic transformation, a wide variation in copy number and insertion patterns exists (Jones et al, 1987; Jorgensen et al., 1987). Moreover, even within a single plant genotype, different patterns of T-DNA integration are possible based on the type of explant and transformation system used (Grevelding et al., 1993). Factors that regulate T-DNA copy number are poorly understood. A reproducible, broadly applicable method to increase the efficiency of producing plants with a low copy number, and preferably a single copy of the T-DNA would be highly desirable to those practicing in the ar
Armstrong Charles L
Rout Jyoti R
Fox David T.
Kruse David H
Monsanto Company
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