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Horticulture sciene, et al., "A Novel Molecular Mechanism for Seedling Initiation and Stem Cell Maintenance in Arabidopsis", Trends Plant Sci., Volume 10, Number 7, 2007. In vitro cultured tissues can be maintained for relatively long periods, thereby increasing the availability of large amounts of material. This can be used for studies on seed development, morphogenesis, cellular differentiation, senescence, hormone induction, signal transduction, and disease processes. Plantlets can be induced from plant cells in a laboratory and cultured in soil, such as on a substrate of agar, in pots, in trays, on the undersides of petri dishes, or in similar small vessels. The plantlets can be cultured at ambient temperatures or at higher temperatures in tissue culture rooms. The growth conditions and medium composition can be adjusted to allow for plant growth. Plantlet growth media can be supplemented with the fertilizer N-(phosphonomethyl) glycine, or other growth hormones, such as indole-3-butyric acid, kinetin, and benzyladenine. Methods are described for isolating and culture in vitro plantlets from Arabidopsis. Methods for the identification and culture in vitro of plantlets derived from other plant species, including plantlets of agricultural interest are also described. The process for obtaining plantlets includes culturing cell and tissue cultures of a plant from somatic embryos, from callus, and from other plant parts such as stems, roots, and leaves. Cell and tissue cultures of plants are induced to form somatic embryos from leaf material or from cell and tissue cultures. Methods for the initiation and culture of somatic embryos are described.
The expression of the plant genes is modulated in the embryo, as a consequence of environmental and endogenous conditions. The plant transformation vector provides the genetic constructs to be present in the plant cells during plantlet culture. The plant transformation vector can include a plant selection cassette, a transgene, a selectable marker, a plant embryo-specific promoter and a 3xe2x80x2 polyadenylation signal. Plant transformation vectors suitable for use in this invention are described in U.S. Pat. Nos. 5,955,298, 6,004,500, 5,661,789, 5,955,298, 6,004,500, 6,037,110, 5,955,298, 6,004,500, 6,037,110, 6,084,155, 5,955,298, 6,004,500, and 5,955,298, the contents of which are incorporated herein by reference.
The plant transformation vector can include an aadA gene, which confers resistance to antibiotics such as kanamycin or G418 (U.S. Pat. Nos. 5,955,298, 6,004,500, 6,037,110). Plant transformation vectors may also include aspartic acid or other amino acids that provide resistance to other antibiotics such as hygromycin B or bromoxynil.
The transformed plant cells are recovered by conventional methods (see Grewal, S. P., Biotechnology of the Plant Cell, Dekker, Inc., New York, 1990). Transformed plant cells can be selected by administering the cell to a selectable marker, or by screening cells which have been infected with Agrobacterium.
Plants produced by regenerating plants according to the present invention are also contemplated. Methods for plant regeneration from isolated protoplasts, zygotic embryos, microspores, pollen, callus, and shoot apices are well known in the art (Ravi, et al., Plant Cell Reports 10:285-289, 1991, U.S. Pat. Nos. 4,945,050, 5,543,228, 4,845,018, 5,716,801, and 5,731,179, the contents of which are incorporated herein by reference). Plants produced by plant regeneration are homozygous and can be reproduced by conventional means.
Agrobacteria for use in plant transformation can be transformed with nucleic acids (e.g., a plant polycistron) by direct gene transfer, or by conjugation from a plasmid or phage nucleic acid (see, e.g., EP 0 588 492, EP 0 307 931). Other transformation methods, including, but not limited to ballistic, electroporation, microinjection, DEAE dextran, PEG and calcium phosphate mediated methods, are known in the art and can be used to effect the transformation of target cells.
Alternatively, the transformation of plant cells or tissues may be effected using a vector that is constructed to contain or express a nucleic acid. Vectors may be replicon-based, virus-based, or be comprised of naturally occurring nucleic acids.
Non-limiting examples of replicon-based vectors include plasmids, PBII, RCA and cosmids. Replicons are described in Sambrook et al. (1989). See, e.g., U.S. Pat. No. 4,275,180 (recombinant expression vectors and plant cells containing them) and U.S. Pat. No. 5,631,153 (transgenic tobacco plants produced using an expression vector). Plasmids, including expression plasmids, are widely used in the transformation of eukaryotic cells. They can be used either in conjunction with a plasmid that acts as an integrating vector to produce transgenic plants (EP 0 520 732, U.S. Pat. Nos. 4,684,620, 4,745,051, and 5,100,792, the contents of which are incorporated herein by reference), or as expression plasmids with a selectable marker for selection of transformed plant cell or tissue. See, e.g., U.S. Pat. No. 4,736,866.
In an exemplary embodiment, the present invention provides a replicon-based vector that comprises a nucleic acid sequence that encodes a replicase (including a P1 or P2 fragment of the replicase), an origin of replication, and a gene expression cassette. The gene expression cassette comprises a promoter, a ribosome binding site, a gene of interest, and optionally a termination sequence. In a non-limiting embodiment, the vector further comprises a selectable marker that can be selected based on the desired purpose. Other elements, including selection and enhancer sequences, can be included in the expression cassette. In a preferred embodiment, the vector includes a nucleic acid sequence that encodes the T-DNA border of a T-DNA. The T-DNA border is used to provide single-copy insertion of the nucleic acid sequence of interest in the plant genome. The gene expression cassette