WO2007050715A2 - Compositions and methods for safe delivery of biologically-active plant transformation agents using non-fibrous silicon carbide powder - Google Patents

Abstract

According to the described invention, a biologically-active nucleic acid may be directly delivered into plant cells in an improved and operator-safe fashion using encapsulated or complexed nucleic acid formed into nanoparticles, using carborundum powder, a nucleic acid binding protein with nuclear localization signals (NLSs), or both. The plant cells are not enzymatically treated, and the delivery is not mediated by any microbial agent. The present invention provides the following: (1) a nucleic acid delivery system (NADS), i.e. macromolecular nanoparticles to impart the novel conditions; (2) a novel carrier medium (CM) used with the nucleic acid delivery system which impart the desired results; (3) novel, specific conditions that impart the desired or improved efficiency of the transformation system into the plant cell without irreversibly damaging it, and (4) a novel method to produce these conditions through the use of a combination of DNA condensing agents, carborundum powder, a nucleic acid binding protein with NLSs, which together imparts improved nucleic acid delivery efficiency and safety as compared to other known systems.

Description

COMPOSITIONS AND METHODS FOR SAFE DELIVERY OF BIOLOGICALLY- ACTIVE PLANT TRANSFORMATION AGENTS USING NON-FIBROUS SILICON

CARBIDE POWDER

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application 60/596,852, filed October 26, 2005, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods for the transformation, transfection or insertion of non-native genes into plants, whether dicots or monocots, resulting in permanent and hereditable alteration of the plant genome.

BACKGROUND OF THE INVENTION

AU publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art. Permanent genetic modification of plants requires the introduction of new genetic material into the genome of a plant cell, a process called transformation. Uniform, non- chimeric, permanent genetic modification of plants requires the introduction of new genetic material into the nuclear genome of a plant cell followed by the regeneration of an entire plant from that one cell. Uniform, non-chimeric, permanent genetic modification of plants can also arise from the introduction of new genetic material into the mitochondria or chloroplasts, but since there are multiple copies of these organelles per cell, considerable additional care must be taken to ensure that all such organelles are direct descendants of the originally altered organelle. Most plant transformations are therefore designed to target the nuclear genome, and require integration of the new genetic material into a chromosome, where it becomes a new, permanent, gene locus. To accomplish this, methods must be developed to introduce DNA past several physical barriers, specifically: the plant cell wall, the cell membrane and the nuclear envelope. The plant cell wall deserves particular mention because unlike animal cell walls, which have extremely thin walls, plant cell walls form an extremely thick (ca. 20 nanometers), rigid and boxlike structure comprised of cellulose fibrils encased in a cement of polysaccharide and proteins. Plant transformation therefore requires specialized methods for plant cell wall penetration that differ from those used for animal cell transformation. The method(s) used must be safe for the technicians or practitioners of transformation method, and the method(s) must not introduce mutations in the donor or transforming DNA nor the recipient DNA. The genetic alteration will be stably inherited by progeny of the transformed plant. Progeny can be obtained either asexually, by taking multiple cuttings of the transformed plant, or sexually, through seed. The preferred method for plant propagation depends on the species; for example, florist's geraniums are nearly always propagated asexually, while tomatoes are nearly always propagated by seed.

By far the most common method for the introduction of new genetic material into a plant genome involves the use of living cells of the bacterial pathogen Agrobacterium tumefaciens to literally inject a piece of DNA, called transfer or T-DNA, into individual plant cells (usually following wounding of the tissue) where it is targeted to the plant nucleus for chromosomal integration. This living plant pathogen thus breaches all three physical barriers: thick cell wall, cell membrane and nuclear envelope, to introduce the DNA. There are numerous patents governing Agrobacterium mediated transformation and particular DNA delivery plasmids designed specifically for use with Agrobacterium — for example, US4536475, EP0265556, EP0270822, WO8504899, WO8603516, US5591616, EP0604662, EP0672752, WO8603776, WO9209696, WO9419930, WO9967357, US4399216, WO8303259, US5731179, EP068730, WO9516031, US5693512, US6051757 and EP904362A1. However, Agrobacterium infection of monocots, including corn, rice, wheat, barley and sugarcane is extremely limited. When such rare infections occur or are forced, the frequency is always much lower than with dicots. However, even dicot transformation frequencies are often extremely low, with some species being highly recalcitrant or impossible to transform (for example, Lee et al, 2004), pointing to a need for more efficient plant transformation methods.

Agrobacterium also infects some plant tissues more efficiently than others. As a result, most of the patents covering use of Agrobacterium are directed to very particular ways to obtain the transformation of embryo tissue, callus tissue, pollen, apical meristems, floral parts, seeds and living plants. Agrobacterium-mediated plant transformation involves as a first step the placement of DNA fragments cloned on plasmids into living Agrobacterium cells, which are then subsequently used for transformation into individual plant cells. Agrobacterium-mediated plant transformation is thus an indirect plant transformation method.

Several direct plant transformation methods using DNA have been reported. As with Agrobacterium, these methods all teach and rely on breaching all three of the three physical barriers to transformation: thick cell wall, cell membrane and nuclear envelope. The first of these to be reported historically is electroporation, which utilizes an electrical current applied to a solution containing plant cells (M. E. Fromm et al., Nature, 319, 791 (1986); H. Jones et al. Plant MoI. Biol, 13, 501 (1989) and H. Yang et al., Plant Cell Reports, 7, 421 (1988). This technique has almost exclusively been done using plant cells treated with enzymes to partially or fully remove the thick cell walls, forming protoplasts. There are a few exceptions (Lee et al., 1989; Chowrira et al. 1998), but these did not result in the regeneration of fully transgenic plants.

The plant cell wall is a particularly thick structural barrier, and therefore the electroporation process itself is usually used to breach only the cell membrane and nuclear envelope, and relies on a first and independent step of removing the cell walls. For example, Marikawa et al., Gene, 41, 121 (1986) used electroporation to transiently transform prepared cell suspensions directly from tobacco leaves using macerozyme. Similarly, pectinolytic enzymes were used prior to electroporation on sugar beet suspension tissue to give transient transformation (Lindsey et al. Plant Molec. Biol, 10, 43 (1987). Stable transformation by electroporation of Zea mays cells treated with wall-degrading polysaccharidase enzymes was disclosed by Krzyzek et al, 1995 (US 5,472,869). Another direct method that has been reported is chemically facilitated endocytosis of

DNA into plant protoplasts using one of several cationic polymers, including polyethylene glycol (PEG), polyethyleneimine (PEI), chitosan, polyamidoamine, lipofectamine and/or polymer micelles. These cationic polymers promote association with the plasma membrane so that the negatively charged DNA molecules can move past through the membrane, which is also negatively charged (Hosseinkhani, H. et al., 2003). Both PEG and PEI are also now known to help condense a DNA molecule and protect it from degradation due to polyion complexation. Again, none of these cationic polymers are known or thought to be capable of penetrating the thick plant cell wall. The first reported use of a cationic polymer, in this case PEG, in plant transformation was with maize protoplasts (G. Donn et al., Abstracts VIIth International Congress on Plant Tissue and Cell Cult., Amsterdam A2-38; Jun. 24-29, 1990). While a number of additional reports have disclosed the introduction of foreign DNA into protoplasts using polymers such as PEG and lipofectin (for example, in tobacco; Sporlein and Koop, 1991), these reports have all involved transformation of protoplasts generated by enzymatic treatment to remove the plant cell wall.

Encapsulation of DNA in cationic polymers has also been used for direct introduction of DNA into animal cells, which by comparison with plant cells have extremely thin cell walls, but efficiencies have been low. Several attempts have been made to use PLL and PEI that have been modified in various ways for improving delivery and expression of genes or transfection of entire viruses in animal cells (Ogris, Steinlein et al. 2001; Wolschek, Thallinger et al. 2002; Varga et al., 2005). Transformation efficiencies remain low, however, which may be due to a number of different factors or rate limiting steps in the process, including overt cell toxicity of the carrier (Spagnou et al., 2004), failed release from the cell membrane following internalization by endocytosis, and/or failed penetration of the nuclear envelope (van der Aa et al., 2006; Varga et al., 2005).

Use of sonication was reported as yet another method to provide direct transformation of plant protoplasts (Joersbo et al. 1990). This method suffers, as do the others requiring use of protoplasts, by the tedious process required to create and preserve plant protoplasts and then regenerate them into whole plants following transformation.

Direct transformation of plants requires dealing with their thick cell walls. Protoplast formation and regeneration is tedious and technically demanding, even in the best of circumstances (Potrykus, 1990) and impossible with many plant species. Even if the tissue is regenerable, often the resulting plants are non-fertile. Clearly, transformation methods that breach the plant cell wall while keeping it intact are needed. The first direct method to do so, called "Holistic bombardment", uses ultrafϊne particles, usually tungsten or gold, that are coated with DNA and then sprayed onto the surface of a plant tissue with sufficient force to cause the particles to penetrate plant cells, including the thick cell wall, membrane and nuclear envelope, but without killing at least some of them (US 5,204,253, US 5,015,580). The method used requires both specialized equipment and expensive reagents. Another problem with this method is that each particle that enters a particular cell is usually coated with multiple copies of the DNA provided for transformation, thereby usually resulting in multiple DNA insertions (Pederson et al.,1997; Kohli et al., 1998; Pawlowski & Somers, 1998; Jackson et al., 2001). Multiple insertions frequently lead to gene silencing, and the greater the number of insertions, the lower the gene expression level (Stoger et al., 1998; Popelka et al., 2003). By contrast, single insert events are frequently observed after Agrobacteriiim mediated gene delivery (Cheng et al., 1997; Fang et al., 2002). In order to make this method work for practical purposes requires a tedious attention to a combination of factors that must be optimized. These include: genotype specific tissue culture (Shimada, 1978) and transformation response (Iser et al., 1999; Rasco- Gaunt et al., 2001), quality and developmental stage of the explants at the time of culture initiation (Armaleo et al., 1990), culture medium composition (Barro et al., 1998) and culture conditions, culture period before and after biolistic gene transfer (Rasco-Gaunt et al., 1999), osmotic treatment of the tissue cultures to reduce tissue damage during biolistic gene transfer (Vain et al., 1993), transgene expression cassettes (Li et al., 1997), biolistic gene transfer system and its specific parameters (Altpeter et al.,1996) and the selection system and its parameters (Christou & Ford, 1995). Clearly, a simpler method is required.

The second direct method used to breach the plant cell wall while keeping it intact uses a very special and carcinogenic form of Beta silicon carbide specifically manufactured using argon gas and a catalyst to form into fibrous or needle-like structures called "whiskers". First invented in 1981 (US4,283,375) as beta silicon carbide, whiskers are manufactured from a variety of metal or ceramic compounds, such as aluminum borate, silicon nitride, and asbestos and consist of finely spun micro-fibers which are used for primarily for insulation (e.g. furnace linings) and as a reinforcing agent in resins, metals and ceramics. These fibrous forms of metal or ceramic consist of sharp, porous or hollow needle-like projections that literally impale the cells, and also the nuclear envelope of cells, which are exposed to them and both silicon carbide and aluminum borate whiskers have been used for plant transformation (Mizuno et al., 2004; Petolino et al., 2000; US5302523 US Application 20040197909) and also for bacterial and animal transformation (Kaepler et al., 1992; Raloff, 1990; Wang, 1995). It is the fibrous, needle-like, "whiskers" form of a material, whether made of metal or ceramic that is claimed and taught as critical for transformation in all publications (Petolino et al., 2000; Mizuno et al., 2004; Kaepler et al., 1992; Raloff, 1990, Wang, 1995) and the inventive step in the plant transformation patent (US5302523).

There is no teaching or suggestion that ordinary silicon carbide powders can be used for plant transformation. Silicon carbide powders, which have a widespread and very long history of use, were first patented in 1893 (US492,767) and are relatively easy to manufacture using silica sand and carbon heated to temperatures of 1600 C to 2500 C. They consist of extremely hard, dark, iridescent crystals that are insoluble in water and other common solvents. This powder is marketed under such familiar trade names as Carborundum and Crystolon. Carborundum powder has long been used as a transfection agent to inoculate or introduce plant viruses into plant tissue (Agrios, 1969), and has more recently been used to create wound sites for Agrobacteήum-meάiateά plant transformation (Curuk et al., 2005; Lim et al. 2005; Cheng et al., 1996). The carborundum particle size traditionally used for viral transfection is a 320 grit, or 32.5-36 micrometers in size, roughly corresponding to the average size of a plant cell. There is no teaching or suggestion in any publications that such powder can be used to directly introduce DNA into plant cells or cell cytoplasm for direct plant transformation. Critically, silicon carbide powders contain no whiskers (Svensson et al., 1997).

Not surprisingly, the fibrous, needle-like "whiskers" form of silicon carbide is a pulmonary health hazard and therefore must be handled much differently from non-fibrous silicon carbide powders because said powders contain no whiskers. Recent studies have demonstrated that the carcinogenicity of silicon carbide whiskers is similar to that of asbestos; 100% of rats developed mesothelioma within one year of intraperitoneal injection (Adachi et al., 2001). Silicon carbide whiskers can be toxic and mutagenic to organisms, including humans; and toxicity may exceed that of crocidolite asbestos fibres. [Johnson et al. 1990; Vaughan et al., 1991, 1993; Svensson et al., 1997]. The two silicon carbide forms, powder and fibrous whiskers, are regulated much differently, with the British Columbian (Canadian) Occupational Health and Safety (OHS) regulating the fibrous form the same as asbestos at 0.1 fiber per cc (f/cc) exposure limit, whereas the ordinary, non-fibrous form has an exposure limit of 3-10 mg/ cubic meter. Silicon carbide whiskers were shown to generate mutagenic reactive hydroxyl radicals in a manner similar to asbestos and to cause DNA strand breakage; silicon carbide powder did not cause such effects because they contain no whiskers (Svensson et al, 1997). Clearly, if silicon carbide powder could be used for plant transformation, such use would be much safer than if silicon carbide whiskers were used.

Silicon carbide powder does not direct any DNA associated with the powder to the plant nucleus, although this may happen at a very low frequency. The size limit for passive diffusion of linear, double stranded DNA through the nuclear envelope is between 200 and 310 base pairs (Ludtke et al., 1999). Since most genes are greater than 1 kilobase in size, the nuclear envelope poses a formidable barrier to direct DNA transformation of eukaryotic cells. This barrier can be overcome, if 1) as in the case of protoplast transformation, one or more DNA condensing agent(s) are used and/or 2) if the DNA is directed to the nucleus in some active process, as occurs in natural plant infections by A. tumefaciens and natural infections of plant and animal cells by certain viruses. For example, when A tumefaciens injects DNA through the plant cell wall, it delivers not naked double stranded DNA, but single stranded DNA (ssDNA) attached to two proteins, VirD2 and VirE2, that both binds ssDNA and provides nuclear localization signal (NLS) sequences (Citovsky et al, 1988; Citovsky et al., 1989; Zupan et al., 1996). Both are evidently needed for effective delivery of ssDNA to the plant nucleus after the DNA is introduced into the plant cell cytoplasm (Ziemienowicz et al., 2001). As a second example, any virus that replicates in the nucleus of a plant or animal cell must both infect the cell by penetrating the cell wall, and also penetrate the nuclear envelope (Whittaker and Helenius, 1998). For geminiviruses, the BRl protein both binds ssDNA and the protein carries nuclear localizing signals (NLSs) in its primary amino acid sequence and is essential for nuclear infection (Pascal et al., 1994). NLSs guide the protein and any associated ssDNA through the nuclear envelope into the plant nucleus. As a third example, the Simian cell virus SV40 virus DNA carries a 72 bp DNA sequence that guides the DNA or any plasmid carrying this DNA sequence through the nuclear envelope and into the nucleus (Dean et al., 2005).

Driven primarily by the promise of human gene therapy, multiple approaches have been developed and used to improve DNA protection and to increase nuclear import of DNA following the delivery of transforming DNA into the cytoplasm of animal cells. These approaches include use of proteins and peptides carrying NLSs complexed to DNA electrostatically, covalently, and by unique chemistries (reviewed by Dean et al., 2005). Such approaches have not been generally researched or developed for use in plants, since breaching the plant cell wall is a much bigger issue and limitation to plant cell transformation and model dicot plants can be efficiently transformed using Agrobacterium. One notable exception is WO 95/05471, which conceptually envisions a method for improving the few non-Agrobacterium methods of plant transformation by use of selected components, such as VirD2 and, optionally, VirE2 that are essential to Agrobacterium transformation. In particular, it discloses a specifically adapted DNA/protein complex comprising a chimeric recombinant nucleic acid specifically containing at least one T-DNA border sequence to which VirD2 specifically and covalently attaches. Delivery through the plant cell wall using one of the few known methods (biolistic, microinjection and protoplast transformation) other than Agrobacterium is briefly mentioned, but as with all approaches developed for animal cells (and where the cell wall is not an issue), the use of carborundum powder to breach the thick plant cell wall is not anticipated.

SUMMARY OF THE INVENTION

The present invention relates to a safe, effective and improved system for delivering genes into plants for the purpose of transformation, or the permanent alteration of a plant's genetic composition. More particularly, the invention relates to a method of plant transformation that includes a delivery system, comprising: 1) a selected DNA condensing agent or group of agents; 2) a nucleic acid, or a nucleic acid complexed with one or more proteins that provide a nuclear localization signal (NLS) sequences; 3) a buffer system conducive to plant cell growth in tissue culture, such as MS-salts, and 4) non-fibrous, silicon carbide powder as an abrasive agent for inserting the selected agent(s) into a target plant tissue or plant cell.

Novel compositions and methods for delivering nucleic acids into or through a plant cell or plant tissue utilizing: 1) a carrier medium (CM); 2) non-fibrous silicon carbide powder to breach the plant cell wall, and 3) one or more DNA binding proteins with NLSs to breach the plant cell nuclear envelope, together operationally providing a nucleic acid delivery system, together with optional DNA condensing agents, and cell wall wetting agents are presented. These techniques provide systems and methods of delivering nucleic acids, including RNA or DNA, proteins complexed or uncomplexed with nucleic acids, or any combination of separate nucleic acids and proteins into or through the tissue surrounding living plant cells and nuclei, or directly into living plant cells and nuclei, efficiently and without producing irreversible biological damage to the plant cell, unnecessary DNA fragmentation or mutation of the introduced gene, and occupational safety for the people performing the plant transformations.

The transformation agent, such as RNA or DNA, may be uncomplexed, complexed with different agents, or incorporated into nanoparticles in the CM to produce the desired efficiency and/or target site. The bulk of the CM is typically an aqueous or oil-based viscous solution, or mixture thereof, which may also include bulking agents, dispersing agents, surface modifiers, and/or permeation enhancers.

Accordingly, the objects of the present invention are to remedy the deficiencies in the prior art and provide both a novel and safe-to-use nucleic acid delivery system consisting of a carrier medium (CM), non-fibrous silicon carbide (carborundum) powder, and/or DNA binding proteins with NLSs.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomelic subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3ΑTTGCC5' and 3'TATGGC share 50% homology.

As used herein, "homology" is used synonymously with "identity." In addition, when the terms "homology" or "identity" are used herein to refer to the nucleic acids and proteins, it should be construed to be applied to homology or identity at both the nucleic acid and the amino acid sequence levels. A first oligonucleotide anneals with a second oligonucleotide with "high stringency" or "under high stringency conditions" if the two oligonucleotides anneal under conditions whereby only oligonucleotides which are at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% complementary anneal with one another. The stringency of conditions used to anneal two oligonucleotides is a function of, among other factors, temperature, ionic strength of the annealing medium, the incubation period, the length of the oligonucleotides, the G-C content of the oligonucleotides, and the expected degree of non-homology between the two oligonucleotides, if known. Methods of adjusting the stringency of annealing conditions are known (see, e.g., Sambrook et al., 1989, In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Thus, for example, the present invention encompasses any oligonucleotide sequences with at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% homology to the nucleotide sequence encoding the PtIiA protein of Xanthomonas, including all known members of the AvrBs3/PthA gene family (Leach and White, 1996). Also, for example, the present invention encompasses any amino acid sequences with at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% homology to the amino acid sequence of the PthA protein of Xanthomonas. The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990, J. MoI. Biol. 215:403- 410), and can be accessed, for example, at the BLAST site of the National Center for Biotechnology Information (NCBI) world wide web site at the National Library of Medicine (NLM) at the National Institutes of Health (NIH). BLAST nucleotide searches can be performed with the NBLAST program (designated "blastn" at the NCBI web site), using the following parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward = 1; expectation value 10.0; and word size = 11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated "blastn" at the NCBI web site) or the NCBI "blastp" program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.

To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402).

Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules {id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used as available on the website of the National Center for Biotechnology Information of the National Library of Medicine at the National Institutes of Health.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted. By the term "specifically binds," as used herein, is meant a compound, e.g. , a protein, a nucleic acid, an antibody, and the like, which recognizes and binds a specific molecule, but does not substantially recognize or bind other molecules in a sample.

A first oligonucleotide anneals with a second oligonucleotide "with high stringency" if the two oligonucleotides anneal under conditions whereby only oligonucleotides which are at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% complementary anneal with one another. The stringency of conditions used to anneal two oligonucleotides is a function of, among other factors, temperature, ionic strength of the annealing medium, the incubation period, the length of the oligonucleotides, the G-C content of the oligonucleotides, and the expected degree of non-homology between the two oligonucleotides, if known. Methods of adjusting the stringency of annealing conditions are known (see, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York

In one embodiment, DNA molecules may be specially formulated into a nucleic acid delivery system (NAD) to achieve transformation without using a nucleic acid binding protein with NLSs. At present, NADs for plant transformations utilize DNA that is: 1) inside a living microbe, such as Agro bacterium sp. that breaches the plant cell wall using its type IV secretion system; 2) coated onto particles, such as gold or tungsten, that breach the plant cell wall by applied biolistic force; 3) encapsulated into PEG and utilized for direct contact with plant cell membranes following enzymatic treatments to remove the plant cell wall prior to plant transformation, with or without the use of applied electroporation or soni cation, and 4) coated onto hollow or pitted, needle-like micro-fibers known as "whiskers" that breach the plant cell wall and nuclear envelope by impaling the cells. The inventors are unaware of DNA or DNA/protein complexes being specifically formulated into nanoparticles and coated onto nonfibrous silicon carbide (carborundum) powder to directly achieve a desired transformation result. The carborundum powder may be in any size range smaller than a plant cell (ie., normally ranging from fine (320 grit or 32.5 to 36 microns) to superfine (600 grit or 13 to 16 microns) to nanopowders of 1000 grit or 6.8 microns. The NAD may include manual rubbing, by vortexing in a solution or by using an artist's air brush. The carrier medium used in the formulation of nanoparticles may be any suitable buffer such as phosphate buffered saline (PBS), MES, HEPES, MS salts or other buffer known to those skilled in the art for use with PEG, PEI, lipofectamine or other DNA condensing agent. hi another embodiment, DNA molecules may be specially formulated into a NADS to achieve a desired transformation result using a DNA binding protein to protect the DNA from nuclease activity. The DNA binding protein may or may not carry NLSs to enhance transfer to the plant nucleus. In another embodiment, DNA molecules may include a nuclear targeting sequence, such as the SV40 72bp enhancer repeat.

In a preferred embodiment, DNA molecules may be specially formulated into a NADS to achieve a desired transformation result using a modular protein, either natural or synthetic, having at a minimum a high-affinity DNA binding module such as a bacterial repressor protein and one or more NLSs to enhance transfer of the bound DNA into the plant nucleus. Said modular protein may be mixed with the DNA as a pure or semi-pure protein preparation, or said modular protein may be expressed in the same cells used for replication of the DNA molecule.

In another embodiment, a polymer is incorporated into the NADS to aid in the delivery of the biologically-active agent. For example, a complexation polymer is used to ionically condense a polynucleotide, enhancing uptake into the cell via endosomes, as well as protect the polynucleotide from nuclease damage, as well as enhance the uptake of the polynucleotide through the nuclear envelope and into the nucleus. In addition, a complexation polymer, such as polyethylene glycol (PEG)₅ poly-1-lysine (PLL), polyethylenimine (PEI) or lipofectamine may be used at different molecular weights and concentrations, and/or be altered to have improved stability in biological solutions or incorporate protonatable groups that serve to buffer the acidic endosome, protecting an endocytosed polynucleotide from degradation. Calcium phosphate particles, or related salts, and/or a peptide and/or a viral agent may also be used to condense DNA and facilitate uptake into plant cells during transformation.

In a preferred embodiment, the invention is directed, but not limited, to the transfer of nucleotides/oligonucleotides and plasmid DNA, as well as other species of nucleic acid including DNA or RNA, into plant cells. Moreover, the nucleic acid may be of any reasonable length and can be single stranded or double stranded. The nucleic acids may also be modified or substituted using synthetic analogs, hi general, this invention maybe used to transfect and/or transfer material to any type of plant cell. The invention is particularly useful with cell types that are difficult to transform using prior art methods, such as monocots generally, and certain dicot species such as geranium. Employing a combination of a nontoxic, readily available silicon carbide powder with a DNA binding protein carrying NLSs to guide the DNA to the nucleus represents a significant departure from the teaching of the prior art. In another embodiment, the concentration ratios of the DNA binding protein to DNA are preferably selected to achieve the most efficient delivery of a single copy of the exogenous DNA in the NADS to the plant of interest.

The goal in many cases may be to maximize transmission of an infectious DNA agent into intact, standing plants. While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. The present invention is not limited to the described compositions and methods, nor is it limited to a particular protein or material, nor is the present invention limited to a particular scale or batch size of production. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows PCR amplification of transgenic P. hortorum cv. 'Tango' transformed using nonfibrous silicon carbide as described. All plant DNA samples taken from leaf tissue still in regeneration medium, 12 weeks after transformation. Lanes 1-5, hygromycin gene screen; Lanes 6-10, gus gene screen. Lane M, molecular weight standard (1 kb ladder).

Lanes 1 & 6, cv. 'Tango' negative control (IPG method, no DNA) leaf tissue; Lanes 2 & 7, cv. 'Avenida' positive control (Agrobacterium method) leaf tissue; Lanes 3 & 8, cv. 'Tango' experimental (IPG method, using DNA) leaf tissue; Lanes 4 & 9, bacterial DNA negative control (no plasmid); Lanes 5 & 10, bacterial DNA positive control (with plasmid).

Figure 2 shows GUS stained petunia cultivar "Whisper Scarlet" transformed using nonfibrous silicon carbide powder as described. Figure 3 shows plasmid pIPG802.

EXAMPLE 1 -TRANSFORMATION OF GERANIUM USING CARBORUNDUM POWDER

Tender healthy looking petioles, ca. 2 cm in length each, of Pelargonium X hortorum were excised from plants grown in a greenhouse. They were soaked in 70% v/v ethanol for one minute, followed by surface sterilization for 5 minutes in 10% (v/v) sodium hypochlorite solution containing two drops of Tween 20 per 100 ml. Approximately 100 segments were rinsed in 500 ml sterile distilled water, repeated 3 X. Both ends of each petiole segment were then cut off and discarded, leaving freshly cut 1 cm pieces.

A 1, 5 or 50 microliter solution of prepared plasmid DNA, in linear or circular form (1 microgram per microliter) was added to filter sterilized CM consisting of 15% PEG 8000 in 4.5 ml MS buffer containing 7.3% mannitol, 0.03% Silwet L-77 and 10 mg/ml carborundum powder (320 or 600 grit). In some experiments, carborundum nanopowder (45 to 55 nm spheres) at 10 mg/ml was used. In some experiments, 2-4% lipofectin reagent (Invtrogen) was used to replace 15% PEG. Approximately 20-25 aseptically prepared petioles were submerged in a plastic tube which was aseptically closed with caps and agitated for 20 seconds using a Genie 2 (Fisher Scientific) vortex machine. The pieces were allowed to soak for 10 minutes, and then vortexed a second time for 20 seconds. In some cases, the CM containing DNA, carborundum powder, mannitol and Silwet L-77 was rubbed across the length of the cut petiole sections using a sterile cotton swab.

Following the vortex treatment, petiole pieces were immediately blotted on sterile paper towels taking care not to let the petioles dry and were then plated on regeneration medium without selection. Plates were sealed with parafilm and incubated at 25 C, in light or dark.

After 2 days, the petiole pieces were transferred to fresh regeneration medium and incubated at 26 C (60% humidity, 16 hour photo period). Selection was made using hygromycin at 10 micrograms per milliliter. Plates were sealed with parafilm and incubated in a chamber maintained at 25 C (60% humidity, 16 hour photo period). After a period of 4-8 weeks, well differentiated shoots emerged.

Shoot pieces were excised from remaining tissue and transferred to magenta boxes containing rooting medium. Magenta boxes were incubated in a growth chamber at 25 C with 16 hour light/ 8 hr darkness. When the plants were well rooted, the lid of the magenta box was opened and covered with plastic wrap. After a day or two, a cut was made in the plastic wrap to facilitate hardening of the plants. After hardening, plants were transferred to pots and grown in a green house.

Plants were determined to be transgenic by PCR, Southern blots and/or marker gene expression. In Fig. 1 is shown PCR amplification of both a uidAl (GUS) marker gene and a hygromycin marker gene in transgenic geranium (Pelargonium X horturum cv. "Tango"). EXAMPLE 2— TRANSFORMATION OF PETUNIA USING CARBORUNDUM POWDER

Tender, healthy looking petunia leaves were surface sterilized by immersing in 70% ethanol for 1 min, followed by a 20 minute immersion in an aqueous 10% (v/v) bleach solution containing 0.1% (v/v) Tween-20 and then rinsed four times in sterile distilled water. Leaves were cut into ca. 2 cm² pieces and used for transformation.

A 1, 5 or 50 microliter solution of prepared plasmid DNA, in linear or circular form (1 microgram/microliter) was added to filter sterilized CM consisting ofl5% PEG 8000 in 4.5 ml MS buffer containing 7.3% mannitol, 0.03% Silwet L-77 and 10 mg/ml carborundum powder (320 or 600 grit). In some experiments, carborundum nanopowder (45-55 nm spheres) at 10 mg/ml was used. In some experiments, 2-4% lipofectin reagent (Invtrogen) was used to replace 15% PEG. Approximately 50-100 petunia leaf pieces were submerged in a plastic tube which was aseptically closed with caps and agitated for 20 seconds using a Genie 2 (Fischer Scientific) vortex machine. Following the vortex treatment, leaf pieces were immediately blotted on sterile paper towels taking care not to let the leaves dry and were then plated on regeneration medium, but without antibiotic selection. Plates were sealed with parafilm and incubated at 26 C (60% humidity, 16 hour photo period).

After 2 days, the leaf pieces were transferred to fresh regeneration medium and incubated at 26 C (60% humidity, 16 hour photo period). Selection was made at this point using kanamycin at 150 micrograms per milliliter. Plates were sealed with parafilm and incubated in a chamber maintained at 26 C (60% humidity, 16 hour photo period). After a period of 4-8 weeks, well differentiated shoots with leaves emerged.

Shoot pieces were excised from remaining tissue and transfered to magenta boxes containing rooting medium. Magenta boxes were incubated in a growth chamber with 16 hour light and 8 hr darkness. When the plants were well rooted, the lid of the magenta box was opened and covered with plastic wrap. After a day or two, a cut was made in the plastic wrap to facilitate hardening of the plants. After hardening, plants were transferred to pots and grown in a green house. Plants were determined to be transgenic by PCR, Southern blots and/or marker gene expression, hi Figure 2 is shown GUS stained petunia cultivar "Whisper Scarlet" transformed using nonfibrous silicon carbide powder as described. The entire leaf section in Fig. 2 is blue (not shown), with veins deeply stained with GUS. EXAMPLE 3---TRANSFORMATION OF RICE USING CARBORUNDUM POWDER

Mature rice seeds were manually dehusked, sterilized with 70% ethanol for 1 min and then with 50% Clorox (2.6% sodium hypochlorite) containing 0.1% Tween 20 for 30 min. on a shaker. After sterilization, seeds were rinsed in sterile distilled water three times and placed on callus induction medium in Petri dishes. Plates were incubated under continuous light at 30 C. Calli became visible in a week. After two weeks, the compact calli derived from the scutella were separated with a scalpel and used for transformation.

A 1, 5 or 50 microliter solution of prepared plasmid DNA, in linear or circular form (1 microgram per microliter) was added to filter sterilized CM consisting of (A) 15% PEG 8000, or (B) 15% PEG 8000 in 5% DMSO, in 4.5 ml MS buffer containing 7.3% mannitol, 0.03% Silwet L-77 and 10 mg/ml carborundum powder (320 or 600 grit). In some experiments, carborundum nanopowder (45-55 nm spheres) at 10 mg/ml was used. In some experiments, 2-4% lipofectin reagent (Invtrogen) was used to replace 15% PEG. Approximately 20-25 aseptically callus pieces were submerged in a plastic tube which was aseptically closed with caps and agitated for 20 seconds using a Genie 2 (Fischer Scientific) vortex machine. The pieces were allowed to soak for 10 minutes, and then vortexed a second time for 20 seconds.

Following the vortex treatment, callus pieces were immediately blotted on sterile paper towels taking care not to let the tissue dry and were then plated on regeneration medium without selection. Plates were sealed with parafϊlm and incubated at 25 C, in light or dark.

After 2 days, the callus pieces were transferred to fresh regeneration medium and incubated at 30 C (60% humidity, continuous light). Selection may be made at this point, or alternatively, screening may be made of regenerated shoots using marker genes, PCR or ELISA. Plates were sealed with parafilm and incubated in a chamber maintained at 30 C (60% humidity, continuous light). After a period of 2-6 weeks, well differentiated shoots emerged.

Shoot pieces were excised from remaining tissue and transferred to magenta boxes containing rooting medium. Magenta boxes were incubated in a growth chamber at 30 C with continuous light. After 2-4 weeks, regenerated plantlets were ready for transplanting to soil in pots and grown in a green house.

Plants were determined to be transgenic by PCR, Southern blots and/or marker gene expression. EXAMPLE 4— PREPARATION AND USE OF A MODULAR PROTEIN CARRYING BOTH DNA-BINDING AND NLS MOTIFS IN PETUNIA TRANSFORMATION.

A translational gene fusion was made that consisted of the entire coding sequence of NuI from phage lambda, encoding the high affinity DNA binding protein gpNul (Yang et al, 1999) fused with the 3 ' terminal region of pthA, encoding three NLSs (Yang and Gabriel, 1995), forming gpNul ::3NLS (SEQ ID No. 1). This protein was expressed in E. coli . Plasmid pIPG802 (Figure 3) is a pUCl 8 based cosmid vector that is 4.9 kb in size and encodes the plant selection antibiotic marker gene kanamycin (800 bp), operationally driven by the 35S plant promoter (860 bp) and ending with the NOS terminator (252 bp). The phage lambda cos site is 355 bp. The crude protein extract containing gpNul ::3NLS was extracted and 10 micrograms of the protein extract was added to 250 nanograms of covalently closed, circular plasmid pIPG802 and incubated at room temperature for two hours. The complex was added to filter sterilized CM in place of the plasmid DNA as described in Example 1 , above.

-IP-

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Claims

WHAT IS CLAIMED IS:

1. A method of preparing a transgenic plant, comprising: (a) obtaining a linear or circular nucleic acid segment; (b) treating the nucleic acid segment with a carrier medium that includes carborundum or non-fibrous silicon carbide powder that has not been specifically produced or formulated to include needle-like structures known as silicon carbide fibers or whiskers; (c) contacting a recipient plant cell with said nucleic acid segment in carrier medium, and (d) regenerating a plant from a plant cell which has been stably transformed with said nucleic acid segment.

2. The method of claim 1 , wherein said carrier medium also includes a nucleic acid binding protein, said protein also encoding one or more nuclear localization signal

(NLS) sequences.

3. The method of claims 1 and/or 2, wherein said nucleic acid segments are further defined as DNA segments.

4. The method of claims 1 and/or 2 or 3, wherein said non-nucleic acid carrier medium includes cationic or neutral liposomes or polymers, such as polyethylene glycol

(PEG), poly-1 -lysine (PLL), or polyethylenimine (PEI).

5. The method of claims 2, 3 or 4, wherein the NLS sequences of said DNA binding protein are obtained from Xanthomonas.

6. The method of claims 2, 3 or 4, wherein the NLS sequences of said DNA binding protein are obtained from an artificial source modeled with 70% or greater amino acid identity to the PthA protein of Xanthomonas .

7. The method of claims 1 and/or 2, 3 or 4, wherein said carrier medium includes a plant growth medium or buffer typically used for plant tissue culture.

8. The method of claim 7 wherein said plant growth medium includes IX Murashige & Skoog's basal salt mixture (MS salts buffer).

9. The method of claims 1 and/or 2, 3 or 4, wherein said carrier medium includes a permeation enhancer, such as DMSO.

10. The method of claims 1 and/or 2, 3 or 4, wherein said carrier medium includes a wetting agent, such as Silwet L-77.

11. The method of claim 3, wherein said DNA segments include a lambda cos site.

12. The method of claim 3, wherein said DNA is cut with an enzyme or any combination of enzymes so as to produce linear DNA segments with just the plant-selectable marker, its promoter and terminator, and the cos site.

13. The method of claim 12, wherein said enzyme is a restriction endonuclease.

14. The method of claim 3, wherein said DNA segments comprise one or more exogenous genes functionally encoding a selectable or a screenable marker trait, or both.

15. The method of claim 3, wherein the recipient cells are transformed with one or more exogenous genes not encoding a selectable or a screenable marker trait.

16. The methods of claims 15 and 16, wherein at least two exogenous genes are positioned on the same DNA segment.

17. A method of transforming a plant comprising: (a) preparing a carrier medium comprising from 1 ng to 5000 ng of DNA per each treatment in IX MS salts buffer, 2% PEG8000 and 320 grit carborundum powder; (b) contacting recipient plant cells with said caπier by immersion of the cells in sterile tubes, followed by agitation, either by mechanical shaking or by swirling; (c) regenerating plants from recipient cells which have received said DNA, and (d) identifying a transgenic plant by selection or by screening, the genome of which has been augmented relative to that of the corresponding nontrans genie recipient plant through the stable introduction of said DNA.

18. A method of transforming a plant comprising: (a) preparing a carrier medium comprising from 1 ng to 5000 ng of DNA per each treatment in IX MS salts buffer, 2% PEG8000 and non-fibrous, silicon carbide nanopowder; (b) contacting recipient plant cells with said carrier by immersion of the cells in sterile tubes, followed by agitation, either by mechanical shaking or by swirling; (c) regenerating plants from recipient cells which have received said DNA, and (d) identifying a transgenic plant by selection or by screening, the genome of which has been augmented relative to that of the corresponding nontransgenic recipient plant through the stable introduction of said DNA.

19. A method of transforming a plant comprising: (a) preparing a carrier medium comprising from 1 ng to 5000 ng of DNA per each treatment in IX MS salts buffer, 2% PEG8000 and 320 grit carborundum powder; (b) contacting recipient plant cells with said carrier by mechanically rubbing the cells with the carrier medium; (c) regenerating plants from recipient cells which have received said DNA, and (d) identifying a transgenic plant by selection or by screening, the genome of which has been augmented relative to that of the corresponding nontransgenic recipient plant through the stable introduction of said DNA.

20. A method of transforming a plant comprising: (a) preparing a carrier medium comprising from 1 ng to 5000 ng of DNA per each treatment in IX MS salts buffer, 2% PEG8000 and non-fibrous, silicon carbide nanopowder; (b) contacting recipient plant cells with said carrier by use of an artist's airbrush; (c) regenerating plants from recipient cells which have received said DNA, and (d) identifying a transgenic plant by selection or by screening, the genome of which has been augmented relative to that of the corresponding nontransgenic recipient plant through the stable introduction of said DNA.

21. A method of transforming a plant comprising: (a) preparing a carrier medium comprising 20 ng of DNA per each treatment in IX MS salts buffer, 2 ug of a crude or purified protein preparation and 320 grit carborundum powder, said DNA containing a protein binding site such as a lambda cos site or repressor region and said protein preparation containing a modular protein with a DNA binding region corresponding to the DNA used such as gpNul and one or more NLSs; (b) contacting recipient plant cells with said carrier by immersion of the cells in sterile tubes, followed by agitation, either by mechanical shaking or by swirling; (c) regenerating plants from recipient cells which have received said DNA, and (d) identifying a transgenic plant by selection or by screening, the genome of which has been augmented relative to that of the corresponding nontransgenic recipient plant through the stable introduction of said DNA.

22. The method of claims 1 and/or 2, 3, 4, 17, 18, 19, 20 or 21, wherein the plant is a monocot.

23. The method of claim 22, wherein the monocot is rice.

24. The method of claims 1 and/or 2, 3, 4, 17, 18, 19, 20 or 21, wherein the plant is a dicot.

25. The method of claim 24, wherein the dicot is geranium.

26. The method of claim 24, wherein the dicot is petunia.

27. All progeny of claims 22, 23, 24, 25, or 26, whether asexually or sexually reproduced, including all produce and seeds, plant tissues and plant parts transformed using the methods claimed herein.