RU2575097C2 - Linear dna molecule delivery using pegylated quantum dots for stable transformation in plants - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly a method for stable integration of a linear nucleic acid molecule of interest into a plant cell having a cell wall, using a positively charged semiconductor nanoparticle. Also disclosed is a method for stable integration of a linear nucleic acid molecule of interest into plant material using a nanoparticle which is a quantum dot.

EFFECT: invention provides efficient and stable integration of a linear nucleic acid molecule of interest into a plant cell having a cell wall.

21 cl, 3 dwg, 5 ex

Description

PRIORITY STATEMENT

This application claims priority on the filing date of a provisional patent application for US "DELIVERY OF A LINEAR DNA MOLECULE IN PLANTS FOR STABLE TRANSFORMATION USING PEGylated QUANTUM DOTS" with serial number 61/362224, filed July 7, 2010.

BACKGROUND

Nanoparticles have unique properties that have been used to deliver DNA to specific animal cells. Nanoparticles have unique properties that were used to deliver DNA to specific animal cells. It has been found that when certain nanoparticles coated with DNA are incubated with cells that do not have a cell wall, the cells absorb the nanoparticles and begin to express genes encoded on the DNA. Semiconductor nanoparticles (e.g., quantum dots (QD)) in the size range of 3-5 nm have also been used as carriers for delivering molecules to cells. DNA and proteins can be associated with some ligands attached to the surface of QD. See, for example, Patolsky, F. et al., J. Am. Chem. Soc. 125: 13918 (2003). Coated with carboxylic acid or amine QD can be crosslinked with molecules containing a thiol group, see, for example, Dubertret B. et al., Science 298: 1759 (2002); Akerman et al., Proc. Natl. Acad. Sci. U.S.A. 99: 12617 (2002); Mitchell et al., J. Am. Chem. Soc. 121: 8122 (1999)), or an N-hydroxysucciniminyl (NHS) ester group, using standard bioconjugation protocols. See, for example, Pinaud, et al., Am. Chem. Soc. 126: 6115 (2004); Bruchez, et al., Science 281: 2013 (1998). An alternative way to attach molecules to the QD surface is to conjugate streptavidin-coated QD to biotinylated proteins, oligonucleotides, or antibodies. See, for example, Dahan, et al., Science 302: 442 (2003); Pinaud, et al., J. Am. Chem. Soc. 126: 6115 (2004); Wu, et al., Nature Biotechnol. 21:41 (2003); Jaiswal, et al., Nature Biotechnol. 21:47 (2003); and Mansson, et al., Biochem. Biophys. Res. Commun. 314: 529 (2004).

The delivery of foreign nucleic acid molecules to plants is a challenge due to the presence of walls in plant cells. The methods used for the genetic transformation of plants are based on invasive delivery. The cell wall in plant cells is a barrier to the delivery of exogenously introduced molecules. Numerous invasive methods have been used to achieve the delivery of genes and small molecules into plant cells with cell walls, such as biolistic delivery (gene gun), microinjection, electroporation and Agrobacterium-mediated transformation, but protein delivery was achieved only using microinjection. When delivery of nucleic acid molecules to plant cells is desired, the cell wall is removed before particles are added to the plant protoplasts (see Torney, et al., Nature Nanotechnol. 2: 295-300 (2007)).

DESCRIPTION OF THE INVENTION

Methods and compositions are described herein for using nanoparticles and linearized nucleic acid molecules to introduce a molecule of interest into a plant cell that has a cell wall. Some embodiments of the disclosed methods can be used to produce stably transformed genetically modified fertile plants. In some embodiments, the characteristic properties of linear nucleic acid molecules provide delivery of specific gene sequences of interest without foreign nucleic acid sequences that may have regulatory functions for the target transgenic organism.

In some embodiments, the nanoparticles may include pegylated linear nucleic acid molecules. In specific embodiments, the nanoparticles may be semiconductor nanoparticles, such as quantum dots (QD). In some embodiments, the linear nucleic acid molecule may be a linearized plasmid DNA. In other embodiments, linear nucleic acid molecules may include phosphinotricin N-acetyltransferase (PAT) and / or yellow fluorescent protein (YFP) coding sequences.

Also disclosed are methods of introducing a molecule of interest into a plant cell having a cell wall, where the methods may include providing a plant cell having a cell wall; coating the surface of the PEG nanoparticles to obtain pegylated nanoparticles; coating PEGylated nanoparticles with at least one linear nucleic acid molecule of interest; bringing into contact with each other a cell of a plant having a cell wall, with pegylated nanoparticles coated with a linear nucleic acid molecule (s) of interest; and enabling absorption of the nanoparticle and linear nucleic acid molecule (s) of interest in a cell having a cell wall.

Additionally disclosed are methods of intogression of a genetic trait into a plant. In some embodiments, the method may include providing a plant cell; coating the surface of PEG nanoparticles to produce PEGylated nanoparticles; coating pegylated nanoparticles by means of expression of a genetic trait in a plant; bringing into contact with each other plant cells with pegylated nanoparticles coated with means of expression of a genetic trait in the plant; and providing absorption by the plant cell of the nanoparticle with means for expressing the genetic trait in the plant to obtain a transformed plant cell; regeneration of a whole plant from a transformed plant cell; and plant reproduction. In some embodiments, a genetic trait that can be introgressed in accordance with the methods of the invention includes a trait selected without limitation from: male fertility; herbicide resistance; insect resistance; and resistance to bacterial, fungal and / or viral diseases.

Also, the disclosed methods of the invention can be used to transform plants in planta. In some embodiments, the plant may be selected from plants of the genus Arabidopsis, for example A. thaliana. In a particular embodiment, the plant transformed in planta may be selected from the A. thaliana plant Colombia ecotype.

Additionally disclosed are genetically modified (GM) plant cells and methods for their preparation, where plant cells have one or more nucleic acids introduced into them by the methods of the present invention. In some embodiments, a plasmid containing at least one gene of interest and a selectable marker can be introduced into a plant cell having a cell wall by means of a nanoparticle in accordance with the present invention. In further embodiments, stable transformants that have a stably integrated at least one gene of interest and / or a selectable marker can be selected. In alternative embodiments, a plant cell that already contains a gene of interest can be propagated to obtain other cells containing the molecule of interest. In other embodiments, the plant cell, already containing the molecule of interest, may be a regenerable cell, which can be used to regenerate the whole plant, including the molecule of interest.

Additionally disclosed are methods for producing regenerated plant cells containing a molecule of interest for use in tissue cultures. This tissue culture may be able to regenerate plants having essentially the same genotype as the regenerated cells. The regenerated cells in such tissue cultures can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods or stems. In addition, some embodiments provide plants regenerated from tissue cultures of this invention.

Additionally disclosed are methods for generating stabilized plant lines containing the desired genetic trait or nucleic acid molecule of interest, where the desired genetic trait or nucleic acid molecule of interest can be introduced first by absorbing the nanoparticle through the cell wall of the plant. Methods for generating stabilized plant lines are well known to those of ordinary skill in the art and may include, without limitation, methods such as self-pollination, backcrossing, producing hybrids, crossbreeding with populations, and the like. Thus, plants and plant cells are also disclosed that contain a desired genetic trait or nucleic acid molecule of interest, first introduced into a plant cell (or its predecessors) by absorption of nanoparticles through the cell wall. Plant cells containing the desired genetic trait or nucleic acid molecule of interest, first introduced into the plant or cell (or its predecessors) by absorbing nanoparticles through the cell wall, can be used in crosses with other, different plant cells to produce hybrid cells of the first generating (F ₁ ) seeds and / or plants with the desired characteristics.

In addition to the illustrative aspects and embodiments described above, additional aspects and embodiments will become apparent in light of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 includes a graphical description of the non-linearized plasmid pDAB3831.

Figure 2 includes a graphical description of the plasmid pDAB3831 linearized by the KpnI restriction enzyme.

FIG. 3 includes sequence alignment between a phosphinotricin N-acetyltransferase (PAT) DNA sequence from nanoparticles with linear DNA of the transformed Arabidopsis genome and a PAT sequence from the NCBI database.

LIST OF SEQUENCES

SEQ ID NO: 1 shows the sequence of the forward primer used to amplify the YFP gene:

TGTTCCACGGCAAGATCCCCTACG.

SEQ ID NO: 2 shows the sequence of the reverse primer used to amplify the YFP gene:

TATTCATCTGGGTGTGATCGGCCA.

SEQ ID NO: 3 shows the sequence of the forward primer used to amplify the PAT gene:

GGAGAGGAGACCAGTTGAGATTAG.

SEQ ID NO: 4 shows the sequence of the reverse primer used to amplify the PAT gene:

AGATCTGGGTAACTGGCCTAACTG.

BEST MODES FOR CARRYING OUT THE INVENTION

I. Overview of some embodiments

Non-invasive gene transfer methods of the invention can be very useful for creating genetically modified plants with desired genetic traits. Non-invasive gene transfer can contribute to the specific targeting and correction of molecular sites within cells for use in areas such as maintaining the desired indicators, achieving the final result, and obtaining the desired agronomic traits of plant cultures. The described methods, as an alternative to GMOs, can also be useful for the transient transformation of plants, for expanding the technology of introgression of genetic traits and making trees or vegetable crops resistant to pathologies where the technology is currently limited.

Recent U.S. Patent Application No. 60/978059 discloses nanoparticle-based non-invasive DNA delivery vehicles using various payload-bearing nanoparticles, including for delivering circular plasmid DNA, and clearly demonstrates the stable integration of transgenes into T ₁ seeds of Arabidopsis plants. Thus obtained transgenic plants containing circular plasmid DNA demonstrate the desired herbicide tolerance phenotypes and show a high level of tolerance while spraying at least four times the amount of ammonium glufosinate in the field. U.S. Application No. 60/978059 showed, inter alia, genetic transformation in Arabidopsis through the use of positively charged gold nanoparticles with circular plasmid DNA. This work describes, in particular, the use of linear nucleic acid molecules for stable genetic transformation of plants.

U.S. Application No. 60/978059 describes, in particular, plasmid DNA delivery mediated by positively charged nanoparticles. However, to date, no stable genomic integration of the transgene has been reported when used to deliver linear plasmids. This invention describes the use of linear nucleic acid molecules using positively charged nanoparticles for stable genetic transformation of plants. Molecular analysis shows the expression of PAT together with YFP in transgenic plants of Arabidopsis T ₁ transformed with the pat gene and yfp gene according to the methods of the invention. Transgenic T ₁ plants are fertile and produce seeds. These seeds can be used for propagation, and segregation analysis can be performed along with molecular and protein analyzes.

Linear nucleic acid molecules have characteristic properties that distinguish them from circular plasmid DNA. For example, linear nucleic acid molecules can have a clearly defined gene cassette without the backbone of the carrier vector and without a selectable marker, a bacterial antibiotic.

Ammonium glufosinate herbicide (GLA) can be sprayed into the field at a concentration level for screening transgenes. Arabidopsis T ₁ seedlings obtained using the methods of the invention showed herbicide resistance at a five-fold dosage level of glufosinate in the field, for example, in a “one day” mode, starting from seven days after emergence. The genomic DNAs of these transgenic plants were analyzed for the presence of pat and yfp by PCR, and the results showed the presence of target pat and yfp sequences in the DNA. Sequencing of the obtained PCR products showed the presence of the correct transgenic pat and yfp sequences in Arabidopsis T ₁ obtained using the methods of the invention.

II. Terms

A number of terms are used in the description and tables that follow. In order to provide a clear and consistent understanding of the description and claims, including the scope of claims to be supported by such terms, the following definitions are provided.

Backcrossing. As used here, the term “backcrossing” can be a process in which a breeder repeatedly crosses hybrid offspring with one of the parents, for example, a hybrid of the first generation F ₁ with one of the parent genotypes of this hybrid F ₁ .

The embryo. As used here, the term "germ" may refer to a small plant contained in a mature seed.

Nanoparticle. As used here, the term "nanoparticle" may refer to a microscopic particle having at least one nanometer size, for example, less than 100 nm. Nanoparticles suitable for use in the present invention may have a size of 1 nm-0.84 microns. One of the classes of nanoparticles is the "quantum dot" (QD). The quantum dot may have a median diameter of 1 nm to 10 nm, preferably 2-4 nm. Other options for nanoparticles include, without limitation: gold nanoparticles; gold-coated nanoparticles; porous nanoparticles; mesoporous nanoparticles; silicon dioxide nanoparticles; polymer nanoparticles, including dendrimers; tungsten nanoparticles; gelatin nanoparticles, nano-shells; nanonuclei; nanospheres; nanorods; magnetic nanoparticles and their combinations.

Among the available nanoparticles, luminescent semiconductor nanocrystals (QDs) provide many well-known applications in biological imaging and sounding. Their usefulness is the result of a combination of unique photophysical characteristics and sizes comparable to the size of a large protein. The hydrodynamic radius of hydrophilic QDs from CdSe-ZnS varies from 5 nm (for nanocrystals capping molecular ligands) to 20 nm for nanocrystals encapsulated in block copolymers. One QD can be conjugated to several biomolecules (for example, antibodies, peptides and nucleic acid molecules), to obtain multifunctional QD bioconjugates with extended avidity. In addition, their strong resistance to chemical and photo degradation can generally provide long-term fluorescence monitoring of specific biological processes. See Nie and Emory, Science 275: 1 102-106 (1997). Many non-covalent conjugation schemes based on self-assembly due to the affinity of metals and biotin-avidin binding can simultaneously be used within the same complex, without the need for additional purification, leading to multifunctional QD bioconjugates that are stable even in intracellular media. See Yezhelyev et al., J. Am. Chem. Soc. 130 (28): 9006-12 (2008). Using an average of 10 YFP plus a nominal amount of 50 penetrating cell peptides (CPP) per QD, intracellular delivery of protein load with a molecular weight of at least 300 kDa and a spatial size of 150 angstroms can be achieved. See there. The delivered load for QD-b-PE conjugates has a much wider range of sizes and molecular weights; for example, delivered assemblies, based on an average of 2.5 streptavidin-b-PE per conjugate, have a molecular weight that generally exceeds 103 kDa and spatial dimensions approach 500 angstroms. Molecular weight and size can be significantly increased if conjugates with higher b-PE valencies are used.

Nucleic acid molecule. Polymeric forms of nucleotides, which can include both sense and antisense strands of RNA, cDNA, genomic DNA, artificial chromosomes (ACE), and synthetic forms and mixtures of the above polymers. A nucleotide refers to a ribonucleotide, deoxyribonucleotide, or a modified form of both types of nucleotides. The term "nucleic acid molecule", as used here, is synonymous with the terms "nucleic acid" and "polynucleotide". A nucleic acid molecule usually has at least 10 bases in length, unless otherwise indicated. This term includes single-stranded and double-stranded forms of DNA. A nucleic acid molecule may include, individually or together, natural and modified nucleotides that are linked by natural and / or non-naturally occurring nucleotide bonds.

Operably linked. The first nucleic acid sequence is operably linked to the second nucleic acid sequence when the first nucleic acid sequence is in functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. In recombinant production, operably linked nucleic acid sequences can be contiguous, and, if necessary, can connect two protein coding regions in the same reading frame. However, in order to be operably linked, nucleic acids may not be contiguous.

Pegylated. As used herein, the term “pegylated” may refer to nanoparticles (eg, quantum dots) when the surfaces of the nanoparticles have been altered using polyethylene glycol (PEG, PEG) to improve biocompatibility. Pegylated nanoparticles can be further coated with various targeting ligands, for example, peptides and antibodies, to improve the efficiency of delivery to specific cells and tissues. PEG was conjugated with nanoparticles and with various medicinal substances, liposomes and polymer micelles, in order, for example, to extend the circulation time of coated nanoparticles in the blood by reducing non-specific adsorption of proteins due to the effect of spatial stabilization.

Quantum dot. As used herein, the term “quantum dot” (QD), also sometimes previously known as nanocrystals, may refer to a semiconductor nanostructure that restricts the movement of electrons from the conduction band and holes of the valence band, or excitons (coupled pairs of electrons from the conduction band and valence holes zones) in all three spatial directions. This limitation can be due, for example, to electrostatic potentials (generated by external electrodes due to doping, deformation, impurities, etc.), the presence of an interface between various semiconductor materials (for example, in nanocrystalline core-shell systems), and the presence of a semiconductor surface (for example, a semiconductor nanocrystal) or combinations thereof. A quantum dot may have a discrete quantized energy spectrum. The corresponding wave functions can be spatially localized at the quantum dot, but extend over many periods of the crystal lattice. A quantum dot contains a small finite number (for example, of the order of 1-100) of electrons in the conduction band, holes of the valence band, or excitons (i.e., has a finite number of elementary electric charges).

Quantum dots are a special class of semiconductor materials, which can be crystals consisting of materials of elements of periodic groups II-VI, III-V or IV-VI. Their sizes can vary, for example, from 2 to 10 nanometers (10-50 atoms) in diameter. In some embodiments, the quantum dots can be made in the form of a core and shell of cadmium selenide and zinc sulfide (CdSe / ZnS), and they have a number of useful electrical and optical properties that differ in character from the characteristics of the original bulk material.

Nanoparticles in the form of quantum dots were investigated as a means for visualization in vivo and in vitro, due to the high quantum yield, high molar absorption coefficient and high resistance to photobleaching.

Resistant to glyphosate. Glyphosate dose resistance refers to the ability of a plant to survive (i.e. a plant cannot die) under the influence of a given dose of glyphosate. In some cases, tolerant plants may temporarily turn yellow or otherwise show some damage induced by glyphosate (for example, excessive shoot formation and / or growth retardation), but then they are restored.

Stabilized. As used herein, the term “stabilized” may refer to plant characteristics that are reproducibly transferred from one generation to the next generation of inbred plants of the same variety.

Transgene. As used herein, the term "transgene" may refer to an exogenous nucleic acid sequence. As one example, a transgene is a gene sequence (e.g., a herbicide resistance gene), a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desired agricultural trait. In yet another example, a transgene is an antisense nucleic acid sequence, where expression of an antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. A transgene may contain regulatory sequences operably linked to a transgene (e.g., promoter). In some embodiments, the nucleic acid molecule of interest that is to be introduced by nanoparticle transformation is a transgene. However, in other embodiments, the nucleic acid molecule of interest is an endogenous nucleic acid sequence for which additional genomic copies of the endogenous nucleic acid sequence, or a nucleic acid molecule that is antisense to the target nucleic acid molecule in the host, are desired.

Absorption. As used herein, the term “absorption” can refer to a translocation of a particle, such as a nanoparticle (eg, a quantum dot), through a cell wall or cell membrane, where translocation takes place not only as a result of an impulse imparted to this particle by something else than the cell into which this particle is absorbed. Non-limiting examples of devices or methods that cause a particle to translocate through the cell wall or cell membrane only as a result of the impulse imparted to the particle are biolystics, a gene gun, microinjection and / or puncture using nanomaterials (impalefection).

III. Delivery of DNA molecules using nanoparticles for stable transformation of plant cells

A. Overview

This invention describes, in particular, new methods for transforming plants using nanoparticle transfer of linearized plasmid DNA for genetic transformation and the development of stable transgenic plants. The methods according to some embodiments of the present invention can provide not only the rapid generation of a transgenic organism, but also additional opportunities for the desired genomic modifications, compared with other transformation methods. Embodiments of the invention have led to a stably transformed, first reported, plant obtained by delivering linearized plasmid DNA mediated by nanoparticles. The disclosed methods of genetic modification represent a rejection of traditional methods of genetic transformation of plants, and can be very useful for the generation of transgenic plant cultures.

B. DNA molecules

With the advent of molecular biological techniques that isolate and characterize genes that encode a particular protein or RNA product (e.g., interfering RNAs (RNAi), plant biologists have been very interested in creating a genome of cells that contain and express foreign genes, or additional or modified variants of native or endogenous genes (possibly under the control of various promoters), in order, for example, to change the genetic characteristics of glu Such foreign additional and / or modified genes are referred to herein by the generic term “transgenes.” Transgenes can, for example, encode a protein of interest, or be transcribed into RNAi. Several methods have been developed over the past fifteen to twenty years to produce transgenic cells, and the present invention in specific embodiments relates to variants of transformed cells, and methods for their production by introducing into the cells of a plant having a cell wall, one or more linear nucleic acid molecules due to the absorption of the nanoparticle through the cell wall. In some embodiments of the invention, the transgene may be contained in a linearized expression vector.

Cell transformation may include the construction of an expression vector that will function in a particular cell. Such a vector may include a nucleic acid sequence that includes a gene under the control of a regulatory element or operably linked to a regulatory element (e.g., a promoter, enhancer, termination sequence, or combination thereof). Thus, the expression vector may contain one or more such operably linked gene / regulatory element combinations. The vector (s) can be in the form of a plasmid, and can be used alone or in combination with other plasmids in order to ensure cell transformation using the transformation methods as described herein to incorporate the transgene (s) into the genetic material of a plant cell containing the cell wall .

In other embodiments, the nucleic acid molecule of interest may be a linear nucleic acid molecule. Linear nucleic acid molecules can be obtained, for example, by cleaving a circular plasmid with a restriction endonuclease. Restriction endonucleases cleave the plasmid at one or more recognition sites within the nucleotide sequence of the plasmid. Thus, plasmids can be created that produce one or more specific linear nucleic acid molecules by cleavage with a specific restriction endonuclease. Alternatively, a given nucleotide sequence of a plasmid can be analyzed to search for recognition sites by one or more specific restriction endonucleases that produce one or more specific linear nucleic acid molecules (nucleic acids). By selecting restriction sites that cleave at specific positions within the ring plasmid or linear nucleic acid molecule, linear nucleic acid molecules can be obtained that lack one or more sequences of the precursor nucleic acid molecule. For example, a linear nucleic acid molecule can be obtained in which there are no foreign nucleic acid sequences (e.g., the backbone of the vector carrier, selection markers, such as bacterial selection markers and unnecessary nucleic acid sequences that are homologous to the genomic DNA of the target cell). Alternatively, a linear nucleic acid molecule can be synthesized so that it does not contain foreign nucleic acid sequences.

In other embodiments, where the molecule of interest includes one or more genes, the gene (s) may (may) be a dominant or recessive allele. For example, the gene (s) can (can) impart characteristics such as resistance to the herbicide, resistance to insects, resistance to bacterial diseases, resistance to fungi, resistance to viral diseases, male fertility, male sterility, improved nutritional quality and industrial use. Genes conferring these and other characteristics are known in the art, and any gene can be introduced into a cell containing a cell wall according to the methods of the invention.

Expression vectors for linearization and absorption through nanoparticles: Marker genes

Expression vectors may optionally include at least one genetic marker, for example, operably linked to a regulatory element that allows transformed cells containing this marker to be removed either by negative selection (i.e., by inhibiting the growth of cells that do not contain this selectable marker gene) or positive selection (i.e., screening for the product encoded by this genetic marker). Many genes of selectable markers for transformation are well known in the art and include, for example, without limitation: genes encoding enzymes that metabolically detoxify a selective chemical agent, which may be an antibiotic or herbicide; or genes encoding an altered target that may be insensitive to the inhibitor. Specific methods for positive selection are also known in the art.

One selectable marker gene, which may be suitable for plant transformation by certain nucleic acid molecules, is the neomycin phosphotransferase II (nptII) gene, optionally under the control of plant regulatory signals, which confers resistance to kanamycin. See, for example, Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80: 4803 (1983). Another selectable marker gene that can be used is the hygromycin transferase gene, which confers antibiotic resistance to hygromycin. See, for example, Vanden Elzen et al., Plant Mol. Biol., 5: 299 (1985).

Additional genes for selectable markers that can be used in the methods of the invention include genes for selectable markers of bacterial origin, for example, those that confer resistance to antibiotics, such as gentamicin acetyltransferase, streptomycin phosphotransferase, aminoglycoside-3'-adenyltransferase and bleomycin. See Hayford et al., Plant Physiol. 86: 1216 (1988), Jones et al., Mol. Gen. Genet, 210: 86 (1987), Svab et al., Plant Mol. Biol. 14: 197 (1990), and Hille et al., Plant Mol. Biol. 7: 171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil. See Comai et al., Nature 317: 741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) and Stalker et al., Science 242: 419-423 (1988).

Other genes for selectable markers that can be used to transform plants include genes of non-bacterial origin. These genes include, for example, without limitation, mouse dihydrofolate reductase, plant 5-enolpyruvyl shikimate-3-phosphate synthase and plant acetolactate synthase. See Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shah et al., Science 233: 478 (1986), and Charest et al., Plant Cell Rep. 8: 643 (1990).

Another class of marker genes suitable for plant transformation may be required for screening presumably transformed plant cells, rather than for direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful for quantifying or visualizing a spatial pattern of gene expression in specific tissues, and they are often referred to as “reporter genes” because they can be fused to a gene or gene regulatory sequence to study gene expression. Genes commonly used for screening transformed cells include, but are not limited to, β-glucuronidase (GUS), β-galactosidase, luciferase, and chloramphenicolacetyltransferase. See Jefferson, R. A., Plant Mol. Biol. Rep. 5: 387 (1987), Teeri et al., EMBO J. 8: 343 (1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84: 131 (1987), and DeBlock et al., EMBO J. 3: 1681 (1984).

More recently, genes encoding fluorescent proteins (e.g., GFP, EGFP, EBFP, ECFP and YFP) have been used as markers for gene expression in prokaryotic and eukaryotic cells. See Chalfie et al., Science 263: 802 (1994). Thus, fluorescent proteins and mutants of fluorescent proteins can be used as markers for screening in some embodiments of the invention.

Expression Vectors for Nanoparticle Absorption: Promoters

Genes included in expression vectors can be triggered by a nucleotide sequence containing a regulatory element, for example, a promoter. Some types of promoters are well known in the art of transformation, as well as other regulatory elements that can be used alone or in combination with promoters.

A promoter is a region of DNA that can be located to the left (upstream) from the start of transcription, and recognition and binding of RNA polymerase and / or other proteins may be included in order to initiate transcription. A "plant promoter" may be a promoter that is capable of initiating transcription in plant cells. Examples of developmentally controlled promoters include promoters that primarily initiate transcription in specific tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are called “tissue preferred”. Promoters that initiate transcription only in specific tissues are called “tissue specific”. A "cell type" specific promoter initially triggers expression in certain types of cells in one or more organs, for example, in vascular cells of roots or leaves. An "inducible" promoter can be a promoter that can be controlled by the environment. Examples of environmental conditions that may affect transcription under inducible promoters include, but are not limited to, anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell-type-specific and inducible promoters form the class of “unconstitutional promoters”. A “constitutive promoter" is a promoter that can be active under most environmental conditions.

1. Inducible promoters

An inducible promoter can be operably linked to a gene expressed in a cell. Optionally, an inducible promoter may be operably linked to a nucleotide sequence encoding a signal sequence that may be operably linked to a gene expressed in the cell. In the case of an inducible promoter, the transcription rate increases in response to the inducing agent.

Any inducible promoter may be used in an embodiment of the present invention. See Ward et al., Plant Mol. Biol. 22: 361-366 (1993). Examples of inducible promoters include, but are not limited to, ACEI promoters that respond to copper (Mett et al., Proc. Natl. Acad. Sci. U.S.A. 90: 4567-4571 (1993)); a maize In2 gene promoter that responds to an antidote to benzenesulfamide herbicides (Hershey et al., Mol. Gen Genetics 227: 229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243: 32-38 (1994)) ; and a Tet repressor promoter from Tn1O (Gatz et al., Mol. Gen. Genetics 227: 229-237 (1991)). A particularly useful inducible promoter may be a promoter that responds to an inducing agent that plants do not normally respond to. An example of such an inducible promoter is the inducible steroid hormone gene promoter, the transcriptional activity of which can be induced by a glucocorticosteroid hormone. See Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88: 0421 (1991).

2. Constitutive promoters

A constitutive promoter can be operably linked to a gene expressed in a cell, or a constitutive promoter can be operably linked to a nucleotide sequence encoding a signal sequence that can be operably linked to a gene expressed in a cell.

Various constitutive promoters may be used in the present invention. Examples of constitutive promoters include, but are not limited to, plant virus promoters, such as the 35S promoter from CaMV (Odell et al., Nature 313: 810-812 (1985)); rice actin gene promoters (McElroy et al., Plant Cell 2: 163-171 (1990)); ubiquitin promoter (Christensen et al., Plant Mol. Biol. 12: 619-632 (1989) and Christensen et al. Plant Mol. Biol. 18: 675-689 (1992)); pEMU promoter (Last et al., Theor. Appl. Genet. 81: 581-588 (1991)); MAS promoter (Velten et al., EMBO J. 3: 2723-2730 (1984)); and the maize histone H3 promoter (Lepetit et al., Mol. Gen. Genetics 231: 276-285 (1992) and Atanassova et al. Plant Journal 2 (3): 291-300 (1992)); and the ALS promoter, an XbaI / NcoI fragment (at the 5 ′ end) of the ALS3 Brassica napus structural gene (or a nucleotide sequence similar to that XbaI / NcoI fragment. See PCT International Publication WO 96/30530.

3. Tissue-specific or tissue-preferred promoters

A tissue-specific promoter can be operably linked to a gene expressed in a cell. Optionally, this tissue-specific promoter may be operably linked to a nucleotide sequence encoding a signal sequence that may be operably linked to a gene expressed in the cell. Plants transformed with a gene of interest operably linked to a tissue-specific promoter can produce the protein product of this transgene exclusively or predominantly in a particular tissue.

Any tissue specific or tissue preferred promoter may be used in the present invention. Examples of tissue-specific or tissue-preferred promoters include, but are not limited to: a root-promoter, for example, a phaseolin gene promoter (Murai et al., Science 23: 476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82: 3320-3324 (1985)); a leaf-specific and light-inducible promoter, for example, a cab or rubisco promoter (Simpson et al., EMBO J. 4 (11): 2723-2729 (1985) and Timko et al., Nature 318: 579-582 (1985)); anther specific promoter, for example, the LAT52 promoter (Twell et al., Mol. Gen. Genetics 217: 240-245 (1989)); a pollen-specific promoter, for example, the ZM13 promoter (Guerrero et al., Mol. Gen. Genetics 244: 161-168 (1993)), or a microspore preferred promoter, for example, the apg promoter (Twell et al., Sex. Plant Reprod. 6 : 217-224 (1993)).

Transport of the protein produced by the transgene into the subcellular compartment, such as chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondria, or for secretion into the apoplast, can be accomplished by operably linking the nucleotide sequence encoding the 5'- and / or 3'- signal sequence end region of a gene encoding a protein of interest. Sequences targeting the 5 ′ and / or 3 ′ end of the structural gene during protein synthesis and processing can determine, for example, where the encoded protein can be finally compartmentalized. Alternatively, such proteins targeting the subcellular compartment can be directly coupled to the nanoparticle to direct the nanoparticle coated with the molecule of interest to the desired subcellular compartment.

The presence of a signal sequence can direct the polypeptide either to the intracellular organelle, or to the subcellular compartment, or for secretion into the apoplast. Many signal sequences are known in the art. See, for example, Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S., Master's Thesis, Iowa State University (1993), Knox, C, et al., Plant Mol. Biol. 9: 3-17 (1987), Lerner et al., Plant Physiol. 91: 124-129 (1989), Fontes et al., Plant Cell 3: 483-496 (1991), Matsuoka et al., Proc. Natl. Acad. Sci. 88: 834 (1991), Gould et al., J. Cell. Biol. 108: 1657 (1989), Creissen et al., Plant J. 2: 129 (1991), Kalderon, et al., Cell 39: 499-509 (1984), Steifel, et al. Plant Cell 2: 785- 793 (1990).

Foreign protein genes and agronomic genes

Transgenic plants, according to embodiments of the present invention, can produce foreign protein in commercial quantities. Thus, the selection and propagation methods of transformed plants produce many transgenic plants that are harvested in a well-known manner. The foreign protein can then be extracted from the tissue of interest or from the total biomass. The extraction of protein from plant biomass can be performed by known methods, which are discussed, for example, in the work of Heney and Orr, Anal. Biochem. 114: 92-96 (1981).

In some aspects of the invention, the plant material providing commercial production of a foreign protein may be a plant, plant tissue, or plant cell. For transgenic plants that exhibit higher levels of expression, a genetic map can be created, for example, using traditional RFLP, PCR, and SSR assays that identify the approximate location of chromosomes in a combined DNA molecule. In this regard, see examples of methodologies in Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton 269: 284 (1993). Mapping information regarding the location of chromosomes may be useful, for example, for patent protection of a transgenic plant, as an object of the invention, or for assessing biological safety. If unauthorized propagation and crossbreeding with another germplasm can be undertaken, this map of the integration region can be compared with similar maps for suspected plants in order to determine whether the latter has a common origin with the plant that is the subject of the invention. Map comparisons may include hybridization, RFLP, PCR, SSR, and sequencing, all of which are common methods.

Similarly, agronomic genes can be expressed in transformed cells or in their progeny. More specifically, plants can be genetically engineered by the methods of this invention to express various phenotypes of agronomic interest. Examples of genes that can be used in this regard include, without limitation, genes grouped below into separate categories.

1. Genes that confer resistance to pests or diseases:

A) Genes for resistance to plant pathologies. Plant protection is often activated by a specific interaction between the product of the plant pathology (R) resistance gene and the product of the corresponding pathogen avirulence (Avr) gene. Plant varieties can be transformed with cloned resistance genes for engineered plants that are resistant to specific strains of pathogens. See, for example, Jones et al., Science 266: 789 (1994) (cloning the Cf-9 gene for tomato resistance to Cladosporium fulvum); Martin et al., Science 262: 1432 (1993) (tomato Pto gene, for resistance to tomato damaging Pseudomonas syringae encoding protein kinase); Mindrinos et al., Cell 78: 1089 (1994) (RSP2 gene for resistance to Pseudomonas syringae).

B) A gene that confers resistance to a pest, such as a soybean cyst nematode (Heterodera glycines). See, for example, PCT International Publication WO 96/30517; PCT International Publication WO 93/19181.

C) A protein of Bacillus thuringiensis, a derivative or synthetic polypeptide thereof, modeling it. See, for example, Geiser et al., Gene 48: 109 (1986) (cloning and nucleotide sequence of the Bt δ-endotoxin gene). Moreover, DNA molecules encoding δ-endotoxin genes can be purchased from the American Type Culture Collection (Manassas, Va.), For example, under accession numbers ATCC 40098, 67136, 31995 and 31998.

D) Lectin. See, for example, Van Damme et al., Plant Molec. Biol. 24:25 (1994) (nucleotide sequences of certain genes that bind clivia miniata lectin mannose.

E) Vitamin Binding Protein, such as Avidin. See PCT International Publication US93 / 06487 (Use of Avidin and Avidin Homologs as Larvicides Against Insect Pests).

F) An enzyme inhibitor, for example, a protease or proteinase inhibitor, or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793 (1987) (nucleotide sequence of a cysteine proteinase inhibitor), Huub et al., Plant Molec. Biol. 21: 985 (1993) (nucleotide cDNA sequence encoding a tobacco proteinase inhibitor I), Sumitani et al., Biosci. Biotech Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus alpha-amylase inhibitor) and US Pat. 5494813.

G) An insect-specific hormone or pheromone, for example, ecdysteroid or juvenile hormone, its variant, a mimetic based on it, or its antagonist or agonist. See, for example, Hammock et al., Nature 344: 458 (1990) (baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone).

H) An insect-specific peptide or neuropeptide that, after expression, disrupts the physiology of the affected pest. See, for example, Regan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNA encoding an insect diuretic hormone receptor), and Pratt et al., Biochem. Biophys. Res. Comm. 163: 1243 (1989) (allostatin can be identified in Diploptera puntata). See also U.S. Pat. 5266317 (genes encoding paralytic neurotoxins specific for insects).

I) Insect-specific poison produced in nature by a snake, wasp or any other organism. See, for example, Pang et al., Gene 116: 165 (1992) (heterologous expression in plants of a gene encoding a peptide that is insectotoxic for scorpion).

J) The enzyme responsible for the over-accumulation of monoterpen, sesquiterpene, steroid, hydroxamic acid, a phenylpropanoid derivative or other non-protein molecule with insecticidal activity.

K) An enzyme involved in the modification, including post-translational modification, of a biologically active molecule, for example: glycolytic enzyme; proteolytic enzyme; lipolytic enzyme; nuclease; cyclase; transaminase; esterase; hydrolase; phosphatase; kinase; phosphorylase; polymerase; elastase; chitinase; or glucanase, whether they are natural or synthetic. See PCT International Publication WO 93/02197 (nucleotide sequence of the callase gene). DNA molecules that contain chitinase-encoding sequences can be obtained, for example, from ATCC under accession numbers 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23: 691 (1993) (nucleotide sequence of cDNA encoding tobacco shredder chitinase) and Kawalleck et al., Plant Molec. Biol. 21: 673 (1993) (nucleotide sequence of the ubi-4-2 parsley polyubiquitin gene).

L) A molecule that stimulates signal transduction. See, for example, Botella et al., Plant Molec. Biol. 24: 757 (1994) (nucleotide sequences for cDNA clones of calmodulin golden bean (Phaseolus aureus)) and Griess et al., Plant Physiol. 104: 1467 (1994) (nucleotide sequence of maize calmodulin cDNA clone).

M) Peptide with hydrophobic moment. See international publication PCT WO 95/16776 (peptide derivatives of tachyplezin that inhibit plant fungi pathogens) and international publication PCT WO 95/18855 (synthetic antimicrobial peptides that confer disease resistance).

N) Membrane permease, channel former or channel blocker. See, for example, Jaynes et al., Plant Sci 89:43 (1993) (heterologous expression of an analog of a cecropin-β-lytic peptide to confer resistance to Pseudomonas solanacearum on transgenic tobacco plants).

O) A virus-invasive protein or a complex toxin derived from it. For example, the accumulation of viral envelope proteins in transformed plant cells confers resistance to viral infection and / or to the development of a disease caused by a virus from which the envelope protein gene can be derived, as well as related viruses. See Beachy et al., Ann. rev. Phytopathol. 28: 451 (1990). Shell-mediated resistance was given to plants after transformation against alfalfa mosaic disease virus, cucumber mosaic disease virus, tobacco streak virus, potato virus X, potato virus Y, tobacco engraving virus, tobacco rattle virus and tobacco mosaic virus. See there.

P) Insect-specific antibody or immunotoxin derived from it. Thus, an antibody aimed at critical metabolic function in the intestine of an insect can inactivate the enzyme that is affected by it, leading to the death of the insect. Wed Taylor et al., Abstract # 497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation in transgenic tobacco by producing single chain antibody fragments).

Q) Virus-specific antibody. See, for example, Tavladoraki et al., Nature 366: 469 (1993) (transgenic plants expressing recombinant antibody genes are protected against viral attack).

R) Inhibition of development of a protein produced in nature by a pathogen or parasite. For example, fungal endo-1,4-D-polygalacturonases facilitate colonization of fungi and release of plant nutrients by solubilization of homo-α-1,4-D-galacturonases of plant cell walls. See Lamb et al., Bio / Technology 10: 1436 (1992). See also Toubart et al., Plant J. 2: 367 (1992) (cloning and characterization of a gene that encodes a protein that inhibits bean endopoligalacturonase).

S) Inhibition of development of a protein naturally produced by a plant. See, for example, Logemann et al., Bio / Technology 10: 305 (1992) (transgenic plants expressing the barley ribosome inactivating gene have increased resistance to fungal diseases).

2. Genes that confer resistance to herbicide:

A) A herbicide that inhibits a growth point or meristem, for example, imidazolinone or sulfonylurea. Examples of genes in this category are genes that encode the mutant protein ALS and AHAS, as described, for example, Lee et al., EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449 (1990), respectively.

B) glyphosate resistance imparted, for example, by mutant genes of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSP) (due to the introduction of recombinant nucleic acids and / or various forms of mutagenesis of native EPSP genes in vivo), aroA genes and glyphosate acetyltrans genes) , respectively); other phosphonic compounds, such as glufosinate (phosphinotricin acetyltransferase (PAT) genes from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes), and pyridinoxy or phenoxy propionic acids and cyclohexones (genes encoding ACC inhibitors). See, for example, US Pat. 4,940,835 and U.S. Pat. 6248876 (nucleotide sequences of forms of EPSP that can confer glyphosate resistance to the plant). A DNA molecule encoding a mutant aroA gene can be obtained under accession number ATCC 39256. See also US patent No. 4769061 (nucleotide sequence of the aroA mutant gene). European Patent Application No. 0333033 and U.S. Pat. 4975374 disclose the nucleotide sequences of glutamine synthetase genes that can confer resistance to herbicides such as L-phosphinotricin. The nucleotide sequence of illustrative PAT genes is provided in European Application No. 0242246 and DeGreef et al., Bio / Technology 7:61 (1989) (production of transgenic plants that express chimeric bar genes encoding PAT activity). Examples of genes conferring resistance to phenoxypropionic acids and cyclohexones, such as setoxydim and haloxifop, include the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall et al., Theor. Appl. Genet. 83: 435 (1992). GAT genes capable of conferring resistance to glyphosate are described, for example, in WO 2005012515. Genes conferring resistance to 2,4-D, fop and pyridyloxy-auxin herbicides are described, for example, in WO 2005107437.

C) A herbicide that inhibits photosynthesis, such as triazine (psbA genes and gs + genes) or benzonitrile (nitrilase gene). See, for example, Przibila et al., Plant Cell 3: 169 (1991) (transformation of Chlamydomonas with plasmids encoding mutant psbA genes). Nucleotide sequences for nitrilase genes are disclosed in US Pat. 4810648, and DNA molecules containing these genes are available under accession numbers ATCC 53435, 67441 and 67442. See also Hayes et al., Biochem. J. 285: 173 (1992) (cloning and expression of DNA encoding glutathione-S-transferase).

3. Genes that give or contribute to a sign of added value, such as:

A) Modified fatty acid metabolism, for example, by transforming a plant with the antisense gene of stearyl ACP desaturase to increase the content of stearic acid in this plant. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89: 2624 (1992).

B) Reduced phytate content. The introduction of a gene encoding phytase can enhance the destruction of phytate, thereby adding additional free phosphate to the transformed plant. See, for example, Van Hartingsveldt et al., Gene 127: 87 (1993) (nucleotide sequence of the Aspergillus niger phytase gene). Perhaps the introduction of a gene to reduce the content of phytate. For example, in maize, this can be accomplished by cloning, followed by re-introducing the DNA associated with a single allele, which may be responsible for maize mutants that are characterized by low levels of phytic acid. See Raboy et al., Maydica 35: 383 (1990).

C) The modified composition of carbohydrates, produced, for example, by the transformation of plants by a gene encoding an enzyme that changes the starch branching pattern. See Shiroza et al., J. Bacteol. 170: 810 (1988) (nucleotide sequence of the fructosyltransferase gene of the Streptococcus mutants), Steinmetz et al., Mol. Gen. Genet. 20: 220 (1985) (Levansaccharase gene), Pen et al., Bio / Technology 10: 292 (1992) (α-amylase), Elliot et al., Plant Molec. Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes), Sogaard et al., J. Biol. Chem. 268: 22480 (1993) (barley β-amylase gene) and Fisher et al., Plant Physiol. 102: 1045 (1993) (enzyme II branching of maize endosperm starch).

C. Nanoparticles

According to some embodiments, the invention provides methods for introducing a linear nucleic acid molecule of interest into a cell including a cell wall (eg, a plant cell). In some embodiments, the method may include placing the nanoparticle coated with the linear nucleic acid molecule of interest in contact with the cell and allowing the nanoparticle to be absorbed through the cell wall. In specific embodiments, the nanoparticle can be reversibly or irreversibly contained, coated, or otherwise linked to and / or carry a linear nucleic acid molecule of interest. In some embodiments, a linear linear nucleic acid molecule of interest may be introduced into the nanoparticles before contact with a plant cell having a cell wall, or simultaneously with the introduction of the nanoparticle into a cell of a plant having a cell wall. Examples of nanoparticles that can be used in embodiments of the present invention include, without limitation, quantum dots, alone or in combination with semiconductor nanoparticles; positively charged nanoparticles; gold nanoparticles; gold-coated nanoparticles, porous nanoparticles, mesoporous nanoparticles, silicon dioxide nanoparticles, polymer nanoparticles, including dendrimers, tungsten nanoparticles, gelatin nanoparticles, nanoshells, nanocores, nanospheres, nanorods and magnetic nanoparticles.

According to embodiments of the present invention, a plant cell having a cell wall may be any plant cell comprising an intact and intact cell wall. Examples of cells having a cell wall include, without limitation, algae, tobacco, carrots, maize, canola, rapeseed, cotton, palm, peanuts, soybeans, sugarcane, Oryza sp., Arabidopsis sp. and Ricinus sp.

Embodiments of the invention may include cells having a cell wall, from any tissue or from any place where they can be found, including without limitation: in germ, meristematic cells, callus, pollen, including haploid and diploid microspores, leaves, anthers, roots, root tips, flowers, seeds, pods, stems and tissue culture.

In embodiments of the invention, the linear linear nucleic acid molecule of interest may be any nucleic acid molecule that can be delivered according to the present invention to a plant cell having a cell wall.

Nucleic acid molecules of interest may include, but are not limited to: DNA; RNA RNAi molecules; genes; plasmids; cosmids; YAC (artificial chromosome of fungi) and BAC (artificial chromosome of bacteria). Nucleic acid molecules of interest can be introduced into a plant cell having a cell wall together with, for example, but not limited to, polypeptides, enzymes, hormones, glycopeptides, sugars, fats, signal peptides, antibodies, vitamins, instant messengers, secondary messengers, amino acids, cAMP, drugs, herbicides, fungicides, antibiotics and / or combinations thereof.

In specific embodiments of the invention, the surface of the nanoparticle can be functionalized, which can, for example, provide targeted absorption or allow reversible or irreversible binding of other substances to the surface of the nanoparticle. As a non-limiting example, the surface of a nanoparticle (e.g., a quantum dot) can be functionalized with a self-assembled monolayer, for example, alkanethiolates, which can be further functionalized or derivatized. In a further non-limiting example, the surface of the nanoparticle can be derivatized with linkers, which themselves can then be further functionalized or derivatized. In one embodiment, the nanoparticle can be pegylated. In another embodiment, the nanoparticle can contain or can be multifunctionalized with one or more nuclei (active or inactive), a steric (spatial) coating (active or inert), a cleavable bond and / or a targeting molecule or ligand.

Nanoparticles, such as quantum dots, can be functionalized by PEG using the protocol of Dubertret et al., Science 298: 1759 (2002), or in accordance with this protocol, amended at the discretion of a qualified specialist. For example, CdSe / ZnS quantum dots coated with TOPO (tri-octylphosphine oxide) can be suspended in chloroform with PEG-PE, followed by evaporation of the solvent and solubilization of the resulting PEGylated quantum dots with water.

In aspects of the invention, the nanoparticle can be absorbed into various parts of the cells. Examples of locations at which the nanoparticle can be absorbed include, but are not limited to, the cytosol, core, tonoplasts, plastids, etioplasts, chromoplasts, leukoplasts, elioplasts, protein plastics, amyloplasts, chloroplasts and double membrane lumen. In other embodiments, uptake of the nanoparticle into a cell containing the cell wall may be via a symplast or apoplastic pathway.

D. Stably transformed plant cells

A stably transformed plant cell of the invention can be any plant cell capable of being transformed by a linear nucleic acid molecule of interest by transformation, mediated by nanoparticles. Accordingly, a plant cell can be isolated or cultured from a dicotyledonous or monocotyledonous plant. A plant cell may also be present in plant tissue or in the whole plant. Non-limiting examples of stably transformed dicotyledonous plant cells of the invention include cells: alfalfa; Beans broccoli; cabbage; carrots; cauliflower; celery; Chinese cabbage; cotton; cucumber eggplant; lettuce; melons peas; pepper; peanuts potatoes; Pumpkins radish / radish; rapeseed; spinach soybeans; Pumpkins sugar beets; sunflower; tobacco tomato and watermelon. Non-limiting examples of stably transformed monocotyledonous plant cells of the invention include cells of cereal, onion, rice, common sorghum, wheat, rye, millet, sugarcane, oats, triticale, millet of barbed and lawn grass.

Transgenic plants according to the invention can be regenerated from stably transformed plant cells obtained by the methods of the invention. Such plants can be used or grown in any way in which the presence of nucleic acid molecules of interest is desired. Accordingly, transgenic plants inter alia can be obtained by genetic engineering in order to have one or more desired genetic traits, being transformed with linear nucleic acid molecules via nanoparticle-mediated transformation, with planting and cultivation by any method known to a person skilled in the art.

The following examples are provided to illustrate individual specific features and / or embodiments. Examples should not be construed as limiting the present invention to particular features and illustrative embodiments.

EXAMPLES

Example 1: Obtaining nanoparticles for transformation of plant cells

Obtaining plasmid DNA

DNA of plasmid pDAB3831, FIG. 1, was isolated and prepared for transformation of a plant mediated by linear PEGylated quantum dot (PQD) / DNA. This plasmid contains the selectable PAT marker gene, which is launched by the 10 Arabidopsis Ubiquitin promoter (AtUbi10), and the Philadium yellow fluorescent protein (JPhiYFP) gene, which is triggered by the cassava vein mosaic virus (CsVMV) promoter. The plasmid containing Escherichia coli strain was inoculated and grown until turbidity appeared in Luria-Bertani broth containing ampicillin at 37 ° C. DNA was isolated using the Qiagen Plasmid Midi-Prep kit (Qiagen, Valencia, CA). The isolated DNA was linearized by restriction enzyme digestion. The KpnI restriction enzyme was used for DNA digestion, thereby obtaining linearized plasmid DNA.

Obtaining linear DNA-PQD complexes

Quantum dots were obtained from Ocean Nanotechnology (Springdale, AZ). CdSe / ZnS quantum dots coated with 2 mg TOPO (trioctylphosphine oxide) were suspended in chloroform with 0.15 mg (5.5 μM) PEG-PE (1,2-diacyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy poly (ethylene glycol)]) (Avanti Polar Lipids, Alabaster, AL), followed by evaporation of the solvent and solubilization with water. PEG conjugation was performed to protect against cytotoxicity.

PQDs were conjugated to linear plasmid DNA. 2 mg PQD with 4 mg HS-PEG-OCH ₃ (Prochimia, Zacisze, Poland) were suspended overnight at approximately 60-70 ° C. The solvent was removed in a vacuum oven. The residue was then suspended in 1 ml of water (18M). The last stage was accompanied by the transition of a red precipitate into an orange, optically transparent, clear solution. To coat H3CO-PEG-SH-QD linearized plasmid DNA for transformation experiments, 0.02 mg of purified linearized plasmid DNA (pDAB3831) was incubated with the resulting PQD conjugates in 2 ml of water for 2 hours at 23 ° C in the dark. Torney, et al., Nature Nanotechnol. 2: 295-300 (2007).

Example 2: Transformation of Arabidopsis Flower Embryos

For genetic transformation in planta, plasmid pDAB3831 was digested with the restriction enzyme KpnI to linearize DNA. After digestion with restriction enzyme, linearized DNA was used at a specific ratio of PEG, QD, and linear DNA.

Linearized DNA was mixed with quantum dots and PEG, and incubated for 30 minutes. Then, the nanocomplex, consisting of nanoparticles and a linearized plasmid DNA solution, was mixed with a working solution containing a 5% solution of sucrose in water with 0.02-0.04% Silwet-L77.

Plant material for transformation in planta

Synchronized seed germination is important to ensure uniform flower development on T ₀ plants. Seeds of A. thaliana cv. Columbia were suspended in a 0.1% agar solution (w / v) and incubated at 4 ° C for 48 hours until complete stratification. 60 mg of seeds were weighed and transferred to a 15 ml tube. For uniform seed distribution, 13 ml of a 0.1% (w / v) agar solution was added to the tube, and it was rotated. This resulted in a suspension with a seed concentration of 4.6 mg per 1 ml of a 0.1% (w / v) agar solution (or approximately 230 seeds / ml). 6 tubes (72 ml of suspension) were prepared for inoculation in 4 pallets containing 18 cups (3 1/2 inches) in each pan. A seed suspension was incubated for 48 hours at 4 ° C to complete stratification. Individually, 1.0 ml of a suspension of stratified seeds per one cup was individually seeded. After seeding all the dishes, the pallets were covered with domes to keep the soil moist when plants were growing. Domes were removed 5 days after the sowing date. After seed germination and the emergence of plants, they were placed for growth in a Conviron® chamber (models CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg, Manitoba, Canada) for a long day (16 hours of light / 8 hours of darkness), at a light intensity of 120 -150 μmol mol / m ² · sec (equivalent to the light energy absorbed by the plant), at a constant temperature (22 ° C) and humidity (40-50%). 10-14 days after sowing the plants, they were irrigated with Hoagland's solution and then DI with water in order to keep the soil moist, but not wet. 4 weeks after the date of planting, they cut flowers to enhance the growth of secondary flowers. Plants were ready for the transformation process in the 5th week after sowing.

Transformations in planta

Transformation of A. thaliana cv. Columbia using linear DNA / PQD was performed using the modified Clough-Bent protocol. See, Clough and Bent, Plant J. 16: 735-43 (1998). A 20 ml suspension of a linear DNA / PQD complex was prepared at a concentration of 0.5 mg linear DNA and 4 nM PQD, which was used to treat Arabidopsis plants (mainly immature inflorescences, including a small number of fertilized pods). Before immersing the plants, Silwet L-77 was added to the suspension of the linear DNA / PQD complex to a concentration of 0.05% (w / v) (250 μl / 500 ml) - 0.005%, and the solution was well mixed. The aerial parts of the plant were immersed for 30 seconds in a linear DNA / PQD solution with gentle mixing. Treated plants were shaded for 30 minutes at 22-24 ° C. Plants were placed in a Conviron chamber under conditions as described above to allow ripening and seed collection.

To screen the total seed yield from T ₀ plants, selection trays of 10.5 × 21 × 1 inches were used, at the rate of approximately 10,000 seeds per tray.

To ensure that the spraying during selection was performed correctly, two control groups were used: Col-0 plants as a negative transform control, and wild-type Columbia Col-0 plants enriched with homozygous seeds for the selectable PAT marker (phosphinotricin-N-acetyltransferase), as a positive transform control. Seeds, for the purpose of synchronization, were stratified in agar solution with a concentration of 0.1% (w / v) for 48 hours before sowing. To ensure a sowing density of 10,000 seeds per selection tray, 200 mg of seeds were added to the agar solution at a concentration of 0.1% (w / v) and the solution was rotated until the seeds were uniformly suspended. Then, stratified seeds were sown on selection trays that were filled with Sunshine LP5 soil mix and were prepared to moisten Hoagland's solution by feeding the solution from below. For effective spraying during plant breeding, it is important that 40 ml of suspended seeds are evenly sown in the selection tray. After sowing, each selection tray was covered with a dome, and plants were grown for selection. Domes were removed approximately 5 days after sowing.

Example 3: Analysis of Transformed Arabidopsis

Transformed Plant Breeding

Freshly harvested T1 seeds were allowed to dry for 7 days at room temperature. T1 seeds were sown in trays of 26.5 × 51 cm in size for germination, each aliquot of 200 mg of stratified T1 seeds (approximately 10,000 seeds) that were previously suspended in 40 ml of 0.1% (w / v) was sown in each. agar solution, and stored at 4 ° C for 2 days, due to requirements for the completion of the dormant period and to guarantee simultaneous germination of seeds.

The Sunshine LP5 primer mixture was coated with pure vermiculite and was wetted with Hoagland's solution by applying the solution from below and then removing excess moisture due to gravity. Each aliquot of stratified seeds with a volume of 40 ml was uniformly pipetted on vermiculite and closed with domes to maintain moisture for 4-5 days. Domes were removed 1 day before the initial selection of transformants using glufosate spraying in the post-emergence period.

Seven days after planting (DAP) (from the cotyledon and stage 2-4 leaves, respectively), T1 plants were sequentially sprayed five times over five days with a 0.2% solution (w / v) Liberty herbicide (200 g glufosinate active ingredient per 1 liter, Bayer Crop Sciences, Kansas City, MO) with a spray volume of 10 ml per tray (703 l / ha) using a DeVilbiss nozzle for spraying with compressed air to ensure delivery of an effective amount of glufosinate at a rate of 280 g / ha glufosinate on the active substance per spray. Surviving plants (actively growing plants) were identified 4-7 days after the final spraying and transplanted into individual 3-inch pots with growth medium in pots (Metro Mix 360). The transplanted plants were covered with domes to maintain moisture for 3-4 days, and they were placed for growth in a chamber at 22 ° C, as before, or were transferred directly to the greenhouse. Subsequently, the domes were removed and the plants were placed in a greenhouse (22 ± 5 ° C; relative humidity 50 ± 30%; light mode: 14 hours of lighting / 10 hours of darkness; illumination (natural + additional light) according to the equivalent of light energy absorbed by the plant - 500 μmol / m ² · sec).

Molecular and biochemical studies

Molecular analyzes were performed to integrate PAT and YFP into the plant genome. PCR amplification products were sequenced and compared with transgenic sequences.

Molecular analysis and evidence of the genomic integration of transgenes into the offspring of T1 Arabidopsis thaliana (cv Columbia)

Genomic DNA from transgenic plants of A. thaliana was extracted from leaf material of 6-week-old plants using the Plant DNAZOL ™ kit (Invitrogen) according to the manufacturer's instructions. To detect PAT and YFP transgenes, PCR primers were designed.

Examples of YFP are presented as SEQ ID NO: 1 and SEQ ID NO: 2. Primers PAT are presented as SEQ ID NO: 3 and SEQ ID NO: 4.

PCR amplification of genomic DNA transgenes

PCR amplification reactions for PAT and YFP were performed using the TaKaRa ExTaq ™ kit (Takara, Otsu, Shiga, Japan). Gene products were amplified in the full reaction volume of 50 μl. The PCR reaction contained 100 nanograms of the genomic DNA template, 1 X ExTaq reaction buffers, 0.2 mM dNTP, 10 pM of each example, and 0.025 U / μl ExTaq. The following PCR conditions were used: 1 cycle at 96 ° C for 5 minutes, and 31 cycles under the following conditions: 94 ° C for 15 seconds, 65 ° C for 30 seconds, 72 ° C for 1 minute, and final amplification at 72 ° C for 7 minutes. The PCR amplification product was analyzed by 0.8% agarose gel electrophoresis and was visualized by ethidium bromide staining. DNA fragments were isolated from agarose gel using a QIAEX ™ II gel purification kit (Qiagen, Valencia, CA).

PCR fragments were sequenced using the forward primer for PAT (SEQ ID NO: 3) and the reverse primer for YFP (SEQ ID NO: 1) using an improved Sanger sequencing technique (MWG Biotech., Huntsville, AL). Sequence data was analyzed using Sequencher software.

The results of sequencing of PAT and YFP amplicons from PCR were consistent with the expected nucleotide sequence for these genes. These results clearly show that the PAT and YFP sequences from pDAB3831 were stably integrated into Arabidopsis genomic DNA using the PEGylated quantum dot and linear DNA transformation protocol.

Gene products for PAT and YFP (yellow fluorescent label, Evrogen) from PCR were amplified in a total reaction volume of 50 μl of a mixture containing 100 nanograms of template genomic DNA, 1X ExTaq reaction buffer (TaKaRa Bio), 0.2 mM dNTP, 10 pM primer and 0.025 U / μl ExTaq. The following PCR conditions were used: 1 cycle at 96 ° C for 5 minutes and 31 cycles according to the following PCR program: 94 ° C, 15 seconds; 65 ° C, 30 seconds; 72 ° C, 1 min. Final amplification was performed at 72 ° C for 7 minutes, for complete synthesis of the PCR product. Gel images were obtained using the BioRad Gel Imagining System. See FIG. 1 and 2. Amplified fragments were isolated from the gel using a gel purification kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.

Fragments from PCR were sequenced using a direct primer for PAT and a direct primer for YFP using an improved Sanger sequencing technique (MWG Biotechnologies, Inc.), and the sequences were analyzed using Sequencher software.

The present results clearly show that the PAT and YFP sequences were delivered by means of a positively charged nanoparticle containing the linearized DNA obtained in Example 1, and thus provide evidence of stable integration of transgenes into the genomic DNA of T1 Arapidopsis plants.

Example 4: Nanoparticle-mediated Delivery of Linear Nucleic Acid Molecules to Cultured Plant Cells

Preparation of plant material in the form of a single cell

For example, both BY2 cells and NT1 cells are used. BY2 cells are green and are a fast-growing line of tobacco cells. NT1 cells are photoautotrophic cells isolated from tobacco. Three to four days prior to transformation, the one-week suspension culture was subcultured in fresh medium by transferring 2 ml of NT1 or BY2 culture to 40 ml of NTIB or LSBY2 medium containing 50 nM DAS-PMTI-1 (a tubular inhibitor) and 0.5-0. 1% (v / v) dimethyl sulfoxide, in a 250 ml flask. Single cells were harvested either four days or seven days after treatment with a microtubule inhibitor. Used BY2 single cells were analyzed using a Beckman Flow flow cytometer to count viable cells. Cells were examined using a differential interference contrast microscope (DIC) equipped with a confocal imaging system to determine that single cells include large amounts of plastids (amyloplasts) distributed throughout the cell cytoplasm. Cells were subcultured once every 14 days by transferring 1 ml of suspension with 3.0 Units. optical density at OD ⁶⁰⁰ . Cultured cells are used as target cells for transformation.

Nanoparticle Preparation and Cell Processing

Plasmid DNA was isolated and prepared for transformation of a plant mediated by linear PEGylated quantum dot (PQD) / DNA. The plasmid contains the selectable PAT marker gene, which is triggered by the 10 Arabidopsis Ubiquitin promoter (AtUbi10), and the Philadium yellow fluorescent protein (JPhiYFP) gene, which is triggered by the promoter of the cassava vein mosaic virus (CsVMV). The plasmid containing Escherichia coli strain was inoculated and grown until turbidity appeared in Luria-Bertani broth containing ampicillin at 37 ° C. DNA was isolated using the Qiagen Plasmid Midi-Prep kit (Qiagen, Valencia, CA). The isolated DNA was linearized by restriction enzyme digestion. The KpnI restriction enzyme was used for DNA digestion, thereby obtaining linearized plasmid DNA.

Quantum dots were obtained from Ocean Nanotechnology (Springdale, AZ). CdSe / ZnS quantum dots coated with 2 mg TOPO (trioctylphosphine oxide) were suspended in chloroform with 0.15 mg (5.5 μM) PEG-PE (1,2-diacyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy poly (ethylene glycol)]) (Avanti Polar Lipids, Alabaster, AL), followed by evaporation of the solvent and solubilization with water. PEG conjugation was performed to protect against cytotoxicity.

PQDs were conjugated to linear plasmid DNA. 2 mg PQD with 4 mg HS-PEG-OCH3 (Prochimia, Zacisze, Poland) were suspended overnight at approximately 60-70 ° C. The solvent was removed in a vacuum oven. The residue was then suspended in 1 ml of water (18M). The last stage was accompanied by the transition of a red precipitate into an orange, optically transparent, clear solution. To coat H ₃ CO-PEG-SH-QD linearized plasmid DNA for transformation experiments, 0.02 mg of purified linearized plasmid DNA (pDAB3831) was incubated with the resulting PQD conjugates in 2 ml of water for 2 hours at 23 ° C in the dark. Torney, et al., Nature Nanotechnol. 2: 295-300 (2007).

PQDs at a concentration of 1-3 μl / ml were added in a 24-well microtiter plate to 500 μl of cells, and the plate was carefully rotated in a shaker for 20 minutes in the dark. Nanoparticles passed through cell walls.

Example 5: Arabidopsis Transformation Mediated by a Multifunctionalized Nanoparticle in Planta

Transformation of Arabidopsis in planta can be performed using the modified Clough-Bent protocol (1998). The concentration of DNA on the multifunctionalized nanoparticle along with the host molecules, such as the protein transduction domain (PTD) and nuclear localization signal units (NLS), is optimized in order to achieve increased transformation efficiency.

Plant material: Healthy Arabidopsis plants are grown on a long day in pots with soil before flowering. The first arrows were trimmed to stimulate the proliferation of many secondary arrows. Plants were ready after about 4-6 days after pruning. Columbia ecotype Arabidopsis thaliana (Col-0) was used as the baseline (T0 plant) to transform flowers in planta. Synchronized seed germination is important in order to guarantee uniform flower development on T0 plants. Wild-type seeds were suspended in a 0.1% agar solution and incubated at 4 ° C for 48 hours to complete stratification. 60 mg of seeds were weighed on paper and transferred to 15 ml tubes. 13 ml of 0.1% agar solution was added and the tubes were rotated until the seeds were uniformly dispersed. This led to a suspension with a seed concentration of 4.6 mg per 1 ml of solution (or approximately 230 seeds / ml). 6 tubes (72 ml of suspension) were prepared for inoculation in 4 pallets containing 18 cups (3 1/2 inches) in each pan and in 2 common cups. A seed suspension was incubated for 48 hours at 4 ° C to complete stratification. Individually, 1.0 ml of a suspension of stratified seeds per one cup was individually seeded. After seeding all the dishes, the pallets were covered with domes to keep the soil moist when plants were growing. Domes were removed 5 days after the sowing date. After seed germination and the emergence of plants, they were placed for growth in a Conviron® chamber (models CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg, Manitoba, Canada) for a long day (16 hours of light / 8 hours of darkness), at a light intensity of 120 -150 μmol mol / m ² · sec (equivalent to the light energy absorbed by the plant), at a constant temperature (22 ° C) and humidity (40-50%). 10-14 days after planting, they were irrigated with Hoagland's solution and then DI water to keep the soil moist, but not wet. 4 weeks after the date of planting, they cut flowers to enhance the growth of secondary flowers. Plants were ready for the transformation process in the 5th week after sowing.

Preparation of nanoconjugates for flower processing: Nanoparticles / quantum dots in the size range 2 - 120 nm were selected for processing and multifunctionalized with a purified pDAB3138 fragment and peptide units for homing according to Derufus et al. (2007).

Quantum dots with emission maxima at 655 or 705 nm, and modified with PEGs with amino groups, were obtained from Quantum Dot Corporation (ITK amino). QD concentrations were measured by optical absorption at 595 nm using extinction coefficients provided by the supplier. The cross-linkers used were sulfo-LC-SPDP (sulfosuccinimidyl 6- (3 '- [2-pyridyldithio] propionamido) hexanoate) (Pierce) and sulfo-SMCC (sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1- carboxylate) (Sigma). Amine-modified QDs were conjugated to thiol-containing plasmid DNA and homing peptides using sulfo-LC-SPDP and sulfo-SMCC as cross-linkers. QD was resuspended in a solution of 50 mM sodium phosphate, 150 nM sodium chloride, pH 7.2, using Amicon Ultra-4 filters (100 kDa cut-off). A cross-linker (1000-fold excess) was added to the QD, allowing reaction for 1 hour. Samples were filtered on a NAP-5 column by gravity (to remove excess cross-linker) into a similar buffer supplemented with 10 mM EDTA. The linearized plasmid pDAB3831 DNA was treated with 0.1 M DTT for 1 hour and filtered on a NAP-5 column in an EDTA-containing buffer. Peptides in the form of lyophilized powder were generally used. Peptides and linearized plasmid DNA were added to the filtered QDs, allowing reaction overnight at 4 ° C. Using three Amicon filters, the product was filtered with Dulbecco phosphate saline (PBS), twice with high saline buffer (1.0 M sodium chloride, 100 mM sodium citrate, pH 7.2), and again twice with PBS. Washing with high salt buffer is necessary to remove electrostatically bound DNA and peptides that are not removed by washing with just one PBS. Sulfo-SMCC has at one end N-hydroxysuccinimide ester (NHS), which reacts with the modified amine QD to form an amide bond, and a maleimide group at the other end, which reacts with the thiolated plasmid DNA to form a thioether. Sulfo-LC-SPDP also contains an amino reactive N-hydroxysuccinimide ester (NHS), which quickly reacts with any molecule containing a primary amine, thus forming a stable amide bond.

In planta transformation and screening of resistant T1 plants: A suspension with a total volume of 250-500 ml was obtained containing nanoparticles, homing peptides and a plasmid DNA conjugate (NHpD) solution, and Arabidopsis plants (mainly immature flower groups with some fertilized pods) were used for further processing. Before plant immersion, Silwet L-77 was added to the NHpD conjugate solution to a concentration of 0.05% (250 μl / 500 ml) - 0.005%, and the solution was well mixed. The aerial parts of the plant were immersed in a solution of NHpD conjugates for 2-30 seconds with gentle stirring. Treated plants were kept under a dome or coating for 16-24 hours at 22-24 ° C. Plants were transferred to a Conviron chamber to allow for plant growth before ripening and seed collection. To screen the total seed yield from T0 plants, selection trays (10.5 × 21 × 1 inch) were used containing approximately 10,000 seeds per tray. To ensure that the spraying during selection was performed correctly, two control groups were used: Col-0 plants as a negative transform control, and wild-type Columbia Col-0 plants enriched with homozygous seeds for the selectable PAT marker (phosphinotricin-N-acetyltransferase), as a positive transform control. Seeds, for the purpose of synchronization, were stratified in agar solution with a concentration of 0.1% (w / v) for 48 hours before sowing. To ensure a sowing density of 10,000 seeds per selection tray, 200 mg of seeds were added to the agar solution at a concentration of 0.1% (w / v) and the solution was rotated until the seeds were uniformly suspended. Then, stratified seeds were sown on selection trays that were filled with Sunshine LP5 soil mix and were prepared to moisten Hoagland's solution by feeding the solution from below. For effective spraying during plant breeding, it is important that 40 ml of suspended seeds are evenly sown in the selection tray. After sowing, each selection tray was closed with a dome, and plants were grown for selection under the conditions indicated above. Domes were removed approximately 5 days after sowing.

The seedlings were sprayed five days after planting and again ten days after planting by spraying the sprouts with a 0.2% (w / v) solution (20 μl / 10 ml dH ₂ O) of ammonium glufosinate (Liberty® herbicide from Bayer Crop Sciences), with a spray volume of 10 ml per tray (703 l / ha), using a DeVilbiss nozzle for spraying with compressed air to deliver an effective amount of glufosinate at a rate of 280 g glufosinate per hectare per spray (GPA). The amount of Liberty® herbicide to be prepared was calculated as follows: (703 L per atomized volume per hectare = 280 GPA). (280 g (acid) per hectare) × (1 ha / 703 l) × (1 l / 200 g glufosinate (acid) = 0.20% solution (or 20 μl / 10 ml). 10 ml of solution for each tray that is sprayed, pipetted into a 20 ml scintillation vial. Spraying was performed using horizontal and vertical movement. Herbicide resistant plants were identified four to seven days after the second spraying.

Claims (21)

1. A method for stably integrating a linear nucleic acid molecule of interest into a plant cell having a cell wall, comprising:
providing a plant cell having a cell wall;
coating a positively charged semiconductor nanoparticle of interest with a linear nucleic acid molecule;
bringing a cell having a cell wall and having a coating of a positively charged semiconductor nanoparticle into contact with each other; and
allowing absorption of a positively charged semiconductor nanoparticle and a linear nucleic acid molecule of interest in a cell containing a cell wall, and
selecting a plant cell that has a stably integrated linear nucleic acid molecule of interest. 2. The method of claim 1, wherein the nanoparticle is coated with polyethylene glycol. 3. The method according to p. 1, further comprising providing the possibility of absorption of the nanoparticles in the compartment of a plant cell containing the cell wall. 4. The method of claim 3, further comprising coating the nanoparticle with a protein targeting the subcellular compartment. 5. The method according to p. 3, where the compartment is selected from the group consisting of cytosol, nucleus, tonoplasts, plastids, etioplast, chromoplast, leukoplast, elioplast, proteinoplast, amyloplast, chloroplast and double membrane lumen. 6. The method of claim 1, wherein the plant cell having a cell wall is a plant cell from commercial types of cereal plants. 7. The method of claim 6, wherein the plant cell having a cell wall is selected from the group consisting of tobacco, carrot, maize, canola, canola, cotton, palm, peanut, soy, Oryza sp., Arabidopsis sp., Ricinus cells sp. and sugarcane. 8. The method of claim 1, wherein the plant cell is derived from tissue selected from the group consisting of an embryo, meristem, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods and stems. 9. The method of claim 1, wherein the plant cell having a cell wall is a cultured cell. 10. The method of claim 1, wherein the semiconductor nanoparticle is a quantum dot. 11. The method of claim 1, further comprising derivatizing the surface of the nanoparticle. 12. The method of claim 1, wherein the linear linear nucleic acid molecule of interest comprises a nucleic acid sequence selected from the group consisting of DNA, RNA, RNAi, and genes. 13. The method of claim 12, wherein the linear linear nucleic acid molecule of interest contains a gene. 14. The method of claim 13, wherein the gene is a foreign protein gene, an agronomic gene, or a marker gene. 15. The method of claim 1, wherein the linear linear nucleic acid molecule of interest is obtained by cleaving the nucleic acid molecule with a nuclease. 16. The method according to p. 15, where the nucleic acid molecule cleaved by a nuclease is selected from the group consisting of plasmid, cosmid, artificial chromosomes, artificial yeast chromosomes and artificial bacterial chromosomes. 17. The method of claim 14, further comprising selecting cells into which the molecule of interest is stably integrated. 18. The method of claim 17, wherein the selected cells are regenerable cells. 19. The method of claim 18, further comprising regenerating the plant from the regenerated cells. 20. A method for stably integrating a linear linear nucleic acid molecule of interest in plant material, wherein the method comprises:
the provision of plant material, where the plant material is selected from the group comprising plant cells, plant tissue and plants;
coating a nanoparticle, which is a quantum dot, with polyethylene glycol;
coating the nanoparticle, which is a quantum dot, a linear nucleic acid molecule of interest;
bringing a plant cell having a cell wall and having a nanoparticle coating, which is a quantum dot, into contact with each other; and
enabling absorption of a nanoparticle, which is a quantum dot, and of interest to a linear nucleic acid molecule in plant material. 21. The method according to p. 20, where the plant material is plant tissue selected from the group consisting of an embryo, meristem tissue, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods and stems.

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