Methods and compositions for producing transformed cells

Abstract

Provided are methods of selecting a transformed cell, including methods comprising delivering an inhibitor of a protein to a plurality of cells, exposing the plurality of cells to a compound, wherein the protein (if present and active) converts the compound to a toxic compound, and selecting viable cells from the plurality of cells not killed by exposure to the compound.

Description

Attorney Docket No. 65864-703601METHODS AND COMPOSITIONS FOR PRODUCING TRANSFORMED CELLSCROSS-REFERENCE TO OTHER APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 681,708, filed on August 9, 2024, the disclosure of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The application contains a Sequence Listing, which is submitted herewith in XML format, and is hereby incorporated by reference in its entirety. The XML copy, created on August 7, 2025, is named 65864-703_601_SL.xml and is 72,299 bytes in size.SUMMARY

[0003] Aspects disclosed herein provide methods of selecting a transformed cell, the method comprising: a. contacting a plurality of cells with a polynucleotide and (1) a chemical inhibitor or (2) a polypeptide inhibitor of an endogenous oligomeric enzyme of the plurality of cells to produce at least a transformed portion of the plurality of cells, wherein the polypeptide is a mutant of the endogenous oligomeric enzyme, and wherein, optionally, the contacting comprises particle bombardment and / or the chemical inhibitor is added at a concentration of no greater than about 200 micromolar (pM) or the polypeptide inhibitor is added at a concentration of about 1-5 micrograms (pg); b. exposing the plurality of cells to a non-toxic compound, wherein the nontoxic compound is a substrate of the endogenous oligomeric enzyme, and wherein the exposing kills an untransformed portion of the plurality of cells, and wherein, optionally, the plurality of cells are exposed to the non-toxic compound for about 30-300 seconds, the plurality of cells are exposed to vapors from the non-toxic compound, the plurality of cells are exposed to about 1- 25% of the non-toxic compound, or any combination thereof; and c. selecting at least a portion of the plurality of cells transformed with the polynucleotide and the inhibitor. In some embodiments, the chemical inhibitor is fomepizole or CNAD. In some embodiments, the concentration of fomepizole is at least about 1 pM. In some embodiments, the concentration of fomepizole is about 1-160 pM. In some embodiments, wherein the concentration of CNAD is at least about 1 nM, and wherein the concentration of CNAD is about 1-100 nM. In some embodiments, the concentration of CNAD is about 1-100 nM. In some embodiments, the mutant is a dominant-negative enzyme of the endogenous oligomeric enzyme. In some embodiments, the concentration of the polypeptide inhibitor is about 2-4 pg. In some embodiments, the endogenous oligomeric enzyme is alcohol dehydrogenase (ADH). In some embodiments, the non-toxic compound is allyl alcohol. In some embodiments, the exposing comprises exposingAttorney Docket No. 65864-703601 the plurality of cells with about 6-11% allyl alcohol. In some embodiments, the exposing comprises exposing the plurality of cells with the non-toxic compound for about 50-200 seconds. In some embodiments, the exposing comprises exposing the plurality of cells with the non-toxic compound for about 105-135 seconds. In some embodiments, the chemical inhibitor or the polypeptide inhibitor reduces activity of the endogenous oligomeric enzyme by at least about 70%. In some embodiments, the polynucleotide confers one or more desirable traits, and wherein the one or more desirable traits comprises herbicide resistance, drought tolerance, salt tolerance, pest resistance, disease resistance, drought stress resistance, high temperature resistance, improved water use efficiency, improved nutrient acquisition efficiency, increased yield, abiotic stress resistance, improved photosynthesis efficiency, or any combination thereof. In some embodiments, the plurality of cells are a plurality of pollen grains or a plurality of embryos. In some embodiments, the plurality of pollen grains or the plurality of embryos are derived from maize, soybean, cotton, rice, wheat, barley, tomato, potato, Pinus, eucalyptus, Populus, citrus, coffee, sugarcane, canola, or oat. In some embodiments, the plurality of pollen grains transformed with the polynucleotide and the chemical inhibitor or the polypeptide inhibitor produce seeds in about 25-40 days. In some embodiments, the method further comprises generating at least 1000 transformed pollen grains in a single experiment. In some embodiments, each of the at least 1000 transformed pollen grains are an independent transformation event.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0005] FIG. 1 shows a schematic representation of a plant transformation method disclosed herein using pollen grains.

[0006] FIG. 2 shows a schematic representation of the plant transformation method disclosed herein using soybean embryo axis.

[0007] FIGS. 3A and 3B show non-limiting examples of stereomicroscope images of inhibition of maize pollen grain germination when exposed to allyl alcohol. FIG. 3A is an image of untransformed pollen grains unexposed to allyl alcohol. FIG. 3B is an image of untransformed pollen grains expressing wildtype ADH1 that did not germinate after exposure to allyl alcohol.Attorney Docket No. 65864-703601

[0008] FIGS. 4A and 4B show non-limiting examples of stereomicroscope images of maize pollen grain germination after exposure to allyl alcohol in the presence of the alcohol dehydrogenase inhibitor fomepizole. FIG. 4A is an image of maize pollen grains on germination medium without allyl alcohol. FIG. 4B is an image of maize pollen grains treated with fomepizole and then exposed to allyl alcohol.

[0009] FIG. 5 shows shoot formation from soybean embryo axes exposed to allyl alcohol after particle bombardment with gold particles coated with the ADH1 inhibitor fomepizole (left panel) and shoot inhibition after particle bombardment without fomepizole (right panel).

[0010] FIG. 6 shows an enzymatic assay to demonstrate the inhibition of recombinant maize ADH1 activity by the ADH inhibitor fomepizole. The recombinant maize ADH1 enzyme was assayed for activity in the presence of varying concentrations of fomepizole.

[0011] FIG. 7 shows an enzymatic assay to demonstrate the inhibition of the oligomeric, native recombinant maize ADH1 (SEQ ID NO: 1) by an inactive, mutated recombinant maize alcohol dehydrogenase (e.g., SEQ ID NOS: 2-8). The top line shows the activity of wild-type ADH1 dimer. The bottom line shows the activity of wildtype-mutant ADH1 dimer.

[0012] FIGS. 8A and 8B show non-limiting examples of polynucleotides used in the method disclosed herein. FIG. 8A shows a polynucleotide designed for using physical methods, such as particle bombardment, comprising a constitutive promoter, such as the ubiquitin promoter, a gene encoding a mutant of an oligomeric enzyme, such as a mutant of ADH1, and a transcriptional terminator. FIG. 8B shows a polynucleotide designed for transformation into a plant cell using the bacterial pathogen Agrobacterium lumefaciens. comprising the right border of the T-DNA, a constitutive promoter, such as the ubiquitin promoter, a gene encoding a mutant of an oligomeric enzyme, such as a mutant of ADH1, and a transcriptional terminator and the left border of the T-DNA.

[0013] FIG. 9 shows a maize ear pollinated with pollen grains transformed by particle bombardment with gold particles of 1 um coated with a DNA polynucleotide comprising of a constitutive promoter, a gene encoding a mutated maize ADH1, and a transcriptional terminator.

[0014] FIG. 10 shows soybean embryo axes transformed by Agrobacterium tumefaciens harboring a DNA construct such as the one depicted in FIG. 8B comprised of a constitutive promotor driven the expression of a mutated ADH1 (FIG. 10A) or a native ADH1 (FIG. 10B). The transformed embryo axes were treated with allyl alcohol and transferred to a shooting medium to recover transformed soybean plants. Embryo axes transformed with the construct harboring the mutated ADH1 construct give rise to transformed soybean plants.Attorney Docket No. 65864-703601

[0015] FIG. 11 shows a schematic representation of the chemical structure of the compounds that can be used in the present invention including allyl alcohol, acrolein, fomepizole, and CNAD. The chemicals work in the present invention when allyl alcohol, a non-toxic molecule, can be converted by ADH1 into the toxic compound acrolein while fomepizole or CNAD, nontoxic compounds, can inhibit the activity of ADH1 preventing the conversion of allyl alcohol into acrolein.DETAILED DESCRIPTION

[0016] Disclosed herein are methods, compositions, and kits for producing transformed cells and selecting transformed cells. The transformed cells can comprise polynucleotides for stable incorporation, transient incorporation, or both. The polynucleotides can include polynucleotides that encode double stranded RNAs that hybridizes with select genes, comprise a sequence complementary to select genes, comprise a mutant of a select gene, encode an exogenous gene, encode a zinc finger nuclease (ZFN), encode a transcription activator-like effector nuclease (TALEN), comprise a clustered regularly interspaced short palindromic repeat (CRISPR)- CRISPR associated protein Cas (CRISPR-Cas) system, or any combination thereof. The methods, compositions, and kits disclosed herein may provide tissue culture-free and / or genotype independent transformation.

[0017] Provided herein are methods, kits, and compositions for transforming cells.

[0018] Aspects disclosed herein provide methods for selecting a transformed cell, the method comprising delivering: a polynucleotide to a plurality of cells, wherein the polynucleotide encodes a mutant of an oligomeric enzyme endogenous to the plurality of cells; a polypeptide to the plurality of cells, wherein the polypeptide is the mutant of the oligomeric enzyme endogenous to the plurality of cells; or a chemical inhibitor molecule of the oligomeric enzyme endogenous to the plurality of cells; quickly exposing the plurality of cells to vapor of a nontoxic compound, wherein the vapor of the non-toxic compound penetrates the plurality of cells; wherein the compound is a substrate of the oligomeric enzyme endogenous to the plurality of cells; wherein the quickly exposure kills untransformed cells of the plurality of cells by the non- toxic compound that is converted by the oligomeric enzyme to a toxic product; and selecting a viable transformed cell from the plurality of cells, wherein the oligomeric enzyme of the viable transformed cell was inhibited by the polynucleotide, the polypeptide, or the chemical inhibitor molecule.

[0019] Aspects disclosed herein comprise methods of cell selection, the methods comprising: (i) at least one cell transiently transformed with a construct comprising an inhibitor of theAttorney Docket No. 65864-703601 endogenous alcohol dehydrogenase present in the plurality of cells (ii) at least one cell that have received a small molecule inhibitor of the endogenous alcohol dehydrogenase (iii) the remaining cells of the plurality of cells that where not transiently transformed with a construct comprising an inhibitor of the endogenous alcohol dehydrogenase present in the plurality of cells or that did not received a small molecule inhibitor of the endogenous alcohol dehydrogenase (iv) exposing the plurality of cells to a non-toxic substrate of the alcohol dehydrogenase protein, wherein at least one cell that where not transiently transformed with a construct comprising an inhibitor of the endogenous alcohol dehydrogenase present in the plurality of cells or that did not received a small molecule inhibitor of the endogenous alcohol dehydrogenase dies because the non-toxic substrate is converted by the endogenous alcohol dehydrogenase into a toxic compound; wherein exposing at least one cell whose alcohol dehydrogenase have been inhibited does not result in cell death; and selecting a viable cell from the plurality of cells after exposing the plurality of cells to the non-toxic substrate, thereby selecting the at least one cell comprising the inhibitor of the alcohol dehydrogenase protein.

[0020] Aspects disclosed herein comprise methods wherein exposing a plurality of pollen grains transformed with inhibitors of alcohol dehydrogenase an exposing said pollen grains to a non- toxic substrate of an alcohol dehydrogenase protein; selecting at least one viable pollen grain from the plurality of pollen grains; pollinating a flower of a plant with the at least one viable pollen grain; and harvesting at least one seed from the plant.

[0021] Aspects disclosed herein comprise kits comprising a polynucleotide encoding an inhibitor of an alcohol dehydrogenase protein; and a substrate of alcohol dehydrogenase.Aspects disclosed herein comprise kits comprising an inhibitor of an alcohol dehydrogenase protein; and a substrate of alcohol dehydrogenase.METHODS

[0022] Provided herein are methods for the transformation and selection of transformed cells. FIG. 1 and FIG. 2 depict non-limiting schematic representations of methods provided herein. FIG. 1 depicts transforming maize pollen grains with a polynucleotide to overexpress in the transformed cell a mutant protein of the oligomeric enzyme alcohol dehydrogenase (ADH). For example, a mutant ADH protein of Table 1 or Table 2. Table 1 shows a non-limiting list of the polynucleotide sequences of the mutants of the oligomeric enzyme alcohol dehydrogenase that may be used in the cell transformation method described herein. Table 2 shows a non-limiting list of the amino acid sequences of the mutants of the oligomeric enzyme alcohol dehydrogenase that may be used in the cell transformation method described herein. In this example, a gene gunAttorney Docket No. 65864-703601 is utilized for transformation, however, other methods of transformation known in the art may be utilized. Following the transient transformation of the cells, the mutant protein, when overexpressed, forms an inactive oligomeric ADH enzyme with the endogenous wildtype ADH through a dominant-negative effect. The cells are exposed to a compound, such as allyl alcohol. The untransformed cells with active ADH convert allyl alcohol into acrolein. Acrolein is toxic to the cell and through exposure to acrolein, the untransformed cell may die. The transformed cells may be unable to convert allyl alcohol to acrolein, given the dominant-negative inactivation of ADH. Without exposure to acrolein, the transformed cells may survive. In this depicted embodiment, the transformed cells can be pollen cells or any meristematic cell that harbors the ability to produce a plant. The cells may be maize pollen grains that have undergone the transformation procedure and are used to pollinate a maize ear. Transformed maize seeds may be produced from the pollinated ear. Transformed immature embryos from the produced seeds may be rescued, placed in a germination medium, and grown to mature plants. Thereafter, the transformed mature plants give rise to seeds.

[0023] FIG. 2 depicts transforming embryo axes removed from soaked soybean seeds with a polynucleotide to overexpress in the transformed cell a mutant protein of the oligomeric enzyme alcohol dehydrogenase (ADH). For example, a mutant ADH protein of Table 1 or Table 2. Following the transformation of the cells, the mutant protein, when transiently overexpressed, forms an inactive oligomeric ADH enzyme with the endogenous wildtype ADH through a dominant-negative effect. The cells are exposed to a compound, such as allyl alcohol. The untransformed cells with active ADH convert allyl alcohol into acrolein. Acrolein is toxic to the cell and through exposure to acrolein, the untransformed cell may die. The transformed cells may be unable to convert allyl alcohol to acrolein, given the dominant negative inactivation of ADH. Without exposure to acrolein, the transformed cells may survive. The cells are meristematic cells from the soybean embryo axis that have undergone the transformation procedure. The transformation procedure may comprise transformation with the gene encoding the mutant protein of ADH and a gene of interest. Soybean plants may be produced by shooting of transformed meristematic cells from the embryo axis. The soybean shoots may develop into plants. Thereafter, the plants give rise to seeds.Attorney Docket No. 65864-703601TABLE 1. Polynucleotide sequences of alcohol dehydrogenasesAttorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601TABLE 2. Polypeptide sequences of alcohol dehydrogenasesAttorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601

[0024] In some embodiments, the cells are stem cells. In some embodiments, the stem cell is an undifferentiated cell. The stem cells may be pluripotent stem cells. A cell can be in vitro. A cell can be in vivo. A cell can be ex vivo. A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture. A cell can be one of a collection of cells. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a pluripotent stem cell, a cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be from a specific organ or tissue. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell. In some embodiments, the cells are from nematodes. In some embodiments, the cell is a haploid cell. In some embodiments, the cell is a sex cell. In some embodiments, the haploid cell is male. In some embodiments, the haploid cell is female. In some embodiments, the haploid cell is a germ cell. In some embodiments, the haploid cell is a gamete. In some embodiments, the cell is a pollen grain. In some embodiments, the cell is an ovule. In some embodiments, the cell is a seed. In some embodiments, the seed is a plant seed. In some embodiments, the cell is an embryo. In some embodiments, the embryo is a plant embryo. In example embodiments, the cell is a pollen grain.

[0025] In some embodiments, the plant is an angiosperm. In some embodiments, the plant is a flowering plant. In some embodiments, the plant is a gymnosperm. In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot. In some embodiments, the plant is an annual. In some embodiments, the plant is a biennial. In some embodiments, the plant is a perennial. In some embodiments, the perennial is herbaceous. In some embodiments, the perennial is a woody plant. In some embodiments, the perennial is a deciduous plant. In some embodiments, the perennial is an evergreen plant. In some embodiments, the perennial is a tender plant. In some embodiments, the perennial is a hardy plant. In some embodiments, the plant is a model plant. Non-limiting examples of model plants include Arabidopsis thaliana. Nicothiana labacam. Nicothiana benlhamiana. Medicago Iriincalula. and Medicago sativa. In some embodiments, the plant is a leguminous plant. Non-limiting examples of leguminous plants include soybean (Glycine max), bean (Phaseolus vulgaris), chickpea (Cicer arietinum), lentil (Lens culinaris), pea (Pisum sativum), peanut (Arachis hypogaea), alfalfa (MedicagoAttorney Docket No. 65864-703601 sativa), mung bean (Vigna radiata), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata). In some embodiments, the plant is a grass. In some embodiments, the grass is a cereal. Nonlimiting examples of cereals include wheat (Triticum spp.), rye (Secale spp.), barley, oat, rice, millet, maize, and sorghum. In some embodiments, the plant is a tree. In some embodiments, the tree is deciduous. In some embodiments, the tree is coniferous. Non-limiting examples of woody trees include poplar, birch, spruce, cedar, ash, oak, willow, eucalyptus, and pine. In some embodiments, the plant is used for textiles. Non-limiting plants used for textiles include cotton, hemp, bamboo, flax, and cork. In some embodiments, the plant is a medicinal herb. Non-limiting examples of medicinal herbs include cannabis (Cannabis spp.), tobacco, kudzu. In some embodiments, the plant is a vegetable. Non-limiting examples of vegetables include tomatoes, potatoes, beet, sugar beet, carrot, and peppers. In some embodiments, the plant is used for oil. Non-limiting examples of plants used for oil include rape, sunflower, and jojoba. In some embodiments, the plant is a fruit tree. In some embodiments, the plant is a citrus tree. In some embodiments, the plant is sugarcane. In some embodiments, the plant is a coffee plant. In some embodiments, the plant is used for the production of produce. In some embodiments, the plant has commercial uses. In some embodiments, the plant is used for research.

[0026] Provided herein are polynucleotides for delivery into a cell. In some embodiments, the polynucleotides comprise a vector. Nucleic acids can be incorporated into vectors. A vector can include any specific DNA segment that is designed to move from a carrier into a target DNA. A vector may be referred to as an expression vector, or a vector system, which is a set of components needed to bring about DNA insertion into a genome, or other targeted DNA sequence such as an episome, plasmid, or even virus / phage DNA segment. In some embodiments vector may be referred to as an expression vector, which is a set of components needed for transient expression in a host cell. In some embodiments, a vector may be referred to a polynucleotide. Vector systems such as viral vectors (e.g., retroviruses, adeno-associated virus and integrating phage viruses), and non-viral vectors (e.g., transposons) used for gene delivery in subjects have two basic components: 1) a vector comprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and a DNA target sequence and inserts the vector into the target DNA sequence. Vectors most often contain one or more expression cassettes that comprise one or more expression control sequences, wherein an expression control sequence is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence or mRNA, respectively.Attorney Docket No. 65864-703601

[0027] Many different types of vectors are known. For example, plasmids and viral vectors, e.g., retroviral vectors, are knownas a vector. Mammalian Expression plasmids typically have an origin of replication, a suitable promoter and optional enhancer, and also any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences. Examples of vectors include: plasmids (which may also be a carrier of another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., HIV-1, SIV or FIV), retrovirus (e.g., ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac).

[0028] Transformation of a cell with a polynucleotide may be accomplished using a physical method. Non-limiting examples of physical methods to deliver nucleic acids include particle bombardment, nanotubes, nanoparticles, viral vectors, electroporation, and microinjection. In some embodiments, the physical method for transformation is particle bombardment. In some embodiments, the particle bombardment uses a gene gun. In some embodiments, particle bombardment is referred to as biolistics. Particle bombardment may be used for nuclear plant transformation and for transformations that require DNA to be delivered to chloroplasts and mitochondria. Particle bombardment may be based on the direct delivery of DNA into plant cells using gold, tungsten, or platinum particles. The gold, tungsten, or platinum particles may be coated with DNA, which may be shot at plant tissue or plant cells at high velocity and may become lodged inside plant cells. Once inside the cell, the DNA may elute off the particles and may become transiently expressed or stably integrated into the host genome. Gene transfer through a gene gun may be part of the biolistic method. During the biolistic method DNA or RNA polynucleotide may adhere to biological inert particles (such as gold, tungsten, or platinum) to form a DNA / particle complex. The DNA / particle complex may be bombarded on a targeted tissue in a vacuum. This bombardment may be achieved by accelerating a powerful shot to the targeted tissue. The high density of the DNA / particle complex may increase the bombardment speed toward the targeted tissue, which may result in effective transformation. Inert metal particles may be bombarded through a solution containing DNA surrounding the cell. Such a complex, once formed, may be directly transferred to the plant cell. In some embodiments, the inhibitors such as small molecule or mutated protein may be embedded into the DNA / particle complex. Delivery of inhibitors embedded into the DNA / particle complex may be used for transformed cell selection.

[0029] Transformation of a cell with a polynucleotide may be accomplished via bacteria delivery. Non-limiting examples of bacteria-mediated transformation methods to deliver nucleic acids comprise use of Agrobacterium lumefacies. Agrobacterium rhizogenes. OchrobactrumAttorney Docket No. 65864-703601 haywardense, and bacteria belonging to the Rhizobiacea family. In some embodiments, the biological method comprises use of Agrobacterium tumefacies. In some embodiments, the biological method comprises use of Agrobacterium rhizogenes. In some embodiments, the biological method comprises use of Ochrobactrum haywardense. In some embodiments, the biological method comprises use of a bacteria belonging to the Rhizobiacea family. Bacteria- mediated transformation may be used for nuclear plant transformation and for transformations that require DNA to be delivered to chloroplasts and mitochondria.

[0030] Transformation of a cell with a polynucleotide may have about 100% efficiency. Transformation of a cell may have about 1% efficiency. Transformation of a cell with a polynucleotide may have about 95% efficiency. Transformation of a cell may have about 5% efficiency. Transformation of a cell with a polynucleotide may have about 90% efficiency. Transformation of a cell may have about 10% efficiency. Transformation of a cell with a polynucleotide may have about 85% efficiency. Transformation of a cell may have about 15% efficiency. Transformation of a cell with a polynucleotide may have about 80% efficiency. Transformation of a cell may have about 20% efficiency. Transformation of a cell with a polynucleotide may have about 75% efficiency. Transformation of a cell may have about 25% efficiency. Transformation of a cell with a polynucleotide may have about 70% efficiency. Transformation of a cell may have about 30% efficiency. Transformation of a cell with a polynucleotide may have about 65% efficiency. Transformation of a cell may have about 35% efficiency. Transformation of a cell with a polynucleotide may have about 60% efficiency. Transformation of a cell may have about 40% efficiency. Transformation of a cell with a polynucleotide may have about 55% efficiency. Transformation of a cell may have about 45% efficiency. Transformation of a cell with a polynucleotide may have about 50% efficiency.

[0031] In some embodiments, a polynucleotide may be designed to increase gene expression for one or more genes. In some embodiments, increased gene expression may result in increased concentration in the cell of the protein encoded by the gene. In some embodiments, a polynucleotide may be designed to express a mutant of an endogenous gene. In some embodiments, the expression of a mutant of the endogenous gene results in the production of a mutated protein. In some embodiments, the mutated protein produced by the expression of the mutant of the endogenous gene may form oligomers with the wild-type protein produced by the endogenous gene. In some embodiments, the oligomers formed by the mutated and wild-type protein may reduce the endogenous protein's activity. In some embodiments, the polynucleotides are stably transformed into a cell. In some embodiments, the polynucleotides are transiently transformed into a cell. In some embodiments, the polynucleotide transformed into a cell harborsAttorney Docket No. 65864-703601 a gene encoding a mutant of an oligomeric enzyme comprising two or more polypeptide chains. The polypeptide chains interact to each other to form oligomers. The individual polypeptide chains of an oligomeric enzyme are termed sub-units and may be identical to or different from one another. In some embodiments, the individual polypeptide encoded by the mutant of the oligomeric enzyme forms oligomeric enzymes with the wild-type polypeptide encoded by the endogenous gene. In some embodiments, the oligomeric enzyme incorporating a mutated polypeptide is inactive. The mutant may be a mutant of ADH, such as exemplified in Tables 1 or 2.

[0032] In some embodiments, the mutated polypeptide incorporated by the oligomeric enzyme act as a dominant-negative. Dominant-negative effects may occur when the active site of the oligomeric enzyme is formed after the oligomerization. The presence of the mutated polypeptide in the oligomeric enzyme prevents the correct formation of the active site of the oligomer. In some embodiments, preventing the correct formation of the active site of the enzyme prevents the ligation of the substrate. Preventing the ligation of the substrate prevents the enzymatic reaction and therefore prevents the conversion of a substrate into product. A dominant-negative polypeptide may comprise a type of genetic mutation that results in a protein that interferes with the normal function of the wild-type protein.

[0033] In some embodiments, the activity of a protein is inhibited by a polypeptide. In some embodiments, the polypeptide may be introduced into a cell by a physical transformation method. In some embodiments, the physical transformation method comprises particle bombardment. In some embodiments, the polypeptide is coated on the particles used in the bombardment. In some embodiments, the polypeptide is transformed into a cell. In some embodiments, the polypeptide is transformed into a cell using carbon nanotubes. In some embodiments, the polypeptide is an alcohol dehydrogenase inhibitor. In some embodiments, the polypeptide is a mutant of alcohol dehydrogenase. In some embodiments, the polypeptide is a mutant of an oligomeric enzyme comprising two or more polypeptide chains. The polypeptide chains interact to each other to form oligomers. The individual polypeptide chains of an oligomeric enzyme are termed sub-units and may be identical to or different from one another. In some embodiments, the individual polypeptide encoded by the mutant of the oligomeric enzyme forms oligomeric enzymes with the wild-type polypeptide. In some embodiments, the oligomeric enzyme incorporating a mutated polypeptide is inactive. The mutant may be a mutant of ADH, such as exemplified in Tables 1 or 2.

[0034] The concentration of the mutant ADH polypeptide may be about 0.90 pg, about 0.95 pg, about 1.0 pg, about 1.2 pg, about 1.4 pg, about 1.5 pg, about 1.6 pg, about 1.8 pg, about 2.0 pg,Attorney Docket No. 65864-703601 about 2.2 pg, about 2.4 pg, about 2.5 pg, about 2.6 pg, about 2.8 pg, about 3.0 pg, about 3.2 pg, about 3.4 pg, about 3.5 pg, about 3.6 pg, about 3.8 pg, about 4.0 pg, about 4.2 pg, about 4.4 pg, about 4.5 pg, about 4.6 pg, about 4.8 pg, about 5.0 pg, about 5.2 pg, about 5.4 pg, about 5.5 pg, about 5.6 pg, about 5.8 pg, or about 6.0 pg. The concentration of the polypeptide may be about0.90 pg. The concentration of the polypeptide may be about 0.90 pg. The concentration of the polypeptide may be about 0.95 pg. The concentration of the polypeptide may be about 1.0 pg. The concentration of the polypeptide may be about 1.2 pg. The concentration of the polypeptide may be about 1.4 pg. The concentration of the polypeptide may be about 1.5 pg. The concentration of the polypeptide may be about 1.6 pg. The concentration of the polypeptide may be about 1.8 pg. The concentration of the polypeptide may be about 2.0 pg. The concentration of the polypeptide may be about 2.2 pg. The concentration of the polypeptide may be about 2.2 pg. The concentration of the polypeptide may be about 2.4 pg. The concentration of the polypeptide may be about 2.5 pg. The concentration of the polypeptide may be about 2.6 pg. The concentration of the polypeptide may be about 2.8 pg. The concentration of the polypeptide may be about 3.0 pg. The concentration of the polypeptide may be about 3.2 pg. The concentration of the polypeptide may be about 3.4 pg. The concentration of the polypeptide may be about 3.5 pg. The concentration of the polypeptide may be about 2.6 pg. The concentration of the polypeptide may be about 3.8 pg. The concentration of the polypeptide may be about 4.0 pg. The concentration of the polypeptide may be about 4.2 pg. The concentration of the polypeptide may be about 4.4 pg. The concentration of the polypeptide may be about 4.5 pg. The concentration of the polypeptide may be about 4.6 pg. The concentration of the polypeptide may be about 4.8 pg. The concentration of the polypeptide may be about 5.0 pg. The concentration of the polypeptide may be about 5.2 pg. The concentration of the polypeptide may be about 5.4 pg. The concentration of the polypeptide may be about 5.5 pg. The concentration of the polypeptide may be about 5.6 pg. The concentration of the polypeptide may be about 5.8 pg. The concentration of the polypeptide may be about 6.0 pg. The concentration of the polypeptide may be from about 0.9 pg to about 6 pg. The concentration of the polypeptide may be from about 1.8 pg to about 4.6 pg. The concentration of the polypeptide may be from about 2.8 pg to about 3.8 pg. In some embodiments, the activity of a protein is inhibited by a chemical inhibitors. In some embodiments, the chemical inhibitor may be introduced into a cell by a physical transformation method. In some embodiments, the physical transformation method comprises particle bombardment. In some embodiments, the chemical inhibitor is coated in the particles used in the bombardment. In some embodiments, the chemical inhibitor is transformed into a cell. In some embodiments, the chemical inhibitor is transformed into a cell using carbon nanotubes. In some embodiments, the chemical inhibitor is an alcohol dehydrogenase inhibitor. In someAttorney Docket No. 65864-703601 embodiments, the chemical inhibitor is an improved derivative of the chemical inhibitor. In some embodiments, the chemical inhibitor is disulfiram, penicillic acid, 4-bromopyrazole, 3- hydroxypropionamide, 4-methylpyrazole hydrochloride, cyanamide, 1,10-phenanthroline, pyrazole, fomepizole, or 5-P-D-ribofuranosylni cotinamide adenine dinucleotide (CNAD). In some embodiments, the chemical inhibitor is fomepizole. In some embodiments, the chemical inhibitor is fomepizole or any improved derivative of fomepizole. Improved derivate is a fomepizole molecule that has been modified to increase the inhibition potency. In some embodiments, the chemical inhibitor is CNAD. In some embodiments, the chemical inhibitor is CNAD or any improved derivative of CNAD. Improved derivate is a CNAD molecule that has been modified to increase the inhibition potency. In some embodiments, the chemical inhibitor is an ADH1 inhibitor.

[0035] In some embodiments, the chemical inhibitor is fomepizole. The concentration of fomepizole may be about 0.70 micromolar (pM), about 0.75 pM, about 0.80 pM, about 0.85 pM, about 0.90 pM, about 0.95 pM, about 1 to 5 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 2 to 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 3 to 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 4 to 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 5 to 25 pM, about 6 to 30 pM, about 35 pM, about 7 to 40 pM, about 45 pM, about 8 to 50 pM, about 55 pM, about 9 to 60 pM, about 65 pM, about 10 to 70 pM, about 75 pM, about 11 to 80 pM, about 85 pM, about 12 to 90 pM, about 95 pM, about 13 to 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 145 pM, about 150 pM, about 155 pM, about 160 pM, about 165 pM, about 170 pM, about 175 pM, about 180 pM, about 185 pM, about 190 pM, about 195 pM, or about 200 pM. The concentration of fomepizole may be about 0.70 pM fomepizole. The concentration of fomepizole may be about 0.75 pM fomepizole. The concentration of fomepizole may be about 0.80 pM fomepizole. The concentration of fomepizole may be about 0.85 pM fomepizole. The concentration of fomepizole may be about 0.90 pM fomepizole. The concentration of fomepizole may be about 0.95 pM fomepizole. The concentration of fomepizole may be about 1 pM fomepizole. The concentration of fomepizole may be about 10 pM fomepizole. The concentration of fomepizole may be about 20 pM fomepizole. The concentration of fomepizole may be about 25 pM fomepizole. The concentration of fomepizole may be about 30 pM fomepizole. The concentration of fomepizole may be about 40 pM fomepizole. The concentration of fomepizole may be about 50 pM fomepizole. The concentration of fomepizole may be about 60 pM fomepizole. The concentration of fomepizole may be about 70 pM fomepizole. The concentration of fomepizole may be about 80 pMAttorney Docket No. 65864-703601 fomepizole. The concentration of fomepizole may be about 90 pM fomepizole. The concentration of fomepizole may be about 100 pM fomepizole. The concentration of fomepizole may be about 110 pM fomepizole. The concentration of fomepizole may be about 120 pM fomepizole. The concentration of fomepizole may be about 125 pM fomepizole. The concentration of fomepizole may be about 130 pM fomepizole. The concentration of fomepizole may be about 140 pM fomepizole. The concentration of fomepizole may be about 150 pM fomepizole. The concentration of fomepizole may be about 160 pM fomepizole. The concentration of fomepizole may be about 170 pM fomepizole. The concentration of fomepizole may be about 180 pM fomepizole. The concentration of fomepizole may be about 190 pM fomepizole. The concentration of fomepizole may be about 200 pM fomepizole. The concentration of fomepizole may be from about 0.7 pM to about 200 pM fomepizole. The concentration of fomepizole may be from about 0.9 pM to about 180 pM fomepizole. The concentration of fomepizole may be from about 1 pM to about 160 pM fomepizole. The concentration of fomepizole may be no less than 1 pM fomepizole.

[0036] In some embodiments, the chemical inhibitor is CNAD. The concentration of CNAD may be about 0.70 nanomolar (nM), about 0.75 nM, about 0.80 nM, about 0.85 nM, about 0.90 nM, about 0.95 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM. , about 105 nM, about 110 nM, about 115 nM, about 120 nM, or about 125 nM. The concentration of CNAD may be about 0.70 nM CNAD. The concentration of CNAD may be about 0.75 nM CNAD. The concentration of CNAD may be about 0.80 nM CNAD. The concentration of CNAD may be about 0.85 nM CNAD. The concentration of CNAD may be about 0.90 nM CNAD. The concentration of CNAD may be about 0.95 nM CNAD. The concentration of CNAD may be about 1 nM CNAD. The concentration of CNAD may be about 10 nM CNAD. The concentration of CNAD may be about 20 nM CNAD. The concentration of CNAD may be about 25 nM CNAD. The concentration of CNAD may be about 30 nM CNAD. The concentration of CNAD may be about 40 nM CNAD. The concentration of CNAD may be about 50 nM CNAD. The concentration of CNAD may be about 60 nM CNAD. The concentration of CNAD may be about 70 nM CNAD. The concentration of CNAD may be about 80 nM CNAD. The concentration of CNAD may be about 90 nM CNAD. The concentration of CNAD may be about 100 nM CNAD. The concentration of CNAD may be about 110 nMAttorney Docket No. 65864-703601CNAD. The concentration of CNAD may be about 120 nM CNAD. The concentration of CNAD may be about 125 nM CNAD. The concentration of CNAD may be from about 0.7 nM to about 125 nM CNAD. The concentration of CNAD may be from about 0.9 nM to about 110 nM CNAD. The concentration of CNAD may be from about 1 nM to about 100 nM CNAD. The concentration of CNAD may be no less than 1 nM CNAD.

[0037] The inhibitor (e.g., polynucleotide, chemical, polypeptide) of the oligomeric enzyme may reduce the activity of the oligomeric enzyme about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, or about 60% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme to about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 0% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme at least about 100%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, or at least about 60% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme to no more than about 40%, to no more than about 35%, to no more than about 30%, to no more than about 25%, to no more than about 20%, to no more than about 15%, to no more than about 10%, to no more than about 5%, or to no more than about 0% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme about 100% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme about 90% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme about 80% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme about 70% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme about 60% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme to about 40% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme to about 30% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme to about 20% asAttorney Docket No. 65864-703601 compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme to about 10% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme to about 5% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor. The inhibitor of the oligomeric enzyme may reduce the activity of the oligomeric enzyme to about 0% as compared to a wild-type oligomeric enzyme not in the presence of an inhibitor.

[0038] In some embodiments, the oligomeric enzyme belongs to the dehydrogenase family. In some embodiments, the dehydrogenase comprises a cytokinin dehydrogenase. In some embodiments, the dehydrogenase comprises an aldehyde dehydrogenase. In some embodiments, the dehydrogenase comprises a succinate dehydrogenase. In some embodiments, the dehydrogenase comprises a malate dehydrogenase. In some embodiments, the dehydrogenase comprises a glutamate dehydrogenase. In some embodiments, the dehydrogenase comprises a dihydroorotate dehydrogenase. In some embodiments, the dehydrogenase comprises an alanine dehydrogenase. In some embodiments, the dehydrogenase comprises a steroid dehydrogenase. In some embodiments, the dehydrogenase comprises a pyruvate dehydrogenase. In some embodiments, the dehydrogenase comprises a lactate hydrogenase. In some embodiments, the dehydrogenase comprises a formate dehydrogenase. In some embodiments, the dehydrogenase comprises an alcohol dehydrogenase. Non-limiting examples of aldehyde dehydrogenase genes that may be used in the method provided herein include ALDH1, ALDH2, ALDH3, ALDH4, ALDH5, ALDH6, ALDH7, ALDH8, ALDH9, ALDH10, ALDH11, ALDH12, ALDH16, ALDH18, ALDH19, ALDH21, ALDH22, ALDH23, ALDH24. Non-limiting examples of alcohol dehydrogenase genes that may be used in the method provided herein include ADH1, ADH2, ADH3, and ADH4.

[0039] As a non-limiting example, a cell is transformed with a polynucleotide for producing a mutant of alcohol dehydrogenase. In some cells alcohol dehydrogenase may be present in high concentrations. The mutant of the alcohol dehydrogenase can form a dominant-negative enzyme complex. In this embodiment, a polynucleotide for generating a mutant of the alcohol dehydrogenase is used. The transformed cells with this polynucleotide may produce a mutated alcohol dehydrogenase. In this depicted embodiment, the mutated alcohol dehydrogenase not only lacks the activity of the wild-type alcohol dehydrogenase, but its monomer also forms inactive oligomers with the wild-type alcohol dehydrogenase monomer. In this depicted embodiment, the mutated alcohol dehydrogenase forms a dominant negative complex lacking alcohol dehydrogenase activity. In this depicted embodiment, the high concentration of alcoholAttorney Docket No. 65864-703601 dehydrogenase in the cell may no longer function. Non-limiting example of mutants alcohol dehydrogenase are provided in Table 1 and Table 2 (SEQ ID NOS: 2-8, 10-16, 24-30, 32-38).

[0040] As a non-limiting example, a cell is transformed by particle bombardment in which the 1 um gold particle is coated with any desirable nucleic acid polynucleotide plus inhibitory concentrations of a mutant of alcohol dehydrogenase. Cells receiving the particles carrying inhibitory concentrations of the mutant of the alcohol dehydrogenase inhibit the endogenous high concentrations of alcohol dehydrogenase. In this embodiment, the transformed cells with survive when exposed to allyl alcohol.

[0041] In some embodiments, an oligomeric enzyme reacts with a non-toxic compound to form a product. In some embodiments, the product is toxic. In some embodiments, the product kills cells that are not transformed. In some embodiments, the transformed cells do not produce the toxic product. In some embodiments, the cells are exposed to the compound following a transformation procedure. In some embodiments, the transformed cells do not produce the toxic product because of a polynucleotide altering activity of the oligomeric enzyme. In some embodiments, the transformed cells do not produce the toxic product because of a polypeptide altering activity of the oligomeric enzyme. In some embodiments, the transformed cells do not produce the toxic product because of a chemical inhibitor altering activity of the oligomeric enzyme. A non-limiting example of a compound used in the method provided herein includes allyl alcohol. A non-limiting reaction comprises alcohol dehydrogenase converting allyl alcohol into acrolein. Acrolein can be toxic to cells.

[0042] In some embodiments, the cells are contacted with allyl alcohol. In some embodiments, the cells are contacted with vapor produced from allyl alcohol. In come embodiments, the cells are contacted with liquid allyl alcohol. In some embodiments, the cells are contacted with gaseous allyl alcohol. The concentration of allyl alcohol may be about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 15.5%, about 16%, about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, about 20%, about 20.5%, about 21%, about 21.5%, about 22%, about 22.5%, about 23%, about 23.5%, about 24%, about 24.5%, or about 25%. The concentration of allyl alcohol may be about 0.8%. The concentration of allyl alcohol may be 0.9%. The concentration of allyl alcohol may be about 1%. The concentration of allyl alcohol may be about 1.5%. The concentration of allyl alcohol may be about 2%. The concentration of allyl alcohol may be about 2.5%. The concentration of allylAttorney Docket No. 65864-703601 alcohol may be about 3%. The concentration of allyl alcohol may be about 3.5%. The concentration of allyl alcohol may be about 4%. The concentration of allyl alcohol may be about 4.5%. The concentration of allyl alcohol may be about 5%. The concentration of allyl alcohol may be about 5.5%. The concentration of allyl alcohol may be about 6%. The concentration of allyl alcohol may be about 6.5%. The concentration of allyl alcohol may be about 7%. The concentration of allyl alcohol may be about 7.5%. The concentration of allyl alcohol may be about 8%. The concentration of allyl alcohol may be about 8.5%. The concentration of allyl alcohol may be about 9%. The concentration of allyl alcohol may be about 9.5%. The concentration of allyl alcohol may be about 10%. The concentration of allyl alcohol may be about 10.5%. The concentration of allyl alcohol may be about 11%. The concentration of allyl alcohol may be about 11.5%. The concentration of allyl alcohol may be about 12%. The concentration of allyl alcohol may be about 12.5%. The concentration of allyl alcohol may be about 13%. The concentration of allyl alcohol may be about 13.5%. The concentration of allyl alcohol may be about 14%. The concentration of allyl alcohol may be about 14.5%. The concentration of allyl alcohol may be about 15%. The concentration of allyl alcohol may be about 15.5%. The concentration of allyl alcohol may be about 16%. The concentration of allyl alcohol may be about 16.5%. The concentration of allyl alcohol may be about 17%. The concentration of allyl alcohol may be about 17.5%. The concentration of allyl alcohol may be about 18%. The concentration of allyl alcohol may be about 18.5%. The concentration of allyl alcohol may be about 19%. The concentration of allyl alcohol may be about 19.5%. The concentration of allyl alcohol may be about 20%. The concentration of allyl alcohol may be about 20.5%. The concentration of allyl alcohol may be about 21%. The concentration of allyl alcohol may be about 21.5%. The concentration of allyl alcohol may be about 22%. The concentration of allyl alcohol may be about 22.5%. The concentration of allyl alcohol may be about 23%. The concentration of allyl alcohol may be about 23.5%. The concentration of allyl alcohol may be about 24%. The concentration of allyl alcohol may be about 24.5%. The concentration of allyl alcohol may be or about 25%. The concentration of allyl alcohol may be from about 0.8% to about 25%. The concentration of allyl alcohol may be from about 1% to about 20%. The concentration of allyl alcohol may be from about 5% to about 15%. The concentration of allyl alcohol may be from about 6% to about 13%. The concentration of allyl alcohol may be from about 8% to about 12%. The concentration of allyl alcohol may be from about 9% to about 11%. The concentration of allyl alcohol may be from about 5% to about 10%. The concentration of allyl alcohol may be from about 6% to about 9%. The concentration of allyl alcohol may be from about 6% to about 8%.Attorney Docket No. 65864-703601

[0043] In some embodiments, the cells are contacted with allyl alcohol. In some embodiments, the cells are contacted with vapor produced from allyl alcohol. In come embodiments, the cells are contacted with liquid allyl alcohol. In some embodiments, the cells are contacted with gaseous allyl alcohol. In some embodiments, the cells are contacted with allyl alcohol for about 10 seconds, about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 75 seconds, about 90 seconds, about 105 seconds, about 120 seconds, about 135 seconds, about 150 seconds, about 165 seconds, about 180 seconds, about 195 seconds, about 210 seconds, about 225 seconds, about 240 seconds, about 255 seconds, about 270 seconds, about 285 seconds, or about 300 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 10 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 15 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 30 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 45 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 60 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 75 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 90 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 105 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 120 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 135 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 150 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 165 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 180 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 195 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 210 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 225 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 240 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 255 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 270 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 285 seconds, or about 300 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 10 seconds to about 300 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 30 seconds to about 240 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 60 seconds to about 180 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 90 seconds to about 150 seconds. In some embodiments, the cells are contacted with allyl alcohol for about 105 seconds to about 135 seconds.Attorney Docket No. 65864-703601

[0044] In some embodiments, a polynucleotide comprises polynucleotides to generate transgenic cells with desirable traits. In some embodiments, a polynucleotide comprises polynucleotides to generate transgenic plants with desirable traits. In some embodiments, a polynucleotide comprises polynucleotides to generate gene-edited plants with desirable traits. In some embodiments, the desirable traits are introduced with exogenous genes. In some embodiments, the desirable traits are introduced through modulation of activity or expression of genes already present in the cells. Any combination of introducing one or more exogenous genes to the cells, downregulating one or more endogenous genes to the cells, and upregulating one or more endogenous genes in the cells may be used to produce a desirable trait or traits. As a nonlimiting example, the desirable trait may be introduced by downregulating one or more endogenous genes in the cells. As another non-limiting example, the desirable trait may be introduced by upregulating one or more endogenous genes in the cells. In yet another nonlimiting example, the desirable trait may be introduced by downregulating one or more endogenous genes in the cells while simultaneously upregulating one or more endogenous genes in the cells. In yet another non-limiting example, the desirable trait maybe introduced by downregulating one or more endogenous genes in the cell while simultaneously introducing an exogenous gene to the cell. In yet another non-limiting example, the desirable trait maybe introduced by upregulating one or more endogenous genes in the cell while simultaneously introducing an exogenous gene to the cell. As a non-limiting example, the cells may be from a plant and the desirable traits may comprise herbicide resistance, drought tolerance, salt tolerance, pest resistance, disease resistance, drought stress resistance, high temperature resistance, improved water use efficiency, improved nutrient acquisition efficiency, increased yield, abiotic stress resistance, and improved photosynthesis efficiency. Pests may include insects, worms, beetles, herbivorous animals. Diseases may include those caused by fungi, bacteria, viruses. Abiotic stress may be caused by heat or drought. In some embodiments, a desirable trait or traits are traits that are commercially desirable. In some embodiments, the commercially desirable traits are introduced to plant cells. Non-limiting examples of commercially desirable traits introduced into plant cells include traits that modulate or introduce oil production in the plant, modulate or introduce biosynthesis of medicinal compounds in the plant, modulate or introduce polymers in the plant such as polymers useful for manufacturing including bioplastics or carbon fibers, or increase or introduce nutritional quality. A desirable trait may be a trait in Table 3.Attorney Docket No. 65864-703601TABLE 3. List of potential desirable traits.Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601Attorney Docket No. 65864-703601

[0045] In some embodiments, a single desirable trait is introduced in the plant using the methods disclosed herein. In some embodiments, more than one trait is introduced in the plant using the method disclosed herein. In some embodiments, two desirable traits are introduced in the plant using the methods disclosed herein. In some embodiments, three desirable traits are introduced in the plant using the methods disclosed herein. In some embodiments, fewer than threeAttorney Docket No. 65864-703601 desirable traits are introduced in the plant using the methods disclosed herein. In some embodiments, fewer than four desirable traits are introduced in the plant using the methods disclosed herein.

[0046] As a non-limiting example, a plasmid is introduced to a cell conferring transient expression of a polynucleotide inhibitor of an oligomeric enzyme at the same time (e.g., cotransformed) another plasmid is introduced to the cell conferring transient expression of a polynucleotide that induces a desirable trait in the plant. As another non-limiting example, a plasmid is introduced to a cell conferring transient expression of a polynucleotide inhibitor of an oligomeric enzyme at the same time (e.g., co-transformed) another plasmid is introduced to the cell conferring stable expression of a polynucleotide that induces a desirable trait in the plant. As yet another non-limiting example, a plasmid is introduced to a cell conferring transient expression of a polynucleotide inhibitor of an oligomeric enzyme at the same time (e.g., cotransformed) another plasmid is introduced to the cell conferring transient expression of a polynucleotide that induces desirable traits in the plant. As yet another non-limiting example, a plasmid is introduced to a cell conferring transient expression of a polynucleotide inhibitor of an oligomeric enzyme at the same time (e.g., co-transformed) another plasmid is introduced to the cell conferring stable expression of a polynucleotide that induces desirable traits in the plant. As yet another non-limiting example, a plasmid is introduced to a cell conferring transient expression of a polynucleotide inhibitor of an oligomeric enzyme at the same time (e.g., cotransformed) two other plasmids are introduced to the cell conferring transient expression of a polynucleotide that induces a desirable trait in the plant. As yet another non-limiting example, a plasmid is introduced to a cell conferring transient expression of a polynucleotide inhibitor of an oligomeric enzyme at the same time (e.g., co-transformed) two other plasmids are introduced to the cell conferring transient expression of a polynucleotide that induces desirable traits in the plant. As yet another non-limiting example, a plasmid is introduced to a cell conferring transient expression of a polynucleotide inhibitor of an oligomeric enzyme at the same time (e.g., cotransformed) two other plasmids are introduced to the cell with one plasmid conferring transient expression of a polynucleotide that induces a desirable trait in the plant and the other plasmid conferring stable expression of a polynucleotide that induces the same or a different desirable trait in the plant.

[0047] The methods disclosed herein may produce about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 6000,Attorney Docket No. 65864-703601 about 7000, about 8000, about 9000, or about 10000 transformed cells in a single experiment. The methods disclosed herein may produce over about 50, over about 100, over about 150, over about 200, over about 250, over about 300, over about 350, over about 400, over about 450, over about 500, over about 550, over about 600, over about 650, over about 700, over about 750, over about 800, over about 850, over about 900, over about 1000, over about 1500, over about 2000, over about 2500, over about 3000, over about 3500, over about 4000, over about 4500, over about 5000, over about 6000, over about 7000, over about 8000, over about 9000, or over about 10000 transformed cells in a single experiment. The methods disclosed herein may produce at least about 50, least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 1000, at least about 1500, at least about 2000, at least about 2500, at least about 3000, at least about 3500, at least about 4000, at least about 4500, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, or at least about 10000 transformed cells in a single experiment. The methods disclosed herein may produce at least 50 transformed cells in a single experiment. The methods disclosed herein may produce at least 100 transformed cells in a single experiment. The methods disclosed herein may produce at least 250 transformed cells in a single experiment. The methods disclosed herein may produce at least 500 transformed cells in a single experiment. The methods disclosed herein may produce at least 750 transformed cells in a single experiment. The methods disclosed herein may produce at least 1000 transformed cells in a single experiment. The methods disclosed herein may produce at least 2500 transformed cells in a single experiment. The methods disclosed herein may produce at least 5000 transformed cells in a single experiment. The methods disclosed herein may produce at least 7500 transformed cells in a single experiment. The methods disclosed herein may produce at least 10000 transformed cells in a single experiment.

[0048] The methods disclosed herein may allow for genotype independent transformation. The methods disclosed herein may allow for the generation of multiple independent transformation events in a single experiment. In this embodiment, each cell transformed in a single experiment may be considered an independent transformation event. The methods disclosed herein may allow for independent transformation events of at least 100 cells in a single experiment. The methods disclosed herein may allow for independent transformation events of at least 500 cells in a single experiment. The methods disclosed herein may allow for independent transformation events of at least 1000 cells in a single experiment. The methods disclosed herein may allow for independent transformation events of at least 5000 cells in a single experiment. The methodsAttorney Docket No. 65864-703601 disclosed herein may allow for independent transformation events of at least 10000 cells in a single experiment.

[0049] The methods disclosed herein may allow for recovery of transformed seeds about 50 days, about 49 days, about 48 days, about 47 days, about 46 days, about 45 days, about 44 days, about 43 days, about 42 days, about 41 days, about 40 days, about 39 days, about 38 days, about 37 days, about 36 days, about 35 days, about 34 days, about 33 days, about 32 days, about 31 days, about 30 days, about 29 days, about 28 days, about 27 days, about 26 days, about 25 days, about 24 days, about 23 days, about 22 days, about 21 days, or about 20 days after a transformation experiment with cells. The methods disclosed herein may allow for recovery of transformed seeds about 45 days after a transformation experiment with cells. The methods disclosed herein may allow for recovery of transformed seeds about 40 days after a transformation experiment with cells. The methods disclosed herein may allow for recovery of transformed seeds about 35 days after a transformation experiment with cells. The methods disclosed herein may allow for recovery of transformed seeds about 30 days after a transformation experiment with cells. The methods disclosed herein may allow for recovery of transformed seeds about 35 days after a transformation experiment with cells. The methods disclosed herein may allow for recovery of transformed seeds about 20 days after a transformation experiment with cells.

[0050] In some embodiments, methods comprise analyzing a sample obtained from a transformed cell, plant, seed, embryo, or pollen. In some embodiments, total DNA is extracted from the sample of the transformed cell, plant, seed, embryo, or pollen. In some embodiments, total RNA is extracted from the sample of the transformed cell, plant, seed, embryo, or pollen. In some embodiments, the total DNA or RNA is used for molecular transformation validation. Validation may be performed by using PCR kits using the total extracted DNA and specific primers to amplify fragments of a polynucleotide sequence present in the polynucleotide. Validation may be performed by using PCR kits using the cDNA prepared from the total extracted RNA and specific primers to amplify fragments of a polynucleotide sequence present in the polynucleotide. The expression level of one or more genes of the contract may be performed through quantitative polymerase chain reaction (qPCR). In some embodiments, the transformed cell, plant, seed, embryo, or pollen are assessed for ADH activity. In some embodiments, the ADH activity is assessed using a colorimetric assay.

[0051] As a non-limiting example of the methods disclosed herein, a pollen cell or plant embryo is transformed using particle bombardment with an inhibitor of alcohol dehydrogenase (ADH) and one or more polynucleotides that confers one or more desirable traits to the pollen cell orAttorney Docket No. 65864-703601 plant embryo. The inhibitor of ADH may be a polynucleotide on a plasmid that is transiently expressed. The polynucleotide inhibitor of AHD may encode a mutant of ADH, such as exemplified in Tables 1 or 2. The mutant of ADH may be a dominant-negative of ADH. The one or more polynucleotides that confers one or more desirable traits may be expressed on another plasmid separate from the plasmid containing the polynucleotide of the inhibitor of ADH. The one or more polynucleotides that confers one or more desirable traits may be expressed transiently or stably. The one or more desirable traits may be one or more traits that help the plant grow and survive, may be one or more traits that are commercially valuable, or may be any combination of the one or more desirable traits. In some embodiments, the inhibitor of ADH is a chemical inhibitor of ADH such as fomepizole or CNAD. In these embodiments, the concentration of fomepizole may be at least 1 micromolar (pM) or the concentration of CNAD may be at least 1 nanomolar (nM). In some embodiments, the concentration of fomepizole is about 1 to 160 pM. In some embodiments, the concentration of CNAD is about 1 to 100 nM. In some embodiments, the concentration of fomepizole is no greater than 160 pM. In some embodiments, the concentration of CNAD is no greater than about 100 nM. In yet another embodiment, the inhibitor of ADH may be a polypeptide mutant of ADH, such as exemplified in Tables 1 or 2. In these embodiments, the concentration of the polypeptide inhibitor of ADH may be about 1-5 pg. In some embodiments, the concentration of the polypeptide inhibitor of ADH may be about 2-4 pg. In some embodiments, the concentration of the polypeptide inhibitor of ADH may be about 2.7-3.7 pg. The inhibitor of ADH (e.g., polynucleotide, chemical inhibitor, polypeptide) may reduce the activity of ADH in the pollen cells or plant embryos by at least about 60%. In some embodiments, the inhibitor of ADH (e.g., polynucleotide, chemical inhibitor, polypeptide) may reduce the activity of ADH in the pollen cells or plant embryos by at least about 70%. In some embodiments, the inhibitor of ADH (e.g., polynucleotide, chemical inhibitor, polypeptide) may reduce the activity of ADH in the pollen cells or plant embryos by at least about 80%. In some embodiments, the inhibitor of ADH (e.g., polynucleotide, chemical inhibitor, polypeptide) may reduce the activity of ADH in the pollen cells or plant embryos by at least about 90%. Following transformation with particle bombardment the pollen cells or plant embryos are exposed to allyl alcohol for 30-300 seconds. In some embodiments, following particle bombardment the pollen cells or plant embryos are exposed to allyl alcohol for 60-300 seconds. In some embodiments, following particle bombardment the pollen cells or plant embryos are exposed to allyl alcohol for 90-150 seconds. In other embodiments, following particle bombardment the pollen cells or plant embryos are exposed to allyl alcohol for 105-135 seconds. The exposing to allyl alcohol may comprise exposing to with allyl alcohol vapors. The concentration of allyl alcohol may be about 1-20% allyl alcohol. In some embodiments, theAttorney Docket No. 65864-703601 concentration of allyl alcohol is about 5-12%. In other embodiments, the concentration of allyl alcohol is 6-11%. Following exposure to allyl alcohol, the pollen cells or plant embryos that were not transformed die and the pollen cells or plant embryos that were transformed survive. The methods disclosed herein may result in greater than 1000 transformed pollen cells from a single transformation experiment. The methods disclosed herein may result in greater than 1000 transformed pollen cells that each have an independent transformation event from a single transformation experiment. The transformed pollen cells may be used to produce transformed seeds in about 25 to 40 days following the transformation experiment. In this embodiment, the transformed seeds will have recovered ADH activity. The chemical structure of compounds used or generated in the example are shown in FIG. 11.DEFINITIONS

[0052] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains.EXAMPLES

[0053] The following example is included for illustrative purposes only and is not intended to limit the scope of the inventive concepts.Example 1. Selection of pollen grains transformed with a polynucleotide designed to overexpress a mutant of the maize ADH1.

[0054] A schematic representation of maize pollen transformation is depicted in FIG. 1 as an example for maize pollen grain transformation. An overview of the method is as follows. Pollen grains were harvested from maize flowers. The pollen grains were transformed with the methods described herein, for example, using particle bombardment. The pollen grains were then treated with allyl alcohol. The treated pollen grains were then used to pollinate the silks of a maize ear. The maize ear was allowed to produce transformed grains. Immature embryos from transformed grains were rescued and germinated in an appropriate growth medium. Plants were recovered, transferred to a greenhouse, and grown to maturity to produce next-generation transformed seeds.

[0055] Pollen grains, collected from the maize panicles, were transformed by particle bombardment with 1 um gold particles coated with a DNA polynucleotide comprising the constitutive promoter Zm-UBI driving the expression of a mutant of maize ADH1 (SEQ ID NO: 2-8) (FIG. 8A). The mutant maize ADH1, when expressed in the pollen grains, allowed theAttorney Docket No. 65864-703601 production of a mutated monomeric polypeptide of ADH1. The mutated ADH1 monomer formed oligomers, in this case, a dimer, with the endogenous wildtype ADH1 monomers. The ADH1 dimers formed with a mutated monomer and a wildtype monomer were inactive. Since mutated ADH1 monomer was in large proportion in relation to the wildtype endogenous monomer, most of the ADH1 dimers were inactive. Mutated ADH1 monomer was shown to inhibit maize ADH1 activity (FIG. 7). Therefore, to select the pollen grains that were successfully transformed with the maize ADH1 mutated polypeptide, the pollen grains are exposed to allyl alcohol. The untransformed pollen grains would have active ADH1 enzyme and convert the allyl alcohol into acrolein, as depicted in FIG. 1. Acrolein is toxic to pollen grains and therefore will kill untransformed pollen grains. To demonstrate that acrolein is toxic, pollen grains were taken from maize flowers. The pollen grains were treated with allyl alcohol and placed in a pollen germination medium. After a few hours, the pollen grains were examined under a stereomicroscope and recorded. Untransformed pollen grains unexposed to allyl alcohol germinated in germination medium (FIG. 3A), whereas untransformed pollen grains which expressed wildtype ADH1 did not germinate (FIG. 3B).Example 2. Selection pollen grains transformed by particle bombardment with sold particles coated with the ADH1 inhibitor fomepizoie.

[0056] Pollen grains were treated by particle bombardment in which the 1 um gold particles were coated with the ADH1 inhibitor fomepizoie. In some examples, the ADH1 inhibitor is an improved derivate is a fomepizoie molecule that has been modified to increase the inhibition potency. FIG. 6 shows the inhibition of wildtype maize ADH1 activity with varying concentrations of fomepizoie. To select pollen grains having successfully been transformed with the 1 um gold particles coated with the ADH1 inhibitor, the pollen grains were exposed to allyl alcohol. The untransformed pollen grains having active ADH1 converted the allyl alcohol into acrolein. After a few hours, a stereomicroscope image of maize pollen grain germination was taken. FIG. 4A shows that maize pollen grains on germination medium without allyl alcohol germinated. FIG. 4B shows that maize pollen grains that were bombarded with 1 um gold particles coated with the ADH1 inhibitor fomepizoie and then exposed to allyl alcohol were able to germinate. This result is contrasted with FIG. 4B where no pollen grains germinated. These results demonstrate that inhibition of ADH1 allowed for germination of only pollen grains successfully bombarded. Therefore, this method could be used to selective germinating pollen grains through inactivation of ADH1 and selection via allyl alcohol.Attorney Docket No. 65864-703601Example 3. Transformation, selection, pollination of maize ear and recovery of transformed plants produced by the pollen transformation method

[0057] As depicted in FIG. 1, the pollen transformation method allows for quick recovery of transformed seeds. Pollen grains were transformed by particle bombardment with gold particles of 1 um coated with a DNA polynucleotide comprising of a constitutive promoter, in this case a ubiquitin promoter, a gene encoding a mutant maize ADH1, and a transcriptional terminator (FIG. 8A). After bombardment, the pollen grains were exposed to allyl alcohol and used to pollinate a maize ear to produce transformed seeds. After pollen transformation by particle bombardment and selection by exposing the pollen grains to allyl alcohol, the pollen was used to pollinate maize ear. About 20 days after pollination, the ear was harvested (FIG. 9). The immature embryos were rescued and placed in a germination medium. Transformed plants were grown to maturity, and T1 seeds were harvested.

[0058] The results demonstrate that the pollen grain were transformed by the DNA polynucleotide to express a mutant ADH1 that dimerized with endogenous ADH1 and formed an inactive dimer. ADH1 dimers comprised of active and inactive monomeric ADH1, in which the inactive ADH1 monomers compete with the active ADH1 monomeric polypeptides resulting in oligomers lacking enzyme activity. As shown in FIG. 7, the wildtype-mutant dimer has less relative activity with allyl alcohol than the wildtype dimer, and would therefore produce less acrolein. Therefore, only the pollen grain that were successfully transformed survived treatment with allyl alcohol because only those pollen grains would convert less of the allyl alcohol to acrolein. These results show the method can be used to quickly transform, select, and then harvest transformed plants.Example 4. Selection of soybean embryo axes transformed by particle bombardment with sold particles coated with the ADH1 inhibitor fomepizoie.

[0059] Soybean embryo axes were bombarded with the gold particles coated with fomepizoie. Soybean embryo axes were treated by particle bombardment in which the 1 um gold particles were coated with the ADH1 inhibitor fomepizoie. FIG. 5 shows on the left, shoot formation from soybean embryo axes bombarded with gold particles coated with the ADH inhibitor fomepizoie, and on the right, shoot inhibition of shoots formation from soybean embryo axes bombarded with gold particles coated without fomepizoie. The results demonstrate that the gold particles coated with fomepizoie could be used via bombardment to treat or transform plants to prevent allyl alcohol conversion to acrolein and allow the soybeans to grow.Attorney Docket No. 65864-703601Example 5. Bacteria-mediated transformation and selection of transformed plants using soybean embryo axes.

[0060] As depicted in FIG. 2, the transformation method was applied to generate transformed soybean plants for quick recovery of transformed plants. The drawing depicts the schematic representation of the plant transformation method disclosed herein using soybean embryo axes. Soybean embryo axes were taken from embedded seeds. The embryo axes were transformed using Agrobacterium tumefaciens comprising a DNA polynucleotide comprising the right border of the T-DNA, a constitutive promoter, such as the ubiquitin promoter, a gene encoding a mutant of an oligomeric enzyme, such as a mutant of ADH1, and a transcriptional terminator and the left border of the T-DNA (FIG. 8B). The embryo axes were then treated with allyl alcohol and placed in a shooting medium to produce transformed plants (FIGs. 10A and 10B). Plants were then transferred to soil and grown to maturity to produce next-generation transformed seeds.Example 6. Particle bombardment transformation of pollen grains and embryonic axes with chemical inhibitors.

[0061] Gold particles of 1 and 1.6 pm diameter are washed with cold, 100% [v / v] ethanol and sterile distilled water. Fifty microliters (pL) of washed gold particles (water solution of 3 mg / 50 pL), 2.5 pL of purified DNA (at Ipg / pL), 50 pL of 2.5 M CaCh, and 20 pL of 0.1 M spermidine are mixed for 10 minutes to precipitate the purified DNA on to the washed gold particles. The DNA-coated gold particles are centrifuged at 6000 rpm for 10 seconds to generate a pellet. The pellets are rinsed with 150 pL of 70% [v / v] ethanol followed by 150 pL of 100% [v / v] ethanol. The pellets are then suspended with 50 pL of 100% [v / v] ethanol containing the chemical inhibitor and briefly sonicated. If the chemical inhibitor is fomepizole, the concentration used is 1-160 pM. If the chemical inhibitor used is CNAD, the concentration used is 1-100 nM. Immediately after sonication, the DNA-coated gold particles are loaded onto the center of a macrocarrier (5 pL of each) and allowed to air dry.

[0062] For transformation of pollen grains, 50 pL of pollen grains are spread evenly over 55- millimeter (mm) filter paper (e.g., Whatman filter paper 1001-055). The filter paper is placed on a 6-centimeter diameter Petri dish and covered with a 127 mm mesh nylon screen attached to the lid of a previously cut Petri dish. The two parts of the Petri dish are joined together so that they fit snugly. The gold particles are shot through the holes of the nylon screen using a rupture pressure of 300-1200 pounds per square inch (psi) with vacuum of 0.03 atmospheres (equivalent to 29 inches Hg). The bombardment conditions are validated in maize coleoptile as a control. The dry bombarded pollen grains are contacted with vapor emanating from 10% allyl alcohol for 2 minutes. After treatment with allyl alcohol, the untransformed pollen grains (e.g., notAttorney Docket No. 65864-703601 containing the ADH inhibitor) do not germinate while the transformed pollen grains (e.g., containing the ADH inhibitor) do germinate.

[0063] For transformation of embryonic axes, the exposed apical meristematic region of the embryonic axes derived from the mature seeds is positioned in the appropriate bombardment culture medium and bombarded with the gold particles using a rupture pressure of 300-1200 pounds per square inch (psi) with vacuum of 0.03 atmospheres (equivalent to 29 inches Hg). The bombardment conditions are validated in maize coleoptile as a control. The bombarded embryos are contacted with vapor emanating from 7% allyl alcohol for 2 minutes.Example 7. In vitro enzymatic assay of ADH in presence of inhibitor.

[0064] Inhibitors of ADH are assessed by conducting in vitro assays using recombinant ADH enzymes with various inhibitors of ADH.

[0065] In the presence of mutated maize ADH (3.2 pg), the activity of the recombinant wildtype maize ADH is reduced to 2.52 pmol of NAD reduced / pg protein / minute. In the absence of the inhibitor, recombinant wild-type maize ADH (0.2 pg) shows 3.91 pmol of NAD reduced / pg protein / minute. The reduced activity of recombinant wild-type maize ADH in the presence of the mutated maize ADH was 64% due to the dominant-negative effect (FIG. 7). In the presence of variable concentrations of fomepizole (0, 10, 20, 40, 80, 160 pM), the activity of a recombinant wild-type maize ADH was 12.19, 9.97, 9.38, 6.18, 4.46, 3.09 pmol of NAD reduced / pg protein / minute, corresponding to 100%, 82%, 77%, 51%, 37%, 25% activity due to the inhibitor fomepizole (FIG. 6).EMBODIMENTS1. A method of selecting a transformed cell, the method comprising: a. delivering: i. a polynucleotide to a plurality of cells, wherein the polynucleotide encodes a mutant of an oligomeric enzyme endogenous to the plurality of cells; ii. a polypeptide to the plurality of cells, wherein the polypeptide is the mutant of the oligomeric enzyme endogenous to the plurality of cells; or iii. a chemical inhibitor molecule of the oligomeric enzyme endogenous to the plurality of cells; b. exposing the plurality of cells to a non-toxic compound, wherein the compound is a substrate of the oligomeric enzyme endogenous to the plurality of cells; whereinAttorney Docket No. 65864-703601 the exposing kills untransformed cells of the plurality of cells by the non-toxic compound that is converted by the oligomeric enzyme to a toxic product; and c. selecting a viable transformed cell from the plurality of cells, wherein the oligomeric enzyme of the viable transformed cell was inhibited by the polynucleotide, the polypeptide, or the chemical inhibitor molecule.2. The method of embodiment 1, wherein the mutant of the oligomeric enzyme endogenous to the plurality of cells is a mutant monomer.3. The method of embodiment 1 or embodiment 2, wherein the mutant of the oligomeric enzyme is a mutated polypeptide.4. The method of embodiment 3, wherein the mutated polypeptide forms an oligomer with the enzyme endogenous to the plurality of cells.5. The method of embodiment 4, wherein the oligomer results in an inactive enzyme endogenous to the plurality of cells.6. The method of any one of embodiments 1-5, wherein the delivering comprises a physical method.7. The method of embodiment 6, wherein the physical method comprises particle bombardment, carbon nanotubes, nanoparticles, viral vectors, electroporation, microinjection, or any combination thereof.8. The method of embodiment 6 or embodiment 7, wherein the physical method comprises particle bombardment.9. The method of any one of embodiments 6-8, wherein the physical method comprises carbon nanotubes.10. The method of any one of embodiments 6-9, wherein the delivering comprises a biological-mediated method.11. The method of any one of embodiments 6-10, wherein the delivering comprises delivering a bacteria to the plurality of cells.12. The method of embodiment 11, wherein the bacteria comprises Agrobacterium tumefaciens, Agrobacterium rhizogenes, Ochrobactrum haywardense, or a bacteria belonging to the Rhizobiacea family.13. The method of embodiment 11 or embodiment 12, wherein the bacteria comprises Agrobacterium tumefaciens.14. The method of any one of embodiments 11-13, wherein the bacteria comprises Agrobacterium rhizogenes.15. The method of any one of embodiments 11-14, wherein the bacteria comprises Ochrobactrum haywardense.Attorney Docket No. 65864-70360116. The method of any one of embodiments 11-15, wherein the bacteria comprises bacteria from the Rhizobiacea.17. The method of any one of embodiments 1-16, wherein the transformed cell undergoes stable transfection.18. The method of any one of embodiments 1-16, wherein the transformed cell undergoes transient transfection.19. The method of any one of embodiments 1-18, wherein the polynucleotide comprises a plasmid vector.20. The method of any one of embodiments 1-19, wherein the polynucleotide encodes a mutant of an oligomeric enzyme endogenous to the plurality of cells.21. The method of any one of embodiments 1-20, wherein the polynucleotide that encodes the mutant of the oligomeric enzyme endogenous to the plurality of cells is a member of the aldehyde dehydrogenase family.22. The method of any one of embodiments 1-21, wherein the polynucleotide that encodes the mutant of the oligomeric enzyme endogenous to the plurality of cells is a member of the alcohol dehydrogenase family (e.g., mutants provided in Table 1 and Table 2, SEQ ID NOS: 2-8, 10-16, 24-30, 32-38).23. The method of any one of embodiments 1-22, wherein the oligomeric enzyme endogenous to the plurality of cells comprises ADH1, ADH2, ADH3, or ADH4.24. The method of any one of embodiments 1-22, wherein the oligomeric enzyme endogenous to the plurality of cells comprises ADH1.25. The method of any one of embodiments 1-24, wherein the non-toxic compound is a substrate of the oligomeric enzyme.26. The method of embodiment 25, wherein the non-toxic compound is converted by the oligomeric enzyme into a toxic product.27. The method of embodiment 25 or embodiment 26, wherein the toxic product is harmful to untransformed cells from the plurality of cells.28. The method of any one of embodiments 1-27, wherein the non-toxic compound is allyl alcohol.29. The method of embodiment 28, wherein the allyl alcohol is converted into acrolein by the oligomeric enzyme.30. The method of embodiment 29, wherein acrolein is toxic to untransformed cells from the plurality of cells.31. The method of any one of embodiments 1-30, wherein the oligomeric enzyme is inactivated by the mutated of the oligomeric enzyme endogenous to the plurality of cells.Attorney Docket No. 65864-70360132. The method of any one of embodiments 1-31, wherein inactivated oligomeric enzyme cannot efficiently use allyl alcohol as substrate.33. The method of any one of embodiments 1-32, wherein inactivated oligomeric enzyme cannot use allyl alcohol as substrate.34. The method of any one of embodiments 1-33, wherein inactivated oligomeric enzyme cannot efficiently convert allyl alcohol into acrolein.35. The method of any one of embodiments 1-34, wherein inactivated oligomeric enzyme cannot convert allyl alcohol into acrolein.36. The method of any one of embodiments 1-35, wherein the activity of the oligomeric enzyme is inhibited by the chemical inhibitor molecule.37. The method of any one of embodiments 1-36, wherein the chemical inhibitor molecule inhibits the oligomeric enzyme from using allyl alcohol as a substrate.38. The method of any one of embodiments 1-37, wherein the chemical inhibitor molecule inhibits the oligomeric enzyme from converting allyl alcohol into acrolein.39. The method of any one of embodiments 1-38, wherein the chemical inhibitor molecule is fomepizole.40. The method of any one of embodiments 1-39, wherein the chemical inhibitor molecule is delivered to the plurality of cells at the concentration of 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM 400 mM, 500 mM, 600 mM.41. The method of any one of embodiments 1-40, wherein the transformed cell is a haploid cell.42. The method of any one of embodiments 1-40, wherein the transformed cell is a gamete.43. The method of embodiment 42, wherein the gamete is male.44. The method of embodiment 42, wherein the gamete is female.45. The method of any one of embodiments 1-40, wherein the transformed cell is a pollen grain.46. The method of embodiment 45, wherein the pollen grain is used to pollinate flowers of a plant.47. The method of any one of embodiments 1-40, wherein the transformed cell is an ovule.48. The method of any one of embodiments 1-40, wherein the transformed cell is a germ cell.49. The method of any one of embodiments 1-40, wherein the transformed cell in an undifferentiated cell.50. The method of any one of embodiments 1-40, wherein the transformed cell is a stem cell.51. The method of embodiment 50, wherein the stem cell is a pluripotent stem cell.Attorney Docket No. 65864-70360152. The method of any one of embodiments 1-51, wherein the method generates a transgenic plant.53. The method of any one of embodiments 1-52, the polynucleotide to a plurality of cells further comprises an exogenous gene.54. The method of any one of embodiments 1-52, the polypeptide to a plurality of cells further comprises an exogenous gene.55. The method of any one of embodiments 1-52, the chemical inhibitor molecule further comprises an exogenous gene.56. The method of any one of embodiments 1-55, wherein the method generates a genome- edited plant.57. A method of cell selection, the method comprising: a. providing a plurality of cells comprising: (i) at least one cell comprising an inhibitor of an alcohol dehydrogenase protein, and (ii) at least one cell that does not comprise the inhibitor of the alcohol dehydrogenase protein; b. exposing the plurality of cells to a substrate of the alcohol dehydrogenase protein, wherein exposing the at least one cell that does not comprise the inhibitor of the alcohol dehydrogenase protein results in cell death, and exposing the at least one cell comprising the inhibitor of the alcohol dehydrogenase protein does not result in cell death; and c. selecting a viable cell from the plurality of cells after exposing the plurality of cells to the substrate, thereby selecting the at least one cell comprising the inhibitor of the alcohol dehydrogenase protein.58. The method of embodiment 57, wherein the inhibitor comprises a gene encoding a mutant alcohol dehydrogenase protein, a mutant dehydrogenase protein, or fomepizole.59. The method of embodiment 57 or embodiment 58, wherein the substrate is allyl alcohol.60. The method of any one of embodiments 57-59, wherein the alcohol dehydrogenase protein is ADH1, ADH2, ADH3 or ADH4.61. The method of any one of embodiments 57-60, wherein the at least one cell comprising the inhibitor of the alcohol dehydrogenase protein comprises an exogenous gene.62. The method of any one of embodiments 57-61, further comprising delivering to two or more cells the inhibitor of the alcohol dehydrogenase, thereby generating the plurality of cells comprising (i) the at least one cell comprising the inhibitor of an alcohol dehydrogenase protein, and (ii) at least one cell that does not comprise the inhibitor of the alcohol dehydrogenase protein.63. The method of embodiment any one of embodiments 57-62, wherein the inhibitor is delivered to the plurality of cells at a concentration of about 1 mM to about 600 mM, orAttorney Docket No. 65864-703601 about 1 mM, 5 mM, lOmM, 20mM, 50mM, lOOmM, 200mM, 300mM 400mM, 500mM, or 600mM, optionally wherein the inhibitor is fomepizole.64. The method of embodiment 62 or embodiment 63, wherein the delivering comprises a physical method.65. The method of embodiment 64, wherein the physical method comprises particle bombardment, carbon nanotubes, nanoparticles, viral vectors, electroporation, microinjection, or any combination thereof.66. The method of embodiment 64, wherein the physical method comprises particle bombardment.67. The method of any one of embodiments 62-66, wherein the delivering comprises delivering a bacteria to the two or more cells.68. The method of embodiment 67, wherein the bacteria comprises Agrobacterium tumefaciens, Agrobacterium rhizogenes, Ochrobactrum haywardense, a bacteria belonging to the Rhizobiacea family, or any combination thereof.69. The method of embodiment 67, wherein the bacteria comprises Agrobacterium tumefaciens.70. The method of embodiment 67, wherein the bacteria comprises Agrobacterium rhizogenes.71. The method of embodiment 67, wherein the bacteria comprises Ochrobactrum haywardense.72. The method of embodiment 67, wherein the bacteria comprises a bacteria from the Rhizobiacea.73. The method of any one of embodiments 62-72, wherein delivering comprises stable transfection.74. The method of any one of embodiments 62-72, wherein the delivering comprises transient transfection.75. The method of any one of embodiments 57-74, wherein the plurality of cells comprises a haploid cell.76. The method of any one of embodiments 57-75, wherein the plurality of cells comprises a gamete.77. The method of embodiment 76, wherein the gamete is male.78. The method of embodiment 76, wherein the gamete is female.79. The method of any one of embodiments 57-78, wherein the plurality of cells comprises a pollen grain.Attorney Docket No. 65864-70360180. The method of embodiment 79, wherein the pollen grain is used to pollinate a flower of a plant.81. The method of any one of embodiments 57-80, wherein the plurality of cells comprises an ovule.82. The method of any one of embodiments 57-81, wherein the plurality of cells comprises a germ cell.83. The method of any one of embodiments 57-82, wherein the plurality of cells comprises an undifferentiated cell.84. The method of any one of embodiments 57-83, wherein the plurality of cells comprises a stem cell.85. The method of embodiment 84, wherein the stem cell is a pluripotent stem cell.86. The method of embodiment 85, wherein the pluripotent stem cell is an induced pluripotent stem cell.87. The method of embodiment 85 or embodiment 86, wherein the pluripotent cell is a cell from a cluster of cells of a plant meristematic region.88. The method of embodiment 87, wherein the cluster of cells of a plant meristematic region is or has been induced from an immature embryo.89. The method of embodiment 87, wherein the cluster of cells of a plant meristematic region is or has been induced from a mature embryo.90. The method of any one of embodiments 57-89, wherein the plurality of cells comprise a cell from a cluster of cells from leaf pieces.91. The method of embodiment 90, wherein the cluster of cells from leaf pieces are from seedlings.92. The method of any one of embodiments 87, wherein the cluster of cells of a plant meristematic region is from an embryo axis of germinated seeds.93. The method of any one of embodiments 57-92, wherein the method generates a transformed plant.94. The method of any one of embodiments 57-93, wherein the method generates a transformed seed.95. The method of any one of embodiments 57-94, wherein the method generates a genome edited plant.96. The method of any one of embodiments 57-95, wherein the method generates a genome edited seed.97. The method of any one of embodiments 57-96, wherein the method generates a transgenic plant.Attorney Docket No. 65864-70360198. A method comprising: a. exposing a plurality of pollen grains to a substrate of an alcohol dehydrogenase protein; b. selecting at least one viable pollen grain from the plurality of pollen grains; c. pollinating a flower of a plant with the at least one viable pollen grain; and d. harvesting a seed from the plant.99. The method of embodiment 98, wherein at least one of the pollen grains of the plurality of pollen grains comprises an alcohol dehydrogenase inhibitor.100. The method of embodiment 99, wherein the alcohol dehydrogenase inhibitor comprises a gene encoding a mutant alcohol dehydrogenase protein (e.g., mutants provided in Table 1, SEQ ID NOS: 2-8, 10-16), a mutant dehydrogenase protein (e.g., mutants provided in Table 2, SEQ ID NOS: 24-30, 32-38), or fomepizole.101. The method of embodiment 99 or embodiment 100, further comprising delivering to two or more pollen grains the alcohol dehydrogenase inhibitor to generate the plurality of pollen grains.102. The method of embodiment 101, wherein the delivering comprises a physical method.103. The method of embodiment 102, wherein the physical method comprises particle bombardment, carbon nanotubes, nanoparticles, viral vectors, electroporation, microinjection, or any combination thereof.104. The method of embodiment 102, wherein the physical method comprises particle bombardment.105. The method of any one of embodiments 102-104, wherein the delivering comprises delivering a bacteria to the two or more pollen grains.106. The method of embodiment 101, wherein the delivering comprises delivering a bacteria to the two or more cells.107. The method of embodiment 106, wherein the bacteria comprises Agrobacterium tumefaciens, Agrobacterium rhizogenes, Ochrobactrum haywardense, a bacteria belonging to the Rhizobiacea family, or any combination thereof.108. The method of embodiment 106, wherein the bacteria comprises Agrobacterium tumefaciens.109. The method of embodiment 106, wherein the bacteria comprises Agrobacterium rhizogenes.110. The method of embodiment 106, wherein the bacteria comprises Ochrobactrum haywardense.Attorney Docket No. 65864-703601111. The method of embodiment 106, wherein the bacteria comprises a bacteria from the Rhizobiacea.112. The method of any one of embodiments 102-111, wherein delivering comprises stable transfection.113. The method of any one of embodiments 102-112, wherein the delivering comprises transient transfection.114. The method of any one of embodiments 98-113, wherein the method generates a transformed plant.115. The method of any one of embodiments 98-114, wherein the method generates a transformed seed.116. The method of any one of embodiments 98-115, wherein the method generates a genome edited plant.117. The method of any one of embodiments 98-116, wherein the method generates a genome edited seed.118. The method of any one of embodiments 98-117, wherein the method generates a transgenic plant.119. The method of any one of embodiments 98-118, wherein the substrate is allyl alcohol.120. The method of any one of embodiments 98-119, further comprising harvesting a seed from the plant, optionally germinating the seed to generate a germinated seed, and further optionally screening a plantlet from the germinated seed for a trait.121. The method of embodiment 120, wherein the trait comprises pest resistance, disease resistance, drought stress resistance, high temperature resistance, improved water use efficiency, improved nutrient acquisition efficiency, or improved photosynthesis efficiency, or any combination thereof.122. The method of any one of embodiments 98-121, wherein the plurality of pollen grains is derived from maize, soybean, cotton, rice, wheat, barley, tomato, potato, Pinus, eucalyptus, Populus, citrus, coffee, sugarcane, canola, or oat.123. The method of any one of embodiments 98-122, wherein the at least one viable pollen grain comprises an exogenous gene.124. A kit compri sing : i. a polynucleotide encoding an inhibitor of an alcohol dehydrogenase protein; and ii. a substrate of alcohol dehydrogenase.125. The kit of embodiment 124, wherein the inhibitor of the alcohol dehydrogenase protein is a dominant-negative alcohol dehydrogenase protein.Attorney Docket No. 65864-703601126. The kit of embodiment 124 or embodiment 125, wherein the inhibitor of the alcohol dehydrogenase protein is a mutant alcohol dehydrogenase protein (e.g., mutants provided in Table 1 and Table 2, SEQ ID NOS: 2-8, 10-16, 24-30, 32-38).127. A kit compri sing : i. an inhibitor of an alcohol dehydrogenase protein; and ii. a substrate of alcohol dehydrogenase.128. The kit of embodiment 127, wherein the inhibitor comprises fomepizole.129. The kit of any one of embodiments 124-128, wherein the substrate is allyl alcohol.130. A method comprising delivering to a plurality of cells the kit of any one of embodiments 124-129.131. The method of embodiment 130, wherein (i) is delivered to the plurality of cells, and then (ii) is delivered to the plurality of cells.132. The method of embodiment 130 or embodiment 131, wherein the delivering comprises delivering an exogenous gene to the plurality of cells.133. The method of any one of embodiments 130-132, wherein the delivering results in a transformed cell within the plurality of cells.134. The method of any one of embodiments 130-133, wherein the delivering comprises a physical method.135. The method of embodiment 134, wherein the physical method comprises particle bombardment, carbon nanotubes, nanoparticles, viral vectors, electroporation, microinjection, or any combination thereof.136. The method of embodiment 134, wherein the physical method comprises particle bombardment.137. The method of any one of embodiments 130-136, wherein the delivering comprises delivering a bacteria to the plurality of cells.138. The method of embodiment 137, wherein the bacteria comprises Agrobacterium tumefaciens, Agrobacterium rhizogenes, Ochrobactrum haywardense, a bacteria belonging to the Rhizobiacea family, or any combination thereof.139. The method of embodiment 137, wherein the bacteria comprises Agrobacterium tumefaciens.140. The method of embodiment 137, wherein the bacteria comprises Agrobacterium rhizogenes.141. The method of embodiment 137, wherein the bacteria comprises Ochrobactrum haywardense.Attorney Docket No. 65864-703601142. The method of embodiment 137, wherein the bacteria comprises a bacteria from the Rhizobiacea.143. The method of any one of embodiments 130-142, wherein delivering comprises stable transfection.144. The method of any one of embodiments 130-142, wherein the delivering comprises transient transfection.145. The method of any one of embodiments 130-144, wherein the inhibitor is a gene encoding a mutant alcohol dehydrogenase protein, a mutant dehydrogenase protein, or fomepizole (e.g., mutants provided in Table 1 and Table 2, SEQ ID NOS: 2-8, 10-16, 24-30, 32-38).146. The method of any one of embodiments 130-145, wherein delivery comprises delivering to the plurality of cells fomepizole at a concentration of about 1 mM to about 600 mM, or about 1 mM, 5 mM, lOmM, 20mM, 50mM, lOOmM, 200mM, 300mM 400mM, 500mM, or 600mM.147. The method of any one of embodiments 130-146, wherein the plurality of cells comprises a haploid cell.148. The method of any one of embodiments 130-147, wherein the plurality of cells comprises a gamete.149. The method of embodiment 148, wherein the gamete is male.150. The method of embodiment 148, wherein the gamete is female.151. The method of any one of embodiments 130-150, wherein the plurality of cells comprises a pollen grain.152. The method of embodiment 151, wherein the pollen grain is used to pollinate a flower of a plant.153. The method of any one of embodiments 130-152, wherein the plurality of cells comprises an ovule.154. The method of any one of embodiments 130-153, wherein the plurality of cells comprises a germ cell.155. The method of any one of embodiments 130-154, wherein the plurality of cells comprises an undifferentiated cell.156. The method of any one of embodiments 130-155, wherein the plurality of cells comprises a stem cell.157. The method of embodiment 156, wherein the stem cell is a pluripotent stem cell.158. The method of embodiment 157, wherein the pluripotent stem cell is an induced pluripotent stem cell.Attorney Docket No. 65864-703601159. The method of embodiment 157 or embodiment 158, wherein the pluripotent cell is a cell from a cluster of cells of a plant meristematic region.160. The method of embodiment 159, wherein the cluster of cells of a plant meristematic region is or has been induced from an immature embryo.161. The method of embodiment 159, wherein the cluster of cells of a plant meristematic region is or has been induced from a mature embryo.162. The method of any one of embodiments 130-161, wherein the plurality of cells comprise a cell from a cluster of cells from leaf pieces.163. The method of embodiment 162, wherein the cluster of cells from leaf pieces are from seedlings.164. The method of embodiment of 159, wherein the cluster of cells of a plant meristematic region is from an embryo axis of germinated seeds.165. The method of any one of embodiments 130-164, wherein the method generates a transformed plant.166. The method of any one of embodiments 130-165, wherein the method generates a transformed seed.167. The method of any one of embodiments 130-166, wherein the method generates a genome edited plant.168. The method of any one of embodiments 130-167, wherein the method generates a genome edited seed.169. The method of any one of embodiments 130-168, wherein the method generates a transgenic plant.

Claims

Attorney Docket No. 65864-703601CLAIMSWHAT IS CLAIMED IS:

1. A method of selecting a transformed cell, the method comprising, a. contacting a plurality of cells with a polynucleotide and (1) a chemical inhibitor or (2) polypeptide inhibitor of an endogenous oligomeric enzyme of the plurality of cells to produce at least a transformed portion of the plurality of cells, wherein the polypeptide is a mutant of the endogenous oligomeric enzyme, and wherein, optionally, the contacting comprises particle bombardment and / or the chemical inhibitor is added at a concentration of no greater than about 200 micromolar (pM) or the polypeptide inhibitor is added at a concentration of about 1-5 micrograms (pg); b. exposing the plurality of cells to a non-toxic compound, wherein the nontoxic compound is a substrate of the endogenous oligomeric enzyme, and wherein the exposing kills an untransformed portion of the plurality of cells, and wherein, optionally, the plurality of cells are exposed to the non-toxic compound for about 30-300 seconds, the plurality of cells are exposed to vapors from the non-toxic compound, the plurality of cells are exposed to about 1-25% of the non-toxic compound, or any combination thereof; and c. selecting at least a portion of the plurality of cells transformed with the polynucleotide and the inhibitor.

2. The method of claim 1, wherein the chemical inhibitor is fomepizole or CNAD.

3. The method of claim 2 wherein the concentration of fomepizole is at least about 1 pM, and wherein the concentration of fomepizole is about 1-160 pM.

4. The method of claim 2, wherein the concentration of CNAD is at least about 1 nM, and wherein the concentration of CNAD is about 1-100 nM.

5. The method of claim 1, wherein the mutant is a dominant-negative enzyme of the endogenous oligomeric enzyme.

6. The method of claim 1, wherein the concentration of the polypeptide inhibitor is about 2-4 pg.

7. The method of any one of claims 1-6, wherein the endogenous oligomeric enzyme is alcohol dehydrogenase (ADH).

8. The method of any one of claims 1-7, wherein the non-toxic compound is allyl alcohol, and wherein the exposing comprises exposing the plurality of cells with about 6-11% allyl alcohol.Attorney Docket No. 65864-7036019. The method of any one of claims 1-8, wherein the exposing comprises exposing the plurality of cells with the non-toxic compound for about 105-135 seconds.

10. The method of any one of claims 1-9, wherein the chemical inhibitor or the polypeptide inhibitor reduces activity of the endogenous oligomeric enzyme by at least about 70%.

11. The method of any one of claims 1-10, wherein the polynucleotide confers one or more desirable traits, and wherein the one or more desirable traits comprises herbicide resistance, drought tolerance, salt tolerance, pest resistance, disease resistance, drought stress resistance, high temperature resistance, improved water use efficiency, improved nutrient acquisition efficiency, increased yield, abiotic stress resistance, improved photosynthesis efficiency, or any combination thereof.

12. The method of any one of claims 1-11, wherein the plurality of cells are a plurality of pollen grains or a plurality of embryos.

13. The method of any one of claims 1-12, wherein the plurality of pollen grains or the plurality of embryos are derived from maize, soybean, cotton, rice, wheat, barley, tomato, potato, Pinus, eucalyptus, Populus, citrus, coffee, sugarcane, canola, or oat.

14. The method of any one of claims 12, wherein the plurality of pollen grains transformed with the polynucleotide and the chemical inhibitor or the polypeptide inhibitor produce seeds in about 25-40 days.

15. The method of any one of claims 12, further comprising generating at least 1000 transformed pollen grains in a single experiment, and wherein each of the at least 1000 transformed pollen grains are an independent transformation event.

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