WO2023214041A1 - Processes, systems and media for delivering a substance to a plant - Google Patents

Abstract

Processes, systems and media for delivering one or more substances to cells of a plant, using nanobubbles, are described. The substance may, for example, be useful in altering physiology and/or gene expression. A method for editing the genome of a plant is described. A method of producing a gene edited plant is also described. Methods of generating a genetically modified plant with abiotic stress tolerance, altered growth, altered yield, disease and insect resistance, herbicide tolerance, modified product quality and/or pollination control; for improving resistance of a plant to pests; and for delivering a plant or crop protection product into a plant are also described. Also described are methods for inducing a change in a phenotype, genotype, chemistry or physiology of a plant; delivering a substance to cells of a plant; and delivering an oligonucleotide to a plant. Plant application media and plant cultivation systems are also described.

Description

PROCESSES, SYSTEMS AND MEDIA FOR DELIVERING A SUBSTANCE TO A PLANT

FIELD OF INVENTION

The present invention relates to processes, systems and media for delivering a substance to a plant. More particularly, the present invention includes methods and associated systems for providing nanobubbles, together with one or more substances, to a plant. For example, providing nanobubbles and a substance at the root of a plant, whereby the substance is delivered to plant cells. The substance may, for example, be useful in altering physiology and/or gene expression.

BACKGROUND

Plants produce a large number of molecules which may be utilised, for example as foods, drugs, colorants, flavourings, comestible additives or crop protection products (for example fungicides, herbicides, insecticides, nematicides, pesticides or the like). These molecules may not be essential to the survival of the plant and thus only expressed under particular conditions and/or only expressed at low levels. Chemical synthesis of such molecules by the plant may be the most efficient synthesis route to generate the molecule(s) for commercial use, for example where the molecules are complex and/or extraction from plants remain the best sources of supply.

Soil-less growth, for example hydroponic growth systems, which allow plant growth under controlled conditions in a greenhouse or outdoors have developed considerably over recent years. Although modulation of growth conditions to allow improved production of secondary metabolites from plants has been provided, further improvements are required.

EP 2 761 993 relates to a method for cultivating a plant using an artificial light-irradiating lamp wherein a plant is irradiated with a red light and then with a blue light, for a predetermined period of time, wherein the cultivation conditions include providing dissolved oxygen in a nutritious liquid. WO 2017/156410 discusses providing a composition containing nanobubbles dispersed in a liquid carrier with another liquid to create an oxygen-enriched composition that is then applied to plant roots. Such a composition can promote germination or growth of plant seedlings.

EP 2 460 582 discusses the production of super-micro bubbles of several hundred nm to several dozen pm in size (diameter) and ways in which such bubbles can be provided.

EP 3 721 979 relates to a charged nanobubble dispersion liquid, a manufacturing method thereof and manufacturing apparatus therefor, and a method to control the growth rate of microorganisms and plants using nanobubble dispersion liquid.

US 2020/0045980 discusses the use of one or more volatile organic compounds produced by Cladosporium sphaerospermum to increase at least one growth characteristic in a plant after exposure of the plant to the volatile organic compound(s) (VOCs) wherein the VOCs from Cladosporium sphaerospermum were provided to the plant’s headspace. Cladosporium sphaerospermum was noted not to be required to grow in the soil with the plant to be treated as; in fact, such growth in soil may result in reduced effects on the plant's phenotype (growth, yield, etc). Methods have been described to provide VOCs into plant cells (Li Zhijian T., Janisiewicz Wojciech J., Liu Zongrang, Callahan Ann M., Evans Breyn E., Jurick Wayne M., Dardick Chris. (2019). Exposure in vitro to an Environmentally Isolated Strain TC09 of Cladosporium sphaerospermum Triggers Plant Growth Promotion, Early Flowering, and Fruit Yield Increase. Frontiers in Plant Science, 9, 1959); but alternative introduction methods are required.

Various methods have been used to introduce short fragments of DNA (antisense oligonucleotides) or small RNAs into plant cells with only limited success.

SUMMARY OF INVENTION

Without proper oxygenation, plants growing in hydroponic solutions die. The application of oxygen to the water in the form of nano- or microbubbles maintains a level of dissolved oxygen in the water that enables roots to absorb nutrients for growth. The use of nanobubbles in the growth of plants to date has been to provide oxygen to promote growth.

The present inventors have determined that nanobubbles generated with an electric field provided in combination with a functional component, such as a compound or substance, wherein the compound or substance is attached to the bubble, in the bubble or in solution with the bubble, allows transport of the compound or substance within a plant/plant cells. The combination of nanobubbles and functional components such as compounds or substances in or attached to such a bubble, or in solution with these nanobubbles can be used for multiple purposes, including to alter, for example, gene expression. It is considered this provides an advantageous way to transport exogenous compounds or substances to cells in the plant.

In particular, it is considered the present technology enables control of plant gene expression during growth, in real-time and in commercial environments. This enables crop production with higher yields, production of new compounds by plants, production of increased yields of compounds in plants, and features like ‘flowering on demand’. For example, production of compounds in the plant may be through the manipulation of latent and active biosynthetic pathways in the plant.

In a first aspect, the present invention provides a method for editing the genome of a plant, the method comprising: generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium; providing at least one gene editing component; and providing the nanobubble-containing carrier medium and the at least one gene editing component to the plant or an explant thereof.

In a second aspect, the present invention provides a method of producing a gene edited plant, the method comprising: generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium; providing at least one gene editing component to an explant of a plant to generate gene edited cells; producing callus from the explant to generate a callused explant; and regenerating shoots from the callused explant to generate the gene edited plant; wherein the method further comprises providing the nanobubble-containing carrier medium to at least one of: cells of the plant, the explant, the callused explant, or the shoots, during at least one of the providing at least one gene editing component. In certain examples of the first and second aspects of the present invention, the gene editing component comprises a CRISPR gene-editing component, optionally a CRISPR reagent, further optionally a CRISPR/Cas9 construct.

In certain examples of the first and second aspects of the present invention, the gene editing component is provided by Agrobacterium-mediated gene transfer, electroporation, PEG-mediated transformation, ribonucleoprotein (RNP) delivery or biolistic bombardment.

In some examples, the nanobubble-containing carrier medium and the at least one gene editing component are mixed prior to providing the nanobubble-containing carrier medium to the plant or explant.

In a third aspect, the present invention provides a method of generating a genetically modified plant having at least one of: abiotic stress tolerance, altered growth, altered yield, disease resistance, herbicide tolerance, insect resistance, modified product quality and pollination control, controlled flowering time, the method comprising the steps of: generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium; providing an explant; and genetically modifying the explant using a transformation vector to produce a genetically modified plant; wherein the method further comprises providing the nanobubble-containing carrier medium to the explant.

In examples of the first, second and third aspects of the present invention, the explant is an immature embryo, mature embryo, plant cell, microspore, protoplast, stem, hypocotyl, cotyledon, internode, node, seed, seedling, flower, pollen, endosperm, microtuber, leaf or root.

In some embodiments of the first, second and third aspect of the present invention, the nanobubbles are generated by application of an electric field or in the presence of an electric field.

In a fourth aspect, the present invention provides a method for delivering a plant or crop protection product or active ingredient into a plant, the method comprising: generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubblecontaining carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field; providing at least one plant or crop protection product or active ingredient of at least one plant or crop protection product; and providing the nanobubble-containing carrier medium and the at least one plant or crop protection product or active ingredient to the plant.

In some examples, the plant or crop protection product or active ingredient comprises at least one pesticide, optionally a herbicide, insecticide, fungicide, molluscicide, plant growth regulator, nematicide or acaricide; or mixtures thereof.

In some examples, the nanobubble-containing carrier medium and the at least one plant or crop protection product or active ingredient are mixed prior to applying to the plant.

In use of the process, the plant or crop protection product is absorbed into a plant tissue, optionally a leaf or root tissue. In certain examples, the plant or crop protection product is translocated from a first tissue of the plant to a second tissue of the plant.

In a fifth aspect, the present invention provides a method for improving the resistance of a plant to pests, the method comprising: generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field; admixing the nanobubble-containing carrier medium with at least one pesticide; and providing the nanobubble-containing carrier medium comprising the pesticide to the plant.

In a sixth aspect, the present invention provides a method for delivering a substance to cells of a plant, the method comprising: generating nanobubbles of at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field; admixing the nanobubble-containing carrier medium with a substance; wherein the substance is at least one of: volatile organic compound (VOCs), plant growth regulators (PGRs), biostimulants, DNA, RNA, viral vectors, expression vectors, peptides or gene editing components and providing the nanobubble-containing carrier medium comprising the substance to a locus or explant of the plant. In certain embodiments, the method comprises, sequentially: pre-mixing the substance with the carrier medium to form a pre-mix; and generating the nanobubbles within the pre-mix.

Advantageously, the substance is at least one substance capable of inducing a change in the phenotype, genotype, chemistry, or physiology of the plant.

In some examples, the substance is at least one compound, vector or nanomaterial.

In further examples, the substance is at least one substance selected from: VOCs; transgenes, nucleic acids, DNA, RNA, siRNA, antisense oligonucleotides, synthetic or native DNA or RNA, synthetic or native DNA or RNA up to 500 nucleotides, optionally up to 200 nucleotides; PGRs, gibberellins, auxins, abscisic acid, cytokinins and ethylene; CRISPR materials; CRISPR/Cas9; RNAi vectors, expression vectors, viral vectors, mono-polysaccharides; polyphenols; terpenoids; proteins or peptides, optionally peptides up to 150 amino acids, optionally up to 50 amino acids; nanomaterials, optionally a nanomaterial selected from: lipid nanoparticles, carbon nanotubes, copper nanoparticles, iron or iron oxide nanoparticles, manganese or manganese oxide nanoparticles, titanium dioxide nanoparticles, and zinc or zinc oxide nanoparticles; and plant protection products.

In some preferred examples, the substance is at least one substance selected from VOCs, RNA, siRNA, antisense oligonucleotides, peptides, CRISPR/Cas9; RNAi vectors, expression vectors and viral vectors. There groups are not mutually exclusive. In other words, the substance may belong to more than one of these groups.

Optionally, the substance is a nanomaterial selected from: lipid nanoparticles, carbon nanotubes, copper nanoparticles, iron or iron oxide nanoparticles, manganese or manganese oxide nanoparticles, titanium dioxide nanoparticles, and zinc or zinc oxide nanoparticles.

The application of phyto-nanotechnology is reviewed in Environ. Sci.: Nano, 2020, 7, 2863-2874, to which further reference should be made. In certain embodiments, the substance is or includes transgenes, synthetic or native DNA or RNA up to 500 nucleotides, optionally up to 200 nucleotides; gibberellins, auxins, abscisic acid, cytokinins, ethylene; peptides up to 150 amino acids, optionally up to 50 amino acids.

In certain embodiments of aspects of the present invention, the step of providing the carrier medium to the plant comprises applying the medium to seeds, roots and/or leaves of the plant, optionally by immersion, spraying, fogging or misting.

Advantageously, the substance and nanobubbles are transported or translocated from the locus of the plant to at least one plant cell, optionally wherein the substance and nanobubbles are transported or translocated from a first plant tissue to a second plant tissue.

In a seventh aspect, the present invention provides a plant application medium, for applying to a locus or explant of a plant, the medium comprising a carrier medium, a substance and nanobubbles of at least one gas, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field, and wherein the substance comprises at least one substance selected from VOCs, plant growth regulators, biostimulants, pesticides, herbicides, insecticides, DNA, RNA, viral vectors, expression vectors, peptides and gene editing components.

In certain examples, the application medium is prepared by mixing the substance with the carrier medium to form a pre-mix; and generating nanobubbles within the pre-mix to form the application medium.

Suitably, the substance is a substance as defined above with respect to the sixth aspect of the present invention.

In an eighth aspect, the present invention provides a method for inducing a change in a phenotype, genotype, chemistry or physiology of a plant by delivering a substance to a plant, the method comprising: providing a substance selected from: VOCs, optionally fungal, microbial or plant VOCs; PGRs, biostimulants, RNA, siRNA; antisense oligonucleotides; peptides; RNAi vectors; expression vectors and viral vectors; generating nanobubbles of at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field; and admixing the carrier medium and the substance and providing the nanobubble-containing carrier medium and the substance to the plant.

Optionally, the substance is a substance which, in use of the method, induces DNA methylation, RNA methylation, histone methylation or histone acetylation, optionally in one or more flowering loci.

In one example, the substance is or includes a nucleic acid; optionally at least one RNAi vector and/or expression vector.

In ninth aspect of the present invention, the present invention provides a seed priming method, the method comprising soaking the seed in a seed priming composition comprising water with nanobubbles, wherein the nanobubbles have been generated by an electric field or in the presence of an electric field.

In certain embodiments, the nanobubbles are nanobubbles of a gas, wherein the gas is, comprises, consists of or consists substantially of air or oxygen.

In a tenth aspect, the present invention provides a plant cultivation system comprising: a nanobubble generating apparatus for generating nanobubbles from at least one gas in a carrier medium, to form a nanobubble-containing carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field; and an applicator system to apply the nanobubble-containing carrier medium to at least one locus of a plant.

In certain examples, the nanobubble-containing carrier medium further comprises at least one substance selected from VOCs, DNA, RNA, PGRs, viral vectors, biostimulants, plant or crop protection products or active ingredients of plant or crop protection products, peptides and gene editing components.

In some embodiments, the applicator system comprises a system for immersion of roots, seeds and/or leaves of the plant in the nanobubble-containing carrier medium; and/or for spraying, fogging or misting the plant with the nanobubble-containing carrier medium and the substance.

Optionally, the applicator system comprises a system for misting leaves of the plant with the nanobubble-containing carrier medium and the substance, wherein the nanobubbles are nanobubbles of a gas, wherein the gas is, comprises, consists of or consists substantially of carbon dioxide.

In certain embodiments, the applicator system is in fluid communication with the nanobubble generating apparatus.

In some examples, the system is a hydroponic plant cultivation system.

In an eleventh aspect, the present invention provides the use of nanobubbles of at least one gas to enhance plant transformation.

In certain embodiments, the nanobubbles are nanobubbles of a gas, wherein the gas is, comprises, consists of or consists substantially of air or oxygen. In some examples, the gas comprises, consists of or consists substantially of carbon dioxide.

In certain embodiments the nanobubbles are generated by application of an electric field or in the presence of an electric field.

In a twelfth aspect, the present invention provides the use of nanobubbles generated by application of an electric field or in the presence of an electric field in plant cultivation, plant growth or plant propagation.

In some embodiments, the use includes delivering a plant or crop protection product or active ingredient to a plant, editing the genome of a plant, improving the resistance of a plant to pests or diseases, or delivering a substance to cells of a plant.

In certain examples of the uses of the present invention, the nanobubbles are nanobubbles in water, an aqueous carrier, a nutrient solution or an agar medium. In a thirteenth aspect, the present invention provides a method for delivering an oligonucleotide to a plant, the method comprising: generating nanobubbles of at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field; providing at least one oligonucleotide; and applying the nanobubble-containing carrier medium and the at least one oligonucleotide to the plant;

In certain embodiments, the nanobubble-containing carrier medium and the at least one oligonucleotide are applied to a root of the plant, optionally wherein the oligonucleotide is translocated, in use of the method, from the root of the plant to a leaf of the plant.

In some examples, the oligonucleotide is a labelled or unlabelled antisense oligonucleotide.

Optionally, in any aspect of the present invention, at least 50%, of the nanobubbles generated have a diameter of less than about 1000 nm, optionally less than about 500 nm, optionally about 20 nm, optionally about 2 nm, optionally in a range from 0.5 nm to 150 nm, or optionally 2 nm or less.

Optionally, in any aspect of the present invention, the at least one gas is selected from the group comprising or consisting of air, oxygen, carbon dioxide, nitrogen, hydrogen, ethylene, ethylene oxide and combinations thereof.

In certain examples, the gas is, comprises, consists of or consists predominantly of air or oxygen.

Advantageously, in any aspect of the present invention, the nanobubbles generated maintain stability for about 2 years or longer.

Optionally, in any aspect of the present invention, the plant is Cannabis sativa, Nicotiana benthamiana, Hordeum vulgare, Solanum lycopersicom, Solanum tuberosum, Nicotiana tabacum, Lactuca sativa, Ocimum basilicum, Zea mays or Glycine max. Suitably, the carrier medium is a liquid medium. The carrier medium may be water or an aqueous carrier medium, for example a liquid growth medium, a sugar-containing solution and/or a nutrient solution.

In some examples, the carrier medium is an aqueous agar medium.

In certain embodiments of any of the aspects of the present invention, the methods further comprise a pre-treatment step wherein rooted plants are incubated in an oxygen nanobubble water for one to two days prior to use of the method. Optionally, the oxygen nanobubble water is generated by generating nanobubbles of oxygen in water. Further optionally, the oxygen nanobubbles are generated by application of an electric field or in the presence of an electric field.

In certain embodiments of any of the aspects of the present invention, the plant is grown from a seed and the method further comprises a step of seed priming comprising soaking the seed in a seed priming composition comprising water and nanobubbles, wherein the nanobubbles have been generated by an electric field or in the presence of an electric field.

Suitably, the above mentioned processes may be carried out such that the step of providing a plant application medium comprises, sequentially: (a) pre-mixing the substance with the carrier medium to form a substance/carrier pre-mix; and (b) generating the nanobubbles within the substance/carrier pre-mix.

In certain embodiments of any of the methods of the present invention, the method further comprises a step of mixing the carrier medium with at least one gene-editing component, transformation vector, substance, plant or crop protection product or active ingredient to form a carrier pre-mix; and a step of generating the nanobubbles within the carrier pre-mix.

In certain embodiments of any of the methods of the present invention, the methods further comprise a step of growing a plant. The present invention further provides plants obtainable or obtained by the method. In certain examples of methods and processes of the present invention, a mixture of a nanobubble water and one or more substance to alter gene expression is provided to a plant at any time in the life cycle of the plant to induce one or more epigenetic changes in real time.

Suitably the one or more substances capable of inducing a change in the phenotype, genotype, chemistry, or physiology of a plant is a specific compound, that can be used to specifically enhance a plant in a desired way. Suitably the compound may alter growth, alter flowering (for example bring forward flowering/provide earlier reproduction), alter crop productivity, for example fruit production (for example, increase yields).

Suitably the compound may increase the amount of a primary or secondary metabolite provided by the plant.

Suitably the compound or compounds may improve the uptake or availability of essential nutrients within the plant to allow for increased plant growth.

Suitably, the compound or compounds may be capable of activating plant defences and/or stimulating pathways which provide protection against biotic and abiotic stresses.

In examples of the aspects of the present invention, the at least one gene-editing component, transformation vector, plant or crop protection product or active ingredient or the substance may be within the nanobubble(s), attached to the nanobubble(s), or may be in solution with (not attached) to the nanobubble(s); or combinations thereof.

It is considered that previous improvements in plant growth are limited to micro- and/or nanobubbles improving uptake of nutrients (mainly nitrogen, phosphorous or potassium) or basic fertilisers within the roots of the plants only. Potentially by upregulating nutrient uptake genes and/or by stimulating growth hormone production. It is not considered there has been any previous teaching or description of nanobubbles generated with an electric field entering the plant and transporting or delivering compounds into the plant cells, in particular to plant cells in the leaf or aerial portions of the plant. In particular it is not considered there has been any previous discussions of nanobubbles generated with an electric field enhancing or enabling the uptake of genetic material, for example nucleic acid, for example RNA, DNA, microRNA, RNAi, CRISPR/Cas9, double stranded DNA or RNA fragments or the like and/or in increasing or enabling the transport of such genetic material within a plant following uptake (for example to the leaf, flowering portions or aerial portions of the plant).

Nitrogen, phosphorus and potassium are typically considered essential for the growth of plants. Other nutrients such as minerals including, for example, calcium, magnesium and iron may be provided in a growth liquid for the plant. Suitably nitrogen fertilizers such as ammonium sulphate, ammonium chloride, ammonium nitrate, urea, nitrogenous lime, potassium nitrate, calcium nitrate and sodium nitrate; phosphate fertilizers such as superphosphate of lime and fused magnesium phosphate; potassium fertilizers such as potassium chloride and potassium sulphate; and minerals such as calcium, magnesium and iron may be provided to a plant growth solution. Suitably, in the present invention, nitrogen, phosphorus and potassium or a mineral such as calcium, magnesium and iron may not be considered to be one or more compounds capable of inducing a change in the phenotype, genotype, chemistry, or physiology of a plant.

Suitably the mixture to be applied to the plant may further comprise one or more further constituents such as surfactants, chelating agents, emulsifiers, antioxidants, reductants, pH buffers and osmotic buffers.

Suitably a nucleic acid construct, for example short interfering RNA (siRNA), antisense oligonucleotide, microRNA or the like may be selected to target a single gene or a gene within a pathway in a plant, for example a pathway responsible for production of a secondary metabolite in a plant. Any suitable secondary metabolite may be selected.

Suitably a secondary plant metabolite may include phenolics, alkaloids, saponins, terpenes, lipids, and carbohydrates.

Suitably the phenolics may be selected from simple phenolics, tannins, coumarins, flavonoids, chromones and xanthones, stilbenes, and lignans.

In certain preferred examples, the substance is at least one oligonucleotide, optionally an antisense oligonucleotide. In certain examples, a PGR is a chemical or biological entity which is applied to a plant of interest to promote an advantageous growth trait. In other examples, particularly when used in crop protection and applied in the field, the PGR may be a plant growth regulator herbicide, to provide control of a weed species.

Suitably a PGR may be selected from auxins, cytokinins, ethylene, gibberellins, brassinosteroids, abscisic acid or other phytohormones. For example a growth regulator may be selected from 1-naphthalenacetic acid (NAA), 2,4-D, 3-indoleacetic acid (IAA), indolebutanoic acid (I BA), dicamba, picloram, gibberellic acid, 6-benzyl aminopurine (BAP), benzyl adenine (BA), 2-iP, kinetin, zeatin, dihydrozeatin, thidiazuron (TDZ), metatopolin, ethylene, florigen, abscisic acid (ABA), brassinosteroids (BR), jasmonic acid (JA), salicylic acid (SA), polyamines, strigolactones (SL) and nitric oxide (NO).

In certain examples, the PGR is gibberellic acid and/or DL-carnitine.

Suitably a peptide may be selected from a plant defence peptide/protein(s), regulatory protein(s), for example regulatory proteins suitably to modulate plant developmental and physiological processes, transcription factor(s), flowering related protein(s), and the like.

Suitably a viral vector may be selected from RNA virus vector based on, for example Potato virus X (PVX), Tobacco rattle virus (TRV), Barley stripe mosaic virus (BSMV) and Cucumber mosaic virus (CMV) vectors, which are able to rapidly induce sequence-specific gene silencing through targeting the coding sequence or the promoter/regulatory sequences of a gene(s).

In certain examples, a volatile organic compound (VOC) is a plant VOC, a fungal VOC, a microbial VOC, combinations of plant VOCs, combinations of fungal VOCs or combinations of microbial VOCs, or combinations of at least two of a plant VOC, a fungal VOC and a microbial VOC.

Suitably a VOC may be selected from small molecules with low boiling point and high vapour pressure, and may be an organic compound, suitably a synthetic organic compound selected from hydrocarbons, terpenes, alcohols, carboxylic acids and esters, ketones, or aromatics. Suitably the VOC may be synthetically produced or a natural product.

Volatile organic compounds (VOCs) include numerous signalling molecules involved in plant-microbial interactions (Junker, R. R., and Tholl, D. (2013). Volatile organic compound mediated interactions at the plant-microbe interface. J. Chem. Ecol. 39, 810- 825, Schulz-Bohm, K., Martin-Sanchez, L., and Garbeva, P. (2017). Microbial Volatiles: small molecules with an important role in intra- and inter-kingdom interactions. Front. Microbiol. 8:2484).

To date, a few thousand VOCs have been described in flowering plants (Knudsen, J. T., Eriksson, R., Gershenzon, J., and Stahl, B. (2006). Diversity and distribution of floral scent. Bot. Rev. 72, 1-120) and microbes (Lemfack, M. C., Gohlke, B.-O., Toguem, S. M. T., Preissner, S., Piechulla, B., and Preissner, R. (2018). mVOC 2.0: a database of microbial volatiles. Nucleic Acids Res. 46, D1261-D1265). These VOCs predominantly include terpenoids, phenylpropanoids/benzenoids, fatty acids, and amino acid derivatives (Dudareva, N., Klempien, A., Muhlemann, J. K., and Kaplan, I. (2013). Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol. 198, 16-32). Suitably the present invention may utilise such a plant VOC.

Suitably a plant VOC may be selected from p-caryophyllene, Ethylbenzene, D-Limonene, Cosmene, Cosmene (isomer) ,o-cymene, Methyl-heptenone, (z)-3-hexen-1-ol, Amyl ethyl carbinol, p-cymenene, Amyl vinyl carbinol, Furfurala-ionene, Dihydroedulan II, Dihydroedulan II, p-linalool, (R)-(+)-menthofuran, 5-methylfurfural, a-ionone, Hotrienol, trans-p-metha-2,8-dienol, Safranal, 3-furanmethanol, Tetramethyl-indane, Ethyl cyclopentenolone, p-menthen-1-ol, 4,7-dibenzofuran, Menthone, Camphor, 2-piperidin methenamine, 1-(1-butenyl)pyrrolidine, Methyl salicylate, trans-geraniol, Teresantalol, p- damascenone, 5-isoproprenyl-2-methylcyclopent-1-enecarboxaldehyde, Calamenene, Piperitenone, p-cymen-8-ol, Exo-2-hydroxy cineole, 3,6-dimethyl-phenyl-1,4-diol, Longipinene Isopiperitenone, Damascenone (isomer), Mint lactone, a,p-dihydro-p- ionone, Seudenone, Dihydroxy-durene, Cinerolon, Carvone, 1-acetoxy-p-menth-3-one, 2,6-diisopropyl naphthalene, (naphtalene derivative), Eugenol, 4-ethylphenol, Thymol, 2- acetyl-4-methylphenol, Carvacrol.

Suitably a fungal VOC may be selected from the Fusarium genus or Trichoderma. Saprophytic fungi, for example Cladosporium and Ampelomyces species (Kaddes A., Fauconnier M. L., Sassi K., Nasraoui B., Jijakli M. H. Endophytic fungal volatile compounds as solution for sustainable agriculture. Molecules. 2019; 24:1065, Morath S. II., Hung R., Bennett J. W. Fungal volatile organic compounds: a review with emphasis on their biotechnological potential. Fungal Biol. Rev. 2012; 26:73-83). Suitably a VOC may be selected from N-1 -naphthylphthalamic acid (NPA). Suitably a VOC or multiple VOCs may be provided by said C. sphaerospermum selected from at least one of C. sphaerospermum Accession No. NRRL 67603, C. sphaerospermum Accession No.NRRL 8131, and C. sphaerospermum Accession No. NRRL 67749.

Suitably a VOC may be selected from y-patchoulene, 3-methyl butanol, 1-octen 3-ol, 2- undecanone, 3-methylbutanoate, 2-methylbutan-1-ol, 4-methyl-2-heptanone, ethanethioic acid, 2-methyl propanal, ethenyl acetate, 3-methyl 2-pentanoene, methyl 2- methylbutanoate, methyl 3-methylbutanoate, 4-methyl 3-penten-2-one, 3-methyl 2- heptanone, myrcene, terpinene, methyl salicylate, 2-pentadecanone, 1 H-pyrrole, ethyl butanoate, chlorobenzene, dimethylsulfone, 2-octanone, 5-dodecanone, 3-methyl-2- pentanone, geosmin, 1 -pentanol, 2-methyl-1 -propanol, dimethyl 2-octanol, disulfide, acetophenone, 2-isobutyl-3-methoxypyrazine, 2-heptanone, 5-methyl-3-heptanone, 2- methyl-2-butanol, 2-pentanol, 3-octanol, ethanol, anisole, 2-isopropyl-3- methoxypyrazine, hexanol, 2-methylfuran, 3-methyl-1 -butanol, 2-pentanone, 3-octanone, 2-ethyl-1 -hexanol, 1 -butanol, isopropanol, 2-hexanone, 3-methylfuran, 3-methyl-2- butanol, 2-pentylfuran, 1-octen-3-ol, 2-ethylfuran, 2-butanone, isopropyl, 3-hexanone, acetate, isobutyrate, 2-methylisoborneol, isovaleraldehyde, a-terpineol, 2-nonanone, ethylfuran, 2r,3r-butanediol, 2-methyl-1 -butanol, citric acid, 1 -octanol, a Nod factor, a flavonoid, a strigalactone, or any combination or derivative thereof.

Suitably a VOC(s) can be injected into a gas flow for incorporation into a nanobubble generated with an electric field or the VOC(s) can be provided in combination with or in solution with a nanobubble generated with an electric field.

Suitably a compound capable of inducing a change in the phenotype, chemistry, or physiology of a plant may enter into the nanobubble as the liquid solution containing the compound(s) passes through a nanobubble generator with an electric field. Suitably a compound capable of inducing a change in the phenotype, chemistry, or physiology of a plant may bind to the surface of a nanobubble in the presence of an electric field.

In methods of the invention which include a plant pre-treatment step, a plant root can be prepared to allow greater uptake of the gas or gases in the nanobubbles generated with an electric field.

Suitably a root portion can be cleaned to allow, promote, enhance or improve uptake.

Suitably a root portion may be pre-oxygenated before the mixture of nanobubbles generated with an electric field with one or more compound discussed herein, for example, a nucleic acid or VOC, is applied.

Suitably a pre-treatment step can comprise incubating rooted shoots in a nanobubble water (or other suitable liquid medium) generated with or in the presence of an electric field formed using a gas for example comprising or consisting of air, oxygen, carbon dioxide, or another suitable gas or combinations thereof, suitably oxygen nanobubble water/ liquid medium.

Suitably, pre-treatment may be provided for at least one minute, at least one hour, at least one day, at least two days, at least one week, at least one month, or several months prior to treatment with the mixture comprising a compound(s).

As will be appreciated, pre-treatment may be suitably applied in view of the size, health, growth stage or other condition of the root zone. It is considered a suitable pre-treatment step may lead to improved uptake of a compound or compounds.

Suitably the methods of the invention can be undertaken in real time, to allow uptake of one or more compounds discussed herein, for example a nucleic acid or VOC, to be provided at any time in the life of the plant.

Suitably, the combination of nanobubble water/liquid medium generated with or in the presence of an electric field, containing at least one gas and a compound or compounds to alter gene expression can be carried out at any time in the plant’s life cycle to effect changes in real time.

Suitably the uptake of the compound may also be monitored in real time to allow control of delivery of the nanobubbles generated with or in the presence of an electric field and compound mixture.

Suitably the nanobubbles generated with or in the presence of an electric field and compound mixture may be provided to the plant via a standard dripper to the root of the plant, for example delivery of the nanobubbles and compound mixture by standard watering or irrigation systems.

Suitably delivery may be to soil, aquaponics systems, standard plant growing media, coco coir, coir, coco peat, compost, standard plant tissue growing substrates or media, or other non-soil substrates.

Suitably the substance may provide covalent modifications of DNA and/or histones, affecting transcriptional activity of chromatin without changing DNA sequence, may induce DNA methylation, RNA methylation, histone methylation or histone acetylation. For example siRNA can induce DNA methylation. The substance can induce transient changes which could last a short time (hours, days, or weeks), or could last the lifetime of the plant.

Suitably a substance may induce DNA methylation, RNA methylation, histone methylation or histone acetylation in one or more flowering loci.

Suitably the substance may be selected from a volatile organic compound (VOC), siRNA, other RNAs, antisense oligonucleotides, plant growth regulators, peptides, RNAi vectors, expression vectors and/or viral vectors.

The mixture of nanobubbles generated with or in the presence of an electric field and compounds can be used along with transgenes, gene editing vectors, RNAi vectors, expression vectors or viral vectors to enhance uptake into recalcitrant plant cells via the roots or other germline tissues. Suitably viral vectors may comprise nucleic acids for gene silencing or to enhance gene expression, for example transient gene expression via exogenous nucleic acids, for example exogenous genes which may be expressed to provide a product of interest.

Suitably the mixture may further comprise one or more further constituents such as surfactants, chelating agents, emulsifiers, antioxidants, reductants, pH buffers and osmotic buffers.

Suitably, the compound may be a substance that induces DNA methylation, RNA methylation, histone methylation or histone acetylation to provide a heritable change.

Suitably a compound may induce DNA methylation, RNA methylation, histone methylation or histone acetylation in one or more flowering loci.

Suitably the compound may be selected from a volatile organic compound(s) (VOC(s)), siRNA, other RNAs, antisense oligonucleotides, plant growth regulators, peptide, RNAi vectors, expression vectors and/or viral vectors.

Suitably the nanobubbles generated with or in the presence of an electric field may be generated using one or a mixture of gases. For example the gas may be selected from the group comprising or consisting of air, oxygen, carbon dioxide, nitrogen, hydrogen, ethylene, ethylene oxide and combinations thereof.

Suitably the nanobubbles generated with or in the presence of an electric field may be generated in the presence of oxygen to provide an oxygen-enriched liquid, which may then be applied to plant roots.

Suitably at least 50% of the nanobubbles generated have a diameter of less than 300 nm, suitably less than 80 nm, optionally 20 nm or less.

Suitably a nanobubble may have a mean diameter less than 500 nm, or less than 200 nm, or ranging from about 0.5 nm to about 500 nm; or from about 20 nm to about 200 nm). Suitably a nanobubble mixture may be provided, for example a nanobubble with a bubble diameter of 20 nm - 10 pm.

Most conventionally-formed bubbles in a liquid easily float to the water surface, burst and the gas contained in the bubble merges with the atmosphere above the liquid. In contrast, nanobubbles may be only slightly affected by buoyancy and exist as they are in the liquid for a longer period of time. Suitably a nanobubble as used in the present invention may have a lifetime of at least one hour, at least 1 day, at least 1 week, at least 1 month or at least 1 year under ambient pressure and temperature.

Suitably the nanobubbles may be positively or negatively-charged nanobubbles. For example the nanobubbles may have a zeta potential of 10 mV to 200 mV, or -10 mV to - 200 mV. Suitably the nanobubbles may have a zeta potential of 1 mV to 150 mV, or -1 mV to -150 mV. Suitably stability of the nanobubbles may be provided due to negatively charged areas of the nanobubble. Suitably pH may be used to generate charged nanobubbles. Suitably electrical fields may be used to provide and/or change the zeta potential of nanobubbles.

Suitably a concentration of nanobubbles in a liquid carrier may be at least 10E+05 bubbles per ml, for example as determined using a Zetasizer (Zetasizer Ultra) or other suitable apparatus.

Suitably, the plant application medium is provided to a plant for an application period of at least 1 hour, at least 4 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 7 days, at least 10 days, at least 14 days, at least 20 days, or over the lifetime of the plant, optionally over the cultivation duration of the plant.

Suitably, the plant application medium is provided to a plant for at least 1 hour each day, at least 4 hours each day, at least 12 hours each day, or continuously each day over the application period.

Suitably, the plant application medium is provided to a plant at less than 1 hour postgermination, at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 hours post-germination, or at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days post-germination, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months post-germination, or at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years post-germination. Germination is considered to occur with the emergence of the root and cotyledonary leaves.

Suitably as discussed herein, a plant may be considered leaf plants, fruit plants, grains, algae, or mosses.

Suitably a plant may be a seed or another plant part, such as a leaf or leaf section, a piece of stem, pollen, anther, embryo, or any other stem cells of the plant from which new plants can be grown.

Suitably a plant tissue (explant) may be incubated in liquid media or on solid media containing nanobubbles made with electric field and compounds to enhance uptake of transformation vectors, etc. into recalcitrant plant species.

Suitably, the plants used may be selected from the group comprising higher or vascular plants adapted to synthesise metabolites in a large quantity or any plant in symbiosis with arbuscular mycorrhiza fungi. Suitably a plant may include Cannabis, hemp, maize/corn, soybean, rice, wheat, potato, sugarcane, tomato, lettuce, microgreens, cabbage, barley, tobacco, pepper, sorghum, cotton, sugar beets, or any other legumes, fruits, nuts, vegetables, pulses, flowers, or other commercial crop not inconsistent with the objectives of this disclosure.

Suitably, a plant may be selected from, without limitation, energy crop plants, plants that are used in agriculture for production of food, fruit, wine, biofuels, fibre, oil, animal feed, plants used in the horticulture, floriculture, landscaping and ornamental industries, and plants used in industrial settings.

Suitably a plant may comprise gymnosperms (non-flowering) or angiosperms (flowering). If an angiosperm, the plant can be a monocotyledon or dicotyledon. Non-limiting examples of plants that could be used include desert plants, desert perennials, legumes, such as Medicago sativa (alfalfa), Lotus japonicas and other species of Lotus, Melilotus alba (sweet clover), Pisum sativum (pea) and other species of Pisum, Vigna unguiculata (cowpea), Mimosa pudica, Lupinus succulentus (lupine), Macroptilium atropurpureum (siratro), Medicago truncatula, Onobrychis, Vigna, and Trifolium repens (white clover), corn (maize), pepper, tomato, Cucumis (cucumber, muskmelon, etc.), watermelon, Fragaria, other berries, Cucurbita (squash, pumpkin, etc.) lettuces, Daucus (carrots), Brassica, Sinapis, Raphanus, rhubarb, sorghum, miscanthus, sugarcane, poplar, spruce, pine, Triticum (wheat), Secale (rye), Oryza (rice), Glycine (soy), cotton, barley, tobacco, potato, bamboo, rape, sugar beet, sunflower, peach (Prunus spp.) willow, guayule, eucalyptus, Amorphophallus spp., Amorphophallus konjac, giant reed (Arundo donax), reed canarygrass (Phalaris arundinacea), Miscanthus giganteus, Miscanthus sp., sericea lespedeza (Lespedeza cuneata), millet, ryegrass (Lolium multiflorum, Lolium sp.), Phleum pratense, Kochia (Kochia scoparia), forage soybeans, Cannabis, hemp, kenaf, Paspalum notatum (bahiagrass), Bermuda grass, Pangola-grass, fescue (Festuca sp.), Dactylis sp., Brachypodium distachyon, smooth bromegrass, orchard grass, Kentucky bluegrass, turf grass, Rosa, Vitis, Juglans, Trigonella, Citrus, Linum, Geranium, Manihot, Arabidopsis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Browaalia, Phaseolus, Avena, Hordeum, and Allium.

Suitably the phenotype, chemistry, or physiology of a plant is altered to enhance the production of a plant-based pharmaceutical and/or industrial product, medicinal and non- medicinal health-related or recreational product, neutraceutical or other functional food product, cosmetical compound, additive, bioceutical, or agricultural product provided by the plant or components used in these fields.

Suitably, engineering the expression of a secretory pathway or parts thereof in plants enables the production of molecules for example biologies that would otherwise accumulate at low levels or in an improperly processed form.

Suitably an enhanced product may be, but is not limited to, a phytohormone, a flavonoid, in particular chaicones, flavones, flavonols, flavandiols, anthocyanins, and proanthocyanidins, condensed tannins or aurones.

Suitably an enhanced product may be a sugar substitute, for example steviol glycosides. For example the enhanced product may comprise or consist of Stevioside, Rebaudioside A, Rebaudioside C, Dulcoside A, Rebaudioside B, Rebaudioside D and/or Rebaudioside E. Suitably an enhanced product may be a plant-derived pharmaceutical, for example a cardiotonic Acetyldigoxin, Adoniside, Convallatoxin, Deslanoside, Digitalin, Digitoxin, Digoxin, Etoposide, Gitalin, Lanatosides A, B, C, Ouabain.

Suitably, the enhanced product may be an anti-inflammatory, for example Aescin.

Suitably, the product may be an anticholinergic - Anisodamine, Anisodine, Atropine, Hyoscyamine. Suitably, the product may be an anti-cancer - Betulinic acid, Camptothecin, Colchicine amide, Colchicine, Demecolcine, Irinotecan, Lapachol, Monocrotaline, or Taxol.

Suitably, the plant product may be selected from Aesculetin, Agrimophol, Ajmalicine, Allantoin, Allyl isothiocyanate, Anabesine, Andrographolide, Arecoline, Asiaticoside, Benzyl benzoate, Berberine, Bergenin, Borneol, Bromelain, caffeine, Camphor, (+)- Catechin, Chymopapain, Cissampeline, Cocaine, Codeine, Curcumin, Cynarin, Danthron, Deserpidine, L-Dopa, Emetine, Ephedrine, Galanthamine, Glaucarubin, Glaucine, Glasiovine, Glycyrrhizin, Gossypol, Hemsleyadin, Hesperidin, Hydrastine, Kaibic acid, Kawain, Kheltin, Morphine, Papavarine, Pilocarpine, Sanguinarine, Scopolamine, Silymarin.

Suitably the product may be a plant-derived cancer drug, for example vinca alkaloids (vinblastine, vincristine and vindesine), epipodophyllotoxins (etoposide and teniposide), taxanes (paclitaxel and docetaxel) or camptothecin derivatives (camptotecin and irinotecan).

Suitably an enhanced product may be a compound naturally formed by a plant, such as cannabis. In particular, one or more substances may be supplied to the plant as described herein to modulate a single or groups of metabolic pathways. This can modify the profile of compounds normally expressed to create a platform for cannabis that provides a route to commercial-scale quantities of the common component cannabidiol and to lesser compounds such as methyl- (Ci), butyl- (C4) and other C_n alkyl cannabinoids.

Suitably modulation of latent biosynthetic pathways in the plant can be utilised to create new pharma based on the aforementioned cannabis molecules but with chemistries altered through glycosylation, o-alkylation, esterification, acetylation, terpene addition and ionisation through addition of inorganic moieties (phosphate, sulphate, nitrate and ammonium).

Suitably an enhanced product may be a colourant. For example, many plants, e.g. Empetrum nigrum, and Isatis tinctoria and Crocus sativus, produce colours used in food, textile, hair dyes etc. Using the processes as described herein, the diversity and proportion of compounds provided by a plant can be modulated to create sustainable colourant feedstocks (crops) with specific (visible spectrum) and reproducible colours. Furthermore, triggering of latent pathways may be utilised to alter the chemistries of the colourants thereby expanding their utility through alkylation, specific oxidation/reduction, glycosylation to provide functional differences such pH stability, photodegradation, water/oil solubility etc.

Suitably an enhanced product may be a functional molecule, for example, a surfactant. Surfactants are organic compounds used to mix two immiscible substances, such as oil and water. They are used in many industries worldwide, most notably those of cosmetic, healthcare, and food and drink. A significant fraction of the market demand for surfactants is met by organo-chemical synthesis using petrochemicals as precursors. The methods disclosed herein may be utilised with seed crops (such as oilseed rape (or other Brassicaceae)), to enhance yield of galactolipids, known as sustainable emulsifiers. Nanobubble/compound application enables new emulsifier/surfactant chemical variants (oligogalactolipids) and an increase in yield, particularly of lesser known/modified galactolipids. Alternatively, emulsifier specific activity, stability/durability, functional pH range etc. may be altered by altering the pathways within the plant utilising the methods disclosed herein.

Suitably an enhanced product may be a functional food molecule, for example egg replacer, such as egg albumin replacer. Such a functional food molecule may act as an emulsifier, clarifier, textural modification, binder, nutritional component, stabiliser, glazing agent etc. Egg albumin is a member of the serpin super family of protease inhibitors. Serpins existing in many plants such as barley where Protein Z is abundant in the grain and the methods of the present invention can be utilised to increase the yield of such serpins. Suitably the phenotype or physiology of a plant may be altered to enhance a structural growth characteristic of the plant. For example a structural growth characteristic may be selected from growth rate, biomass weight (whole plant, aerial portion of plant, root, tuber), plant height, number of branches, branch thickness, branch length, branch weight, number of leaves, leaf size, leaf weight, leaf thickness, leaf expansion rate, petiole size, petiole diameter, petiole thickness, stem thickness, trunk thickness (caliper), stem length, trunk length, stem weight, trunk weight, canopy/branching architecture, root biomass, root extension, root depth, root weight, root diameter, root robustness, root anchorage, or root architecture.

Suitably the phenotype or physiology of a plant may be altered to enhance a growth characteristic of the plant in response to environmental conditions for example selected from abiotic stress tolerance such as cold, heat, salinity and/or drought), or in response to biotic stress from for example microbial or fungal attack or infestation or predation.

Suitably the phenotype or physiology of a plant may be altered to enhance a growth characteristic of the plant selected from: anthocyanin pigment production, anthocyanin pigment accumulation, plant oil quality and quantity, secondary metabolite accumulation, sensory and flavour compound production, content of phytopharmaceutical or phytochemical compounds, protein content, fibre hypertrophy and quality, quantity of chlorophyll, photosynthesis rate, photosynthesis efficiency, leaf senescence retardation rate, early and efficient fruit set, early fruit maturation, fruit yield, yield of vegetative parts, root and tubers, fruit/grain and/or seeds, size of fruit, grain and/or seeds, firmness of fruit, grain and/or seeds, weight of fruit, grain and/or seeds, starch content of vegetative parts, root and tuber, fruit, grain, and/or seeds, sugar content of fruit, grain and/or seeds, content of organic acids in fruit and seeds, early flowering (flowering precocity), or harvest duration.

Suitably the phenotype or physiology of a plant is altered to enhance a combination of growth characteristics of the plant.

Suitably the plant may be grown on or in soil-less culture on a porous support, by continuous soaking in the nutrient solution, by temporary immersion in said nutrient solution (sub-irrigation, hydroponics, nutrient film, etc.), by use of a standard dripper, or by contacting with a nutrient solution in the form of a mist or fog. Suitably, in the processes of the invention, the mixture is applied to an organ of the plant.

Optionally, the organ is the plant root system. Suitably, the mixture is applied to the plant root system via a liquid plant growth medium.

Nanobubbles generated using an electric field as discussed herein are considered to provide several unique physical and mechanical characteristics. For example they provide longevity, which provides a highly useful extension of shelf-life and storage life for a crop; virtual disappearance of buoyancy; high internal pressure; extremely large surface/volume ratio; and a high oxygen dissolution rate.

Suitably VOCs may be injected into the air/gas channel going into the nanobubble generator to provide these in combination with the nanobubble. Suitably, other compounds may be introduced into the nanobubble water/liquid carrier before or after generation of nanobubbles with an electric field to provide such compounds in the methods of the invention.

Suitably a compound may be delivered into liquid. This liquid may be in contact with an electric field to generate nanobubbles. At that stage the compounds may be taken up inside the nanobubble or attached to its surface. Suitably, a compound is pre-mixed with a carrier and nanobubbles are generated within the carrier, such that, as the nanobubbles form, the compound is adsorbed onto the surfaces of the nanobubbles.

Suitably nanobubbles may be provided under sterile conditions. The apparatus (gas supply, recirculating liquid culture media, water, sugar solution or other liquid medium and electric field nanobubble generator) may be housed in, and the processes carried out in, a laminar flow cabinet. Suitably liquid or solid media containing nanobubbles generated by an electric field are produced for plant tissue culture.

Suitably solid media may include pre-culture media, co-cultivation media, callus induction media, shoot regeneration media, rooting media or tissue culture media for micropropagation of plants.

In certain examples, the system is a sterile and/or automated system. In the context of the present application, a gene editing component includes any component, reagent or material, or combination of components, reagents or materials, that can be delivered to a plant to induce transformation, such as: CRISPR reagents or constructs, gene editing cassettes in bacterial, viral and free DNA forms, Transcription activator-like effector nucleases (TALENs), Cas proteins, Cas mRNA, guide RNA and any other extensions and helpers like peptides and nanoparticles of any kind.

In the context of the present invention, a carrier medium is an agriculturally-acceptable solvent. In most examples, the carrier medium is an aqueous medium, which may be water or water containing additional components, such as nutrients, gelling agents, and/or agriculturally-acceptable excipients, such as preservatives, anti-freeze, adjuvants, surfactants, wetting agents, buffers, anti-foams and other formulation components. The carrier medium will, in certain examples, be fluid and used as a fluid. In other examples, for example an agar gel carrier medium, the carrier medium may be gelled in use.

In the context of the present invention, the term nanobubble water refers to a combination of nanobubbles and water as a carrier for the nanobubbles. Except where otherwise indicated, nanobubble water is generated by generating nanobubbles of the specified gas in water and is substantially free of other components. In certain examples, the nanobubbles are generated by application of an electric field or in the presence of an electric field.

In the context of the present invention, an application medium is a medium including nanobubbles as described above, a carrier medium as described above and at least one functional component as described above that can be applied to a plant or explant.

In the context of the present invention, an explant means a plant tissue that has been removed from a plant, such as an immature embryo, mature embryo, plant cell, callus, shoot or root meristem, microspore, pollen, anther, petiole, hypocotyl, cotyledon, protoplast, stem, internode, node, leaf, leaf pieces or root.

In the context of the present invention, a plant or crop protection product refers to any agriculturally-acceptable (including horticulturally-acceptable) chemical or biological treatment for plants to improve pest resistance. Such products include pesticides, such as herbicides, insecticides, nematicides, plant growth regulator herbicides, fungicides and acaricides. The term as used herein encompasses both the active ingredient and formulated compositions comprising the active ingredient and agriculturally-acceptable adjuvants, diluents and so on.

In the context of the present invention, a biostimulant refers to a substance or microorganism (or mixtures thereof) that stimulate natural plant processes, including efficiency of nutrient use, desired quality traits and tolerance to environmental stress.

In the context of the present invention, a transformation vector is an entity, such as a plasmid or viral vector, which is used to carry a nucleic acid sequence into a cell.

The above and other aspects of the present invention will now be described in further detail, by way of example only, with reference to following examples and the accompanying figures, in which:

Figure 1 illustrates biomass in cannabis and lettuce plants with electric field-generated nanobubbles (EFNBs);

Figure 2 illustrates biomass in lettuce plants grown in mechanically-generated oxygen nanobubble (ONB) water and with electric field-generated nanobubble (EFNB) water with 50% less fertiliser;

Figure 3 illustrates biomass in lettuce shoots with CO2 fogging to leaves;

Figure 4 illustrates average shoot biomass in basil plants grown with dilutions of volatile compound (2,3-BDO) in mechanical-generated oxygen nanobubble (ONB) water or electric field-generated nanobubble (EFNB) water;

Figure 5 illustrates fluorescence in barley roots after 24 hr incubation with CY3-labelled oligos mixed with oxygen nanobubble (ONB) water or electric field-generated nanobubble (EFNB) water; Figure 6 illustrates fluorescence in Nicotiana benthamiana leaves after 30 hr incubation of roots with CY3-labelled oligos post- and pre-mixed before generating electric fieldgenerated nanobubbles (EFNBs);

Figure 7 illustrates texture analysis of plant tissue culture media prepared with electric field nanobubbles;

Figure 8 illustrates the effect of electric field-generated nanobubbles (EFNBs) on rooting of potato nodes and apical shoots;

Figure 9 illustrates the effect of mechanical-generated nanobubbles (NBs) and electric field nanobubbles (EFNBs) on Agrobacterium-based transient expression in potato;

Figure 10 illustrates the effect of mechanical-generated nanobubbles (NBs) and electric field nanobubbles (EFNBs) on Agrobacterium-based transient expression in barley immature embryos;

Figure 11 illustrates the effect of electric field-generated nanobubbles (EFNBs) on seed priming of beetroot;

Figure 12 illustrates the effect of mechanical-generated nanobubbles (NBs) and electric field nanobubbles (EFNBs) on CRISPR/Cas9-based gene editing efficiency in seedlings of Nicotiana tabacum transgenic line N1 DC4; and

Figure 13 is a schematic diagram of a system of generation and use of nanobubbles.

DETAILED DESCRIPTION

Electric field nanobubble (EFNB) generator apparatus

Suitable apparatus for generating nanobubbles in the presence of an electric field is described, for example, in WO 2020/079020, to which further reference should be made. In broad terms, the apparatus includes a vessel containing a first liquid and having means for supplying a gas or liquid medium, from which the nanobubbles will be formed, to the vessel and means for distributing the medium within the first liquid. An electric field is generated in the proximity of the vessel using an electrode, where the electrode and the first liquid are not in direct electrical contact. The prevention of direct electrical contact prevents electrolysis occurring within the vessel but facilitates the generation of nanobubbles of the medium within the first liquid.

In preferred examples, nanobubbles were generated using a nanobubble generator apparatus constructed in accordance with Figure 5 of WO 2020/079020.

In mechanical nanobubble generation, relatively large bubbles are generated and then subjected to a process which ‘explodes’ the large bubbles into a plurality of smaller bubbles. In contrast, in electric field nanobubble generation, gas dissolved in the liquid is caused to form bubbles which grow in size under the influence of the electric field.

The gas medium and flow rates are given below in respect of individual examples.

It will be appreciated by those skilled in the art that modifications can be made to this arrangement to suit particular uses of the apparatus.

Generation of nanobubbles showing inputs and outputs (Figure 13).

Figure 13 shows an example of a nanobubble generation arrangement in accordance with the present invention that can be used with either a mechanical or electric field nanobubble generator.

The main inputs to a nanobubble generator are gas (air, oxygen, carbon dioxide, nitrogen, etc.) and water (or other liquid solution). The generation of mechanical oxygen nanobubbles (ONBs) requires input of oxygen. The inventors have determined that electric field-generated nanobubbles (EFNBs) do not require a gas line. It has been determined that atmospheric air can be used successfully as the medium for EFNBs in the context of the present invention.

As determined by the inventors, compounds can be added (i) into the gas flow (e.g., VOCs, PGRs/hormones) and/or (ii) into the water (e.g., DNA, RNA, proteins, pesticides, nutrients, hormones) as a pre-mixing step. Following EFNB generation, the output solution (nanobubble water with a variety of compounds) can be used as a treatment to the roots or leaves (as a foliar spray) of a plant.

Alternatively, EFNB water can be generated with a gas and aqueous solution only and the compounds or substances for delivering to the plant cells, can be added afterwards, as a post-mix, before application to the plant.

Examples

Example 1:

Biomass in cannabis and lettuce plants is increased when using Electric Field Nanobubbles (EFNBs) compared to other treatments in hydroponics (Figure 1).

Small, freshly rooted cannabis cuttings or newly germinated lettuce seedlings were introduced into hydroponics and grown with four different watering treatments on day 1. The treatments contained 1. EFNB water made with a single pass through an electric field nanobubble generator using atmospheric air as gas supply and 9L/min water flow; 2. Mechanical oxygen nanobubble (ONB) water made with oxygen at 1.5 bar pressure and 12L/min water flow; 3. Oxygenated water made with a portable oxygen generator running constantly at 92% (no nanobubbles); 4. Tap water (no nanobubbles). After 27 days shoot and root biomass were harvested and weighed. All 3 plants for each treatment were weighed together to give total biomass for shoot or root material. For both crops the weight of shoots or roots was greatest when using the EFNB treatment.

In particular, the results of this example demonstrate improved growth can be obtained using electric field-generated nanobubbles generated with air rather than requiring a pure oxygen supply, with associated higher supply costs and handling precautions, even at a lower flow rate.

Example 2:

Increased biomass in lettuce plants grown in Electric Field Nanobubble (EFNB) water with 50% less fertiliser (Figure 2).

Lettuce seeds were germinated and 5-day old uniform seedlings with fully expanded cotyledons were selected and introduced into different hydroponic systems. Eight or nine plants were used per treatment. Fresh water was prepared every 2 days to top up the buckets in hydroponic system. The different waters contained: 1. Tap water control (no nanobubbles); 2. Mechanically generated ONBs recirculating with oxygen at 1.5 bar gas pressure and 12L/min water flow; 3. EFNBs made with a single pass through an electric field nanobubble generator using an air pump as gas supply and 9L/min water flow. Fertiliser (Canna Coco A+B) was diluted to give 50% of the manufacturer’s recommended dosage of 4 ml/L each. After 19 days plants were harvested and shoot biomass measured in each plant. The highest biomass was found in the plants grown with EFNB water.

This example confirmed the observations in Example 1, that improved growth can be obtained using electric field-generated nanobubbles generated with air rather than requiring a pure oxygen supply (as required by mechanically generated nanobubbles), and at a lower flow rate.

Example 3:

Increased biomass in lettuce shoots grown in Electric Field Nanobubble (EFNB) water with additional CO2 fogging to leaves (Figure 3).

Lettuce seeds were germinated and 5-day old uniform seedlings with fully expanded cotyledons were selected and grown in hydroponic systems with EFNBs for 22 days. Half of the plants were additionally treated with fog to the shoots. The fog was generated by passing CO2 from a gas cylinder through an electric field generator to make EFNBs with CO2. Water in hydroponics was topped up every 2 days with freshly prepared water. There were 10 plants per treatment. After 22 days the shoots were harvested, and biomass weighed. The average biomass was greater with additional CO2 fog to the leaves supplementing EFNB hydroponic treatment.

Example 4:

Shoot biomass in basil plants grown with dilutions of a volatile compound (2,3- Butanediol) and nanobubbles (Figure 4).

A dilution series of a volatile compound, 2,3-Butanediol (2,3-BDO) purchased from Sigma Aldrich, UK was mixed with water before generating mechanical oxygen nanobubbles (ONBs) or electric field nanobubbles (EFNBs). Dilutions from 1:5000 up to 1 : 1000000000 were used to grow basil seedlings for 28 days. The delivery of 2,3-BDO was improved with EFNBs (highest shoot biomass at 1:100000, white stars) compared to ONBs (highest shoot biomass at 1:50000, white stars).

All plants treated with 2,3-BDO had improved growth, with a requirement for higher dilution when using EFNBs compared to ONBs.

Example 5:

Fluorescence in barley roots after 24 hr incubation with CY3-labelled oligos and nanobubble waters (Figure 5).

Nanobubble water was prepared with mechanical oxygen nanobubbles (ONBs) or electric field nanobubbles (EFNBs). The size and concentrations of nanobubbles were measured on a Zetasizer Ultra; ONB size 39.99 nm, concentration 1.4E+10 particles per ml and EFNB size 43.0 nm, concentration 6.0E+09 particles per ml. CY3-labelled oligos (5'-CY3-GCTATTTCATCAGGA-3’) were added to the nanobubble waters and ultrapure water (UPW) as a control, then barley seedlings roots were immersed in the different water treatments in the dark. After 24 hr, root samples were visualised under a confocal microscope at 10X magnification to compare signals from both treatments and control.

The highest signal was visualized in the roots incubated with EFNB water and CY3 oligos, indicating higher uptake with the use of EFNBs.

Example 6:

Fluorescence in Nicotiana benthamiana leaves after 30 hr incubation of roots with CY3-labelled oligos post- and pre-mixed with Electric Field Nanobubbles (Figure 6).

Ultrapure water was used to generated nanobubbles averaging 4.6 nm at a concentration of 3.5E+14 particles per ml. 1 uM CY3-labelled oligos were added to EFNB water and used to incubate Nicotiana benthamiana plant roots. Ultrapure water was also pre-mixed with CY3-labelled oligos before generating EFNBs. The combined solution measured 80 nm particle size with a concentration of 3.3E+6 particles per ml. As with Example 5, fluorescence was higher in leaves using electric field-generated nanobubbles. Furthermore, fluorescence was significantly higher in the example in which the solution is pre-mixed prior to making EFNBs, indicating an even higher uptake. We conclude, without wishing to be bound by any theory, that compounds adsorb or otherwise attach to the electric field-generated nanobubbles as they grow under the influence of the electric field.

Example 7:

Texture analysis of plant tissue culture media prepared with electric field nanobubbles (Figure 7).

Liquid MS30 was prepared, then run through a mechanical nanobubble generator to make oxygen nanobubbles (ONBs) or an electric field generator to make electric field nanobubbles (EFNBs). Melted agar was added to the liquid media to a final concentration of 0.8% and 100 ml was poured into 3 Magenta containers per sample. Agar texture was measured using a CT3 Texture Analyser (AMETEK Brookfield) with TA7 Knife Edge clear acrylic probe (8 g; 60 mm wide) in compression mode. Figure 7 shows maximum force required to pierce surface of agar made with ONBs or EFNBs compared with no nanobubbles control.

Agar gelling is reduced in the presence of both oxygen nanobubbles and EFNBs.

Example 8: i) Effect of electric field-generated nanobubbles (EFNBs) on rooting of potato nodes and apical shoots (Figure 8).

4X MS30 solid media was prepared and mixed 1 :3 with electric field nanobubble (EFNB) water or ultrapure water (NoNB). Nodes and shoots from tissue culture potato cv. Desiree were cut and placed on media for 8 days before rooting was scored. Rooted plantlets were photographed on Day 11.

A higher percentage of rooting was present on both nodes and shoots incubated on media containing EFNBs. ii) Effect of nanobubbles (NBs) on Agrobacterium-based transient expression in potato (Figure 9).

A T-DNA construct constitutively expressing the reporter b-glucuronidase (GUS) and selectable neomycin phosphotransferase II (NPTII) marker genes was introduced into Agrobacterium AGL1 for transient transformation of potato cv. Desiree. To prepare media, mechanical oxygen nanobubble (ONB) or electric field nanobubble (EFNB) water was added to MS40 at 3:1 ratio.

Four-week-old potato plantlets were inoculated with Agrobacterium culture with and without nanobubbles. After inoculation, the plantlets were blotted on filter paper to remove excess liquid then transferred to deep 0.5% agar plates and incubated for two days in dark at 24°C followed by a heat shock treatment at 37°C for 30 mins. The plantlets were kept an additional two days under 16 h light and 8 h dark photoperiod. GUS histochemical staining was used to detect transient expression spots and score their total number.

Transient expression was shown to be higher in the presence of oxygen nanobubbles, and considerably higher with EFNBs.

Example 9:

Effect of nanobubbles (NBs) on Agrobacterium-based transient expression in barley immature embryos (Figure 10).

A T-DNA construct constitutively expressing the reporter b-glucuronidase (GUSPIus) and selectable hygromycin B phosphotransferase (hph) marker genes (Fig 10a) was introduced into Agrobacterium AGL1 for transient transformation of barley embryos. To prepare nanobubbles, ultrapure water was run through a mechanical nanobubble generator to make oxygen nanobubbles (ONBs). Electric field nanobubbles (EFNBs) were prepared by running ultrapure water or co-cultivation medium through an electric field generator in the presence or absence of Agrobacterium and barley embryos.

Immature embryos of 14-week-old barley cv. Golden Promise were collected and inoculated with Agrobacterium with and without nanobubbles. After inoculation, the embryos (Fig 10b) were transferred on to solid co-cultivation medium and incubated for 3 days in dark at 24°C. GUS histochemical staining was used to detect transient expression spots and score their total number (Fig 10c).

Highest expression was detected when EFNBs were prepared directly in the cocultivation medium prior to adding Agrobacterium and barley embryos.

Example 10:

Effect of nanobubbles in seed priming of beetroot (Figure 11).

Beetroot seeds cv. Detroit Dark Red (Premier Seeds Direct) were soaked either in tap water or electric field nanobubbles (EFNBs), unsoaked seeds were used as additional controls. Seeds (2 seeds in one universal tube) were soaked in 30 mL universal tubes with 25 mL water treatment, at 25°C for 3h (6 changes of tap or EFNBs water every 30 minutes). Primed beetroot seeds were drained on paper towels and seeded into V 9x9x10 cm PP TEKLI Poppelmann pots (one seed per pot) with general compost mix (70% peat, 30% wood fibre, limestone, Osmocote start 12+14+24, Osmocote standard 16+9+12, H2GRO wetting agent, perlite). All plants were hand watered with tap water only.

Plants from seeds soaked in EFNBs had biggest whole plants and beetroot biomass.

Example 11:

Effect of nanobubbles on CRISPR/Cas9 based gene editing efficiency in seedlings of Nicotiana tabacum transgenic line N1DC4 (Figure 12).

The effect of mechanical oxygen nanobubbles (ONBs) and electric field nanobubbles (EFNBs) used in inoculation media with Agrobacterium was compared to determine the effect on CRISPR/Cas9-based gene editing in Nicotiana tabacum.

A T-DNA construct containing two transgenes expressing tomato-codon-optimised Cas9 (LeCas9) and a guide RNA (gRNA) to target p-Glucosidase (GUS) transgene. This construct was introduced into Agrobacterium AGL1 for delivery into plant cells.

The target gene was a non-functional p-Glucosidase (GUS) made of two defective partial GUS fragments missing the 5’ or 3’ end but sharing homology of 567 bp. This transgene was stably integrated as a single locus in a transgenic tobacco line N1DC4. Upon DNA break induction by Cas9/gRNA, homologous recombination (HR) between the two partial GUS fragments restores the functional GUS gene.

Four-week-old transgenic N1 DC4 seedlings were collected and inoculated with Agrobacterium clone with ONBs or EFNBs and without nanobubbles. After inoculation, the seedlings were transferred to deep 0.5% agar plates and incubated for two days in dark at 24°C followed by a heat shock treatment at 37°C for 30 mins. The plantlets were kept an additional two days under 16 h light and 8 h dark photoperiod. GUS histochemical staining was used to detect transient expression spots and score their total number. Strong HR was consistently detected as blue staining in the presence of Agrobacterium with ONBs or EFNBs.

Strong HR was consistently detected as blue staining in the presence of EFNBs and Agrobacterium containing CRISPR construct targeting GUS gene.

Utility of NBs in gene editing

CRISPR technology has the potential to revolutionise crop improvement. Agrobacterium- mediated transformation is the most efficient system for editing plants but the technology is genotype-dependent; it does not work in all crops, or all varieties within a crop species. However, gene constructs delivered without Agrobacterium are subject to degradation via plant nucleases.

A further challenge arises since tissue culture parameters need to be optimised for every crop/variety to enable good regeneration of gene edited shoots.

The present inventors have determined that use of nanobubbles, in particular EFNBs, in accordance with the present invention at various stages in the process of Agrobacterium- mediated transformation (AMT) produces significant advantages.

AMT comprises the steps of: (i) an optional pre-culture treatment of explant material, (ii) liquid or solid co-culture, (iii) elimination of Agrobacterium, (iv) callus induction, (v) selection, (vi) shoot regeneration, and (vii) rooting. In some crop species or varieties the callus step can be skipped and go straight to shoot regeneration. Cultivation of plant cells with Agrobacterium leads to cell death in many crop varieties. Within most crop species, very few varieties will recover and produce transformed plants after co-cultivation with Agrobacterium. The present inventors have determined that by using EFNBs in accordance with the present invention to deliver vector DNA with Agrobacterium, uptake of DNA into the plant cells is increased 20-fold. In other words, Agrobacterium delivery becomes more efficient. Furthermore, the inventors have observed that EFNBs do not have any negative impact on the rate of growth of Agrobacterium and may result in reduced cell death after introducing Agrobacterium with EFNBs in accordance with the present invention, showing the potential to transform recalcitrant crops & varieties.

The steps of callus initiation and shoot regeneration can also be inefficient in recalcitrant crop varieties; even if they survive Agrobacterium infection, they may not develop good callus or regenerate shoots. By utilising EFNBs in accordance with the present invention in solid or liquid at various stages in the process it is possible to reduce the recalcitrance, develop callus and regenerate transformed shoots.

Transformation without Agrobacterium is difficult since DNA is easily degraded by nucleases in plant cells. The present inventors have introduced small fragments of DNA using EFNBs in accordance with the present invention and have achieved efficient gene silencing. Without being bound by theory, it is thought that use of the systems, methods and media of the present invention generates nanobubbles that have a particularly thick skin, which acts to form a barrier, protecting DNA from degradation. In this way, larger genetic fragments can also be introduced to plant cells, permitting introduction of components for CRISPR gene editing into plant cells, enabling editing of plant genes without use of Agrobacterium.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in its entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.

Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.

Figures Legends

Figure 1. Increased biomass in cannabis and lettuce plants with Electric Field Nanobubbles (EFNBs).

Cannabis (Fig 1a, b) and lettuce (Fig 1 c, d) plants photographed after 15 days growing in hydroponics with different watering treatments: electric field nanobubbles (EFNBs), mechanical oxygen nanobubbles (ONB), oxygenated water (O2 water) and tap water (control). Three plants per treatment were combined to measure the total shoot biomass and total root biomass in cannabis (Fig 1e) and lettuce (Fig 1f) for each water treatment after 27 days.

Figure 2. Increased biomass in lettuce plants grown in Electric Field nanobubble water with 50% less fertiliser.

Lettuce plants (Fig 2a) photographed at harvest after 19 days in different water treatments: tap water (control), mechanical oxygen nanobubbles (ONB) and electric field nanobubbles (EFNBs) with 50% less fertiliser (2 ml/L Canna Coco A+B). Average shoot biomass (Fig 2b) was measured for each treatment, 8 or 9 plants per treatment.

Figure 3. Increased biomass in lettuce shoots grown in Electric Field Nanobubble (EFNB) water with additional CO2 fogging to leaves.

Lettuce plants photographed at harvest after 22 days (Fig 3a) growing in EFNB water with and without CO2 fogging to the leaves. Average shoot biomass was measured in 10 plants per treatment (Fig 3b). Figure 4. Shoot biomass in basil plants grown with dilutions of a volatile compound (2,3- Butanediol) and nanobubbles.

Basil plants after 28 days (Fig 4a) growing in a dilution series of 2,3-Butanediol (2,3- BDO) and either mechanical oxygen nanobubbles (ONBs) or electric field nanobubbles (EFNBs) water. The average shoot biomass measurements (Fig 4b) were calculated. White stars in Fig 4a show optimal dilutions for each nanobubble treatment.

Figure 5. Fluorescence in barley roots after 24 hr incubation with CY3-labelled oligos and nanobubble waters.

Relative fluorescence in roots incubated with UPW-no oligos (Fig 5a), ONB-no oligos (Fig 5b), EFNB-no oligos (Fig 5c), UPW+CY3 oligos (Fig 5d), ONB+CY3 oligos (Fig 5e) and EFNB+CY3 oligos (Fig 5f).

Figure 6. Fluorescence in Nicotiana benthamiana leaves after 30 hr incubation of roots with CY3-labelled oligos post- and pre-mixed before generating EFNBs.

Relative fluorescence in leaves after incubation of roots with UPW-no oligos (Fig 6a), EFNBs-no oligos (Fig 6b), UPW+CY3 oligos (Fig 6c), EFNBs post-mixed with CY3 oligos (Fig 6d) and UPW+CY3 oligos pre-mixed before making EFNBs (Fig 6e).

Figure 7. Texture analysis of plant tissue culture media prepared with mechanical and electric field nanobubbles (EFNB).

The graph shows maximum force required to pierce the surface of MS30 media made with oxygen nanobubbles (ONBs) or electric field nanobubbles (EFNBs). Control sample contains no nanobubbles. Three samples per treatment.

Figure 8. Effect of electric field nanobubbles (EFNBs) on rooting of potato nodes and shoots. Fig 8a shows a box plot of percentage of nodes and shoots that developed roots after 8 days on media made with or without EFNBs; Fig 8b shows a representative selection of nodes and shoots after 11 days on media with or without nanobubbles.

Figure 9. Effect of nanobubbles (NBs) on Agrobacterium-based transient expression in potato.

Fig 9a shows plasmid construct constitutively expressing the reporter GUS and selectable NPTII marker genes. Fig 9b shows number of blue spots in potato plantlets following GUS transient expression with oxygen nanobubbles (ONBs), electric field nanobubbles (EFNBs) or no nanobubbles (No NBs).

Figure 10. Effect of nanobubbles (NBs) on Agrobacterium-based transient expression in barley immature embryos.

Fig 10a shows plasmid construct constitutively expressing the reporter GUSPIus and selectable hph marker genes. Fig 10b shows barley immature embryos transiently transformed with GUS expressing Agrobacterium then stained for GUS activity (white arrowheads indicate GUS staining spots). Fig 10c shows the number of GUS spots per seedling scored and plotted for each treatment. NC, negative control without Agrobacterium and nanobubbles; No NBs, presence of Agrobacterium and no nanobubbles; ONBs, Agrobacterium and oxygen nanobubbles in ultrapure water; EFNBs_Postmix, EFNBs made in ultrapure water then added to Agrobacterium cocultivation medium; EFNBs_Premix, EFNBs prepared directly in Agrobacterium cocultivation medium; EFNBs_Premix (+Embryos), EFNBs prepared in co-cultivation medium in the presence of both Agrobacterium and barley embryos.

Figure 11. Effect of nanobubbles in seed priming of beetroot.

Fig 11a shows plants photographed after 49 days. Whole plant biomass (Fig 11b) and beetroot biomass (Fig 11c) were measured at 49 days old.

Figure 12. Effect of nanobubbles on CRISPR/Cas9-based gene editing efficiency in seedlings of Nicotiana tabacum transgenic line N1DC4. Fig 12a shows CRISPR/cas9 construct expressing tomato-codon-optimised Cas9 (LeCas9) and a guide RNA (gRNA) to target p-Glucosidase (GUS) transgene. Fig 12b shows target GUS gene is made of two defective partial GUS fragments missing the 5’ or 3’ end. Upon DNA break at target sites (black arrowhead), homologous recombination (HR) between the two fragments restores the functional GUS gene. Histochemical GUS staining detects these events as blue spots. Fig 12c shows total number of blue spots scored in each treatment. No NBs, ultrapure water without Agrobacterium or nanobubbles; No NBs/Agro; ultrapure water with Agrobacterium and no nanobubbles; ONBs/Agro, oxygen nanobubbles with Agrobacterium; EFNBs/Agro, Agrobacterium was added in the presence of electric field nanobubbles (EFNBs).

Figure 13. Generation of nanobubbles showing inputs and outputs.

Diagram shows gas and water inputs to nanobubble generator. Other compounds are added prior or during nanobubble generation as a pre-mix or after nanobubble generation as a post-mix. Output nanobubble water used to water plants at roots or as a leaf spray.

Claims

1. A method for editing the genome of a plant, the method comprising:

(i) generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium;

(ii) providing at least one gene editing component;

(iii) providing the nanobubble-containing carrier medium and the at least one gene editing component to the plant or an explant thereof.

2. A method of producing a gene edited plant, the method comprising:

(i) generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium;

(ii) providing at least one gene editing component to an explant of a plant to generate gene edited cells;

(iii) producing callus from the explant to generate a callused explant;

(iv) regenerating shoots from the callused explant to generate the gene edited plant; wherein the method further comprises providing the nanobubble-containing carrier medium to at least one of: cells of the plant, the explant, and the callused explant, during at least one of steps (ii) and (iii).

3. A method as claimed in claim 1 or claim 2 wherein the gene editing component comprises a CRISPR reagent, optionally a CRISPR/Cas9 construct.

4. A method as claimed in any preceding claim wherein the gene editing component is provided by Agrobacterium-mediated gene transfer, electroporation, PEG-mediated transformation, ribonucleoprotein (RNP) delivery or biolistic bombardment.

5. A method as claimed in any preceding claim wherein the nanobubble-containing carrier medium and the at least one gene editing component are mixed prior to providing the nanobubble-containing carrier medium to the plant or explant.

6. A method of generating a genetically modified plant having at least one of: abiotic stress tolerance, altered growth, altered yield, disease resistance, herbicide tolerance, insect resistance, modified product quality and pollination control, controlled flowering time, the method comprising the steps of:

(i) generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium;

(ii) providing an explant;

(iii) genetically modifying the explant using a transformation vector to produce a genetically modified plant; wherein the method further comprises providing the nanobubble-containing carrier medium to the explant during step (iii). A method as claimed in any preceding claim wherein the explant is an immature embryo, mature embryo, plant cell, microspore, protoplast, stem, hypocotyl, cotyledon, internode, node, seed, seedling, flower, pollen, endosperm, microtuber, leaf or root. A method as claimed in any one of the preceding claims wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field. A method for delivering a plant or crop protection product or active ingredient of a plant or crop protection product into a plant, the method comprising:

(i) generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field;

(ii) providing at least one plant or crop protection product or active ingredient; and

(iii) admixing the nanobubble-containing carrier medium and the at least one plant or crop protection product or active ingredient; and

(iv) providing the mixture to the plant. A method as claimed in claim 9 wherein the plant or crop protection product comprises at least one herbicide, insecticide, fungicide, molluscicide, plant growth regulator herbicide, nematicide or acaricide; or mixtures thereof. A method for improving the resistance of a plant to pests, the method comprising:

(i) generating nanobubbles from at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field;

(ii) admixing the nanobubble-containing carrier medium with at least one pesticide; and

(iii) providing the nanobubble-containing carrier medium comprising the pesticide to the plant. A method for delivering a substance to cells of a plant, the method comprising:

(i) generating nanobubbles of at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field;

(ii) admixing the nanobubble-containing carrier medium with a substance; wherein the substance is at least one of: VOCs, plant growth regulators, DNA, RNA, viral vectors, expression vectors, biostimulants, peptides or gene editing components and

(iii) providing the nanobubble-containing carrier medium comprising the substance to a locus or explant of the plant. A method as claimed in claim 12 further comprising, sequentially:

(a) pre-mixing the substance with the carrier medium to form a pre-mix; and

(b) generating the nanobubbles within the pre-mix. A method as claimed in claim 12 or claim 13 wherein the substance is or includes transgenes, synthetic or native DNA or RNA up to 500 nucleotides, optionally up to 200 nucleotides; gibberellins, auxins, abscisic acid, cytokinins, ethylene; peptides up to 150 amino acids, optionally up to 50 amino acids. A method according to any preceding claim wherein the step of providing the carrier medium to the plant comprises applying the medium to roots, seeds and/or leaves of the plant, optionally by immersion, spraying, fogging or misting. A plant application medium for applying to a locus or explant of a plant, the medium comprising a carrier medium, a substance and nanobubbles of at least one gas, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field, and wherein the substance comprises at least one substance selected from VOCs, plant growth regulators, DNA, RNA, viral vectors, expression vectors, biostimulants, peptides, gene editing components and pesticides. A plant application medium as claimed in claim 16 wherein the application medium is prepared by mixing the substance with the carrier medium to form a pre-mix; and generating nanobubbles within the pre-mix to form the application medium. A method for inducing a change in a phenotype, genotype, chemistry or physiology of a plant by delivering a substance to a plant, the method comprising:

(i) providing a substance selected from: VOCs, optionally fungal, microbial or plant VOCs; RNA, siRNA; antisense oligonucleotides; biostimulants, peptides; RNAi vectors; expression vectors; viral vectors; and plant growth regulators;

(ii) generating nanobubbles of at least one gas in a carrier medium, to produce a nanobubble-containing carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field; and

(iii) admixing the carrier medium and the substance and providing the nanobubble-containing carrier medium and the substance to the plant. A method according to claim 18 wherein, in use of the method, the substance induces DNA methylation, RNA methylation, histone methylation or histone acetylation, optionally in one or more flowering loci. A method as claimed in any one of claims 1 to 15, 18 or 19 further comprising a pretreatment step wherein rooted plants are incubated in an oxygen nanobubble water for one to two days prior to use of the method. A method as claimed in any one of claims 1 to 15 or 18 to 20 wherein the plant is grown from a seed and wherein the method comprises a step of seed priming comprising soaking the seed in a seed priming composition comprising water and nanobubbles, wherein the nanobubbles have been generated by an electric field or in the presence of an electric field. A method as claimed in any one of claims 1 to 15 or 18 to 21 comprising a step of mixing the carrier medium with at least one gene editing component, transformation vector, plant or crop protection product or active ingredient of a plant or crop protection product, or substance to form a carrier pre-mix; and a step of generating the nanobubbles within the carrier pre-mix. A method as claimed in any one of claims 1 to 15 or 18 to 22 further comprising a step of growing a plant. A plant obtainable or obtained by the method of claim 23. A plant cultivation system comprising:

(i) a nanobubble generating apparatus for generating nanobubbles from at least one gas in a carrier medium, to form a nanobubble-containing carrier medium, wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field; and

(ii) an applicator system to apply the nanobubble-containing carrier medium to at least one locus of a plant. A plant cultivation system as claimed in claim 25 wherein the nanobubble-containing carrier medium further comprises at least one substance selected from VOCs, DNA, RNA, plant growth regulators, viral vectors, biostimulants, plant or crop protection products or active ingredients of plant or crop protection products, peptides and gene editing components. A system as claimed in claim 25 or claim 26 wherein the applicator system comprises a system for immersion of roots and/or leaves of the plant in the nanobubblecontaining carrier medium. A system as claimed in any one of claims 25 to 27 wherein the applicator system comprises a system for spraying, fogging or misting the plant with the nanobubble- containing carrier medium and the substance, optionally wherein the at least one gas comprises carbon dioxide and the applicator system comprises a system for misting leaves of the plant. A system as claimed in any one of claims 25 to 28 wherein the applicator system is in fluid communication with the nanobubble generating apparatus. A system as claimed in any one of claims 25 to 29 comprising a hydroponic plant cultivation system. A system as claimed in any one of claims 25 to 30, a method as claimed in any one of claims 1 to 15 or 18 to 23, or a medium as claimed in claim 16 or claim 17, wherein the plant is Cannabis sativa, Nicotiana benthamiana, Hordeum vulgare, Solanum lycopersicom, Solanum tuberosum, Nicotiana tabacum. Lactuca sativa or Ocimum basilicum, Zea mays or Glycine max. A system as claimed in any one of claims 25 to 30, a method as claimed in any one of claims 1 to 15, 18 to 23 or 31 , or a medium as claimed in claim 16, claim 17 or claim 31 , wherein the at least one gas comprises, consists of, or consists predominantly of, oxygen, carbon dioxide or air, or mixtures thereof. A system as claimed in any one of claims 25 to 32, a method as claimed in any one of claims 1 to 15, 18 to 23, 31 or 32, or a medium as claimed in claim 16, claim 17, claim 31 or claim 32, wherein the carrier medium is water, an aqueous carrier medium, a nutrient solution, or an aqueous agar medium. Use of nanobubbles of at least one gas to enhance plant transformation, optionally wherein the gas is air or oxygen. Use as claimed in claim 34 wherein the nanobubbles are generated by application of an electric field or in the presence of an electric field. Use of nanobubbles generated by application of an electric field or in the presence of an electric field in plant cultivation, plant growth or plant propagation. Use as claimed in any one of claims 34 to 36 wherein the nanobubbles are nanobubbles in water, an aqueous carrier, a nutrient solution or an agar medium. Use as claimed in any one of claims 34 to 37 wherein the nanobubbles are nanobubbles of a gas comprising, consisting of, or consisting essentially of, air, oxygen or carbon dioxide, or mixtures thereof.