WO2022245725A2 - Lignées cellulaires, variétés et procédés de production in vitro de fibres de coton colorées - Google Patents

Lignées cellulaires, variétés et procédés de production in vitro de fibres de coton colorées Download PDF

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Publication number
WO2022245725A2
WO2022245725A2 PCT/US2022/029428 US2022029428W WO2022245725A2 WO 2022245725 A2 WO2022245725 A2 WO 2022245725A2 US 2022029428 W US2022029428 W US 2022029428W WO 2022245725 A2 WO2022245725 A2 WO 2022245725A2
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Prior art keywords
expression
cotton
pigment
chromoprotein
different
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PCT/US2022/029428
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English (en)
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WO2022245725A3 (fr
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Jung Hun PARK
Paula Maria ELBL
Jarrett Andrew MAN
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Galy Co.
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Publication of WO2022245725A2 publication Critical patent/WO2022245725A2/fr
Publication of WO2022245725A3 publication Critical patent/WO2022245725A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/60—Malvaceae, e.g. cotton or hibiscus
    • A01H6/604—Gossypium [cotton]

Definitions

  • the present invention relates to in vitro methods of cotton fiber production using cotton cells that express selected genes.
  • Cotton is the most widespread non-food crop in the world. However, cotton production is expensive both in terms of money and resources required for its successful cultivation. For example, cotton is a water-intensive crop, with an estimated 9,000-17,000 liters of water required for each kilogram of cotton fiber produced. This equates to enough drinking water to sustain 5,000 people for a day used in order to produce enough cotton to make two t-shirts. Similarly, cotton cultivation requires land, which must be otherwise diverted from other crop production, such as food production. It is estimated that for every acre of cotton grown, only about 500 kilograms of cotton fiber is produced. Cotton cultivation is also a net-emitter of greenhouse gasses, with approximately between .75 and 2.25 kilograms of carbon dioxide gas emitted per kilogram of cotton fiber produced. Moreover, because cotton is a plant, its cultivation can lead to failed crops, mistimed crops, and even excess production. Every year, billions of dollars are spent on logistics to overcome unexpected cotton harvest results.
  • the present invention provides methods and compositions for the in vitro production of colored cotton fiber, without growing cotton plants and without the need to dye or otherwise color the resulting fiber.
  • the invention employs cotton cells that express selected genes of interest, which cause the cells to produce colored cotton fibers.
  • Cotton cells of the invention include independently activatable genes and/or expression constructs. When activated, each different gene/construct leads to the generation of a different colored pigment or chromoprotein in fiber produced from the cells. Since the genes and/or expression constructs can be independently activated, during the disclosed methods for in vitro cotton fiber production, the cotton cells can produce fiber with a desired combination of pigments/chromoproteins of selected colors. Moreover, the activity of the genes and/or expression constructs can be tuned, such that selected colors are generated in the fibers at varying levels of intensity and saturation. This provides the ability to produce cotton fiber of any desired color, using wholly in vitro methods.
  • a cotton cell can be modified to include a number of activatable expression constructs.
  • one of the constructs When activated, one of the constructs causes the cotton cell to produce a red pigment/chromoprotein, a second a blue pigment/chromoprotein, and a third a green pigment/chromoprotein.
  • RBG red, blue and green
  • CMYK optionally black
  • cotton fiber can be produced using approximately 77% less water, 80% less land, and producing approximately 84% less carbon dioxide emissions than traditional in planta methods. Concurrently, the methods completely avoid the costs, transportation requirements, and environmental impacts associated with dyeing cotton fiber.
  • the methods of the invention produce cotton fiber much faster that in planta methods.
  • Cotton traditionally requires 5-6 months from planting to harvest, after which it must be transported to a textile mill for dyeing.
  • the in vitro methods of the present disclosure can produce a colored cotton fiber harvest in approximately 45 days or less. Additionally, because the disclosed methods are in vitro as opposed to in planta, they can be more rigidly controlled. Therefore, the propensity for failed, mistimed, or excess crops can be reduced, if not completely, eliminated. Further, the colored cotton fiber can be produced with a consistent and desired coloration. As the fiber is inherently colored, there is less variation amongst individual fibers than when dyed using traditional methods, which require all fibers receive equal dye contact and saturation.
  • the invention includes cotton cells with multiple, different activatable genes and/or expression constructs that each generate different pigments/chromoproteins in the cotton fiber.
  • the genes and/or expression constructs can be independently activated and tuned such that any color of cotton can be produced.
  • the cotton cells include inducible genes/constructs, which are activated to produce pigments/chromoproteins upon contact with an inducer.
  • the cells can include constitutively activated genes/constructs.
  • the constitutively activated genes/constructs can produce chromoproteins/pigments upon contact with a substrate, such as an enzymatic substrate for a protein encoded by a gene/construct.
  • These Lac cells can be maintained in culture prior to inducement of the genes/constructs and elongation of the Lac fiber. As such, they can provide continually available sources of cotton cells that can be induced and elongated to produce cotton of any desired color. This further shortens the required time to produce color Lac fiber, as the initial steps of modifying and expanding the Lac cells have been completed. The cells do not need to be modified to include a gene/construct that produces a desired color of Lac, and subsequently expanded for a harvest.
  • the present invention also includes a vector, which can be introduced into Lac cells in order to produce one or more pigments or chromoproteins that color the Lac fiber produced by the cell.
  • the invention provides a vector that includes at least one introduced transgenic construct having a plurality of different inducible expression constructs. Inducement of each different construct leads to production of a pigment or chromoprotein in a Lac cell.
  • inducement of each of the different expression constructs leads to the production of a different pigment or chromoprotein.
  • the different expression constructs may be differently induced from one another. For example, each different expression construct can be induced upon contact with a different inducer.
  • each expression construct is variably induced such that contact with an increasing concentration of the inducer leads to a corresponding increase in the amount of the pigment or chromoprotein produced.
  • Coton cells comprising an exemplary vector of the invention produce Lac fiber that includes visually detectable amounts of the pigments and/or chromoproteins produced from inducement of one or more of the expression constructs.
  • the Lac fiber may have a specific and/or desired coloration.
  • the color of Lac fiber produced from Lac cells with a vector of the invention can be modulated by contacting one or more of the different expression constructs with an inducer.
  • the color can be further modulated by contacting one or more of the different expression constructs with a specific concentration of the inducer such that a specific concentration of the pigment or chromoprotein is produced.
  • the transgenic construct includes three expression constructs.
  • inducement of a first expression leads to the production of a red pigment or chromoprotein
  • inducement of a second expression construct leads to the production of a green pigment or chromoprotein
  • inducement of a third expression construct leads to the production of a blue pigment or chromoprotein.
  • the transgenic construct includes four expression constructs.
  • inducement of a first expression construct leads to the production of a cyan pigment or chromoprotein
  • inducement of a second expression construct leads to the production of a yellow pigment or chromoprotein
  • inducement of a third expression construct leads to the production of a magenta pigment or chromoprotein
  • inducement of a fourth expression construct leads to expression of black pigment or chromoprotein.
  • each different expression construct is operably connected to a different inducible promotor.
  • contacting the inducible promotor with an inducer causes expression construct to express one or more products, such as a pigment or chromoprotein or one or more proteins that produce the pigment/chromoprotein.
  • the invention also provides a composition including cotton cells having a vector as disclosed herein.
  • the invention also provides cotton fiber produced by cotton cells having a vector as disclosed herein.
  • the invention also provides methods for generating colored cotton in vitro.
  • An exemplary method for generating colored cotton in vitro includes tuning in a cotton cell, via one or more genetic elements, expression of each of a plurality of different expression constructs associated with production of a pigment or chromoprotein.
  • tuning results in a different level of activation of each of the plurality of different expression constructs, to thereby produce different concentrations of pigments or chromoproteins in the cotton cell.
  • the expression constructs are each operably connected to an inducible promoter. Inducement of an inducible promoter with an inducer may cause selective expression of the expression construct in one or more specific organelles of the cotton cell.
  • the specific organelles can include, for example, vacuoles.
  • inducement of the expression construct leads to production of a pigment selected from a melanin, an anthocyanin, a violacein, a betalain, b-carotene, and indigo.
  • the pigment is a melanin and the expression construct comprises a melA gene or a gene expressing a tyrosinase protein and an ORF-438 gene.
  • the pigment is an anthocyanin and the expression construct comprises an anthocyanin regulatory Lc gene.
  • the pigment is a violacein and the expression construct comprises genes that cause the production of a VioA protein, a VioB protein, a VioC, protein, a VioD protein, and/or a VioE protein.
  • the pigment is a betalain and the expression construct comprises genes that cause expression of a P450 oxygenase and/or L-DOPA 4,5- dioxygenase.
  • the pigment is a b-carotene and the expression construct comprises a cotton phytoene synthase gene (GhPSY2D).
  • the expression construct includes genes that lead to production of a chromoprotein selected from amajLime and cjBtlue.
  • the present invention also provides method for producing colored cotton fiber.
  • An exemplary method for producing colored cotton fiber includes modifying cotton cells such that they comprise a transgenic construct that includes an inducible expression construct. The method further includes inducing the expression construct. Inducement of the expression construct leads to production of a pigment or chromoprotein in the cotton cells. Before, after, and/or during inducement, the method further includes culturing the modified cotton cells in vitro to produce cotton fiber, wherein the cotton fiber includes visually detectable amounts of the pigment or chromoprotein produced from inducement of the expression construct.
  • the method further includes harvesting the cotton fiber without growing a cotton plant.
  • the expression construct is operably connected to an inducible promoter.
  • the promoter may cause selective expression of the expression construct in one or more specific organelles of the cotton cells.
  • the specific organelles may include, for example, vacuoles.
  • inducement of the expression construct leads to production of a pigment selected from a melanin, an anthocyanin, a violacein, a betalain, b-carotene, and indigo.
  • the pigment is a melanin and the expression construct comprises a melA gene or a gene expressing a tyrosinase protein and an ORF-438 gene.
  • the pigment is an anthocyanin and the expression construct comprises an anthocyanin regulatory Lc gene.
  • the pigment is a violacein and the expression construct comprises genes that cause the production of a VioA protein, a VioB protein, a VioC, protein, a VioD protein, and/or a VioE protein.
  • the pigment is a betalain and the expression construct comprises genes that cause expression of a P450 oxygenase and/or L-DOPA 4,5- dioxygenase.
  • the pigment is a b-carotene and the expression construct comprises a cotton phytoene synthase gene (GhPSY2D).
  • the expression construct includes genes that lead to production of a chromoprotein selected from amajLime and cjBtlue.
  • the modifying step includes modifying cotton cells such that they comprise a transgenic construct that includes a plurality of different inducible expression constructs.
  • inducement of each different expression construct leads to production of a different pigment or chromoprotein in the cotton cells causing the cells to produce cotton fiber that includes visually detectable amounts of the pigment or chromoprotein produced from inducement of the expression construct.
  • An exemplary method includes inducing one or more of the different expression constructs, wherein each different expression construct is induced upon contact with a different inducer.
  • Each expression construct can be variably induced such that contact with an increasing concentration of an inducer leads to a corresponding increase in the amount of the pigment or chromoprotein produced.
  • An exemplary method further includes modulating a color of the cotton fiber produced by the cotton cells by selectively inducing one or more of the different expression constructs to produce selected pigments and/or chromoproteins in the cotton cells. Modulating the color of the cotton fiber may further include contacting one or more of the different expression constructs with specific concentrations of the inducers such that each expression construct produces a pigment or chromoprotein at a specific concentration.
  • the transgenic construct includes three expression constructs.
  • inducement of a first expression leads to the production of a red pigment or chromoprotein
  • inducement of a second expression construct leads to the production of a green pigment or chromoprotein
  • inducement of a third expression construct leads to the production of a blue pigment or chromoprotein.
  • the transgenic construct includes four expression constructs.
  • inducement of a first expression construct leads to the production of a cyan pigment or chromoprotein
  • inducement of a second expression construct leads to the production of a yellow pigment or chromoprotein
  • inducement of a third expression construct leads to the production of a magenta pigment or chromoprotein
  • inducement of a fourth expression construct leads to expression of black pigment or chromoprotein.
  • the present invention also includes a vector with at least one introduced transgenic construct having a number of different expression constructs that each express a different expression product. Upon contact with a cognate expression product substrate, each different expression product leads to the generation of a different pigment/chromoprotein in fiber produced by cotton cells comprising the vector.
  • each different expression product leads to the production of a pigment or chromoprotein upon contact with a different expression product substrate.
  • each different expression construct is constitutively active, such that it continually expresses its expression product. Contacting each different expression product with an increasing concentration of its substrate leads to a corresponding increase in the amount of the pigment or chromoprotein produced in fiber of the cotton cell.
  • the different expression products include enzymes and the different expression product substrates are enzymatic substrates for those enzymes.
  • One or more of the different expression product substrates may not be endogenous to the cotton cell.
  • cotton cells having the vector produce cotton fiber that with visually detectable amounts of the pigments and/or chromoproteins produced as a result of contacting the cotton cells with the expression product substrates.
  • the cotton fiber produce by cells with the vector may have a specific coloration.
  • the specific coloration can be modulated by contacting one or more of the different expression products with a substrate.
  • the specific coloration can be further modulated by contacting one or more of the different expression products with a specific concentration of the substrate, such that a corresponding, specific concentration of the pigment or chromoprotein is produced.
  • the transgenic construct of the vector includes three expression constructs that each produce a different expression product.
  • contact with a first expression product substrate leads to production of a red pigment or chromoprotein
  • contact with a second expression product substrate leads to the production of a green pigment or chromoprotein
  • contact with a third expression product substrate leads to the production of a blue pigment or chromoprotein.
  • the transgenic construct includes four expression constructs.
  • contact with a first expression product substrate leads to production of a cyan pigment or chromoprotein
  • contact with a second expression product substrate leads to production of a yellow pigment or chromoprotein
  • contact with a third expression product substrate leads to production of a magenta pigment or chromoprotein
  • contact with a fourth expression product substrate leads to production of a black pigment or chromoprotein.
  • one or more of the expression constructs is operably connected to an inducible promotor. Contacting the inducible promotor with an inducer may cause expression of the expression product.
  • the present invention also includes a composition comprising cotton cells that have a vector with at least one introduced transgenic construct having a number of different expression constructs that each express a different expression product.
  • the present invention also includes a method for generating colored cotton in vitro.
  • An exemplary method includes tuning in a cotton cell via contact with a plurality of different expression product substrates, production of pigments or chromoproteins in the cotton cell.
  • the cotton cell includes a plurality of different expression constructs that each produce a different expression product, which in turn generates a different pigment or chromoprotein upon contact with an expression product substrate.
  • tuning includes contacting the cell with a different concentration of each different expression product substrate, thereby producing different concentrations of pigments or chromoproteins in the cotton cell.
  • the present invention further includes a method for producing colored cotton fiber.
  • An exemplary method includes modifying cotton cells such that they comprise a transgenic construct, which includes an expression construct that expresses an expression product.
  • An exemplary method further includes contacting the expression product with an expression product substrate. Contacting the expression product with the substrate leads to production of a pigment or chromoprotein in the cotton cells.
  • An exemplary method also includes culturing the modified cotton cells in vitro to produce cotton fiber, which possesses visually detectable amounts of the pigment or chromoprotein produced from contacting the expression product with the substrate.
  • the method further includes harvesting the cotton fiber without growing a cotton plant.
  • the modifying step includes modifying cotton cells such that they comprise a transgenic construct that with a plurality of different expression constructs.
  • Each different expression construct expresses a different expression product.
  • Contacting each different expression product with a corresponding expression product substrate leads to production of a different pigment or chromoprotein in the cotton cell.
  • the cotton fiber produced from the method includes visually detectable amounts of different pigments or chromoproteins produced from contacting the expression products with expression product substrates.
  • each of the different expression products are contacted with an increasing concentration of their respective substrate, which leads to a corresponding increase in the amounts of the different pigments/chromoproteins produced.
  • the method further includes comprising the color of the cotton fiber produced by the cotton cells by selectively providing one or more of the different expression product substrates to produce selected pigments and/or chromoproteins in the cotton cells at desired concentrations. In doing so, the methods of the invention allow cotton fiber of any color to be produced.
  • the methods of the present invention can be used to produce at least 1 kilogram of colored cotton fiber for every 4,000 liters of water used in the method. In some instances, the methods of the invention can produce at least 1 kilogram of colored cotton fiber for between every 2,000 and 4,000 of water used in the method.
  • FIG. 1 shows an exemplary method of the invention
  • FIG. 2 provides a schematic showing how the color of fiber is tuned.
  • FIG. 3 shows varying color schemes to produce cotton fiber of any color.
  • FIG. 4 shows an exemplary expression construct.
  • FIG. 5 shows a plurality of expression constructs in a single cell.
  • FIG. 6 provides a schematic of an exemplary vector.
  • FIG. 7 shows a computer system that is programmed or otherwise configured to implement methods provided herein.
  • FIG. 8 shows a flowchart of the concept of a commercial scale process for the cotton fiber in vitro production.
  • the present invention provides methods and compositions for the in vitro production of colored cotton fiber. Due to the ubiquity of cotton production around the world, there has long been interest in modifying cotton plants, through genetic alterations or selective breeding, in order to produce cotton plants with commercially-relevant characteristics.
  • the present invention includes, for the first time, methods and compositions in which cotton cells express selected genes that cause the cells to produced colored cotton fibers without growing cotton plants and without the need to dye or otherwise color the resulting fiber.
  • the present invention provides cotton cells that are modified to include independently inducible genes and/or expression constructs. When induced, each different gene/construct leads to the generation of a different colored pigment or chromoprotein in fiber produced from the cells. These genes and/or expression constructs can be independently activated. As a result, during the disclosed methods for in vitro cotton fiber production, the cotton cells can produce fiber with a desired combination of selected colors. Moreover, the activity of the genes and/or expression constructs can be tuned, such that selected colors are generated in the fibers at varying levels of intensity and saturation. This provides the ability to produce cotton fiber of any desired color, using wholly in vitro methods.
  • the present invention also provides cotton cells that are modified to include genes and/or expression constructs, that each produce an expression product, such as an enzyme.
  • a substrate e.g., an enzymatic substrate
  • contacting each different gene product with its substrate leads to the generation of a different colored pigment or chromoprotein.
  • each different gene product is contacted with a different substrate.
  • Chromoprotein/pigment generation can be controlled by providing substrates at varying amounts and/or for varying times.
  • the color of cotton fiber produced by the cells can be tuned by adjusting the amounts and/or times substrates are provided to cotton cells. I As a result, during the disclosed methods for in vitro cotton fiber production, the cotton cells can produce fiber with a desired combination of selected colors at desired intensities.
  • the present invention includes producing modified cotton cells by introducing the genes and/or expression constructs into cotton cells as transgenes.
  • the modified cotton cells can then be used in the presently disclosed in vitro methods for colored cotton fiber production.
  • methods of the invention can be used to produce colored cotton fiber without the need to grow cotton plants.
  • the methods and compositions of the invention can be scaled up, thereby allowing industrial scale production of cotton fiber.
  • genes and/or gene expression patterns leading to improved in vitro colored cotton fiber production can be determined. Once determined, these genes and/or gene expression patterns can be introduced into other cotton plants/cells, for example, as a transgene.
  • FIG. 1 provides an exemplary method 101 of the invention for the in vitro production of colored cotton fiber.
  • the method 101 begins with modifying or selecting cotton cells 103.
  • the cotton cells are modified 103 to express, or selected 103 for expression, of one or inducible genes or expression constructs.
  • the inducible genes/constructs express gene products that lead to the production of one or more pigments and/or chromoproteins in the cotton cells, such that cotton fiber produced from the cotton cells includes visible levels of the pigments and/or chromoproteins.
  • the cotton cells are modified 103 to express, or selected 103 for expression of one or more gene constructs that each produce a gene product, such as an enzyme.
  • a gene product such as an enzyme.
  • the method 101 requires multiplying 107 the cells in the bioreactor.
  • the multiplied cells are then elongated 109 to produce colored cotton fibers.
  • the resulting colored cotton fiber is harvested 111.
  • one or more of the inducible genes or expression constructs is induced before the inoculating 105 step. In certain aspects, one or more of the inducible genes or expression constructs is induced after the inoculating 105 step. In certain aspects, one or more of the inducible genes or expression constructs is induced after the multiplying step 107. In certain, preferred embodiments, one or more of the inducible genes or expression constructs is induced during the elongation 109 step.
  • one or more of the expression products is contacted with an appropriate substrate before the inoculating 105 step. In certain aspects, one or more of the expression products is contacted with an appropriate substrate after the inoculating 105 step. In certain aspects, one or more of the expression products is contacted with an appropriate substrate after the multiplying step 107. In certain, preferred embodiments, one or more of the expression products is contacted with an appropriate substrate during the elongation 109 step.
  • An exemplary method for generating colored cotton in vitro includes tuning in cotton cells, via one or more genetic elements, the expression of each of a plurality of different expression constructs associated with production of a different pigment or chromoprotein.
  • Tuning results in a different level of activation of each of the plurality of different expression constructs, to thereby produce different concentrations of pigments or chromoproteins in the fibers of the cotton cells.
  • Tuning generally includes inducing one or more of the genes/constructs, such that their activity leads to the generation of a specific concentration of one or more chromoproteins/pigments in the cotton fiber produced by the cells. This can be accomplished, for example, by providing varying concentrations of inducers, activating multiple genes and/or expression constructs that produce the same pigment/chromoprotein, activating genes/constructs for a specified period of time, and halting or repressing one or more genes/constructs from producing additional pigment.
  • tuning can include providing a substrate for gene product expressed in a modified cotton cell.
  • the cotton cell can be modified such that it expresses a gene that produces the gene product.
  • a gene/construct that expresses the gene product can be constitutively active, such that it continually produces the gene product.
  • the gene product substrate is not endogenous to the cotton cell, or is produced at low levels.
  • a gene/construct may be introduced into the cotton cell that expresses an enzyme such as cytochrome P450 mono-oxygenase (CYP2A6).
  • CYP2A6 cytochrome P450 mono-oxygenase
  • Indole is a substrate for CYP2A6, which converts indole into indoxyl, a molecular precursor to indigo.
  • the expressed CYP2A6 is able to produce indoxyl, which leads to indigo generation.
  • the substrate such as indole, is absent or present in the cotton cell at low levels. Thus, in the absence of the substrate, no pigment/chromoprotein is produced.
  • a repressor when production of a particular pigment/chromoprotein is undesired, can be introduced to the cotton cell, which prevents or stops a particular gene/construct from producing an expression product that leads to generation of the pigment/chromoprotein.
  • an expression construct is under the control of a repressor. Contact with an inducer may deactivate the repressor.
  • the repressor is a genetic element. The genetic element may express a gene product, e.g., a protein, which prevents expression of a pigment/chromoprotein gene. Contact with an inducer can prevent expression of the repressor gene product, thereby permitting expression of a pigment/chromoprotein gene.
  • FIG. 2 is a schematic showing an exemplary method of tuning in accordance with the invention.
  • a cotton cell has three inputs - A, B, and C. These inputs represent different inducers, which each activate a different gene(s) and/or expression construct(s). When induced, each gene/construct produces a different pigment/chromoprotein. Varying the concentrations of the inducers (Inputs A, B, and C), causes the genes and/or constructs induced to generate a corresponding varying concentration of the pigment/chromoprotein.
  • Input A induces a gene/construct that produces a red pigment/chromoprotein in the fiber of the cotton cells.
  • input B induces production of a green pigment/chromoprotein
  • input C induces a blue pigment/chromoprotein.
  • each input can induce production of more than one pigment/chromoprotein.
  • the cells can produce fiber of any color.
  • the three inputs may be, or may include, different expression product substrates.
  • the substrates may be provided to expression products respectively expressed by the different genes/constructs. Providing the substrates to the expression products leads to the generation of pigments/chromoproteins. By varying the concentration of these substrates, the concentrations of different pigments/chromoproteins generated can be modulated.
  • the RGB color model used in FIG. 3 is an additive color model in which red, green, and blue light reflected by the chromoproteins/pigments are added together in various intensities to produce different colors.
  • Cells can also employ constructs that, when induced, produce cyan, magenta, yellow and optionally black/key (CMYK).
  • CMYK color model is a subtractive color model, based on the CMY color model.
  • the CMY color model is subtractive in the sense that mixtures of pigments that subtract specific wavelengths from the spectral power distribution of the illuminating light.
  • the CMYK and CMY models work using pigments partially or entirely preventing certain colors of a lighter (usually white) colors on a lighter, usually white, background from reflecting.
  • the present invention includes methods and compositions in which the cotton cells include only a single gene or expression construct that leads to the production of a single pigment/chromoprotein.
  • cotton cells of the invention include genes/constructs as described herein to produce a number of different chromoproteins/pigments.
  • the production of each different chromoprotein/pigment can be independently activatable and/or tunable.
  • the cotton cells of the invention include genes/constructs that independently lead to the production of at least 2 different pigments, at least 3 different pigments, at least 4 different pigments, at least 5 different pigments, at least 6 different pigments, at least 7 different pigments, at least 8 different pigments, at least 9 different pigments, at least 10 different pigments, at least 15 different pigments, or at least 20 different pigments.
  • the cotton cells of the invention include genes/constructs that independently lead to the production of at least 2 different chromoproteins, at least 3 different chromoproteins, at least 4 different chromoproteins, at least 5 different chromoproteins, at least 6 different chromoproteins, at least 7 different chromoproteins, at least 8 different chromoproteins, at least 9 different chromoproteins, at least 10 different chromoproteins, at least 15 different chromoproteins, or at least 20 different chromoproteins.
  • the cotton cells of the invention include genes/constructs that independently lead to the production of at least one chromoprotein and at least one pigment.
  • one or more genes, the expression of which leads to pigment/chromoproteins in the cotton fiber may be encoded by an exogenous gene.
  • An exogenous gene is a nucleic acid introduced into a cell.
  • the exogenous gene may encode an expression product, i.e., an RNA and/or protein expressed in a cell.
  • the exogenous gene may include a heterologous gene from, or derived from, a different species (including a non-plant species), cotton varietal, and/or be partially or completely synthetic.
  • the exogenous gene may include a homologous gene from, or derived from, the same species or varietal (homologous), relative to the cell being modified.
  • the exogenous gene can include a homologous gene that occupies a different location in the genome of the cotton cell or is under different control, relative to the endogenous copy of the gene.
  • An exogenous gene may be present in more than one copy in the cell.
  • An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
  • one or more of these exogenous genes may be introduced into the cell as part of an expression construct.
  • FIG. 4 provides a schematic of an exemplary expression construct 401, which is introduced into cotton cells in accordance with the invention.
  • the construct 401 includes one or more genes 403, that when activated, lead to the production of a particular pigment/chromoprotein.
  • the gene(s) 403 produce a blue pigment/chromoprotein when activated.
  • the construct also includes a regulatory element or repressor (RA).
  • Inducer A acts on regulatory element or repressor RA, which permits expression of the one or more genes 403.
  • the construct may also include promoter site 405.
  • Promoter site 405 may be constitutively promoted or by a specific promoter added to the cell. Promoter site 405 can be blocked, impeded, or otherwise deactivated as a site to which a promoter can successfully bind to initiate expression of gene(s) 403 due to the activity of RA. The blocking activity of RA can be reduced or eliminated upon contact with Inducer A.
  • regulatory element or repressor RA encodes a gene product that is expressed when RA is activated using, in this example, a 35S promoter. Expression of the RA gene product represses expression of gene(s) 403. Contact with Inducer A prevents expression of the RA product, which in turn permits expression of gene(s) 403.
  • the gene(s) 403 are constitutively activated.
  • the expression constructs may not include a regulatory element or repressor (RA) that regulates expression of gene(s) 403.
  • production of the pigment/chromoprotein is activated and/or modulated using an expression product substrate.
  • the gene(s) 403 express one or more gene product, e.g., a protein.
  • the expression product can, for example, be an enzyme and the expression product substrate a substrate for the enzyme. When contacted with the substrate, the enzyme may convert the substrate into a pigment/chromoprotein, or precursor thereof. Varying the concentration of the substrate provided can allow production of corresponding varying concentrations of pigment/chromoprotein.
  • a single cotton cell of the invention can include multiple expression constructs, each independently inducible using a different inducer. As shown in FIG. 5, inducement of each separate construct leads to the production of different pigment/chromoprotein. In this example, when all three illustrated constructs are induced, a blue, a green, and a red chromoprotein/pigment are produced by the cotton cell.
  • different repressors RA, RB, RC
  • RA, RB, RC can repress activity through expression of different gene products.
  • the different repressors are activated (i.e., have their blocking activity initiated) in a common manner, e.g., by using the same promoter for each repressor.
  • the repressing activity of the repressors is activated, thus repressing the pigment/chromoprotein gene(s), until the cotton cell is contacted with an inducer.
  • a cotton cell is modified with a genetic construct that includes a plurality of different, inducible expression constructs.
  • each different expression construct produces a different pigment/chromoprotein when induced.
  • Each expression construct may be independently inducible and tunable.
  • the multiple expression constructs may produce different expression products.
  • a different expression product substrate is provided for each different expression product, e.g., substrates for various enzymes.
  • Production of different pigments/chromoproteins from the different expression constructs can thus be activated by providing the appropriate substrates.
  • the production of different pigments/chromoproteins can be independently tuned by providing varying concentrations of the substrates for each respective expression product.
  • the production of different pigments/chromoproteins from different expression constructs is activated and/or tuned using a combination of inducible expression constructs and expression product substrates.
  • the gene construct comprises a nucleic acid vector into which the expression vectors are inserted.
  • the vector is then introduced into cotton cells, thereby providing the expression constructs to generate pigments/chromoproteins.
  • FIG. 6 provides an exemplary vector 601 into which three expression constructs 603,
  • each expression construct is independently inducible and tunable when induced, a first expression construct 603 produces a red pigment/chromoprotein, a second 605 produces a green pigment/chromoprotein, and a third 607 produces a blue pigment/chromoprotein.
  • this vector of the present invention can be introduced into a cotton cell and used for the in vitro production of cotton fiber of any color.
  • the invention provides plant vectors, plasmids, or constructs.
  • the term vector refers broadly to any plasmid or virus encoding an exogenous nucleic acid, such as the inducible genes and expression constructs of the invention.
  • Vectors used in accordance with the invention also include non-plasmid and non-viral compounds which facilitate transfer of nucleic acids into cells or other particles for transfer, such as, polylysine compounds.
  • the vector may be a viral or viral -based vector that is suitable as a delivery vehicle for delivery of the genes and/or expression constructs described herein.
  • the vector can be a non-viral vector which is suitable for the same purpose. Examples of vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746), which is incorporated by reference.
  • Genes and expression constructs can be introduced into cotton cells using any known method.
  • the genes/constructs can be introduced using a variety of different transformation methods including, but not limited to, microinjection; electroporation; biolistic bombardment; viral-mediated transformation; and Agrobaclerium-mediaied transformation.
  • any of the genes/constructs described herein can introduced into cotton cells using a transient vector.
  • Transient vectors introduce the genes/constructs into the nucleus of a host cell, where there are transiently.
  • any of the genes/constructs described herein can be stably transfected, such that they are integrated into the genome of the cotton cells. When stably transfected, the introduced genes/constructs can be passed to successive generations of cells and cultures.
  • An expression product of interest may include, for example, at least one peptide, protein, and/or nucleic acid.
  • the nucleic acid may be an RNA molecule.
  • the RNA molecule may be capable of interaction with one or more genes, nucleic acids and/or proteins in the cotton cell.
  • the RNA may inhibit one or more gene, nucleic acid, and/or protein in the cotton cell.
  • Inhibitory RNAs may include, for example, miRNA, siRNA, mRNA, tRNA, sense RNA, antisense RNA, hairpin RNA, and/or ribozymes.
  • the expression product of interest is a protein, which produces or leads to the production of a chromoprotein/ pigment.
  • the cotton cells may be modified by the inclusion of a chimeric gene.
  • a chimeric gene may include a pigment- or chromoprotein-related gene of interest operably linked to a promoter or promoter region.
  • a chimeric gene may further include one or more functional elements and/or regulatory elements, which may alter the expression or activity of the chimeric and/or selected gene of interest or a linked promoter.
  • a chimeric gene may also include a transcription termination sequence and/or a polyadenylation sequence.
  • the chimeric gene may operably link a selected gene of interest with an unrelated nucleic acid sequence, such as a promoter, or another gene.
  • the chimeric gene may be a gene that is not normally found in a cotton plant.
  • the cotton cells are modified by the inclusion of a transgene/chimeric gene that, by its inclusion, modulates the level of an expression product endogenous to the cotton cell.
  • the transgene/chimeric gene may encode a protein endogenous to the cotton, and expression of the gene increases the level of protein that would otherwise be produced in the cotton cell.
  • the transgene/chimeric gene may modulate the temporal and/or spatial expression and/or levels of an expression product.
  • the gene or expression construct is operably linked to a promoter or promoter region.
  • the promoter region may by homologous to a portion of the gene/construct.
  • the promoter and gene/construct are not normally associated in nature.
  • the promoter can be used to modulate or tune the expression of a gene or expression construct, such that a specific concentration of a pigment/chromoprotein is generated in the fiber of the cotton cells. Modulated expression may lead to an increase or decrease in the expression of the gene or expression construct, and consequently pigment/chromoprotein production. Modulated expression may also or alternatively modulate the spatial and/or temporal expression pattern of the gene or expression construct and the corresponding pigment/chromoprotein.
  • the gene or expression construct can be synthesized to include a promoter that assures expression at a certain level, under specific growth conditions, in certain tissues and tissue types, and/or under specified spatial and/or temporal constraints.
  • one or more of the genes and or expression constructs are inducible.
  • the genes/constructs may be induced through the use of an inducible promoter.
  • the inducible promoter may also be a repressible promoter.
  • Inducible and/or repressible promoters includes promoters that are under either environmental or exogenous control.
  • such promoters may be induced to cause expression or repressed to reduce expression by contacting the cotton cells with particular inducers/repressors, including specific environmental conditions, such as anaerobic conditions, certain chemicals, the presence of light, and any other means known in the art.
  • inducers/repressors including specific environmental conditions, such as anaerobic conditions, certain chemicals, the presence of light, and any other means known in the art.
  • Exogenous control of an inducible or repressible promoter can be affected by providing a suitable chemical or other agent that via interaction with target polypeptides result in induction or repression of the promoter.
  • an inducible or repressible promoter as used in the invention may be tissue-specific, such that they only promote expression of genes/constructs in a particular cotton cell type, tissue and/or organelle.
  • an inducible or repressible promoter is a developmental promoter, which is induced or repressed in response to a particular developmental stage in the cotton cells, such as cotton fiber elongation.
  • an inducible promoter of the invention is a non-constitutive promoter, meaning it does not initiate expression, or initiates expression at a low level, until induction.
  • a constitutive promoter can be used.
  • the inducer is an enzyme.
  • the enzyme is a nuclease, which modifies a nucleic acid (e.g., via cleavage) containing a gene/construct, such that the gene/construct can be activated.
  • the nuclease is an endonuclease.
  • the endonuclease is a zinc-finger nuclease.
  • the endonuclease is a CRISPR-associated protein, such as a Cas9 protein.
  • CRISPR stands for clustered regularly interspaced short palindromic repeats, which are used by a CRISPR-associated protein to guide it to a sequence of interest, which can then be modified, e.g., by cleavage.
  • pigment/chromoprotein production is activated and/or tuned by providing an expression product substrate.
  • An expression construct/gene as described herein produces an expression product, e.g., an enzyme.
  • the expression product substrate can be, for example, a molecular precursor of a pigment and a substrate for the enzyme. Providing the molecular precursor to the enzyme allows it to catalyze a reaction that converts the precursor into the pigment or another pigment precursor.
  • Substrates can be provided by any appropriate method.
  • the substrate can be provided to cotton cells in a culture to activate/tune pigment/chromoprotein production.
  • Substrates can also be provided, for example, by activating or increasing expression of a gene encoding the substrate.
  • pigment/chromoprotein production is activated/modulated using a combination of inducible constructs and substrates.
  • Pigment/chromoprotein production can also be activated/modulated by a variety of other methods known in the art including, for example, addition of additional enzymes and enzyme co factors, altering environmental conditions to cause a particular response, and the like.
  • a gene or expression construct of the invention leads to the production of a pigment in the cotton fiber produced from the cell.
  • a pigment is any substance produced in the fiber of a cotton cell that provides a visually perceived color as a result selective spectral absorption and/or reflection.
  • Exemplary pigments used in the invention are plant pigments and/or flower pigments, which may be endogenous to plants that are not cotton plants.
  • Other exemplary pigments include those produced by other organisms, such as algae, fungi, chromists, protozoa, archaea, and bacteria.
  • Exemplary pigments produced in cotton fiber using the disclosed methods and compositions include melanins. Melanins are generally dark pigments. There are a variety of melanins produced by a number of plants and animals, which include, for example eumelanins and pheomelanins. Many melanins are produced in cells using a biosynthetic pathway, which includes a number of enzymes. Certain pathways, any component of which can be included in a genetic construct/vector of the invention, include a tyrosinase that converts tyrosine into L- DOPA, which is subsequently converted to dopaquinone. Depending on the pathway employed, dopaquinone is eventually converted to a pheomelanin or eumelanin.
  • An exemplary genetic construct/vector of the invention includes the genes TYRA and ORF438, which are activated to produce an expression product upon contact with an inducer.
  • the genes TYRA and ORF438 from S. antibioticus have been shown to induce production of melanin pigments in the fiber of cotton plants when included in an expression consent under the control of a cotton-specific LTP3 promoter as shown in Xu et ak, “Designing and transgenic expression of melanin gene in tobacco trichome and cotton fiber”, Plant Biol, 2007 Jan;9(l):41-8 (2007), which is incorporated by reference.
  • An exemplary genetic construct/vector of the invention includes the Me/A gene from, for example, a S. colwelliana bacterium, which causes production of melanin when expressed as a transgene in an E. coli host as shown in Fuqua et ak, “The melA gene is essential for melanin biosynthesis in the marine bacterium Shewanella colwelliana”, Microbiology, vok 139, issue 5 (1993), which is incorporated by reference.
  • Exemplary pigments used in accordance with the invention also include anthocyanins. Depending on the concentration, saturation, and identity of anthocyanin, these pigments can produce a red, purple, blue, or black coloration in cotton fiber.
  • Exemplary pigments used in accordance with the invention also include violacein, a purple pigment generally produced in certain types of bacteria.
  • violacein is produced from the five protein violacein operon, which include genes encoding the VioA, VioB, VioC, VioD, and VioE proteins.
  • Exemplary genetic constructs/vectors can include one or more components of the violacein operon.
  • Exemplary pigments used in accordance with the invention include betalains, which are a class of red and yellow tyrosine-based pigments, often found in plants of the order Caryophyllales .
  • Exemplary betalains used in accordance with the invention include betacyanins, which are reddish to violet betalain pigments.
  • Betacyanins found in plants include betanin, isobetanin, probetanin, and neobetanin.
  • Exemplary betalains used in accordance with the invention also include betaxanthins, which are yellow to orange. Betaxanthins present in plants include, for example, vulgaxanthin, miraxanthin, portulaxanthin, and indicaxanthin.
  • Biosynthesis of betalains requires three enzymatic reactions to convert tyrosine into betalain.
  • Tyrosine is hydroxylated by the P450 oxygenase CYP76AD1 on the benzene ring to produce 1-3,4-dihydroxyphenylalanine (1-DOPA).
  • 1-DOPA can be further oxidized into cyclo- DOPA by CYP76AD1.
  • 1-DOPA is catalyzed by 1-DOPA 4,5-dioxygenase (DODA) into betalamic acid, which is condensed with cyclo-DOPA into betanidin.
  • DODA 1-DOPA 4,5-dioxygenase
  • a sugar moiety is added to betanidin by a glucosyltransferase to generate betalain.
  • Exemplary pigments used in accordance with the invention include carotenes, which are terpenoids, synthesized from eight isoprene units.
  • An exemplary carotene useful in the invention is b-carotene, an orange/yellow pigment.
  • Exemplary constructs/vectors of the invention include the cotton phytoene synthase gene ( GhPSY2D ), which was shown in Yao et al., “Specific Upregulation of a Cotton Phytoene Synthase Gene Produces Golden Cottonseeds with Enhanced Provitamin A”, Scientific Reports (2018)8:1348, incorporated by reference, to produce colored cotton seeds when expressed.
  • the constructs/vectors of the invention produce one or more non pigment coloring agents in cotton fiber of cells contacted with an appropriate inducer and/or substrate.
  • Non-pigment coloring agents include, for example, chromoproteins. Chromoproteins are made in certain bacteria and zooxanthellae. Exemplary chromoproteins include, for example, meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue, aeBlue, amilCP (magenta), tsPurple, and gfasPurple.
  • one or more constructs are introduced into cotton cells, such that when induced and/or contacted with appropriate substrates, they lead to the production of a combination of pigments and chromoproteins as described herein.
  • construct/vectors are introduced into cotton cells.
  • the constructs/vectors include activatable/tunable genes that produce a betalain (red), an anthocyanin (purple), b-carotene (orange/yellow), amajLime (green), and cjBtlue (blue).
  • the cotton cell produces a combination of pigments and chromoproteins of unique colors. These colors can be combined at varying concentrations to produce cotton fiber of any desired color.
  • the cotton cells of the invention can also be modified to include a gene selected for improvement and/or modulation of cotton fiber development in vitro, such as those disclosed by the present inventors in U.S. Provisional Application No. 63/137,952, which is incorporated by reference.. These genes may improve/modulate cotton fiber development in cotton cells and/or cotton plants. However, genes that do not modulate/improve cotton fiber development in plants may nevertheless modulate/improve cotton fiber development using the in vitro methods disclosed herein.
  • the gene selected for improvement and/or modulation of cotton fiber development may be endogenous to one or more species or varietal of cotton plant, may be a gene from, or derived from, another species, or may be a synthetic gene.
  • the gene may be, or derived from, a cotton fiber development gene.
  • Expression or overexpression of genes such as GhHOX3, GhHD-1, SPL5, GaMYB2, iaaM, GhPIN3a, GhWlim5, and GhFPl has been shown to have a positive impact on cotton fiber growth (Cai, C., et ak, 2018; Liu, Z. et ak, 2020; Shan, C. et ak, 2014; Walford, S. et ak, 2012; Wang, S. et ak, 2004; Zhang, M., et ak, 2011; Mei, G. et ak, 2019; Iqbal, A. et ak, 2020).
  • the cotton cells of the invention can also be modified to include a developmental regulator gene, such as those disclosed by the present inventors in U.S. Provisional Application No. 63/137,952, which is incorporated by reference.
  • the cotton cells may be selected or modified to express at least one developmental regulator gene and/or at least one gene selected for improvement and/or modulation of cotton fiber development.
  • Developmental regulator genes such as Wus2 and Bbm have been shown to have a positive impact in com, increasing plant cell growth and transformation efficiency dramatically (Hoerster, G., 2020; Gordon-Kamm, B., 2019; Lowe, K., 2015; Lowe, K., 2018). In cotton.
  • these developmental growth regulators When transformed into cotton cells, such as ovule epidermal cells, these developmental growth regulators shorten the time and/or increase the number of cells that produce fiber when transformed either alone or in combination with one or more genes selected for improvement and/or modulation of cotton fiber development in vitro, such as GhHOX3, GhHD-1, SPL5, GaMYB2, iaaM, GhPIN3a, GhWlim5, and GhFPl.
  • Tissue specific expression of developmental regulator genes Wus2 and Bbm has been shown to be essential for normal plant development in prior studies in com (Hoerster, G., 2020; Gordon- Kamm, B., 2019; Lowe, K., 2015; Lowe, K., 2018). Therefore, tissue specific protomers were identified in cotton to ensure cotton fibers develop normally in vitro.
  • Exemplary promoters that can be used in accordance with the invention include cotton- specific promoters or promoter regions of endogenous cotton genes that have temporally and/or spatially regulated expression, including late embryogenesis-abundant gene D113 from cotton accumulates at high levels in mature seeds, leaves, embryos, and callus (Luo et al., 2008), GbPDFl, cotton PROTODERMAL FACTOR1 gene ( GbPDFl ) which is predominantly expressed in the epidermis of ovules and developing fibers during fiber initiation and early elongation (Deng et al., 2012), and GhMYB109, a cotton fiber specific R2R3 MYB gene (Pu et al., 2008).
  • transgenic reporter gene analysis has shown that a 2-kb GhMYB109 promoter was sufficient to confirm its fiber-specific expression via GUS staining.
  • a GhSCFP promoter was shown to specifically activate transcription in seed coat and fiber associated genes.
  • non-cotton specific constitutive protomers such as 35S promoter (such as CaMV 35 S), an FBP7 (petunia floral binding protein 7) promoter, a tobacco TA29 promoter, would function best, in the in vitro methods of the disclosure.
  • 35S promoter such as CaMV 35 S
  • FBP7 floral binding protein 7
  • tobacco TA29 promoter a tobacco TA29 promoter
  • One or more of the promoters used in the methods and compositions of the invention may be a PDF1 promoter, such as a GbPDFl promoter from G. barbadense or a GhPDFl promoter from G. hirsutum.
  • the cotton PROTODERMAL FACTOR1 gene PDF! is predominantly expressed in the epidermis of ovules and developing fibers during fiber initiation and early elongation (Deng et al., 2012). In cotton plants, GbPDFl was found to be preferentially expressed during fiber initiation and elongation, with a highest accumulation in fiber cells five days post anthesis.
  • GbPDFl promoter :GUS constructs in transgenic cotton predominantly expressed in the epidermis of ovules and developing fibers. (Deng et al., 2012). A 236 basepair promoter fragment of GbPDFl was shown to promote GbPDFl transcription in cotton. The temporally preferential expression of GbPDFl during fiber initiation and elongation makes GbPDFl promoters useful for controlling expression of a differently expressed gene in the in vitro methods of cotton production disclosed herein. Table 1 provides two exemplary cotton PDF1 promoters, which include the 236 base pair promoter fragment, and the full CDS of GbPDFl and GhPDFl.
  • GhACT genes Due to the tissue and temporal preferential expression of GhACT genes in plantae, their promoter sequences are useful as promoters for the differentially expressed genes used in the in vitro methods of cotton production disclosed herein. Exemplary promoter nucleic acid sequences of GhACT 1 are provided in Table 1.
  • GhMYB109 is a cotton fiber specific R2R3 MYB gene. Reporter gene analysis showed that a 2-kb GhMYB109 promoter provided fiber-specific expression using a GUS staining construct. (Pu et al., 2008). Due to this fiber-specific expression, GhMYB109 promoters are useful as promoters for controlling expression of a differently expressed gene in the in vitro methods of cotton production disclosed herein.
  • An exemplary GbMYB109 promoter is provided in Table 1.
  • G. hirsutum being fiber-specific promoter activates transcription in seed coat and fiber associated genes. (Yaqoob et al. 2020). Due to this preferential regulation of seed coat and fiber associated genes, GhSCFP promoters are useful as promoters for controlling expression of a differently expressed gene in the in vitro methods of cotton production disclosed herein. Exemplary GhSCFP promoters are provided in Table 1.
  • the promoter includes a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to anyone of the genes listed in Table 1, one or more fragments thereof, or an endogenous promoter sequence for such a gene.
  • Derivatives may include, one or more mutations, such as deletions, point mutations, restriction site alterations, nucleotide substitutions, additions and/or codon modifications. Derivatives may also include, for example, regulatory elements, and/or functional elements or modified functional elements.
  • Percent sequence identity refers to the percentage of identical nucleotides between two segments of a window of optimally aligned DNA.
  • Tools and methods for alignment are well known in the art, for example, the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman. These algorithms may be implemented as, or included in, computer programs, for example, GAP, BESTFIT, and FASTA.
  • An identity fraction for aligned sequence segments refers to the number of identical components that are shared by aligned test and reference sequences divided by the number of components in the reference sequence segment. Percent sequence identity is shown herein as the identity fraction multiplied by 100. The comparison of one or more DNA sequences may of an entire or full-length sequence or a portion thereof, or to a longer DNA sequence.
  • modifying the cotton cell may involve the use of a vector.
  • Vectors can be used to produce, transfer or manipulate transgenes, chimeric genes, and/or genetic constructs.
  • a vector is a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage or plant virus, into which a nucleic acid sequence may be inserted into a cotton cell plant, explant or cell.
  • a vector may include one or more unique restriction sites, capable of autonomous replication in a selected cotton cell, tissue, explant, or plant, or integrated into the genome of a cotton cell, plant, or explant such that the cloned sequence is reproducible.
  • the vector may be an autonomously replicating vector, which exists as an extrachromosomal entity, its replication independent of chromosomal replication, for example, a linear or closed circular plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector may be one which, when introduced into a cell, is integrated into the genome of the recipient cell and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system may include a single vector or plasmid, or two or more vectors or plasmids, which contain the DNA to be introduced into the genome of a cell.
  • a vector may also comprise a selection marker, for example, an antibiotic resistance gene that can be used for selection of suitable transformants.
  • a vector is used to introduce pigment- and/or chromoprotein-related genes into cotton cells.
  • the cells may be obtained or derived from the tissue from any meristematic part of a cotton plant or explant, including apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flower, flower stalks, floral whorls, roots, bulbs, germinated seeds, somatic and zygotic embryo, and/or cambial meristematic cells (CMC).
  • CMC cambial meristematic cells
  • the vector is used to introduce the gene(s)/construct(s) of the invention into a cotton plant, cotton seed, cotton explant, and/or cotton plant tissue.
  • cotton plants, explants, and/or cotton plant tissue can be grown. Once grown, cotton cells can be selected.
  • cotton plants are grown for one or more generations before cells are selected. The cotton plants can be backcrossed or crossed with other cotton plants to introduce desirable genetic backgrounds before the cells are selected.
  • cotton cells and/or plants can be modified with an induced mutation.
  • An induced mutation is an artificially induced genetic variation, for example, using chemical, radiation or biologically-based mutagenesis.
  • the resulting mutations may include nonsense mutations, frameshift mutations, additions deletions, insertional mutations or splice-site variants.
  • the mutations may modulate the activity of the selected gene.
  • the cotton cells used in the methods for producing colored cotton fiber in vitro may include the use of cotton cells that have a differently expressed gene (DEG).
  • DEG differently expressed gene
  • a differently expressed gene may have increased or decreased expression when compared to an endogenous and/or wild type gene.
  • the an endogenous and/or wild type gene may have no naturally occurring expression.
  • a DEG may also have a different spatial and/or temporal expression pattern when compared to an endogenous and/or wild type gene.
  • the DEG can be synthesized to include a promoter that assures the DEG is expressed at a certain level, under specific growth conditions, in certain tissues and tissue types, and/or under specified spatial and/or temporal constraints.
  • Cotton cells or plants of the present disclosure can be subject to a mutagenic process to give rise the DEG. This process can occur in vitro and without ever growing a whole cotton plant with the DEG.
  • Mutagenesis can be achieved by radiation and/or chemical means, including EMS or sodium azide treatment of seed, or gamma irradiation. Chemical mutagenesis favors nucleotide substitutions rather than deletions. Heavy ion beam (HIB) irradiation is a known technique for mutagenesis. Ion beam irradiation has two physical factors, the dose (gy) and LET (linear energy transfer, keV/um) for biological effects that determine the level of DNA damage and the size of any DNA deletion(s), and these can be adjusted according to change the extent of mutagenesis.
  • Biological agents can also be used to create site-specific mutations in cotton cells. These agents may include enzymes that cause double stranded breaks in DNA, which stimulate endogenous repair mechanisms. These enzymes include endonucleases, zinc finger nucleases, transposases and site-specific recombinases.
  • Isolation of cotton cells or plants with a selected gene may be achieved by screening mutagenized cotton plants or cells.
  • a mutagenized population of cotton plants may be screened directly for a particular genotype or indirectly by screening for a desired phenotype. Screening directly for the genotype may include assaying for the presence of mutations, e.g., using PCR- or sequencing-based assays.
  • cotton cells are selected from cotton plants produced using the process known as TILLING (Targeting Induced Local Lesions IN Genomes).
  • TILLING Targeting Induced Local Lesions IN Genomes
  • introduced mutations are induced in a population of plants by treating seeds or pollen with a chemical or radiation mutagen, and then advancing plants to a generation where mutations will be stably inherited, typically an M2 generation where homozygotes may be identified.
  • DNA is extracted, and seeds are stored from all members of the population to create a resource that can be accessed repeatedly over time.
  • PCR primers are designed to specifically amplify a single gene target of interest. PCR products from pooled DNA of multiple individuals are amplified using the primers.
  • mismatched base pairs are denatured and reannealed to allow the formation of mismatched base pairs.
  • Mismatches or heteroduplexes, represent both naturally occurring single nucleotide polymorphisms (SNPs) (i.e., several plants from the population are likely to carry the same polymorphism) and induced SNPs (i.e., only rare individual plants are likely to display the mutation).
  • SNPs single nucleotide polymorphisms
  • induced SNPs i.e., only rare individual plants are likely to display the mutation.
  • the use of an endonuclease that cleaves mismatched DNA is the key to discovering novel SNPs within a TILLING population.
  • the cotton cells, plants, and/or explants (or engineered cotton) described herein can be derived from a Gossypium species.
  • the Gossypium species can be selected from the group consisting of G. arboreum, G. anomalum, G. armourianum, G. klotzchianum, and G. raimondii.
  • the cotton (or engineered cotton) can be derived from a Gossypium species selected from the group consisting of G. hirsutum, G. arboreum, G. barbadense, G. anomalum, G. armourianum, G. klotzchianum, and G. raimondii.
  • the cotton (or engineered cotton) can be Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum, Gossypium herbaceum, or another species of cotton.
  • a bioreactor is inoculated 105 with a small number of the modified and/or selected cotton cells.
  • the bioreactor may be inoculated with a small number of cotton ovule cells, which may include ovule epidermal cells.
  • the bioreactor will be inoculated with a small number of cotton cells from a proliferating cell aggregate. As shown in the examples below, milligram quantities of cotton cells from a proliferating cell aggregate are sufficient to eventually inoculate a bioreactor.
  • Inoculating 105 may include preparing a growth medium in a vessel, such as a flask or plate, and introducing a small number of cotton cells from a proliferating cell aggregate into the medium. The vessel may then be left for inoculum growth. Alternatively, inoculum growth may occur inside the bioreactor.
  • a vessel such as a flask or plate
  • the Inventors found that, surprisingly, inoculum growth under dark conditions provided superior growth.
  • the vessel may be shaken or agitated during inoculum growth, for example, at a rate of 80-180 rpm.
  • inoculum growth occurs at a temperature of about 30 °C to about 35 °C.
  • the medium is a solution that comprises plant hormones, plant growth regulators, and/or sucrose and/or glucose.
  • Inoculum growth generally takes about 16 days, but may be more or less as desired or due to conditions or individual cotton cell lines.
  • the inoculum may be a cell suspension in a liquid or semi-solid medium.
  • the suspension may be optionally homogenized to provide a fine cell suspension culture.
  • the present Inventors discovered that a homogenous cell suspension can provide more reproducible and reliable results when inoculating a bioreactor.
  • Homogenizing may include any methods known in the art, including one or more of subculturing the suspension, filtering, pipetting/decanting, and/or addition of a low concentration of pectinase.
  • the resulting inoculum is then introduced into a bioreactor.
  • the resulting inoculum can be preserved, e.g., by freezing, for later use in inoculating a bioreactor.
  • the inoculum or homogenous cell suspension which includes cells that include one or more selected genes of interest, may be cryopreserved indefinitely, for example, in liquid nitrogen. This generally requires suspending cells from the inoculum/homogenous cell suspension in a cryoprotectant solution, for example a solution of glycerol and sucrose.
  • the cryoprotectant solution can be supplement, for example, using proline.
  • Cryopreserved cells can be recovered, for example, using a recovery media, before their use in inoculating a bioreactor.
  • the proliferating cell aggregate may be a callus.
  • the proliferating cell aggregate is a friable callus, which is not sticky or soft, but is also not so hard or dense that it cannot be physically broken or crumbled.
  • a friable callus thus differs “a hard callus”, which is compact and brittle, and thus not amenable to being broken or crumbled.
  • the Inventors discovered that a friable callus allows for simple mechanical manipulation to easily disassociate individual cells from the friable callus for use in inoculating 105 a bioreactor and/or preparing an inoculum.
  • the method 101 requires multiplying 107 the cells in the bioreactor. This phase generally lasts for between 5 and 12 days, with duplication for the cotton cells taking approximately 1 to 3 days depending on cotton lineage.
  • the cells may be duplicated, for example, by culturing the cells in a cell culture medium.
  • the multiplied cells are then elongated 109 to produce cotton fibers.
  • This may include using an elongation medium to induce elongation in the multiplied cells.
  • the elongation medium facilitates release of a phenolic compound from a vacuole of an elongated cotton cell.
  • the elongated cells may include cotton pre-fibers, which will mature into cotton fibers with a desired coloration.
  • a semi-solid elongation medium is used to elongate the cotton cells.
  • the Inventors made the surprising discovery that superior results are achieved when using a semi-solid medium as opposed to a liquid medium.
  • the elongated cotton cells are separated from any non- elongated cotton cells.
  • the non-elongated cotton cells will not mature into cotton fibers. However, they may be recycled and used in subsequent iterations of the method. Separating the elongated cotton cells from the non-elongated cells may include one or more of filtering, sieving, decanting, and centrifuging the cells.
  • the elongated cotton cells which at this point may have cotton pre-fibers, are matured.
  • Maturing the cells may include the use of a maturation medium.
  • sugars are combined in the cells to produce cellulose, which is the main component of cotton fiber (natural glucose polymerization) that occurs inside the cell forming a secondary wall.
  • the cotton pre-fibers increase in number, density, and/or length.
  • cotton fiber harvested 111 from the cotton cells by for example, separating the fibers from the cells in a solution/buffer.
  • the harvested cotton fiber is then dried to a moisture content of less than 5% by, for example, passing air through the cotton fiber.
  • the method 101 can produce cotton fibers from cotton cells without growing cotton plants. These methods allow quick and efficient cultivation of cotton fiber in a controlled environment.
  • the method 101 may also include preparing a friable callus.
  • a friable callus can be made, for example, by obtaining cells from a cotton explant and contacting the cells with a callus induction medium. Surprisingly, the Inventors discovered that tissue from any meristematic part of a cotton plant can be used to produce a friable callus.
  • the cells from the cotton explant can from cotton apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flower, flower stalks, floral whorls, roots, bulbs, germinated seeds, somatic and zygotic embryo, and/or cambial meristematic cells (CMC).
  • CMC cambial meristematic cells
  • Preparing a friable callus may include contacting the cells of a cotton explant with a callus induction medium.
  • the callus induction medium may facilitate the division of at least a subset of cells of a plant explant. Using the callus induction medium results in dedifferentiated cell masses. The cells in these masses can be subsequently cultured, which may include the use of a callus growth medium.
  • hormones and/or growth regulator(s) including auxins, gibberilins, etc.
  • the hormones/regulators can be used, for example, in the mediums described herein for culturing cotton cells.
  • Plant hormones and/or growth regulators can be derived from naturally occurring sources, synthetically produced, or semi-synthetically produced, i.e. starting from naturally derived starting materials then synthetically modifying said materials. These modifications can be conducted using conventional methods as envisioned by a skilled worker.
  • plant hormones and/or growth regulators used in the present invention are exemplified by those in Table A.
  • Table A Exemplary plant hormones or plant growth regulators and exemplary applications in plant cell engineering.
  • Y indicates that the corresponding plant hormone or plant growth regulator in the row can be used for the application indicated in the column heading.
  • “Inhibitor” indicates that the corresponding plant hormone or plant growth regulator in the row can be used for inhibiting the activity indicated in the column heading. “ND” indicates that effect(s) of the corresponding plant hormone or plant growth regulator for the application indicated in the column heading is not yet determined (at least to some extent).
  • the invention uses an induction medium or callus induction medium.
  • the callus induction medium described herein can be configured to facilitate division of at least a subset of cells of a plant explant.
  • the callus induction medium can facilitate or promote induction of a cotton plant callus.
  • the callus induction medium can comprise a diluted basal medium (i.e., from 1:1.5 to 1:5, from 1:1.5 to 1:4, from 1:1.5 to 1:3, etc.).
  • the callus induction medium can comprise one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones (such as those that can facilitate or promote induction).
  • the callus induction medium can be a liquid at about 25 °C.
  • the callus induction medium can be not a liquid at a specified temperature.
  • the callus induction medium is not a liquid at about 25 °C.
  • the callus induction medium can be a semi- solid medium (such as gelled) at 25 °C.
  • Non-limiting examples of a semi-solid medium include soft agar, soft agarose, soft methylcellulose, xantham gum, gellan gum, carrageenan, isabgol, guar gum, other soft polymeric gels, or any other gelling agent known in the art.
  • the callus induction medium can comprise agar.
  • the callus induction medium can be agar-free.
  • the callus induction medium is free of any gelling agent.
  • the callus induction medium that is agar- or gelling agent-free can be a liquid.
  • the callus induction medium that is agar- or gelling agent-free can be a solid.
  • the callus induction medium that is agar-free can be a gel. In some embodiments, the callus induction medium that is agar-free can comprise an agar-substitute. In some embodiments, the callus induction medium can have a pH. The pH of the callus induction medium can be appropriate for induction of a plant callus. In some embodiments, the pH of the callus induction medium can be optimized for induction of a plant callus. In some embodiments, the pH of the callus induction medium can be from 5.3 to 6.3.
  • the pH of the callus induction medium can be, or be about, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or a range between any two foregoing values.
  • the present disclosure includes callus mediums or callus growth mediums, and their use in the in vitro methods for producing cotton.
  • the callus growth medium described herein can facilitate or promote growth of a plant callus and/or produce a proliferating cell aggregate.
  • the callus growth medium can be a gel medium, and in some embodiments, can comprise agar and/or another gelling agent and a mixture of macronutrients and micronutrients for the plant type of the plant callus.
  • the callus medium can be enriched with nitrogen, phosphorus, or potassium.
  • a callus growth medium can be a liquid medium.
  • the callus growth medium can comprise at least one plant hormone or growth regulator (including auxins, gibberilins, etc.), or at least two plant hormones or growth regulators, or at least three plant hormones or growth regulators, or at least four plant hormones or growth regulators, or at least five plant hormones or growth regulators, or at least six plant hormones or growth regulators, or at least seven plant hormones or growth regulators, or at least eight plant hormones or growth regulators.
  • plant hormone or growth regulator including auxins, gibberilins, etc.
  • the at least one plant hormone or plant growth regulator (or at least two, at least three, at least four, at least five, or at least six plant hormones or plant growth regulators) (including auxins, gibberilins, etc.) can be any one or combination selected from the group consisting of indole acetic acid (IAA), Indoy 1-3 -acrylic acid, 4-C1- Indoy 1-3 -acetic acid, Indoyl-3-acetylaspartate, indole-3-acetaldehyde, indole-3 -acetonitrile, indole-3-lactic acid, indole-3-propionic acid, indole-3-pyruvic acid, indole butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4 D), tryptophan, phenylacetic acid (PAA), Glucobrassicin, naphthaleneacetic acid (NAA), picloram (PIC), Dicamba, ethylene
  • the at least one plant hormone or plant growth regulator (or at least two, at least three, at least four, at least five, or at least six plant hormones or plant growth regulators) (including auxins, gibberilins, etc.) can be any one or combination selected from the group consisting of indoy 1-3 -acetic acid, indoyl-3-acrylic acid, indoyl-3-butyric acid, 4-Cl-Indoyl-3-acetic acid, Indoy 1-3 -acetylaspartate, indole-3-acetaldehyde, indole-3-acetonitrile, indole-3-lactic acid, indole-3-propionic acid, indole-3-pyruvic acid, tryptophan, phenylacetic acid, Glucobrassicin, 2,4-Dichlorophenyoxyacetic acid, 1- naphthaleneacetic acid, Dicamba, Pichloram, ethylene, benzo(b)se
  • the at least one plant hormone or plant growth regulator (or at least two, at least three, at least four, at least five, or at least six plant hormones or plant growth regulators) (including auxins, gibberilins, etc.) can be any one or combination selected from the group consisting of indole acetic acid (IAA), indole butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4 D), naphthaleneacetic acid (NAA), para-chlorophenoxyacetic acid (pCPA), b-naphthoxyacetic acid (NOA), 2-benzothiazole acetic acid (BTOA), picloram (PIC), 2,4,5,-trichlorophenoxyacetic acid (2,4,5-T), phenylacetic acid (PAA), kinetin (KIN), 6-benzylaminopurine (6BA), N6-(2- isopentenyl) adenine (2iP), zeatin (ZEA), gibberellin
  • the callus growth medium can be a liquid at about 25 °C. In some embodiments, the callus growth medium can be not a liquid at about 25 °C. In some embodiments, the callus growth medium can be a semi-solid medium (such as gelled) at 25 °C. Non-limiting examples of a semi-solid medium include soft agar, soft agarose, soft methylcellulose, xantham gum, gellan gum, carrageenan, isabgol, guar gum, other soft polymeric gels, or any other gelling agent known in the art. In some embodiments, the callus growth medium can comprise agar. In some embodiments, the callus growth medium can be agar-free.
  • the callus growth medium is free of any gelling agent.
  • the callus growth medium that is agar- or gelling agent-free can be a liquid.
  • the callus growth medium that is agar- or gelling agent-free can be a solid.
  • the callus growth medium that is agar-free can be a gel. In some embodiments, the callus growth medium that is agar-free can comprise an agar-substitute.
  • the callus growth medium can have a pH.
  • the pH of the callus growth medium can be appropriate for growing a plant callus and/or producing a proliferating cell aggregate.
  • the pH of the callus growth medium can be optimized for growing a plant callus and/or producing a proliferating cell aggregate.
  • the pH of the callus growth medium can be from 5.3 to 6.3.
  • the pH of the callus growth medium can be, or be about, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or a range between any two foregoing values.
  • the present invention includes cell culture mediums (e.g., amultiplication/duplication mediums), and their use in the in vitro methods for producing cotton described herein.
  • the cell culture medium described herein can facilitate or promote proliferation of a cell population, or a proliferating cell aggregate.
  • the cell culture medium can comprise one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones (such as those that can facilitate or promote proliferation).
  • the cell culture medium can be configured to proliferate a cell population, such as a proliferating cell aggregate.
  • the cell culture medium can comprise an enzyme that can degrade a plant cell wall of a plant cell of a cell population, or a proliferating cell aggregate.
  • the enzyme can be a pectocellulolytic enzyme.
  • the enzyme can comprise cellulase, hemicellulose, cellulysin, or a combination thereof.
  • the cell culture medium can have a pH. The pH of the cell culture medium can be appropriate for culturing a cell population, or a proliferating cell aggregate.
  • the pH of the cell culture medium can be optimized for culturing a cell population, such as a proliferating cell aggregate. In some embodiments, the pH of the cell culture medium can be optimized for cell division. In some embodiments, the pH of the cell culture medium can be from 5.3 to 6.3. In some embodiments, the pH of the cell culture medium can be, or be about, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or a range between any two foregoing values.
  • the cell culture medium can have a different pH than a callus growth medium. In some embodiments, the cell culture medium can have a same pH as a callus growth medium. In some embodiments, the pH of the cell culture medium can differ from a pH of a callus growth medium by less than 0.1, less than 0.2, or less than 0.3 units. For example, the pH of a cell culture medium can differ from a pH of a callus growth medium by less than 0.2 units.
  • a cell culture medium of the present disclosure includes one or more of MS, B5, glucose, sucrose, Kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D), NAA, and coconut water.
  • the cell culture medium comprises 2,4-D.
  • the cell culture medium includes MS, B5, glucose/sucrose, and 2,4-D.
  • the present invention also includes recovery mediums, and their use in the in vitro methods for producing cotton fiber.
  • a recovery medium can be used, for example, for recovery of cotton cell inoculum after cryopreservation.
  • Some embodiments described herein are related to a recovery medium.
  • the recovery medium described herein can be a medium that can facilitate or promote recovery of cotton cells.
  • the recovery medium can comprise one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones that can facilitate or promote elongation.
  • the present invention includes elongation mediums, and their use in the in vitro methods for producing cotton fiber.
  • the elongation mediums described herein can facilitate or promote elongation of cells capable of being elongated, for example, elongation of cotton cells.
  • the elongation mediums described herein can comprise one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones (such as those that can facilitate or promote elongation).
  • the elongation mediums can be configured to facilitate a release of a phenolic compound from a vacuole from a cotton cell.
  • the phenolic compound (such as O-diphenol) is configured to initiate fiber differentiation by inhibiting indoleacetic acid (IAA) oxidase and/or increase an intracellular auxin level.
  • the elongation medium can comprise at least one plant hormone or growth regulator (including auxins, gibberilins, etc.), or at least two plant hormones or growth regulators, or at least three plant hormones or growth regulators, or at least four plant hormones or growth regulators, or at least five plant hormones or growth regulators, or at least six plant hormones or growth regulators, or at least seven plant hormones or growth regulators, or at least eight plant hormones or growth regulators.
  • the at least one plant hormone or plant growth regulator (or at least two, at least three, at least four, at least five, or at least six plant hormones or plant growth regulators) (including auxins, gibberilins, etc.) can be any one or combination selected from the group consisting of indole acetic acid (IAA), Indoy 1-3 -acrylic acid, 4-C1- Indoyl-3-acetic acid, Indoyl-3-acetylaspartate, indole-3-acetaldehyde, indole-3 -acetonitrile, indole-3 -lactic acid, indole-3-propionic acid, indole-3-pyruvic acid, indole butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4 D), tryptophan, phenylacetic acid (PAA), Glucobrassicin, naphthaleneacetic acid (NAA), picloram (PIC), Dicamba, ethylene
  • the at least one plant hormone or plant growth regulator (or at least two, at least three, at least four, at least five, or at least six plant hormones or plant growth regulators) (including auxins, gibberilins, etc.) can be any one or combination selected from the group consisting of indoyl-3-acetic acid, indoyl-3-acrybc acid, indoyl-3-butyric acid, 4-Cl-Indoyl-3-acetic acid, Indoyl-3-acetylaspartate, indole-3-acetaldehyde, indole-3-acetonitrile, indole-3-lactic acid, indole-3-propionic acid, indole-3-pyruvic acid, tryptophan, phenylacetic acid, Glucobrassicin, 2,4-Dichlorophenyoxyacetic acid, 1- naphthaleneacetic acid, Dicamba, Pichloram, ethylene, benzo(b)s
  • the at least one plant hormone or plant growth regulator (or at least two, at least three, at least four, at least five, or at least six plant hormones or plant growth regulators) (including auxins, gibberilins, etc.) can be any one or combination selected from the group consisting of indole acetic acid (IAA), indole butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4 D), naphthaleneacetic acid (NAA), para-chlorophenoxyacetic acid (pCPA), b-naphthoxyacetic acid (NOA), 2-benzothiazole acetic acid (BTOA), picloram (PIC), 2,4,5,-trichlorophenoxyacetic acid (2,4,5-T), phenylacetic acid (PAA), kinetin (KIN), 6-benzylaminopurine (6BA), N6-(2- isopentenyl) adenine (2iP), zeatin (ZEA), gibberellin
  • the callus growth medium can be a liquid at about 25 °C. In some embodiments, the callus growth medium can be not a liquid at about 25 °C. In some embodiments, the callus growth medium can be a semi-solid medium (such as gelled) at 25 °C. The present Inventors discovered that a semi-solid medium provides beter results than a liquid medium. Non-limiting examples of a semi-solid medium include soft agar, soft agarose, soft methylcellulose, xantham gum, gellan gum, carrageenan, isabgol, guar gum, other soft polymeric gels, or any other gelling agent known in the art. In some embodiments, the callus growth medium can comprise agar. In some embodiments, the callus growth medium can be agar-free.
  • the callus growth medium is free of any gelling agent.
  • the callus growth medium that is agar- or gelling agent-free can be a liquid.
  • the callus growth medium that is agar- or gelling agent-free can be a solid.
  • the callus growth medium that is agar-free can be a gel. In some embodiments, the callus growth medium that is agar-free can comprise an agar-substitute.
  • the elongation medium can have a pH.
  • the pH of the elongation medium can be appropriate for producing/inducing an elongated cell, such as an elongated cotton cell or a plurality of elongated cotton cells.
  • the pH of the elongation medium can be optimized for cell elongation (such as cotton cell elongation).
  • the pH of the elongation medium can be from 5.3 to 6.3.
  • the pH of the elongation medium can be, or be about, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or a range between any two foregoing values.
  • the present invention includes elongation mediums, and their use in the in vitro methods for producing cotton fiber.
  • the maturation mediums described herein can facilitate or promote maturation of cells, such as maturation of cotton cells.
  • a maturation medium can comprise one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones (such as those that can facilitate or promote maturation).
  • the maturation medium can comprise a maturation reagent.
  • the maturation reagent of the maturation medium can be a wall-regeneration reagent.
  • the present invention includes the use of proliferating cell aggregates, and their creation, for use in the in vitro methods of cotton fiber production.
  • the plant cell composition as described hereinbelow or described anywhere else herein can be derived from the proliferating cell aggregate.
  • the proliferating cell aggregate can be an aggregate of plant cells that are proliferating. Proliferating cells in an aggregate can be attached or connected to each other, for example, via cell to cell interactions.
  • the proliferating cell aggregate can be a friable callus is friable, which is not sticky or soft, but is also not so hard or dense that it cannot be physically broken or crumbled.
  • a friable callus thus differs “a hard callus”, which is compact and brittle, and thus not amenable to being broken or crumbled.
  • the callus is a friable callus.
  • the present Inventors discovered that a friable callus can have individual cells dissociated from the callus using simple mechanical manipulation.
  • Proliferating cells can be of one type (a homogenous aggregate) or of two or more types (a heterogeneous aggregate).
  • the proliferating cell aggregate can be a mixed aggregate (e.g., where cell types are mixed together), a clustering aggregate (e.g., where cells of different types are tending toward different parts of the aggregate), or a separating aggregate (where cells of different types are pulling apart from each other).
  • Cells of the proliferating cell aggregate can divide at a rate greater than a cell division rate of remaining cells in said plant callus. In some embodiments, cells of the proliferating cell aggregate can divide at a rate that can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 times greater than a cell division rate of plant callus cells.
  • the present invention includes the use of cells from a cotton plant cell callus and methods for preparing such a callus.
  • the plant callus can be a growing mass of plant parenchyma cells.
  • the Inventors discovered that, surprisingly, cells from any meristematic part of a cotton plant are sufficient for callus induction.
  • the plant callus can be created using cells obtained or derived from cotton apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flower, flower stalks, floral whorls, roots, bulbs, germinated seeds, somatic and zygotic embryo, and/or cambial meristematic cells (CMC).
  • CMC cambial meristematic cells
  • the mass of plant parenchyma cells can be unorganized.
  • the plant callus can be collected from cells covering the wound of a plant or plant part.
  • the plant callus is created by inducing a plant tissue sample (e.g., an explant) with a callus induction medium.
  • induction of an explant can occur after surface sterilization and plating onto a medium in vitro (e.g., in a closed culture vessel such as a Petri dish).
  • Induction can comprise supplementing the medium with plant growth regulators, such as auxins, cytokinins, or gibberellins to initiate callus formation.
  • plant growth regulators such as auxins, cytokinins, or gibberellins to initiate callus formation.
  • Induction can be performed at a temperature of, or of about, 20 °C, 25 °C, 28 °C, 30 °C, 35 °C, or 40 °C, or a range between any two foregoing values.
  • compositions comprising cotton plant cells are included in the present invention.
  • the plant cell compositions described herein can be a final product of a method for preparation of cell bank stocks provided herein.
  • the plant cell compositions can be compositions of engineered cells, or a compositions of wildtype cells.
  • the plant cell compositions can be cell bank stocks.
  • the plant cell compositions can comprise a plurality of plant cells obtained by growing a callus in a growth medium to produce a proliferating cell aggregate followed by culturing the proliferating cell aggregate.
  • the plant cell compositions described herein can be in a growth phase.
  • the growth phase can comprise cell division, cell enlargement, and/or cell differentiation.
  • the growth phase comprising cell division can be an exponential growth phase (e.g., dowaiting).
  • the exponential growth phase can occur as cells are mitotic.
  • each generation of cells can be twice as numerous as the previous generation. In some embodiments, not all cells may survive in a given generation.
  • each generation of cells can be less than twice as numerous as the previous generation.
  • the exponential growth phase can be determined (e.g., quantified or identified) by a cell viability assay.
  • another aspect of the plant cell composition can be determined by a cell viability assay.
  • the cell viability assay can be an assay that can determine the ability of a cell to maintain or recover viability.
  • the cells of the plant cell composition can be assayed for their ability to divide or for active cell division.
  • the cell viability assay can be an ATP test, calcein AM, clonogenic assay, ethidium homodimer assay, Evans blue, fluorescein diacetate hydrolysis / propidium iodide staining (FDA/PI staining), flow cytometry, formazan- based assays (e.g., MTT or XTT), green fluorescent protein based assays, lactate dehydrogenase (LDH) based assays, methyl violet, neutral red uptake, propidium iodide, resazurin, trypan blue, or a TUNEL assay.
  • the cell viability assay can determine a cytoplasmic level of diphenol compounds in the plant cell composition.
  • bioreactors configured to produce any one or more compositions associated with the in vitro production of fiber as disclosed herein
  • a bioreactor can be configured to produce a cell bank stock.
  • a bioreactor can be configured to carry out a method for preparing a cell bank stock.
  • a bioreactor can be configured to utilize components of a kit for preparation of a cell bank stock, such as a callus growth medium and/or a multiplication medium.
  • FIG. 7 provides a flow chart illustrating an example of different processes that can be performed by a bioreactor, and how these processes can be interconnected.
  • a bioreactor can be configured to produce a cotton fiber. In some embodiments, a bioreactor can be configured to carry out a method for large scale cotton fiber production. In some embodiments, a bioreactor can be configured to carry out a method for rapid cotton fiber production. In some embodiments, a bioreactor can be configured to utilize components of a kit for large scale fiber production. In some embodiments, a bioreactor can be configured to utilize components of a kit for rapid fiber production.
  • a bioreactor can be configured to produce engineered cotton.
  • a bioreactor can be configured to utilize components of a kit for production of engineered cotton, which can comprise elements of kits provided herein.
  • the present invention includes computer systems that are programmed to implement methods of the disclosure.
  • FIG. 7 shows a computer system 701 that is programmed or otherwise configured to provide and/or implement instructions for or means of implementation of induction, callus growth, cell culture, elongation, or maturation.
  • the computer system 701 can regulate various aspects of induction, callus growth, cell culture, elongation, or maturation of the present disclosure.
  • the computer system 701 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system 701 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 705, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 701 also includes memory or memory location 710 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 715 (e.g., hard disk), communication interface 720 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 725, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory 710, storage unit 715, interface 720 and peripheral devices 725 are in communication with the CPU 705 through a communication bus (solid lines), such as a motherboard.
  • the storage unit 715 can be a data storage unit (or data repository) for storing data.
  • the computer system 701 can be operatively coupled to a computer network (“network”) 730 with the aid of the communication interface 720.
  • the network 730 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network 730 in some cases is a telecommunication and/or data network.
  • the network 730 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network 730, in some cases with the aid of the computer system 701, can implement a peer-to-peer network, which may enable devices coupled to the computer system 701 to behave as a client or a server.
  • the CPU 705 can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 710.
  • the instructions can be directed to the CPU 705, which can subsequently program or otherwise configure the CPU 705 to implement methods of the present disclosure. Examples of operations performed by the CPU 705 can include fetch, decode, execute, and writeback.
  • the CPU 705 can be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the system 701 can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • the storage unit 715 can store files, such as drivers, libraries and saved programs.
  • the storage unit 715 can store user data, e.g., user preferences and user programs.
  • the computer system 701 in some cases can include one or more additional data storage units that are external to the computer system 701, such as located on a remote server that is in communication with the computer system 701 through an intranet or the Internet.
  • the computer system 701 can communicate with one or more remote computer systems through the network 730. For instance, the computer system 701 can communicate with a remote computer system of a user.
  • remote computer systems examples include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants.
  • the user can access the computer system 701 via the network 730.
  • Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 701, such as, for example, on the memory 710 or electronic storage unit 715.
  • the machine executable or machine-readable code can be provided in the form of software.
  • the code can be executed by the processor 705.
  • the code can be retrieved from the storage unit 715 and stored on the memory 710 for ready access by the processor 705.
  • the electronic storage unit 715 can be precluded, and machine-executable instructions are stored on memory 710.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.
  • “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • a machine readable medium such as computer-executable code
  • a tangible storage medium such as computer-executable code
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory, such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
  • Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.
  • RF radio frequency
  • IR infrared
  • Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data.
  • Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the computer system 701 can include or be in communication with an electronic display 735 that comprises a user interface (UI) 740 for providing, for example, instructions for or means of implementation of induction, callus growth, cell culture, elongation, or maturation.
  • UI user interface
  • Examples of UFs include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • Methods and systems of the present disclosure can be implemented by way of one or more algorithms.
  • An algorithm can be implemented by way of software upon execution by the central processing unit 705.
  • the algorithm can, for example, provide and/or execute instructions for or means of implementation of inducing expression of pigments/chromoproteins at selected concentrations, callus growth, cell culture, elongation, or maturation.
  • Example 1 Preparation of a plant cell composition
  • cells are isolated by placing sterilized explants from apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flower, flower stalks, floral whorls, roots, bulbs, germinated seeds, somatic and zygotic embryo, and cambial meristematic cells (CMC) on a callus induction medium (e.g., a semi-solid basal salts medium) for induction.
  • a callus induction medium e.g., a semi-solid basal salts medium
  • the dedifferentiated masses formed are conditioned by passing three up to five subculturing at intervals of 21-26 days on a callus growth medium (e.g., a semi-solid basal salts medium) for growth.
  • Example 2 Crvopreservation of suspension-cultured cells
  • Cryovials containing cell suspension in LS are cooled to -35 °C at a rate of -0.5, -1, or -2 °C min _1 using a programmable freezer. After reaching -35 °C, cells are kept at -35 °C for 0, 30, or 60 minutes, and then plunged into liquid nitrogen.
  • the vials containing cryopreserved cells are transferred from the liquid nitrogen storage vessel into a Dewar flask containing liquid nitrogen. Each vial is transferred (one by one) to a clean 35-40 °C water bath and gently flipped several times until thawed (the last piece of ice disappears). Immediately, each vial is placed on ice again. Each vial is centrifuged at 100 g, at 4 °C for 1-2 min. The outside of each vial is wiped with 70% (vol/vol) ethanol and the supernatant from each vial is removed using a sterile Pasteur pipette. A sterile 3.5-ml transfer pipette is used to transfer two-thirds’ volume of the cells by spreading or placing them as a few clusters onto the filter paper. The dish is closed and sealed with Parafilm.
  • the dish(es) are covered with one or two sheets of filter paper to reduce the light intensity then placed in the culture room in regular conditions (24-26 °C). After 2 days of recovery, a spatula (width of 4 mm) is used to collect some cell mass (about 100-200 mg FW) from the plate and place into a microtube for viability testing. The remaining cells are transferred with the upper filter paper to a fresh recovery dish containing recovery medium. The dishes are closed and sealed, covered with filter paper, and then returned to the culture room.
  • cells are allowed to grow for an additional number of days in the same culture room, in regular conditions (24-26 °C).
  • the cell mass is transferred to a fresh dish containing recovery medium without filter paper for a further 1-2 weeks under standard conditions (at this recovery stage, agarose may be replaced by agar or another gelling agent). After a recovery period of 3-9 weeks, cells are transferred to a liquid medium to initiate suspension culture.
  • DI deionized
  • FIG. 8 An illustrative schematic of the bioreactor can be found in FIG. 8.
  • the bioreactor is fed with in vitro cells, with sterilized medium, and air compression.
  • the bioreactors are connected to the controller prior to inoculation, to stabilize pH 5.8 ( ⁇ 0.2) and to control and calibrate the flow of C .
  • the first vessel of the inoculum train occurs at a temperature from about 30 °C to about 35 °C with a lOOg L of cells at an exponential phase.
  • the sterilization of the culture medium occurs at approximately 125 to approximately 140 °C and returns (stream 16) to the heat exchanger (stream 13) to cooling the medium at a temperature from about 30 °C to about 35 °C (E-103).
  • the sterile medium is ready to feed the reactors of the multiplication area (reactors R-101 to R-104).
  • the air for cell oxygenation is also adjusted to the process temperature in the heat exchanger (E-105) and thus is split into four different streams (streams 27, 28, 29 and 31) that feed the inoculum train (reactors R-101 to R-104).
  • the multiplication occurs in a duration from 5 to 12 days for cells, and the duplication time is approximately 1 day to 3 days (depending on linage(s)). These times conclude when the cell amount increases, for example, 64 times.
  • the content is loaded to the next reactor (R-102) and so on.
  • the last reactor (R-104) has an adjacent lung tank, where after the reaction the contents are discharged in the batch feeding tank (Tq-101) with continuous output (stream 5).
  • Tq-101 batch feeding tank
  • Table B below, provides experimental results showing the success of inoculating a bioreactor using cotton cells in accordance with the methods disclosed herein. Table B provides details regarding the cotton varieties from which the cells were obtained, the cell growth medium which was inoculated in the bioreactor, and other relevant conditions.
  • the present disclosure provides methods of inoculating a bioreactor using a cotton cell culture, wherein the inoculation occurs under dark conditions.
  • the composition of the growth medium used when inoculating a bioreactor has an impact on cell growth.
  • the present disclosure provides methods of inoculating a bioreactor with cotton cells, wherein the growth medium comprises plant hormones or growth regulators.
  • the growth medium included 2,4- dichlorophenoxyacetic acid (2,4-D), growth improved.
  • the present disclosure provides methods of inoculating a bioreactor with cotton cells, wherein the growth medium comprises 2,4-dichlorophenoxyacetic acid (2,4-D).
  • the methods of the present disclosure allowed successful cell growth when inoculating a bioreactor with cells from all cotton varieties tested.
  • the present disclosure provides methods of inoculating a bioreactor with any of the cotton varieties disclosed herein.
  • the cotton cells used to inoculate a bioreactor in accordance with the methods disclosed herein are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from PAYMASTER HS26, PD 2164, SA 2413, SEALAND #1 (G.B. X G.H.), SOUTHLAND Ml, STATION MILLER, TASHKENT 1, TIDEWATER 29 (G.B.
  • the cotton cells used to inoculate a bioreactor in accordance with the methods disclosed herein are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from PD 2164, Acala MAXXA, FJA, Pima S-7, or a progeny of any thereof.
  • Pima S-7 provided good growth when inoculating a bioreactor. This included when using milligram quantities of cotton cells to form an inoculum and across a range of growth mediums. Unexpectedly, Pima S-7 provided superior growth/inoculation compared to Acala MAXXA and FJA. Moreover, this superior growth occurred even when using the same growth medium. For example, as shown in Table B, Pima S-7 provided good growth, while Acala MAXXA and FJA showed poor growth when all were cultured using a growth medium with the same concentrations MS, B5, glucose, Kinetin, and 2,4-D. Accordingly, the present disclosure provides inoculating a bioreactor with cells derived and/or obtained, in whole or in part, from a cotton plant of the Pima S-7 variety, or a progeny thereof.
  • Example 5 Elongation of cells
  • plant cells are separated from the medium using a decanter vessel (S-101) (stream 6) and the medium can be relocated for water treatment (stream 45), as illustrated in the flowchart in FIG. 8.
  • the elongation growth medium is added to the reactors to sterilization by autoclaving at same conditions used in multiplication step and cooling at a temperature from about 30 °C to about 35 °C for cell differentiation.
  • Each reactor receives a third of the cells and the reaction volume comprises the cells (stream 6), medium (stream 38), and air (stream 32) flows.
  • Example 6 Separation and isolation of elongated cells [0218] After elongation according to Example 5, 3 tanks (Tq-102, Tq-103, and Tq-104) are fed, which in the flowchart in FIG. 8 are represented only by block Tq-102. Each tank, with volume slightly larger than those of the reactors, receives the substantially same volume of the three reactors.
  • the output of the elongation tanks (stream 7) is routed to the second decanter (S-102).
  • the bottom product (stream 8), comprising elongated and unelongated cells, is routed to the sieve (S-103), while the medium (stream 46) is removed to the effluent treatment.
  • the function of the sieve is to remove unelongated and smaller cells that are not pre-fibers.
  • the sieve (S-103) retains the elongated cells (pre-fibers) and releases all nonelongated cells (which will not become cotton fibers).
  • a sterilized medium is used in the maturation stage, as well as in the multiplication and elongation stages. Maturation is recognized by secondary cell wall deposition. Sugars are combined to produce cellulose, which is the main component of cotton fiber (natural glucose polymerization) that occurs inside the cell forming the secondary wall. In this process, the density of pre-fiber increases from 1.05 to 1.55 g/ml, which is the density of cotton fiber.
  • the R-108 output is directed to the buffer tank Tq-105 (FIG. 8) to enable a continuous downstream process.
  • the mid-fiber mixture (stream 10) is routed to the third decanter (S-104), where the cotton fibers (stream 11) are separated from the medium (stream 48).
  • the fibers produced have moisture content above acceptable level (10 to 20% in water mass).
  • a drying process working with air is implemented. This air passes through the cotton fibers and part of the water is removed until a moisture content of at most 5% is reached.
  • Example 8 Recycling
  • a composition created via a method described herein can be recycled.
  • an aliquot of a composition is reserved and re-introduced into an earlier step in a method.
  • an aliquot of cells unsuccessful in induction, growth, elongation, or maturation is reserved and re-introduced into an earlier step in a method.
  • Tables C, D, and E, below, show the results of growth and elongation of cotton cell cultures in accordance with the methods disclosed herein.
  • cotton ovule cells which may include ovule epidermal cells, were mechanically extracted from a cotton boll. The extracted cotton ovule cells were cultured, multiplied, and in some cases, elongated.
  • Each table provides the genotype/cultivar and variety name from which the cotton cell cultures were originally obtained. The tables also provide the ovule location from which the cells were taken from a parental cotton plant for the cell cultures.
  • Table C Cotton cell culture results from cells cultured from an upper ovule location.
  • Table D Cotton cell culture results from cells cultured from a middle ovule location.
  • Table E Cotton cell culture results from cells cultured from a bottom ovule location.
  • the present disclosure provides cotton, methods of growing cotton in accordance with any of the method/protocols provided herein, and persistent cell lines, wherein the cotton cells are derived and/or obtained, in whole or in part, which can be used across a range of cotton species and varietals.
  • the in vitro methods of cotton production can use cotton cells derived from a cotton plant of any varietal, including one selected from PAYMASTER HS26, PD 2164, SA 2413, SEALAND #1 (G.B.
  • the cotton cells are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from PD 2164, SOUTHLAND Ml, ACALA 5, CD3HCABCUH-1-89, FJA, TASHKENT 1, WESTERN STORMPROOF, PAYMASTER HS200, Pima S-7, and Acala MAXXA, or a progeny of any thereof. Certain varieties produced excellent growth.
  • the cotton cells are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from PD 2164, ACALA 5, SOUTHLAND Ml, CD3HCABCUH-1-89, FJA, Pima S-7, and Acala MAXXA, or a progeny of any thereof.
  • the cotton cells are derived and/or obtained, in whole or in part, from ovule cells and/or ovule epidermal cells obtained from the top third of a cotton boll from at least one cotton plant of a variety selected from PD 2164, SOUTHLAND Ml, ACALA 5, and CD3HCABCUH-1-89, or a progeny of any thereof.
  • the cotton cells are derived and/or obtained, in whole or in part, from ovule and/or ovule epidermal cells obtained from the top third of a cotton boll from at least on cotton plant of a variety selected from PD 2164 and ACALA 5, or a progeny of any thereof.
  • the cotton cells are derived and/or obtained, in whole or in part, from ovule cells and/or ovule epidermal cells obtained from the middle third of a cotton boll from a cotton plant of a variety selected from at least one cotton plant of a variety selected from PD 2164, SOUTHLAND Ml, CD3HCABCUH-1-89, FJA, or a progeny of any thereof.
  • the cotton cells are derived and/or obtained, in whole or in part, from ovule cells and/or ovule epidermal cells obtained from the middle third of a cotton boll from at least on cotton plant of a variety selected from PD 2164 and FJA, or a progeny of any thereof.
  • the cotton cells are derived and/or obtained, in whole or in part, from ovule cells and/or ovule epidermal cells obtained from the bottom third of a cotton boll from a cotton plant of a variety selected from at least one cotton plant of a variety selected from PD 2164, SOUTHLAND Ml, TASHKENT 1, WESTERN STORMPROOF, ACALA 5, CD3HCABCUH- 1-89, FJA, Pima S-7, Acala MAXXA, or a progeny of any thereof.
  • the cotton cells are derived and/or obtained, in whole or in part, from ovule cells and/or ovule epidermal cells obtained from the bottom third of a cotton boll from at least on cotton plant of a variety selected from PD 2164, SOUTHLAND Ml, ACALA 5, CD3HCABCUH-1-89, FJA, Pima S-7, and Acala MAXXA, or a progeny of any thereof.
  • the cotton cells are derived and/or obtained, in whole or in part, from ovule cells and/or ovule epidermal cells obtained from at least one cotton plant of a variety selected from PD 2164, SOUTHLAND Ml, ACALA 5, FJA, or a progeny of any thereof.
  • the cotton cells are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from SEALAND #1 (G.B. X G.H.), ACALA 5, SA 2413, TOOLE, M.U.8B UA 7-44, DIXIE KING, or a progeny of any thereof.
  • SEALAND #1 G.B. X G.H.
  • ACALA 5 SA 2413
  • TOOLE M.U.8B UA 7-44
  • DIXIE KING DIXIE KING
  • Example 10 Producing Colored Cotton Fiber
  • the present invention includes the transformation and selection of transformed cells, and induction of those cells to elongate to form fiber.
  • cotton cells are obtained from the cotton varietals listed in Table F in accordance with the methods described herein.
  • cotton cells obtained from any cotton varietal including those specifically listed herein (such as those listed in Tables B-E), can be used in accordance with the methods of the invention.
  • the obtained cells are from cotton plants of the varietals in Table F and/or are the progeny of cells obtained from plants of these varietals.
  • the cotton cells are obtained or are the progeny of cotton cells obtained from cotton plant or explant apical meristems, cotyledons, young leaves, hypocotyls, ovules, stems, mature leaves, flower, flower stalks, floral whorls, roots, bulbs, germinated seeds, somatic and zygotic embryo, and/or cambial meristematic cells (CMC).
  • CMC cambial meristematic cells
  • the cells derived from suspension cultures or calli are then subject to a transformation to introduce a plurality of expression constructs into the cotton cells
  • the transformation method used is either an Agrobacterium or biolistic transformation method based on prior published studies of cotton transformation to regenerate whole plants, however, in this case, cotton cells are transformed and elongated to form fiber rather than whole plants (Jin, 2005; Leelavathi, 2004; Finer, 1990).
  • the cells are co-cultivated with an Agrobacterium carrying vector that includes three expression constructs.
  • One of the constructs produces a red chromoprotein/pigment, one a green chromoprotein/pigment, and one a blue chromoprotein/pigment.
  • Each construct is independently inducible and tunable.
  • each expression construct is operably connected to an inducible promoter.
  • the promoters include, for example, promoters that show preferential tissue and/or temporal expression in cotton plants or cotton cells.
  • the cells are subject to bombardment with the genes of interest and the selection gene(s).
  • the cells are grown with media containing hormones to induce cell growth, a selection agent to inhibit the growth of untransformed cells due to the selection gene(s).
  • media also includes antibiotic(s) to inhibit the growth of excess Agrobacterium.
  • a desired color for the cotton fiber produced from the cells is chosen.
  • the cotton cells are contacted with varying concentrations of Inducers A, B, and C.
  • Each different inducer induces a separate gene construct, which leads to the production of a particular pigment/chromoprotein in the cotton fiber with saturation that corresponds to the concentration of inducer used for the construct.
  • concentrations of each inducer required to produce cotton fiber of a particular color are stored in a database, such that cotton fiber of a particular color can quickly be replicated using the disclosed in vitro methods of colored cotton production.
  • the cells are cultured, the constructs induced, and the cells caused to elongate in order to produce cotton fiber of a desired color.
  • AtWuschel promotes formation of the embryogenic callus in Gossypium hirsutum.
  • PloS one, 9(1), e87502.

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Abstract

La présente invention concerne des procédés in vitro de production de fibrse de coton colorées.
PCT/US2022/029428 2021-05-17 2022-05-16 Lignées cellulaires, variétés et procédés de production in vitro de fibres de coton colorées WO2022245725A2 (fr)

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