CN117120626A

CN117120626A - Cell lines, varieties and methods for in vitro cotton fiber production

Info

Publication number: CN117120626A
Application number: CN202180083163.9A
Authority: CN
Inventors: L·L·布埃诺; P·M·厄尔布尔; D·多兹; A·特瓦里; L·S·德苏萨卢兹
Original assignee: Gary Co
Current assignee: Gary Co
Priority date: 2020-10-26
Filing date: 2021-10-26
Publication date: 2023-11-24

Abstract

The present disclosure provides in vitro methods for producing cotton fibers, including methods using cotton varieties found to be particularly suitable for use in the presently disclosed in vitro cotton fiber production methods.

Description

Cell lines, varieties and methods for in vitro cotton fiber production

Background

Cotton is the most widely used non-food crop in the world. However, both the money and resources required for successful planting of cotton production are costly. For example, cotton is a water-dense crop where production of cotton fibers per kilogram is estimated to require 9,000 to 17,000 liters of water. This corresponds to the use of drinking water sufficient to sustain 5,000 people for a day to produce cotton sufficient to make two t-shirts. Similarly, cotton planting requires land that must be otherwise transferred from other crop production (e.g., food production). It is estimated that only about 500 kg cotton fibers are produced per acre of cotton grown. Cotton planting is also a net emitter of greenhouse gases, with an increase in carbon dioxide gas of about 0.75 to 2.25 kg per kg cotton fiber produced. Furthermore, since cotton is a plant, its planting can result in a harvest, untimely crop, and even an excess yield. Billions of dollars are spent on streams annually to overcome unexpected cotton harvesting results.

The in vitro production of plant cell compositions can overcome a variety of limitations associated with plant in situ production of plant derived products, thereby providing a reliable, energy efficient and eco-friendly alternative to traditional agriculture. For example, in vitro produced plant cell compositions may be continuously available, while crops grown in situ of plants are often constrained by the availability of circulation.

However, there is currently no known method for producing cotton fibers in vitro, especially on an industrial scale. The in vitro rate and scale of production of plant cell compositions is currently limited by a variety of engineering constraints, such as difficulty in preparing sufficient quantities of cell inoculum with sufficient cell homogeneity, or lack of streamlined protocols for the in vitro plant cell production cycle.

Thus, there is a need for in vitro methods of producing cotton.

Disclosure of Invention

The present invention provides methods and compositions for producing cotton fibers in vitro. The methods and compositions of the present invention can be scaled up, thereby allowing cotton fibers to be produced on an industrial scale. The inventors of the present invention have surprisingly found that while cells derived from most, but not all, cotton varieties can be used in these in vitro cotton production methods, certain varieties possess properties that make them particularly suitable for in vitro cotton fiber production. When used in the in vitro methods of the invention, some of these varieties show, for example, unexpectedly rapid cell growth, rapid cell multiplication/replication, early cotton fiber/pre-formed fiber growth, and/or efficient bioreactor inoculation.

Furthermore, the inventors of the present invention have surprisingly found that cotton cells from or derived from any meristem of a cotton plant can be used in the disclosed in vitro cotton fiber production methods. Unexpectedly, however, within certain meristematic tissues, the location on the tissue from which the cells are obtained may have a profound effect on cell growth, culture and fiber development in the presently disclosed in vitro cotton production methods. For example, the inventors of the present invention have surprisingly found that cotton ovule cells (including cotton ovule epidermal cells) obtained from different locations on a cotton boll (e.g., the top, middle, or bottom third of the boll) provide different levels of cell growth and fiber development when used in the presently disclosed methods. This change occurs even when cells are obtained from cotton plants of the same variety. The inventors of the present invention have also surprisingly found that the ideal location on the tissue for cell growth, culture and/or fiber development varies between cotton varieties.

By using the presently disclosed in vitro cotton production method, cotton fibers can be produced using approximately 77% less water and 80% less land area than traditional plant in situ methods. At the same time, these processes can produce cotton fibers and produce about 84% less carbon dioxide emissions than conventional processes. Although less costly in resources, the process of the present invention produces cotton fibers faster than the in-situ process of plants. Traditionally, cotton takes 5-6 months from cultivation to harvest, whereas the in vitro methods of the present disclosure may harvest cotton fibers in about 45 days or less. Furthermore, because the disclosed methods occur in vitro rather than in situ in plants, these methods can be more tightly controlled. Therefore, if the crop cannot be completely eliminated, the tendency of the crop to be polluted, untimely or superfluous may be reduced.

Surprisingly, the inventors of the present invention overcome many of the obstacles associated with in vitro crop production. The inventors of the present invention have unexpectedly found that nearly all cotton explant tissue can be used to produce proliferating cell aggregates to seed bioreactors for ex-donor cotton production. Furthermore, unexpectedly, cells derived from certain cotton varieties have been shown to produce inoculums to rapidly and effectively inoculate bioreactors. In addition, the proliferated cell aggregates can be stably refrigerated. In addition, a bioreactor seeded with proliferating cell aggregates can rapidly double cells, and the doubled cells can be elongated to produce cotton fibers.

Accordingly, the present invention comprises a method for producing cotton fibers. In an exemplary method, the method comprises: inoculating the bioreactor with cotton cells; doubling the cells in the bioreactor; elongating the multiplied cells; and harvesting cotton fibers from the elongated cells. In certain methods, the cotton cells used to seed the bioreactor are from or derived from cotton plants of a variety selected from the group consisting of: PAYMASTER HS26, PD2164, SA 2413, SEALAND#1 (G.B.X G.H.), SOUTHLAND Ml, status MILLER, TASHKENT 1, TIDEWATER (G.B.X.G.H.), TOOLE, WESTERN STORMPROOF, ACALLA 5, ALLEN 33, CD3HCABCUH-1-89, DELTAPINE 14, DES 24, DIXIE KING, FJA, M.U.8B UA 7-44, NC 88-95, PAYMASTER HS200, pima S-7, acala and MAXXA, or progeny of any of these. In some methods, the variety is selected from PD2164, WESTERN STORMPROOF, CD3HCABCUH-1-89, TASHKENT 1, SOUTHLAND Ml, ACALA 5, FJA, PAYMASTER HS200, pima S-7, and Acala MAXXA or progeny of any of these. In some methods, the variety is selected from PD2164, SOUTHLANDMl, ACALA 5, CD3HCABCUH-1-89, FJA, pima S-7 and Acala MAXXA or progeny of any of these. In certain methods, the variety is selected from PD2164, SOUTHLAND Ml, and CD3HCABCUH-1-89 or progeny of any of these. In addition, in some methods, the variety is PD 2164.

In certain methods, the methods comprise seeding the bioreactor with cotton ovule cells, which may comprise cotton ovule epidermal cells. The cotton ovule cells and ovule epidermal cells may be obtained from cotton bolls. In certain methods, the cotton ovule cells and/or the ovule epidermal cells are obtained from the bottom third, middle third, or top third of the cotton boll, wherein the bottom is the location on the boll where growth begins from the cotton plant stem. In certain methods, the cotton ovule cells and/or the ovule epidermal cells are obtained from the top third of the cotton boll, and the variety is selected from PD 2164 and ACALA 5 or progeny of either. In certain methods, the cotton ovule cells and/or the ovule epidermal cells are obtained from the middle third of the cotton boll, and the variety is selected from PD 2164 and FJA or progeny of either. In certain methods, the cotton ovule cells and/or the ovule epidermal cells are obtained from the bottom third of the cotton boll, and the variety is selected from PD 2164, SOUTHLAND Ml, ACALA 5, CD3HCABCUH-1-89, FJA, pima S-7, and ACALA MAXXA or progeny of any one of them.

The method for producing cotton fibers may comprise seeding the bioreactor with the cotton ovule cells. Alternatively or additionally, the methods may comprise seeding the bioreactor with cells from the proliferating cell aggregates. In certain methods, the cells of the proliferating cell aggregate are obtained and/or derived from cotton plants of the variety Pima S-7.

Preferably, the proliferating cell aggregate is friable callus. Thus, the methods may further comprise: obtaining cells from cotton explants; and contacting the cells from the cotton explant with a callus induction medium to produce the friable callus. Surprisingly, these cells from cotton explants were from cotton apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flowers, pedicel, flower wheel, roots, corms, germinated seeds, somatic and zygotic embryos and/or Cambium Meristem Cells (CMC).

The methods may further comprise: isolating cells from the friable callus; and culturing the isolated cells. The isolated cells after culture can be used to inoculate the bioreactor. Culturing the isolated cells may further comprise culturing the isolated cells in a liquid or semi-solid medium to form a cell suspension. The methods may further comprise: cryopreserving the cell suspension; and inoculating the bioreactor with the cryopreserved cell suspension, the method further comprising homogenizing the cell suspension to form a fine cell suspension. The homogenizing may comprise one or more of the following: subculturing the cell suspension; filtering the cell suspension; pipetting and/or decanting the cell suspension; and adding pectase to the suspension.

The methods may further comprise: isolating elongated cells from any non-elongated cells; and harvesting cotton fibers from the separated elongated cells. Additionally, the method may comprise recirculating any unextended cells for use in a subsequent iteration of the method.

The method for producing cotton may further comprise: maturing the elongated multiplied cells using a maturation medium; and harvesting cotton fibers from the mature elongated cells. Harvesting may comprise separating cotton fibers from the maturation medium; and air is passed through the separated cotton fibers until the moisture content of the fibers is less than 5%. In certain aspects, a method for producing cotton may comprise seeding a bioreactor with cotton cells from and/or derived from cotton plants of the cotton (Gossypium) species selected from the group consisting of: upland cotton (g.hirsutum.), asian cotton (g.arbor), sea island cotton (g.barbaidense), abnormal cotton (g.anomalum), horseradish cotton (g.armourian), clauz cotton (g.klotzchianium), redmond cotton (g.raimondii), herb cotton (g.herbaceae), or progeny of any of these. In certain aspects, the cotton plant belongs to a variety selected from the group consisting of: PAYMASTER HS26, PD 2164, SA 2413, SEALAND#1 (G.B.X G.H.), SOUTHLAND Ml, status MILLER, TASHKENT 1, TIDEWATER (G.B.X.G.H.), TOOLE, WESTERN STORMPROOF, ACALLA 5, ALLEN 33, CD3HCABCUH-1-89, DELTAPINE 14, DES 24, DES 56, DIXIE KING, FJA, M.U.8B UA7-44, NC 88-95, PAYMASTER HS200, pima S-7, acala MAXXA, coassland 320 or progeny of any of these.

In certain aspects, the methods of the present invention may be used to produce at least 1 kilogram of cotton fibers per 4,000 liters of water used in the method. In some cases, the process of the present invention may produce at least 1 kilogram of cotton fibers for every 2,000 to 4,000 water used in the process.

In certain aspects, the invention comprises an in vitro method for producing cotton fibers using cells obtained and/or derived from cotton plants of a variety selected from the group consisting of: PD2164, WESTERN STORMPROOF, CD3HCABCUH-1-89, TASHKENT 1, SOUTHLAND Ml, ACALA 5, FJA, PAYMASTER HS200, pima S-7 and Acala MAXXA or progeny of any of these.

In certain aspects, the invention comprises an in vitro method for producing cotton fibers using cells obtained and/or derived from cotton plants of a variety selected from the group consisting of: PD2164, SOUTHLAND Ml, FJA, PAYMASTER HS200, TIDEWATER, TASHKENT 1, DIXIE KING and Acala MAXXA or progeny of any of these.

Drawings

Fig. 1 illustrates an exemplary method of the present invention.

Figure 2 shows a flow chart of the concept of a commercial scale process for producing cotton fibers in vitro.

FIG. 3 illustrates a computer system programmed or otherwise configured to implement the methods provided herein.

Detailed Description

The present invention provides methods and compositions for producing cotton fibers in vitro. The methods of the present disclosure may be cell-based and do not require growing the entire cotton plant to plant cotton fibers. These methods allow cotton fibers to be planted in a controlled environment quickly and efficiently.

Surprisingly, while cells derived from most, but not all, cotton varieties can be used in these in vitro cotton production methods, some varieties possess characteristics that make them particularly suitable for the presently disclosed in vitro cotton fiber production methods. When used in the methods of the invention, some of these varieties show, for example, unexpectedly rapid cell growth, rapid cell multiplication/replication, early cotton fiber/pre-fiber growth, and/or efficient bioreactor inoculation.

Furthermore, the inventors of the present invention have unexpectedly found that cotton cells from and/or derived from any meristem of cotton plants can be used in the disclosed in vitro cotton fiber production methods. Surprisingly, however, within certain meristematic tissues, the location on the tissue from which the cells are obtained may affect cell growth, culture and fiber development in the presently disclosed in vitro cotton production methods. For example, the inventors of the present invention found that cotton ovule cells and/or ovule epidermal cells obtained from different locations on the cotton boll (e.g., the top, middle, or bottom third of the boll) provide different levels of cell growth and fiber development when used in the presently disclosed methods. This change occurs even when cells are obtained from cotton plants of the same variety. The inventors of the present invention have also surprisingly found that the ideal location on the tissue for cell growth, culture and/or fiber development varies from cotton variety to cotton variety.

Advantageously, the methods and compositions of the present invention can be scaled up, thereby allowing cotton fibers to be produced on an industrial scale. By using these in vitro cotton production methods, approximately 77% less water and 80% less land can be used to produce cotton fibers than traditional plant in situ methods. These methods can also produce cotton fiber harvesting that produces about 84% less carbon dioxide emissions than traditional methods. Although less costly in resources, the process of the present invention produces cotton fibers faster than the in-situ process of plants. Traditionally, cotton takes 5-6 months from cultivation to harvest, whereas the in vitro methods of the present disclosure may harvest cotton fibers within about 45 days.

Furthermore, because the disclosed methods occur in vitro rather than in situ in plants, these methods can be more tightly controlled. Therefore, if the crop cannot be completely eliminated, the tendency of the crop to be polluted, untimely or superfluous may be reduced. These methods can be practiced indoors using automated machinery and even professional cotton cell lines to ensure cotton fiber harvesting of the desired quality.

Surprisingly, the inventors of the present invention overcome many of the obstacles associated with in vitro crop production. The inventors of the present invention have unexpectedly found that nearly all cotton explant tissue can be used to produce proliferating cell aggregates to seed bioreactors for ex-donor cotton production. In addition, the proliferated cell aggregates can be stably refrigerated. Thus, once produced, the cell aggregates can be used to produce cotton fibers without reliance on living cotton plants. Bioreactor seeded with a small amount of cells from the proliferating cell aggregates doubles the cells rapidly in the bioreactor and the doubled cells can be elongated to produce cotton fibers.

Fig. 1 provides an exemplary method 101 of the present disclosure. First, the bioreactor is seeded 103 with a small amount of cotton cells. The bioreactor may be seeded with a small number of cotton ovule cells, which may comprise ovule epidermal cells. Typically, the bioreactor will be seeded with a small amount of cotton cells from the proliferating cell aggregates. As shown in example 4, a milligram amount of cotton cells from the proliferating cell aggregates was sufficient to ultimately inoculate the bioreactor.

Inoculation 103 may comprise preparing a growth medium in a vessel (e.g., flask or plate) and introducing a small amount of cotton cells from the proliferating cell aggregate into the medium. The container may then be left for inoculum growth. Alternatively, inoculum growth may occur inside the bioreactor.

The inventors of the present invention have surprisingly found that inoculum growth under dark conditions provides superior growth. The vessel may be shaken or shaken during the growth of the inoculum, for example at a rate of 80-180 rpm. Preferably, inoculum growth occurs at a temperature of about 30 ℃ to about 35 ℃. Preferably, the medium is a solution comprising plant hormones, plant growth regulators and/or sucrose and/or glucose. Inoculum growth typically takes about 16 days, but may be more or less depending on the requirements or due to conditions or cotton cell lines alone.

The inoculum may be a suspension of cells in a liquid or semi-solid medium. The suspension may optionally be homogenized to provide a fine cell suspension culture. The inventors of the present invention have found that a homogeneous cell suspension can provide more reproducible and reliable results when inoculating a bioreactor.

Homogenization may comprise any method known in the art, including one or more of the following: subculturing the suspension; filtration, pipetting/decanting; and/or adding a low concentration of pectase.

The resulting inoculum is then introduced into a bioreactor. Alternatively, the resulting inoculum may be stored, for example by freezing, for later use in inoculating the bioreactor. The inoculum or homogeneous cell suspension may be cryopreserved indefinitely, for example in liquid nitrogen. This typically requires suspending cells from an inoculum/homogenous cell suspension in a cryoprotectant solution, such as a solution of glycerol and sucrose. The cryoprotectant solution may be a supplement, for example using proline. The cryopreserved cells may be recovered, for example, using recovery medium, after which the cells are used when inoculating the bioreactor.

The proliferating cell aggregate may be a callus. Preferably, the proliferating cell aggregate is a friable callus that is neither sticky nor soft nor so hard or dense that it cannot be physically destroyed or crushed. Friable callus is thus distinguished from "hard callus" which is compact and friable and thus not easily destroyed or comminuted. The inventors of the present invention found that friable callus allows for simple mechanical manipulation to easily separate individual cells from friable callus for use in seeding 103 a bioreactor and/or preparing an inoculum.

After inoculation 103, method 101 entails multiplying 105 the cells in a bioreactor. This period generally lasts from 5 days to 12 days, with replications of cotton cells taking about 1 to 3 days, depending on cotton lineages. Cells can be replicated, for example, by culturing the cells in a cell culture medium.

The multiplied cells are then elongated 107 to produce cotton fibers. This may comprise using an elongation medium to induce multiplied cell elongation. In certain aspects, the elongation medium facilitates release of phenolic compounds from vacuoles of elongated cotton cells. The elongated cells may comprise cotton pre-fibers that will mature into cotton fibers.

In certain aspects, the semi-solid elongation medium is used to elongate cotton cells. The inventors of the present invention have surprisingly found that good results are achieved when a semi-solid medium is used instead of a liquid medium.

Optionally, after elongation, the elongated cotton cells are separated from any non-elongated cotton cells. The unextended cotton cells will not mature into cotton fibers. However, the non-elongated cotton cells may be recycled and used in subsequent iterations of the method. Separating the elongated cotton cells from the non-elongated cells may comprise one or more of filtering, sieving, decanting, and centrifuging the cells.

Once isolated, the elongated cotton cells (which may now have cotton pre-fibers) mature. Maturation of the cells may comprise the use of maturation media. During maturation, sugars are incorporated into the cells to produce cellulose, the major component of cotton fibers (natural glucose polymerization), which occurs within the cells forming the secondary wall. Cotton pre-formed fibers increase in number, density and/or length.

After maturation, cotton fibers are harvested from the cotton cells, for example, by separating the fibers from the cells in solution/buffer. The harvested cotton fibers are then dried to a moisture content of less than 5%, for example by passing air through the cotton fibers.

Thus, the method 101 can produce cotton fibers from cotton cells without growing the cotton plant. These methods allow cotton fibers to be planted in a controlled environment quickly and efficiently.

Method 101 may further comprise preparing friable callus. Friable callus may be prepared, for example, by obtaining cells from cotton explants and contacting the cells with callus induction medium. Surprisingly, the inventors of the present invention found that tissue from any meristematic portion of a cotton plant can be used to produce friable callus. Thus, these cells from cotton explants may be from cotton apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flowers, pedicel, flower wheel, roots, corms, germinated seeds, somatic and zygotic embryos, and/or Cambium Meristem Cells (CMC).

Preparation of friable callus may comprise contacting cells of cotton explants with callus induction medium. The callus induction medium may promote division of at least a subset of cells of the plant explant. The callus induction medium was used to generate dedifferentiated cell mass. Cells in these clusters may then be cultured, which may include the use of callus growth medium.

Certain aspects of the present invention require the use of plant hormones and/or growth regulators (including auxins, gibberellins, etc.). Hormone/modulator may be used, for example, in the media described herein to culture cotton cells. The plant hormones and/or growth regulators (including auxins, gibberellins, etc.) may be derived from naturally occurring sources, synthetically produced or semi-synthetically produced, i.e., starting from naturally derived starting materials and then synthetically modifying said materials. These modifications may be made using conventional methods as contemplated by the skilled artisan. The following references contain plant hormones and/or growth regulators (including auxins, gibberellins, etc.) for use in plant cell compositions as described below or elsewhere herein: gaspar et al, in Vitro cytodevelopmental biology-Plant (In Vitro cell. Dev. Biol Plant), 32,272-289,1996, 10-12 months and Zhang et al,

instructions for agricultural science (Journal of Integrative Agriculture), 2017,16 (8): 1720-1729; the content of each of these documents (in particular, all plant hormones and/or plant growth regulators) is incorporated herein by reference. In particular, those skilled in the art will appreciate that certain gibberellins are capable of promoting plant cell elongation.

In some aspects, the plant hormones and/or growth regulators used in the invention are illustrated 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 may be used for the application indicated in the column heading.

"inhibitor" indicates that the corresponding plant hormone or plant growth regulator in the row may be used to inhibit the activity indicated in the column heading.

"ND" indicates that the effect of the corresponding plant hormone or plant growth regulator on the application indicated in the column heading has not been determined (at least to some extent).

In certain aspects, the invention uses induction medium or callus induction medium. The callus induction media described herein may be configured to promote division of at least a subset of cells of a plant explant. For example, the callus induction medium may facilitate or promote induction of callus of cotton plants. The callus induction medium may comprise a diluted basal medium (i.e., 1:1.5 to 1:5, 1:1.5 to 1:4, 1:1.5 to 1:3, etc.). The callus induction medium may include one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones (such as those that may facilitate or promote induction). The callus induction medium may be liquid at about 25 ℃. Alternatively, the callus induction medium may not be liquid at the indicated temperature. In some embodiments, the callus induction medium is not liquid at about 25 ℃. In some embodiments, the callus induction medium may be a semi-solid medium (e.g., gelled) at 25 ℃.

Non-limiting examples of semi-solid media include soft agar, soft agarose, soft methylcellulose, xanthan gum, gellan gum, carrageenan, isabgol, guar gum, other soft polymer gels, or any other gelling agent known in the art. The callus induction medium may comprise agar. In some embodiments, the callus induction medium may be agar-free. In some embodiments, the callus induction medium may be free of any gelling agent. In some embodiments, the callus induction medium without agar or without gelling agent may be a liquid. In some embodiments, the callus induction medium may be solid without agar or without gelling agent. In some embodiments, the agar-free callus induction medium may be a gel. In some embodiments, the agar-free callus induction medium may comprise an agar substitute. In some embodiments, the callus induction medium may have a pH. The pH of the callus induction medium may be suitable for inducing plant callus. In some embodiments, the pH of the callus induction medium may be optimized to induce plant callus. In some embodiments, the pH of the callus induction medium may be 5.3 to 6.3. In some embodiments, the pH of the callus induction medium may be at or 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 of the foregoing values.

The present disclosure comprises callus culture media or callus growth media and their use in an in vitro method for producing cotton. The callus growth media described herein may facilitate or promote plant callus growth and/or produce proliferating cell aggregates. The callus growth medium may be a gel medium and in some embodiments may include agar and/or another gelling agent and a mixture of macronutrients and micronutrients for the plant type of plant callus. In some cases, the callus culture medium may be enriched with nitrogen, phosphorus, or potassium. In some cases, the callus growth medium may be a liquid medium. In some embodiments, the callus growth medium may beIncluding at least one plant hormone or growth regulator (comprising auxin, gibberellin, 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 phytohormone or plant growth regulator (or at least two, at least three, at least four, at least five or at least six phytohormones or plant growth regulators) (comprising auxins, gibberellins, etc.) may be any one or a combination selected from the group consisting of: indoleacetic acid (IAA), indolyl-3-acrylic acid, 4-Cl-indolyl-3-acetic acid, indolyl-3-acetoacetate, indole-3-acetaldehyde, indole-3-acetonitrile, indole-3-lactic acid, indole-3-propionic acid, indole-3-pyruvic acid, indolebutyric acid (IBA), 2, 4-dichlorophenoxyacetic acid (2, 4D), tryptophan, phenylacetic acid (PAA), brassica glucosinolate, naphthalene Acetic Acid (NAA), picloram (PIC), dicamba, ethylene, p-chlorophenoxyacetic acid (pCPA), p-naphthyloxy acetic acid (NOA), benzo (b) selenious acyl-3 acetic acid, 2-benzothiazoloacetic acid (BTOA), N6- (2-isopentenyl) adenine (2 iP), zeatin (ZEA), dihydrozeatin, zeatin riboside, kinetin (N), 6- (benzyladenine) -9- (2-tetrahydropyranyl) -9H-purine, 2,4, 5-trichlorophenoxy acetic acid (2, 4, 5-phenylurea (6, 6-phenylthiourene), 6-2-phenylurea (KIN-2-P), 2-phenylurea (KIN-phenylurea) and 2 '-phenylurea (6, 4' -dichlorophenoxyurea (N-phenylurea), gibberellin A ₅ Gibberellin A1 (GA 1), gibberellic acid (GA 3), gibberellin A4 (GA 4), gibberellin A7 (GA 7), brassinolide (BR), jasmonic Acid (JA), gibberellin A ₈ Gibberellin A ₃₂ Gibberellin A ₉ 15-beta-OH-gibberellin A ₃ 15-beta-OH-gibberellin A ₅ 12-beta-OH-gibberellin A ₅ 12-alpha-gibberellin A ₅ Salicylic acid, (-) jasmonic acid, (+) -7-isojasmonic acid, putrescine, spermidine, spermine, oligosaccharin and stigmasterol. The at least one phytohormone or plant growth regulator (or at least two, at least three, at leastFour, at least five, or at least six plant hormones or plant growth regulators) (comprising auxins, gibberellins, etc.) may be any one or a combination selected from the group consisting of: indolyl-3-acetic acid, indolyl-3-acrylic acid, indolyl-3-butyric acid, 4-Cl-indolyl-3-acetic acid, indolyl-3-acetoacetate, indol-3-acetaldehyde, indol-3-acetonitrile, indol-3-lactic acid, indol-3-propionic acid, indol-3-pyruvic acid, tryptophan, phenylacetic acid, brassinosteroids, 2, 4-dichlorophenoxyacetic acid, 1-naphthylacetic acid, dicamba, picloram, ethylene, benzo (b) selenioyl-3 acetic acid, trans-zeatin, N ⁶ - (2-isopentenyl) adenine, dihydrozeatin, zeatin riboside, kinetin, benzylamide, 6- (benzyladenine) -9- (2-tetrahydropyranyl) -9H-purine, 1, 3-diphenylurea, N- (2-chloro-4-pyridinyl) -N ' -phenylurea, (2, 6-dichloro-4-pyridinyl) -N ' -phenylurea, N-phenyl-N ' -1,2, 3-thiadiazol-5-yl urea, gibberellin A ₁ Gibberellic acid A ₃ Gibberellin A ₄ Gibberellin A ₅ Gibberellin A ₇ Gibberellin A ₈ Gibberellin A ₃₂ Gibberellin A ₉ 15-beta-OH-gibberellin A ₃ 15-beta-OH-gibberellin A ₅ 12-beta-OH-gibberellin A ₅ 12-alpha-gibberellin A ₅ Salicylic acid, jasmonic acid, (-) jasmonic acid, (+) -7-isojasmonic acid, putrescine, spermidine, spermine, oligosaccharins, brassinolide, and stigmasterol. The at least one phytohormone or plant growth regulator (or at least two, at least three, at least four, at least five or at least six phytohormones or plant growth regulators) (comprising auxins, gibberellins, etc.) may be any one or a combination selected from the group consisting of: indoleacetic acid (IAA), indolebutyric acid (IBA), 2, 4-dichlorophenoxyacetic acid (2, 4D), naphthaleneacetic acid (NAA), p-chlorophenoxyacetic acid (pCPA), p-naphthyloxy acetic acid (NOA), 2-benzothiazoloacetic acid (BTOA), picloram (PIC), 2,4, 5-trichlorophenoxyacetic acid (2, 4, 5-T), phenylacetic acid (PAA), kinetin (KIN), 6-benzylaminopurine (6 BA), N6- (2-isopentenyl) adenine (2 iP), zeatin (ZEA), gibberellin A1 (GA 1), gibberellic acid (GA 3), gibberellin A4 (GA 4), gibberellin A7 (GA 7), ethylene, brassinolide (BR) and Jasmonic Acid (JA).

In certain aspects, the callus growth medium may be liquid at about 25 ℃. In some embodiments, the callus growth medium may not be liquid at about 25 ℃. In some embodiments, the callus growth medium may be a semi-solid medium (e.g., gelled) at 25 ℃. Non-limiting examples of semi-solid media include soft agar, soft agarose, soft methylcellulose, xanthan gum, gellan gum, carrageenan, isabgol, guar gum, other soft polymer gels, or any other gelling agent known in the art. In some embodiments, the callus growth medium may comprise agar. In some embodiments, the callus growth medium may be agar-free. In some embodiments, the callus growth medium may be free of any gelling agent. In some embodiments, the callus growth medium may be liquid without agar or without gelling agent. In some embodiments, the callus growth medium may be solid without agar or without gelling agent. In some embodiments, the agar-free callus growth medium may be a gel. In some embodiments, the agar-free callus growth medium may include an agar substitute.

In some embodiments, the callus growth medium may have a pH. The pH of the callus growth medium may be suitable for growing plant callus and/or producing proliferating cell aggregates. In some embodiments, the pH of the callus growth medium may be optimized to grow plant callus and/or produce proliferating cell aggregates. In some embodiments, the pH of the callus growth medium may be 5.3 to 6.3. In some embodiments, the pH of the callus growth medium may be at or 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 of the foregoing values.

The invention encompasses cell culture media (e.g., multiplication/replication media) and their use in the in vitro methods described herein for producing cotton. In some embodiments, the cell culture media described herein can facilitate or promote proliferation of a population of cells or a proliferating cell aggregate. The cell culture medium may include one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones (such as those that may facilitate or promote proliferation). In some cases, the cell culture medium may be configured to proliferate a population of cells (e.g., a proliferating cell aggregate). The cell culture medium may include enzymes that can degrade plant cell walls or proliferating cell aggregates of plant cells of the cell population. In some embodiments, the enzyme may be a pectolyase. In some embodiments, the enzyme may include cellulase, hemicellulose, cellose (celluysin), or a combination thereof. In some embodiments, the cell culture medium may have a pH. The pH of the cell culture medium may be suitable for culturing a population of cells or proliferating cell aggregates.

In some embodiments, the pH of the cell culture medium may be optimized to culture a population of cells (e.g., a proliferating cell aggregate). In some embodiments, the pH of the cell culture medium may be optimized for cell division. In some embodiments, the pH of the cell culture medium may be 5.3 to 6.3. In some embodiments, the pH of the cell culture medium may be at or 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 of the foregoing values. In some embodiments, the cell culture medium may have a different pH than the callus growth medium. In some embodiments, the cell culture medium may have the same pH as the callus growth medium. In some embodiments, the pH of the cell culture medium may differ from the pH of the callus growth medium by less than 0.1 units, less than 0.2 units, or less than 0.3 units. For example, the pH of the cell culture medium may differ from the pH of the callus growth medium by less than 0.2 units.

Preferably, the cell culture medium of the present disclosure comprises one or more of the following: MS, B5, glucose, sucrose, kinetin, 2, 4-dichlorophenoxyacetic acid (2, 4-D), NAA and coconut water. Preferably, the cell culture medium comprises 2,4-D. In certain aspects, the cell culture medium comprises MS, B5, glucose/sucrose, and 2,4-D.

The invention also comprises a recovery medium and its use in an in vitro method for producing cotton fibers. The recovery medium may be used, for example, to recover cotton cell inoculum after cryopreservation. Some embodiments described herein relate to recovery media. In some embodiments, the recovery medium described herein can be a medium that can facilitate or promote recovery of cotton cells. The recovery medium may include one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones that may facilitate or promote elongation.

The invention comprises an elongation medium and its use in an in vitro method for producing cotton fibers. The elongation media described herein can facilitate or promote elongation of cells capable of elongation, e.g., cotton cells. The elongation media described herein may include one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones (such as those that may facilitate or promote elongation). In some embodiments, the elongation medium may be configured to facilitate release of the phenolic compound from the vacuoles from cotton cells. In some embodiments, the phenolic compound (e.g., O-diphenol) is configured to initiate fiber differentiation and/or increase intracellular auxin levels by inhibiting indoleacetic acid (IAA) oxidase. In some embodiments, the elongation medium may include at least one plant hormone or growth regulator (including auxin, gibberellin, 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 phytohormone or plant growth regulator (or at least two, at least three, at least four, at least five or at least six phytohormones or plant growth regulators) (comprising auxins, gibberellins, etc.) may be any one or a combination selected from the group consisting of: indoleacetic acid (IAA), indolyl-3-acrylic acid, 4-Cl-indolyl-3-acetic acid, indolyl-3-acetoacetate, indole-3-acetaldehyde, indole-3-acetonitrile, indole-3-lactic acid, indole -3-propionic acid, indole-3-pyruvic acid, indolebutyric acid (IBA), 2, 4-dichlorophenoxyacetic acid (2, 4 d), tryptophan, phenylacetic acid (PAA), brassinosteroid, naphthylacetic acid (NAA), picloram (PIC), dicamba, ethylene, p-chlorophenoxyacetic acid (pCPA), p-naphthyloxy acetic acid (NOA), benzo (b) selenious acyl-3 acetic acid, 2-benzothiazoloacetic acid (BTOA), N6- (2-isopentenyl) adenine (2 iP), zeatin (ZEA), dihydrozeatin, zeatin riboside, kinetin (KIN), 6- (benzyladenine) -9- (2-tetrahydropyranyl) -9H-purine, 2,4, 5-trichlorophenoxyacetic acid (2, 4, 5-T), 6-benzylaminopurine (6 BA), 1, 3-diphenylurea, N- (2-chloro-4-pyridinyl) -N '-phenylurea, (2, 6-dichloro-4-pyridinyl) -N' -phenylurea, N-phenylthiourea, N-2, 5-phenylurea, 2, 3-diazole ₅ Gibberellin A1 (GA 1), gibberellic acid (GA 3), gibberellin A4 (GA 4), gibberellin A7 (GA 7), brassinolide (BR), jasmonic Acid (JA), gibberellin A ₈ Gibberellin A ₃₂ Gibberellin A ₉ 15-beta-OH-gibberellin A ₃ 15-beta-OH-gibberellin A ₅ 12-beta-OH-gibberellin A ₅ 12-alpha-gibberellin A ₅ Salicylic acid, (-) jasmonic acid, (+) -7-isojasmonic acid, putrescine, spermidine, spermine, oligosaccharin and stigmasterol. The at least one phytohormone or plant growth regulator (or at least two, at least three, at least four, at least five or at least six phytohormones or plant growth regulators) (comprising auxins, gibberellins, etc.) may be any one or a combination selected from the group consisting of: indolyl-3-acetic acid, indolyl-3-acrylic acid, indolyl-3-butyric acid, 4-Cl-indolyl-3-acetic acid, indolyl-3-acetoacetate, indol-3-acetaldehyde, indol-3-acetonitrile, indol-3-lactic acid, indol-3-propionic acid, indol-3-pyruvic acid, tryptophan, phenylacetic acid, brassinosteroids, 2, 4-dichlorophenoxyacetic acid, 1-naphthylacetic acid, dicamba, picloram, ethylene, benzo (b) selenioyl-3 acetic acid, trans-zeatin, N ⁶ - (2-isopentenyl) adenine, dihydrozeatin, zeatin riboside, kinetin, benzylamide, 6- (benzyladenine) -9- (2-tetrahydropyranyl) -9H-purine, 1, 3-diphenylurea, N- (2-chloro-4-pyridinyl) -N ' -phenylurea, (2, 6-dichloro-4-pyridinyl) -N ' -phenylurea, N-phenyl-N ' -1,2, 3-thiadiazole-5-Ureido, gibberellin A ₁ Gibberellic acid A ₃ Gibberellin A ₄ Gibberellin A ₅ Gibberellin A ₇ Gibberellin A ₈ Gibberellin A ₃₂ Gibberellin A ₉ 15-beta-OH-gibberellin A ₃ 15-beta-OH-gibberellin A ₅ 12-beta-OH-gibberellin A ₅ 12-alpha-gibberellin A ₅ Salicylic acid, jasmonic acid, (-) jasmonic acid, (+) -7-isojasmonic acid, putrescine, spermidine, spermine, oligosaccharins, brassinolide, and stigmasterol. The at least one phytohormone or plant growth regulator (or at least two, at least three, at least four, at least five or at least six phytohormones or plant growth regulators) (comprising auxins, gibberellins, etc.) may be any one or a combination selected from the group consisting of: indoleacetic acid (IAA), indolebutyric acid (IBA), 2, 4-dichlorophenoxyacetic acid (2, 4D), naphthaleneacetic acid (NAA), p-chlorophenoxyacetic acid (pCPA), p-naphthyloxy acetic acid (NOA), 2-benzothiazoloacetic acid (BTOA), picloram (PIC), 2,4, 5-trichlorophenoxyacetic acid (2, 4, 5-T), phenylacetic acid (PAA), kinetin (KIN), 6-benzylaminopurine (6 BA), N6- (2-isopentenyl) adenine (2 iP), zeatin (ZEA), gibberellin A1 (GA 1), gibberellic acid (GA 3), gibberellin A4 (GA 4), gibberellin A7 (GA 7), ethylene, brassinolide (BR) and Jasmonic Acid (JA).

In certain aspects, the callus growth medium may be liquid at about 25 ℃. In some embodiments, the callus growth medium may not be liquid at about 25 ℃. In some embodiments, the callus growth medium may be a semi-solid medium (e.g., gelled) at 25 ℃. The inventors of the present invention found that semi-solid media provided better results than liquid media. Non-limiting examples of semi-solid media include soft agar, soft agarose, soft methylcellulose, xanthan gum, gellan gum, carrageenan, isabgol, guar gum, other soft polymer gels, or any other gelling agent known in the art. In some embodiments, the callus growth medium may comprise agar. In some embodiments, the callus growth medium may be agar-free. In some embodiments, the callus growth medium may be free of any gelling agent. In some embodiments, the callus growth medium may be liquid without agar or without gelling agent. In some embodiments, the callus growth medium may be solid without agar or without gelling agent. In some embodiments, the agar-free callus growth medium may be a gel. In some embodiments, the agar-free callus growth medium may include an agar substitute.

In some embodiments, the elongation medium may have a pH. The pH of the elongation medium may be adapted to produce/induce elongated cells, such as an elongated cotton cell or a plurality of elongated cotton cells. In some embodiments, the pH of the elongation medium may be optimized for cell elongation (e.g., cotton cell elongation). In some embodiments, the pH of the elongation medium may be from 5.3 to 6.3. In some embodiments, the pH of the elongation medium may be at or 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 of the foregoing values.

The invention comprises an elongation medium and its use in an in vitro method for producing cotton fibers. In some embodiments, the maturation media described herein can facilitate or promote maturation of cells, such as cotton cells. The maturation medium can include one or more salts, macronutrients, micronutrients, organic molecules, and/or hormones (such as those that can facilitate or promote maturation). In some embodiments, the maturation medium can include maturation agents. In some embodiments, the maturation reagent of the maturation medium can be a wall regeneration reagent.

The invention comprises the use of proliferating cell aggregates and their production for use in an in vitro cotton fiber production method. In some embodiments, the plant cell composition as described below or elsewhere herein may be derived from a proliferating cell aggregate. The proliferating cell aggregates may be aggregates of proliferating plant cells. The proliferating cells in the aggregate may be linked to each other, for example, by intercellular interactions. The proliferating cell aggregate may be a friable callus that is neither sticky nor soft, nor so hard or dense that it cannot be physically destroyed or crushed. Friable callus is thus distinguished from "hard callus" which is compact and friable and thus not easily destroyed or comminuted. Preferably, the callus is friable callus. The inventors of the present invention found that friable calli can have individual cells isolated from the calli using simple mechanical manipulation.

The proliferating cells may be of one type (homogeneous aggregates) or of two or more types (heterogeneous aggregates). The proliferating cell aggregates can be mixed aggregates (e.g., where cell types are mixed together), clustered aggregates (e.g., where different types of cells tend to be different parts of the aggregate), or segregated aggregates (where different types of cells are pulled away from each other). Cells that proliferate the cell aggregates can divide at a greater rate than the rate of cell division of the remaining cells in the plant callus. In some embodiments, cells of the proliferating cell aggregate can divide at a rate that can be at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold the rate of cell division of plant callus cells.

The invention encompasses the use of cells from cotton plant cell calli and methods for preparing such calli. Plant callus may be growing plant parenchyma cell clusters. However, the inventors of the present invention found that cells from any meristematic portion of cotton plants were sufficient for callus induction. Thus, plant calli may be generated using cells obtained or derived from: cotton apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flowers, pedicel, flower wheel, roots, corms, germinated seeds, somatic and zygotic embryos and/or Cambium Meristem Cells (CMC). In some cases, the plant parenchyma cell mass may be unstructured. Plant calli may be collected from cells covering a wound of a plant or plant part. Preferably, plant callus is produced by inducing a plant tissue sample (e.g., an explant) with a callus induction medium. In some cases, induction of the explants may occur after surface sterilization and plating onto in vitro media (e.g., in closed culture vessels such as petri dishes). Induction may include supplementation of the culture medium with plant growth regulators such as auxins, cytokinins or gibberellins to initiate callus formation. The induction may be carried out at or about 20 ℃, 25 ℃, 28 ℃, 30 ℃, 35 ℃ or 40 ℃ or at a temperature in the range between any two of the above values.

Compositions comprising cotton plant cells are encompassed by the present invention. The plant cell compositions described herein may be the end product of the methods provided herein for preparing cell bank stock. The plant cell composition may be a composition of engineered cells or a composition of wild-type cells. The plant cell composition may be a cell bank stock. The plant cell composition may include a plurality of plant cells obtained by growing callus in a growth medium to produce a proliferation cell aggregate followed by culturing the proliferation cell aggregate.

The plant cell compositions described herein may be in a growing phase. The growth phase may include cell division, cell expansion, and/or cell differentiation. The growth phase including cell division may be an exponential growth phase (e.g., waiting). In some embodiments, an exponential growth phase may occur when the cell is mitotic. In some embodiments, during exponential growth, the number of cells of each generation may be twice the number of cells of the previous generation. In some embodiments, not all cells may survive a given generation. In some embodiments, the number of cells of each generation may be less than twice the number of cells of the previous generation. In some embodiments, the exponential growth phase may be determined (e.g., quantified or identified) by a cell viability assay. In some embodiments, another aspect of the plant cell composition may be determined by a cell viability assay. In some embodiments, the cell viability assay may be an assay that can determine the ability of a cell to maintain or restore viability. In some embodiments, the cell division capacity or active cell division of a plant cell composition can be determined. In some embodiments, the cell viability assay may be an ATP test, calcein AM, a clone formation assay, an ethidium homodimer assay, evans blue, a fluorescein diacetate hydrolysis/propidium iodide staining (FDA/PI staining), flow cytometry, a formazan-based assay (e.g., MTT or XTT), a green fluorescent protein-based assay, a Lactate Dehydrogenase (LDH) -based assay, a methyl violet, neutral red absorption, propidium iodide, resazurin, trypan blue, or TUNEL assay. In some embodiments, the cell viability assay can determine the cytoplasmic level of the diphenolic compound in the plant cell composition.

In some embodiments, cotton (or engineered cotton) described herein may be derived from cotton species. The cotton species may be selected from the group consisting of: asian cotton, abnormal cotton, horseradish cotton, clauz cotton and raymond cotton. The cotton (or engineered cotton) may be derived from a cotton species selected from the group consisting of: upland cotton, asian cotton, island cotton, abnormal cotton, horseradish cotton, clauz cotton, and raymond cotton. The cotton (or engineered cotton) may be upland cotton, gossypium barbadense, asian cotton, herbaceous cotton or another cotton species.

The cotton cells used in the method for producing cotton fibers in vitro may comprise using cotton cells having a Differentially Expressed Gene (DEG). Cotton cells of the present disclosure can be subjected to a mutagenesis process to produce DEG. This process can occur in vitro and without growing the entire cotton plant with the DEG.

Mutagenesis may be achieved by irradiation and/or chemical methods, EMS or sodium azide treatment or gamma irradiation of the seed containing. Chemical mutagenesis favors nucleotide substitutions over deletions. Heavy Ion Beam (HIB) irradiation is a known mutagenesis technique. Ion beam irradiation has two physical factors, namely dose (gy) and LET (linear energy transfer, keV/um) that determine the level of DNA damage and the biological effect of the size of any DNA deletion, and these can be adjusted according to the variation in the degree of mutagenesis.

Biopharmaceuticals can also be used to create site-specific mutations in cotton cells. These agents may contain enzymes that cause DNA double strand breaks, which stimulate endogenous repair mechanisms. These enzymes include endonucleases, zinc finger nucleases, transposases and site-specific recombinases.

In certain aspects, the cotton cells can be transgenic. Transgenic cells are genetically modified plant cells in which an endogenous genome or gene is complemented or modified by an introduced foreign or exogenous gene or sequence. Typically, these genes are under the control of their operably linked promoters. Transgenes are foreign exogenous genes or sequences that are introduced into plants.

Also provided herein are bioreactors configured to produce any one or more compositions associated with in vitro production of fibers as disclosed herein.

In some embodiments, the bioreactor may be configured to produce a cell bank stock solution. In some embodiments, the bioreactor may be configured to perform a method for preparing a cell bank stock solution. In some such cases, the bioreactor may be configured to utilize components of a kit for preparing a cell bank stock, such as callus growth medium and/or multiplication medium.

Fig. 2 provides a flow chart showing examples of different processes that may be performed by the bioreactor and how these processes are interconnected.

In some embodiments, the bioreactor may be configured to produce cotton fibers. In some embodiments, the bioreactor may be configured to perform a method for large scale cotton fiber production. In some embodiments, the bioreactor may be configured to perform a method for rapid cotton fiber production. In some embodiments, the bioreactor may be configured to utilize components of a kit for large-scale fiber production. In some embodiments, the bioreactor may be configured to utilize components of a kit for rapid fiber production.

In some embodiments, the bioreactor may be configured to produce engineered cotton. In some embodiments, the bioreactor may be configured to utilize components of a kit for producing engineered cotton, which may include elements of the kits provided herein.

The present invention encompasses a computer system programmed to implement the methods of the present disclosure. FIG. 3 illustrates a computer system 301 programmed or otherwise configured to provide and/or implement instructions or modes of embodiments for inducing, callus growth, cell culture, elongation, or maturation. Computer system 301 can regulate various aspects of induction, callus growth, cell culture, elongation, or maturation of the present disclosure. The computer system 301 may be the user's electronic device or a computer system remotely located relative to the electronic device. The electronic device may be a mobile electronic device.

The computer system 301 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 305, which may be a single-core or multi-core processor or multiple processors for parallel processing. Computer system 301 also includes memory or memory location 310 (e.g., random access memory, read only memory, flash memory), electronic storage unit 315 (e.g., a hard disk), communication interface 320 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 325 such as cache, other memory, data storage devices, and/or electronic display adapters. The memory 310, storage unit 315, interface 320, and peripheral devices 325 communicate with the CPU 305 through a communication bus (solid lines), such as a motherboard. The storage unit 315 may be a data storage unit (or data repository) for storing data. Computer system 301 may be operatively coupled to a computer network ("network") 330 by way of a communication interface 320. The network 330 may be the internet, the internet and/or an extranet, or an intranet and/or an extranet in communication with the internet. In some cases, the network 330 is a telecommunications network and/or a data network. Network 330 may include one or more computer servers that may implement distributed computing, such as cloud computing. In some cases, network 330 may implement a peer-to-peer network with the aid of computer system 301, which may enable coupling of devices to computer system 301 to act as clients or servers.

The CPU 305 may execute a series of machine readable instructions which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 310. The instructions may relate to the CPU 305, which may then program or otherwise configure the CPU 305 to implement the methods of the present disclosure. Examples of operations performed by the CPU 305 may include fetch, decode, execute, and write back.

The CPU 305 may be part of a circuit, such as an integrated circuit. One or more other components of system 201 may be included in the circuit. In some cases, the circuit is an Application Specific Integrated Circuit (ASIC).

The storage unit 315 may store files such as drivers, libraries, and saved programs. The storage unit 315 may store user data, such as user preferences and user programs. In some cases, computer system 301 may include one or more additional data storage units located external to computer system 301, such as on a remote server in communication with computer system 301 via an intranet or the Internet.

Computer system 301 may communicate with one or more remote computer systems over network 330. For example, computer system 301 may communicate with a user's remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), tablet or tablet PCs (e.g., iPad、Galaxy Tab), phone, smart phone (e.g.)>iPhone, android enabled device, < ->) Or the number of individualsA word assistant. A user may access computer system 301 through network 330.

The methods as described herein may be implemented by machine (e.g., a computer processor) executable code stored on an electronic storage location of computer system 301, such as memory 310 or electronic storage unit 315. The machine executable code or machine readable code may be provided in the form of software. During use, code may be executed by processor 305. In some cases, the code may be retrieved from the storage unit 315 and may be stored on the memory 310 for immediate access by the processor 305. In some cases, electronic storage unit 315 may be eliminated and machine executable instructions stored on memory 310.

The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled during runtime. The code may be supplied in a programming language that may be selected to effect execution of the code in a precompiled or class compiled (as-loaded) manner.

Aspects of the systems and methods provided herein, such as computer system 301, may be embodied in programming. Aspects of the technology may be viewed as an "article of manufacture" or "article of manufacture" generally in the form of machine (or processor) executable code and/or associated data embodied on or in a type of machine readable medium. The machine executable code may be stored on an electronic storage unit such as a memory (e.g., read only memory, random access memory, flash memory) or hard disk. A "storage" type medium may comprise any or all of the tangible memory of a computer, processor, etc., or associated modules thereof, such as various semiconductor memories, tape drives, hard drives, etc., that may provide non-transitory storage for software programming at any time. All or part of the software may sometimes communicate over the internet or various other telecommunications networks. Such communication may, for example, enable loading of software from one computer or processor into another computer or processor, such as from a management server or host computer into a computer platform of an application server. Accordingly, another type of medium that may carry software elements includes light waves, electric waves, and electromagnetic waves, such as those used on physical interfaces between local devices through wired and optical landline networks, and through various air links. Physical elements carrying such waves, such as wired or wireless links, optical links, etc., may also be considered as media carrying software. As used herein, unless defined as a non-transitory tangible "storage" medium, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

Accordingly, a machine-readable medium (e.g., computer-executable code) may take many forms, including but not limited to, tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media includes, for example, optical or magnetic disks, any of the storage devices, etc., in any computer, such as any storage device that may be used to implement the databases, etc., shown in the figures. Volatile storage media include dynamic memory, the 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 can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during Radio Frequency (RF) and Infrared (IR) data communications. Thus, common forms of computer-readable media 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, RAM, ROM, PROM and EPROM, a flash-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, a cable or link transporting such a carrier wave, or any other medium from which a computer can 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 301 may include or be in communication with an electronic display 335 that includes a User Interface (UI) 340 for providing instructions or means for the implementation of, for example, induction, callus growth, cell culture, elongation, or maturation. Examples of UIs include, but are not limited to, graphical User Interfaces (GUIs) and web-based user interfaces.

The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithm may be implemented by software when executed by the central processing unit 305. The algorithm may, for example, provide and/or execute instructions or embodiments for inducing callus growth, cell culture, elongation or maturation.

The invention is further described by the following non-limiting examples.

Examples

Example 1: preparation of plant cell compositions

Cells are isolated from a selected plant (e.g., cotton) by placing sterile explants from the following on callus induction medium (e.g., semi-solid basal salt medium) for induction: apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flowers, pedicel, flower wheel, roots, corms, germinated seeds, somatic and zygotic embryos, and Cambium Meristem Cells (CMC). The resulting dedifferentiation groups were regulated by three to five subcultures at intervals of 21-26 days on callus growth medium (e.g., semi-solid basal salt medium) for growth.

After stabilization of the cell culture, cells from the soft or friable callus are transferred to liquid medium to form suspension cells. The suspension is subcultured at 15-20 day intervals by filtration, pipetting/decanting or adding low concentration of pectase to provide a fine cell suspension culture. The homogenous nature of the cells in these cultures produced reproducible and reliable results.

Example 2: cryopreservation of suspension-cultured cells

Cryopreservation techniques eliminate the need for frequent culture and thus reduce the chance of microbial contamination. The protocol provided below allows for the simultaneous cryopreservation of more than 100 cell lines during a day.

Cells from suspension cultures of cotton in exponential growth phase (Gossypium spp.) and other species were transferred to 15ml tubes and centrifuged at 100xg for 1 min. The cell suspension was treated using a micropipette with a large orifice tip. The supernatant was removed and the cells were then suspended in a cryoprotectant solution (LS: 2M glycerol, 0.4M sucrose) supplemented with up to 100mM L-proline at a cell density of 10% (v/v) and incubated at room temperature for 0-120 min with or without shaking at 60 rpm. An aliquot of the cell suspension (0.5 ml) was dispensed into a frozen vial (feishier technologies company (Fisher Scientific)). Using a programmable refrigerator at-0.5deg.C, -1deg.C or-2deg.C for min ^-1 The frozen vials containing the cell suspension in LS were cooled to-35 ℃. After reaching-35 ℃, the cells were held at-35 ℃ for 0, 30 or 60 minutes and then plunged into liquid nitrogen.

In vitro dedifferentiated plant cell suspension cultures are more convenient for large scale production as they offer the advantage of a simplified model system for plant research. Cell suspension cultures contain relatively homogeneous cell populations, allowing for rapid and uniform acquisition of cell nutrients, precursors, growth hormones, and signaling compounds.

Example 3: cell recovery

Vials containing cryopreserved cells were transferred from liquid nitrogen storage containers to Dewar flasks (Dewar flash) containing liquid nitrogen. Each vial (one by one) was transferred to a clean 35-40 ℃ water bath and gently flipped several times until thawed (the last piece of ice disappeared). Immediately, each vial was again placed on ice. Each vial was centrifuged at 100g for 1-2 min at 4 ℃. The outside of each vial was wiped with 70% (vol/vol) ethanol and the supernatant from each vial was removed using a sterile Pasteur pipette (Pasteur pipette). Two-thirds of the volume of cells were transferred using a sterile 3.5-ml pipette by spreading or placing them as several clusters on filter paper. The dishes were closed and sealed with a Parafilm sealing film.

The dish was covered with one or two filter papers to reduce the light intensity, and then placed in a culture chamber under conventional conditions (24-26 ℃). After 2 days of recovery, some cell mass (about 100-200mg FW) was collected from the plate using a spatula (width 4 mm) and placed in microtubes for viability testing. The remaining cells were transferred with the upper filter paper to a new recovery dish containing recovery medium. The dishes were closed and sealed, covered with filter paper, and then returned to the culture chamber.

Depending on their growth rate, cells were allowed to grow in the same culture chamber under conventional conditions (24-26 ℃) for additional days. When most filters are covered with a thick layer of cells, the cell mass is transferred to a new dish without filter paper containing recovery medium and left under standard conditions for an additional 1-2 weeks (during this recovery phase agarose can be replaced by agar or another gelling agent). After a recovery period of 3-9 weeks, the cells were transferred to liquid medium to initiate suspension culture.

Example 4: bioreactor inoculation

For inoculum, the medium was prepared with Deionized (DI) water to make a total volume of 200mL (1L flask) and sterilized by autoclaving at 121 ℃ for 15 minutes. After cooling to room temperature, plant growth regulators and amino acids were added using a 0.2 μm pore size membrane filter. Twenty grams of cells were seeded and maintained in a shaker at 80rpm in the dark at a temperature of about 30 ℃ to about 35 ℃ and then left for inoculum growth. After 16 days (7-day lag phase and 9-day exponential phase), the density of the suspension was sufficient for bioreactor feed (titer=100 g L ^-1 Equivalent to concentrated applesauce without visible free medium).

An illustrative schematic of a bioreactor can be found in fig. 2. The bioreactor is fed with in vitro cells, sterile medium and air compression. The bioreactor was connected to a controller prior to inoculation to stabilize the pH 5.8 (+ -0.2) and control and calibrate O ₂ Is a flow rate of (a). As illustrated by the flow chart in fig. 2The first vessel of the inoculum train is shown at a temperature of about 30℃to about 35℃with 100g L ^-1 The cells are in the exponential phase. In parallel, sterilization of the medium occurs at about 125 ℃ to about 140 ℃, and (stream 16) is returned to the heat exchanger (stream 13) for cooling the medium at a temperature of about 30 ℃ to about 35 ℃ (E-103). In this case, the sterile medium is ready for feeding into the reactors of the multiplication zone (reactors R-101 to R-104).

The air used for cell oxygenation is also adjusted in the heat exchanger (E-105) according to the process temperature and is thus split into four different streams (streams 27, 28, 29 and 31) which are fed to the inoculum train (reactors R-101 to R-104).

For cells, doubling occurred over a duration of 5 to 12 days, and replication time was approximately 1 to 3 days (depending on lineage). These times end when the number of cells increases, for example 64 times. Finally, the contents are loaded into the next reactor (R-102), and so on. The last reactor (R-104) has an adjacent lung tank, wherein after the reaction the contents are discharged into a batch feed tank (Tq-101), continuously output (stream 5). Thus, during the doubling time of the R-104 reactor, tq-101 continuously unloaded cells at a continuous flow rate for the next stage of separation.

Table B below provides experimental results showing the success of seeding a bioreactor with cotton cells according to the methods disclosed herein. Table B provides details regarding cotton varieties from which cells were obtained, cell growth media inoculated in bioreactors, and other relevant conditions.

Table B

As shown in table B, seeding the bioreactor was more efficient when performed in dark conditions rather than bright conditions. Thus, the present disclosure provides methods of inoculating a bioreactor using a cotton cell culture, wherein the inoculation occurs under dark conditions.

As shown in table B, the composition of the growth medium used in seeding the bioreactor had an effect on cell growth. Accordingly, the present disclosure provides a method of inoculating a bioreactor with cotton cells, wherein the growth medium comprises a plant hormone or growth regulator. As shown in Table B, when the growth medium contained 2, 4-dichlorophenoxyacetic acid (2, 4-D), growth was promoted. Accordingly, the present disclosure provides a method of inoculating a bioreactor with cotton cells, wherein the growth medium comprises 2, 4-dichlorophenoxyacetic acid (2, 4-D).

As shown in table B, the methods of the present disclosure allow for successful cell growth when the bioreactor is seeded with cells from all cotton varieties tested. Accordingly, the present disclosure provides methods of inoculating a bioreactor with any cotton variety disclosed herein. In some embodiments, cotton cells for seeding a bioreactor according to the methods disclosed herein are derived and/or obtained, in whole or in part, from at least one cotton plant selected from the following varieties: PAYMASTER HS26, PD 2164, SA 2413, SEALAND#1 (G.B.X G.H.), SOUTHLAND Ml, status MILLER, TASHKENT 1, TIDEWATER (G.B.X.G.H.), TOOLE, WESTERN STORMPROOF, ACALLA 5, ALLEN 33, CD3HCABCUH-1-89, DELTAPINE 14, DES 24, DES 56, DIXIE KING, FJA, M.U.8B UA7-44, NC 88-95, PAYMASTER HS200, pima S-7, acala MAXXA, coassland 320 or progeny of any of these. In certain embodiments, cotton cells for seeding a bioreactor according to the methods disclosed herein are derived and/or obtained, in whole or in part, from at least one cotton plant selected from the following varieties: PD 2164, acala MAXXA, FJA, pima S-7 or a progeny of any of these.

As shown in table B, surprisingly, cells from cotton variety Pima S-7 provided good growth when inoculated into a bioreactor. This includes the case when microgram amounts of cotton cells are used to form an inoculum and across a range of growth media. Unexpectedly, pima S-7 provided superior growth/inoculation compared to Acala MAXXA and FJA. Furthermore, this superior growth occurs even when the same growth medium is used. For example, as shown in Table B, pima S-7 provided good growth while Acala MAXXA and FJA showed poor growth when cultured with all growth media at the same concentrations of MS, B5, glucose, kinetin and 2, 4-D. Thus, the present disclosure provides for seeding a bioreactor with cells derived and/or obtained in whole or in part from cotton plants of the Pima S-7 variety or progeny thereof.

Example 5: elongation of cells

For elongation, plant cells are separated from the medium using a decanter vessel (S-101) (stream 6), and the medium can be repositioned for water treatment (stream 45), as illustrated in the flow chart in fig. 2. The elongated growth medium is added to the reactor to be sterilized by autoclaving under the same conditions as used in the multiplication step and cooling at a temperature of about 30 ℃ to about 35 ℃ at which cell differentiation is performed.

Thus, cells from the three multiplying (stream 6) feed elongation reactors (R-105, R-106 and R-107) are represented by the reactor box (R-105) in the flow diagram of FIG. 2. Each reactor contains one third of the cells and the reaction volume includes a cell stream (stream 6), a medium stream (stream 38) and an air stream (stream 32).

Example 6: isolation and isolation of elongated cells

After elongation according to example 5, 3 tanks (Tq-102, tq-103 and Tq-104) are fed, which is represented only by the box Tq-102 in the flow chart of FIG. 2. Each tank, which has a volume slightly greater than the volume of the reactors, accommodates a volume substantially the same as the volume of the three reactors. The output of the elongation tank (stream 7) is routed to a second decanter (S-102). The bottom product (stream 8), comprising elongated cells and non-elongated cells, is routed to a screen (S-103) and the medium (stream 46) is removed for effluent treatment. The function of the screen is to remove non-elongated cells and smaller cells that are not pre-fibers. The screen (S-103) retains the elongated cells (pre-fibers) and releases all the non-elongated cells (which will not become cotton fibers).

Example 7: maturation and drying of cells

During the maturation stage and during the multiplication and elongation stages, sterile medium is used. Maturation is identified by secondary cell wall deposition. The sugars are combined to produce cellulose, the main component of cotton fibers (natural glucose polymerization), which occurs in the cells forming the secondary wall. In the process, the density of the preformed fibers was increased from 1.05g/ml to 1.55g/ml, which is the density of the cotton fibers.

After the maturation time, the R-108 output is directed to buffer tank Tq-105 (FIG. 2) to achieve a continuous downstream process. In sequence, the intermediate fiber mixture (stream 10) is routed to a third decanter (S-104) where cotton fibers (stream 11) are separated from the medium (stream 48). At this stage, the moisture content of the produced fibers is above an acceptable level (10% to 20% by mass). In order to reduce the moisture content, a drying process working with air is carried out. This air passes through the cotton fibers and part of the water is removed until the moisture content reaches at most 5%.

Example 8: recycle of

In some embodiments, the composition produced by the methods described herein may be recycled. For example, in such cases, after the completion of the method or steps of the method, an aliquot of the composition is retained and reintroduced into the earlier steps of the method. In some cases, an aliquot of cells that were unsuccessful in induction, growth, elongation, or maturation is retained and reintroduced into an earlier step of the method.

Example 9: production of cotton fibers from cotton ovule cells

Tables C, D and E below show the results of producing and elongating cotton cell cultures according to the methods disclosed herein. For each growth result, cotton ovule cells, which may comprise ovule epidermal cells, were mechanically extracted from cotton bolls. 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 culture was originally obtained. The table also provides the ovule location where cells were obtained from the parent cotton plant for cell culture.

Table C: cotton cell culture results from cells cultured from the upper ovule location.

Variety name	Ovule position	Growth results
			PAYMASTERHS26	Upper part	Some of the following
PD2164	Upper part	Excellent in
			SA2413	Upper part	Poor quality
SEALAND#1(G.B.XG.H.)	Upper part	Some/fibres
			SOUTHLANDM1	Upper part	Excellent in
STATIONMILLER	Upper part	Some of the following
			TASHKENT1	Upper part	Excellent in
TIDEWATER29(G.B.XG.H.)	Upper part	Excellent in
			TOOLE	Upper part	Poor quality
WESTERNSTORMPROOF	Upper part	Some of the following
			ACALA5	Upper part	Excellent/fiber
ALLEN33	Upper part	Some of the following
			CD3HCABCUH-1-89	Upper part	Good quality
DELTAPINE14	Upper part	Some of the following
			DES24	Upper part	Some of the following
DES56	Upper part	Poor quality
			DIXIEKING	Upper part	Excellent in
FJA	Upper part	Excellent in
			M.U.8BUA7-44	Upper part	Some of the following
NC88-95	Upper part	Some of the following
			PAYMASTERHS200	Upper part	Excellent in
PimaS-7	Upper part	Some of the following
			AcalaMAXXA	Upper part	Excellent in
Coasland320	Upper part	Poor quality

Table D: cotton cell culture results from cells cultured from the location of the intermediate ovule.

Table E: cotton cell culture results from cells cultured from the bottom ovule location.

As shown in table C, D, E, all varieties were successfully grown according to the methods of the present disclosure. Accordingly, the present disclosure provides cotton, methods of growing cotton according to any of the methods/protocols provided herein, and durable cell lines in which cotton cells are derived and/or obtained in whole or in part, which can be used across a range of cotton species and varieties. Thus, the in vitro cotton production method may use cotton cells derived from cotton plants of any variety, including those selected from the group consisting of: PAYMASTER HS26, PD 2164, SA 2413, SEALAND#1 (G.B.X G.H), SOUTHLAND Ml, status MILLER, TASHKENT 1, TIDEWATER (G.B.X G.H), TOOLE, WESTERN STORMPROOF, ACALLA 5, ALLEN 33, CD3HCABCUH-1-89, DELTAPINE, DES 24, DES 56, DIXIE KING, FJA, M.U.8B UA 7-44, NC 88-95, PAYMASTER HS200, pima S-7, acala MAXXA, coasland 320 and/or progeny of any of these.

As shown in tables C, D and E, certain varieties produced good or excellent growth. Thus, in certain embodiments, the cotton cells are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from the group consisting of: PD 2164, SOUTHLAND Ml, ACALA 5, CD3HCABCUH-1-89, FJA, TASHKENT 1, WESTERN STORMPROOF, PAYMASTER HS200, pima S-7, TIDEWATER 29, DIXIE KING, FJA, SOUTHLAND Ml and Acala MAXXA or progeny of any of these. Some varieties produce excellent growth. Thus, in certain embodiments, the cotton cells are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from the group consisting of: PD 2164, ACALA 5, SOUTHLAND Ml, CD3HCABCUH-1-89, FJA, pima S-7 and Acala MAXXA or progeny of any of these.

As shown in table C, some varieties produced good or excellent growth using cells obtained from ovules, which may comprise ovule epidermal cells, which may be located on the upper/top third of the bell, for example distal to the location on the bell where they are connected or connected to the stem of a cotton plant. Thus, in certain embodiments, 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 the bolls of at least one cotton plant of a variety selected from the group consisting of: PD 2164, SOUTHLAND Ml, ACALA 5, acala MAXXA, FJA, TASHKENT 1, PAYMASTER HS200, TIDEWATER, DIXIE KING and CD3HCABCUH-1-89 or progeny of any of these. As shown in table C, some varieties produced excellent growth using ovule cells and/or ovule epidermal cells obtained from the top third of the boll. Thus, in certain embodiments, 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 the bolls of at least one cotton plant of a variety selected from the group consisting of: PD (potential difference) device

2164. SOUTHLAND Ml, ACALA 5, acala MAXXA, FJA, TASHKENT 1, PAYMASTER HS, TIDEWATER and DIXIE KING or the progeny of any of these.

As shown in table D, some varieties produced good or excellent growth using ovule cells and/or ovule epidermal cells obtained from the middle third of the bolls. Thus, in certain embodiments, 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 the bolls of at least one cotton plant selected from the varieties of cotton plants selected from the varieties: PD 2164, SOUTHLAND Ml, CD3HCABCUH-1-89, acala MAXXA, FJA, TASHKENT 1, PAYMASTER HS, TIDEWATER and DIXIE KING or progeny of any of these.

As shown in table D, some varieties produced excellent growth using ovule cells and/or ovule epidermal cells obtained from the middle third of the boll. Thus, in certain embodiments, 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 the bolls of at least one cotton plant of a variety selected from the group consisting of: PD 2164, SOUTHLAND Ml, acala MAXXA, FJA, TASHKENT 1, PAYMASTER HS, TIDEWATER and DIXIE KING or the progeny of any of these.

As shown in table E, some varieties produced good or excellent growth using ovule cells and/or ovule epidermal cells obtained from the bottom third of the bell. Thus, in certain embodiments, 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 the bolls of at least one cotton plant selected from the varieties of cotton plants selected from the varieties: PD 2164, SOUTHLAND Ml, TASHKENT 1, WESTERN STORMPROOF, ACALA 5, CD3HCABCUH-1-89, FJA, pima S-7, acala MAXXA, PAYMASTER HS200, TIDEWATER 29 and DIXIE KING or progeny of any of these. As shown in table E, some varieties produced excellent growth using ovule cells and/or ovule epidermal cells obtained from the bottom third of the bolls. Thus, in certain embodiments, 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 the bolls of at least one cotton plant of a variety selected from the group consisting of: PD 2164, SOUTHLAND Ml, ACALA 5, TASHKENT 1, PAYMASTER HS, TIDEWATER 29, DIXIE KING, CD3HCABCUH-1-89, FJA, pima S-7 and Acala MAXXA or progeny of any of these.

As shown in tables C, D and E, certain varieties produced good or excellent growth using the material from more than one ovule location. Thus, in certain embodiments, 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 the group consisting of: PD 2164, SOUTHLAND Ml, ACALA 5, FJA, acala MAXXA, DIXIE KING, TIDEWATER, PAYMASTER HS200, and TASHKENT 1 or progeny of any of these.

As shown in tables C, D and E, certain varieties rapidly produced detectable levels of fiber in growing cells. Thus, in certain embodiments, the cotton cells are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from the group consisting of: SEALAND #1 (g.b.x G.H), ACALA 5, SA 2413, tolle, m.u.8b UA 7-44, DIXIE ping or progeny of any of these. As shown in tables C, D and E, certain varieties rapidly produced detectable levels of fiber in growing cells. As shown in tables C and E, ACALA 5 shows both rapidly detectable levels of fiber and excellent growth.

Table F below shows the results of growing and elongating cotton cell cultures derived from a particular variety according to the methods disclosed herein. For each growth result, cotton ovule cells, which may comprise ovule epidermal cells, were mechanically extracted from cotton bolls. 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 culture was originally obtained.

Table F: results of cotton cell culture

Variety name	Growth results	Fiber
			PD2164	Excellent in	Is that
SOUTHLANDM1	Excellent in	Is that
			TASHKENT1	Excellent in	Is that
TIDEWATER29	Excellent in	Is that
			DIXIEKING	Excellent in	Is that
FJA	Excellent in	Is that
			PAYMASTERHS200	Excellent in	Is that
AcalaMAXXA	Excellent in	Is that

As shown in Table F, using cells from ovules, the selected varieties produced excellent growth. Thus, in certain embodiments, 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 the group consisting of: PD2164, SOUTHLAND M1 FJA, PAYMASTER HS200, TIDEWATER, TASHKENT 1, DIXIE KING and Acala MAXXA or progeny of any of these.

As shown in Table F, cells from these selected varieties rapidly produced detectable levels of fiber in the growing cells. Thus, in certain embodiments, the cotton cells are derived and/or obtained, in whole or in part, from at least one cotton plant of a variety selected from the group consisting of: PD2164, SOUTHLAND M1, FJA, PAYMASTER HS200, TIDEWATER, TASHKENT 1, DIXIE KING and Acala MAXXA or progeny of any of these.

Claims

1. A method for producing cotton fibers, the method comprising:

Inoculating the bioreactor with cotton cells;

doubling the cells in the bioreactor;

elongating the multiplied cells; and

cotton fibers are harvested from the elongated cells,

wherein the cotton cells are derived and/or obtained from cotton plants of a variety selected from the group consisting of: PAYMASTER HS26, PAYMASTERHS200, PD2164, SA2413, sealand#1 (g.b. xg.h.), SOUTHLANDMl, STATIONMILLER, TASHKENT1, TIDEWATER29 (g.b. xg.h.), TOOLE, WESTERNSTORMPROOF, ACALA, ALLEN33, CD3HCABCUH-1-89, DELTAPINE14, DES24, DIXIEKING, FJA, m.u.8bua7-44, NC88-95, PAYMASTER HS200, pimaS-7, acala, and MAXXA or progeny of any of them.

2. The method of claim 1, wherein the variety is selected from PD2164, WESTERN STORMPROOF, CD3HCABCUH-1-89, TASHKENT1, SOUTHLANDMl, ACALA, FJA, PAYMASTERHS200, pimaS-7, and acaalamaxxa or progeny of any one thereof.

3. The method of claim 2, wherein the variety is selected from PD2164, SOUTHLANDMl, ACALA5, CD3HCABCUH-1-89, FJA, pimaS-7, and acaalamaxxa or progeny of any one thereof.

4. The method of claim 3, wherein the variety is selected from PD2164, SOUTHLANDMl, and CD3HCABCUH-1-89 or progeny of any one thereof.

5. The method of claim 4, wherein the variety is PD2164 or progeny thereof.

6. The method of claim 1, wherein the bioreactor is seeded with cotton ovule cells and/or ovule epidermal cells.

7. The method of claim 6, wherein the cotton ovule cells and/or the ovule epidermal cells are obtained from cotton bolls.

8. The method of claim 6, wherein the cotton ovule cells and/or the ovule epidermal cells are obtained from a bottom third, middle third, or top third of the cotton boll, wherein the bottom is a location on the boll where growth begins from cotton plant stems.

9. The method of claim 8, wherein the cotton ovule cells and/or the ovule epidermal cells are obtained from the top third of the cotton boll, and the variety is selected from PD2164 and ACALA5 or progeny of either.

10. The method of claim 8, wherein the cotton ovule cells and/or the ovule epidermal cells are obtained from the middle third of the cotton boll, and the variety is selected from PD2164 and FJA or progeny of either.

11. The method of claim 8, wherein the cotton ovule cells and/or the ovule epidermal cells are obtained from the bottom third of the cotton boll, and the variety is selected from PD2164, SOUTHLANDMl, ACALA5, CD3HCABCUH-1-89, FJA, pimaS-7, and acaalamaxxa or progeny of any one of them.

12. The method of claim 1, wherein the bioreactor is seeded with cells from a proliferating cell aggregate.

13. The method of claim 12, wherein the variety is PimaS-7 or progeny thereof.

14. The method of claim 1, wherein the proliferating cell aggregate is friable callus.

15. The method as recited in claim 14, further comprising:

obtaining cells from cotton explants; and

contacting said cells from said cotton explant with callus induction medium to produce said friable callus.

16. The method of claim 15, wherein the cells from cotton explants are from cotton apical meristems, cotyledons, young leaves, hypocotyls, ovules, ovule epidermal cells, stems, mature leaves, flowers, pedicel, flower wheels, roots, corms, germinated seeds, somatic and zygotic embryos, and/or Cambium Meristem Cells (CMC).

17. The method of claim 15, wherein the method further comprises:

isolating cells from the friable callus;

culturing the isolated cells; and

inoculating the bioreactor with the cultured isolated cells.

18. The method of claim 17, wherein the method further comprises:

culturing the isolated cells in a liquid or semi-solid medium to form a cell suspension;

cryopreserving the cell suspension; and

the bioreactor is seeded with a cryopreserved cell suspension.

19. The method of claim 18, wherein the method further comprises homogenizing the cell suspension to form a fine cell suspension.

20. The method of claim 1, wherein the method produces at least 1 kilogram of cotton fibers for every 4,000 liters of water used in the method.

21. An in vitro method for producing cotton fibers using cotton plant cells obtained and/or derived from cells of a cotton plant of a variety selected from the group consisting of: PD2164, WESTERNSTORMPROOF, CD3HCABCUH-1-89, TASHKENT1, SOUTHLANDMl, ACALA, FJA, PAYMASTERHS200, pimaS-7 and Acala MAXXA or progeny of any of these.

22. The method of claim 1, wherein the variety is selected from PD2164, SOUTHLANDMl, FJA, PAYMASTERHS200, TIDEWATER, TASHKENT1, DIXIEKING, and Acala MAXXA or progeny of any one thereof.

23. The method of claim 8, wherein the cotton ovule cells and/or the ovule epidermal cells are obtained from the top third of the cotton boll, and the variety is selected from PD2164, SOUTHLANDMl, FJA, PAYMASTERHS200, TIDEWATER, TASHKENT1, DIXIEKING, and acaalamaxxa or progeny of any one of them.

24. An in vitro method for producing cotton fibers using cotton plant cells obtained and/or derived from cells of a cotton plant of a variety selected from the group consisting of: PD2164, SOUTHLANDMl, FJA, PAYMASTERHS200, TIDEWATER, TASHKENT1, DIXIEKING and AcalaMAXXA or progeny of any of these.