Self-Fertilization as Methods for Stabilizing Ploidy after Colchicine Treatment Model

Exemplary methods include a method of stabilizing ploidy including doubling a plant's ploidy, self-fertilizing the doubled plant, and creating an offspring with a germline ploidy of a desired doubled ploidy targeted when doubling the plant's ploidy. Another exemplary method of stabilizing ploidy includes self-fertilizing a haploid plant, and creating an offspring plant with a germline ploidy that is diploid. A further method of stabilizing ploidy includes treating a female microtubule inhibitor treated plant with an ethylene blocker, producing male reproductive organs and pollen by the female microtubule inhibitor treated plant, fertilizing a clone of the female microtubule inhibitor treated plant with the pollen, and producing seeds and/or offspring.

BRIEF SUMMARY OF EXEMPLARY EMBODIMENTS

Exemplary methods include a method of stabilizing ploidy including doubling a plant's ploidy, self-fertilizing the doubled plant, and creating an offspring with a germline ploidy of a desired doubled ploidy targeted when doubling the plant's ploidy. Another exemplary method of stabilizing ploidy includes self-fertilizing a haploid plant, and creating an offspring plant with a germline ploidy that is diploid. A further method of stabilizing ploidy includes treating a female microtubule inhibitor treated plant with an ethylene blocker, producing male reproductive organs and pollen by the female microtubule inhibitor treated plant, fertilizing a clone of the female microtubule inhibitor treated plant with the pollen, and producing seeds and/or offspring.

In various exemplary embodiments, the plant may be from aCannabisgenus, including marijuana and hemp. Some exemplary embodiments may include fertilizing the female microtubule inhibitor treated plant itself with the pollen it produces. Various exemplary embodiments may include identifying a plant with spontaneously doubled haploids.

A leaf ploidy, according to various exemplary embodiments, may be approximately 52.69% diploid and approximately 11.83% tetraploid, or approximately 51.46% diploid and approximately 10.26% tetraploid, or approximately 38.37% diploid and approximately 7.69% tetraploid.

Certain exemplary embodiments may include the offspring plant with the germline ploidy being 100% diploid.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

TheCannabisplant is unique in that it is rarely able to spontaneously convert haploid genomes (In) to diploid genomes (2n) when haploidy has been induced by artificial means. This rare characteristic of theCannabisplant presents challenges when attempting to produce so-called “doubled haploids,” which have been shown to be valuable tools in plant breeding programs. Specifically, doubled haploid plants of a given species may be mated to one another to produce F1-hybrid seeds that give rise to identical plants. Consistency across individuals of a crop is a valuable agronomic trait—something that is non-existent inCannabiscrops cultivated from seed.

Haploid plants can be treated with colchicine or other chemicals (any chemical that will achieve increasing a ploidy level such as a doubling of the chromosomes with Oryzalin) to double the ploidy level to achieve a more stable and useful diploid (2n) state. In some exemplary embodiments, a colchicine concentration (in water) of 0.25% may be used in addition to 0.5%, 1% and 5%. An issue arises withCannabisin that not all the cells or tissues of a colchicine treated plant will double their ploidy. Colchicine treated haploidCannabisplants very rarely, if ever, achieve a fully diploid state.

On the single-cellular level, colchicine treatment can arrest mitosis just before anaphase by destabilizing microtubules, which are normally required to separate the doubled chromosomes synthesized during S-phase. Depending on the local dose, the cell either completes mitosis and gives rise to two cells with non-doubled chromosomes, or incomplete mitosis occurs, resulting in one cell with doubled chromosomes. If the entire plant can be derived from the latter single doubled event, the basal ploidy will be uniform throughout the plant.

On the multi-cellular level, doubling the ploidy of plants requires colchicine treatment applied to the shoot apical meristem (SAM). The SAM is composed of three distinct cell layers: L1 (outermost layer), L2 (middle layer), and L3 (innermost layer). Within the SAM, stem cell division takes place along the peripheral zone where new leaves emerge (Heidstra, 2014). The L1 gives rise to the epidermis or the protective covering of the leaf and stem, L2 to the sub epidermal photosynthetic mesophyll tissue, and L3 to the vascular tissue including the xylem and phloem. L1 and L2 divide anticlinally or parallel to each other and thus remain distinct layers (Meyerowitz, 1997). On occasion, a cell in the SAM can divide periclinally whereby the daughter cell invades and acquires the identity one layer out and brings its ploidy with it. This organizational dynamic creates a stable yet mutable developmental path. In fact, the discovery of the stability of different layers within a meristem was discovered by creating a chimera using colchicine onDatura. The researchers were able to follow the developmental lineage of the differently sized cells due to the different ploidy levels within the SAM (Satina, 1940).

Aside from a scenario in which one ploidy has a growth advantage over another, to double the entire plant completely and thoroughly, each layer of the SAM would need an independent doubling event. Doubling the ploidy with uniformity of the entire SAM is unlikely due to the complexity of the SAM's multicellular three-dimensional dome-like structure and the requirement for doubling events in each layer of every cell column. Indeed, several report mixaploid or chimeric plants after colchicine treatment (Sakhanokho, 2004).

The fact thatCannabisis very stable in a haploid state (the inventors have 12 independent exemplary embodiments of stable haploidy inCannabis) suggests diploid tissue may have little to no competitive growth advantage over haploid tissue, which means they can coexist. For example, if a successful conversion of both the L1 and L2 layer to a diploid state but the L3 layer remains haploid after colchicine treatment, L3 layer may unpredictably contribute haploid cells to the L2 layer and if this happens in the anther it could negatively affect gamete development. These unstable scenarios are not practical for a commercial seed producer that requires consistency.

According to exemplary embodiments, colchicine treatedCannabisplants can be “selfed” to produce seeds. Self-fertilizing a doubled haploid will form a diploid zygote from male and female gametes, each contributing identical haploid chromosome content. Because this single celled zygote will give rise to the entire plant (including all three layers of the SAM) seeds give rise to stable plants with uniform basal ploidy (De Rybel, 2016). Alternative methods of producing diploid plants from haploid plants require laborious analytical methods to ensure that all cells and tissue layers are fully diploid. Our method for establishing diploidy through self-fertilization is an elegant alternative to previous methods.

FIG.1Ashows exemplary leaves from the cultivar “Standing Liberty” either (left) in a haploid (n) state, or (middle) mixaploid (n+2n) state, and (right) from the diploid (2n) parent of the haploid. A “mixaploid state” refers to a condition in which a plant or organism has cells with varying ploidy levels, meaning some cells have different numbers of chromosome sets.

A “mixaploid state” refers to a condition in which a plant or organism has cells with varying ploidy levels, meaning some cells have different numbers of chromosome sets.

Haploid (n): Cells with a single set of chromosomes.

Diploid (2n): Cells with two sets of chromosomes, typical of most organisms, including humans.Polyploid: Cells with more than two sets of chromosomes. Examples include:Triploid (3n): Three sets of chromosomes.Tetraploid (4n): Four sets of chromosomes.Hexaploid (6n): Six sets of chromosomes.Mixaploidy: The condition of having different ploidy levels within the same organism. This can occur naturally or be induced artificially. It might result from irregular cell division, somatic mutation, or experimental treatments.

Possible Scenarios of Mixaploidy:

Somatic Mosaicism: An organism may have cells with different ploidy levels due to endoreduplication during development. This results in a mosaic pattern where some cells are diploid while others are polyploid.

Chimeras: An organism composed of genetically distinct cells. For example, grafting can result in a plant with tissues that have different ploidy levels.

Experimental Induction: Researchers might create mixaploid conditions to study the effects of different ploidy levels on plant development and physiology.

FIG.1Bshows exemplary flow cytometer data from the cultivar “Standing Liberty” in a haploid (n) state, in which approximately 65.11% of the cells counted are haploid, approximately 9.2% are diploid. The diploid cells are likely a result of naturally occurring endoreduplication or spontaneous doubling, whereas the triploid and tetraploid cell counts do not have a distinct peak apart from the background noise.

FIG.1Cshows an exemplary mixaploid (n+2n) state plot shows a ploidy of approximately 49% diploid, approximately 12.48% haploid, and approximately 4.39% tetraploid. The mixaploid state has a pattern distinct from the haploid and diploid, which show mostly diploid cells, which likely accounts for a mostly successful doubling, but also a stubbornly stable subset of haploid cells, which do have their own distinct peak.

FIG.1Dshows an exemplary diploid (2n) parent of the haploid shows approximately 52.69% diploid, approximately 11.83% tetraploid, which is most likely from naturally occurring endoreduplication. This diploid parent also has counts from haploid and triploid cells that do not have their own distinct peak apart from the noise.

FIG.2Ashows exemplary leaves from the cultivar “Herbaceous Sting” either (left) in a haploid (n) state, or (middle) mixaploid (n+2n) state, and (right) from the diploid (2n) parent of the haploid. A “mixaploid state” refers to a condition in which a plant or organism has cells with varying ploidy levels, meaning some cells have different numbers of chromosome sets.

FIG.2Bshows exemplary flow cytometer data from the cultivar “Herbaceous Sting” in a haploid (n) state, in which approximately 61.06% of the cells counted are haploid, approximately 14.4% are diploid. The diploid cells are likely a result of naturally occurring endoreduplication or spontaneous doubling, whereas the triploid and tetraploid cell counts do not have a distinct peak apart from the background noise.

FIG.2Cshows an exemplary mixaploid (n+2n) state plot shows a ploidy of approximately 45.82% diploid, approximately 12.15% haploid, and approximately 3.93% tetraploid The mixaploid state has a pattern distinct from the haploid and diploid, which show mostly diploid cells, which likely accounts for a mostly successful doubling, but also a stubbornly stable subset of haploid cells, which do have their own distinct peak.

FIG.2Dshows an exemplary diploid (2n) parent of the haploid shows approximately 51.46% diploid, approximately 10.26% tetraploid, which is most likely from naturally occurring endoreduplication. This diploid parent also has counts from haploid and triploid cells that do not have their own distinct peak apart from the noise.

FIG.3Ashows exemplary leaves from the cultivar “Operation Green Harvest Haze” either (left) in a haploid (n) state, or (middle) mixaploid (n+2n) state, and (right) from the diploid (2n) parent of the haploid. A “mixaploid state” refers to a condition in which a plant or organism has cells with varying ploidy levels, meaning some cells have different numbers of chromosome sets.

FIG.3Bshows exemplary flow cytometer data from the cultivar “Operation Green Harvest Haze” in a haploid (n) state, in which approximately 56.4% of the cells counted are haploid, approximately 8.63% are diploid. The diploid cells are likely a result of naturally occurring endoreduplication or spontaneous doubling, whereas the triploid and tetraploid cell counts do not have a distinct peak apart from the background noise.

FIG.3Cshows exemplary mixaploid (n+2n) state plot shows a ploidy of approximately 22.8% diploid, approximately 32.4% haploid, and approximately 3.1% tetraploid. The mixaploid state has a pattern distinct from the haploid and diploid, which show mostly diploid cells, which likely accounts for a mostly successful doubling, but also a stubbornly stable subset of haploid cells, which do have their own distinct peak.

FIG.3Dshows an exemplary diploid (2n) parent of the haploid shows approximately 52.69% diploid, approximately 11.83% tetraploid, which is most likely from naturally occurring endoreduplication. This diploid parent also has counts from haploid and triploid cells that do not have their own distinct peak apart from the noise.

FIG.4Ashows exemplary leaves from the cultivar “Cryptic Lineage Legacy” either (left) in a haploid (n) state, or (middle) mixaploid (n+2n) state, and (right) from the diploid (2n) parent of the haploid. A “mixaploid state” refers to a condition in which a plant or organism has cells with varying ploidy levels, meaning some cells have different numbers of chromosome sets.

FIG.4Bshows exemplary flow cytometer data from the cultivar “Cryptic Lineage Legacy” in a haploid (n) state, in which approximately 59.11% of the cells counted are haploid, approximately 12.43% are diploid. The diploid cells are likely a result of naturally occurring endoreduplication or spontaneous doubling, whereas the triploid and tetraploid cell counts do not have a distinct peak apart from the background noise.

FIG.4Cshows an exemplary mixaploid (n+2n) state plot shows a ploidy of approximately 20.65% diploid, approximately 17.65% haploid, and approximately 2.63% tetraploid. The mixaploid state has a pattern distinct from the haploid and diploid, which show mostly diploid cells, which likely accounts for a mostly successful doubling, but also a stubbornly stable subset of haploid cells, which do have their own distinct peak.

FIG.4Dshows an exemplary diploid (2n) parent of the haploid shows approximately 52.69% diploid, approximately 11.83% tetraploid, which is most likely from naturally occurring endoreduplication. This diploid parent also has counts from haploid and triploid cells that do not have their own distinct peak apart from the noise.

FIG.5Ashows exemplary leaves from the (left) self-fertilized doubled haploid cultivar “Mary Jane Manhunt” in a diploid (2n) state, or (middle) haploid (n) state, and (right) from the diploid (2n) parent of the haploid. The mixaploid data for the colchcine treated haploid was not gathered.

FIG.5Bshows exemplary flow cytometer data from the self-fertilized doubled haploid cultivar “Mary Jane Manhunt” in a diploid (2n) state, in which approximately 58.33% of the cells counted are diploid, approximately 7.4% are tetraploid. This self-fertilized doubled haploid cultivar also has counts from haploid and triploid cells that do not have their own distinct peak apart from the noise.

FIG.5Cshows an exemplary haploid (n) state plot shows a ploidy of approximately 61.06% haploid, approximately 14.4% diploid. The diploid cells are likely a result of naturally occurring endoreduplication or spontaneous doubling, whereas the triploid and tetraploid cell counts do not have a distinct peak apart from the background noise.

FIG.5Dshow an exemplary diploid (2n) parent of the haploid shows approximately 38.37% diploid, approximately 7.69% tetraploid, which is most likely from naturally occurring endoreduplication. This diploid parent also has counts from haploid and triploid cells that do not have their own distinct peak apart from the noise.

At step601, a plant is treated with colchicine (or any chemical that will achieve increasing a ploidy level such as a doubling of the chromosomes with Oryzalin). In various exemplary methods, the plant is from the genusCannabis(hemp or marijuana).

Oryzalin belongs to the dinitroaniline class of herbicides and works by inhibiting the formation of microtubules, which are essential for cell division and growth. Colchicine is a naturally occurring alkaloid derived from the plantColchicum autumnale, commonly known as autumn crocus or meadow saffron. Colchicine works by inhibiting microtubule polymerization, which disrupts various cellular functions, including mitosis (cell division).

Micro tubule polymerization is a cellular process involving the assembly of microtubules, which are dynamic, cylindrical structures made of tubulin protein subunits. Microtubules are a critical component of the cytoskeleton, providing structural support, facilitating intracellular transport, and playing key roles in cell division and motility.

Microtubules are composed of α-tubulin and β-tubulin dimers. These dimers polymerize to form protofilaments, which then associate laterally to create the hollow, tube-like structure of a microtubule.

Nucleation: The initial step where a small number of tubulin dimers come together to form a stable nucleus, which serves as a template for further growth.

Elongation: The addition of tubulin dimers to the growing ends of the microtubule. Microtubules have a dynamic nature, with a “plus” end that grows faster and a “minus” end that grows more slowly.

Dynamic Instability: Microtubules constantly switch between phases of growth (polymerization) and shrinkage (depolymerization), a phenomenon known as dynamic instability. This allows the cell to rapidly reorganize its cytoskeleton in response to various stimuli.

Role of Microtubules:

Cell Division: Microtubules form the mitotic spindle, which is crucial for the segregation of chromosomes during mitosis and meiosis.

Intracellular Transport: Microtubules serve as tracks for the movement of organelles and vesicles, with motor proteins like kinesin and dynein transporting cargo along these tracks.

Cell Shape and Motility: Microtubules help maintain cell shape and enable cellular movements, including cilia and flagella beating and amoeboid movement.

Regulation of Microtubule Dynamics:

Microtubule-Associated Proteins (MAPs): These proteins bind to microtubules and regulate their stability and dynamics. Examples include tau protein and MAP2.

GTP Hydrolysis: Tubulin dimers bind to GTP before adding to the microtubule. GTP hydrolysis to GDP after incorporation affects the stability of the microtubule, promoting dynamic instability.

Inhibitors of Microtubule Polymerization:

Certain drugs and natural compounds can inhibit microtubule polymerization by binding to tubulin and preventing its assembly. These include:

Colchicine: Binds to tubulin, inhibiting microtubule polymerization, and is used to treat gout.

Vincristine and Vinblastine: Chemotherapy agents that disrupt microtubule dynamics, inhibiting cell division.

Taxanes (e.g., Paclitaxel): Stabilize microtubules and prevent their depolymerization, also used in cancer therapy.

At optional step602, identify doubled haploids.

Spontaneously doubled haploids are plants that naturally undergo chromosome doubling from the haploid state to the diploid state. This process results in plants that are homozygous for all genes, meaning they carry identical alleles for every gene.

At step603, the plant from step601or602is self-fertilized.

Self-fertilization, or selfing, in plants is a reproductive process where a plant's sperm cells (pollen) fertilize its own egg cells (ovules). This leads to the production of seeds that develop into new plants. Self-fertilization can occur in plants that possess both male (anthers) and female (stigma) reproductive structures. Self-fertilization can also occur if a clone is used to fertilize another clone of the same genetic background.

At step604, create/germinate offspring with a stable doubled haploid germline.

At step701, a female colchicine (or any chemical that will achieve increasing a ploidy level such as a doubling of the chromosomes with Oryzalin) treated plant is treated with a colloidal silver spray or other ethylene response blocker.

Colloidal silver spray is a suspension of microscopic silver particles dispersed in water. It is often used for its purported antibacterial, antiviral, and antifungal properties. The silver particles in colloidal silver are typically between 1 and 100 nanometers in size. These particles are suspended in a liquid, usually distilled water.

An ethylene response blocker is a substance that inhibits the action or production of ethylene, a plant hormone involved in regulating various physiological processes such as fruit ripening, flower wilting, leaf abscission, and senescence. By blocking ethylene's effects, these substances can extend the shelf life of fruits and flowers, delay senescence, and manage other ethylene-related processes in plants. Ethylene response blockers prevent the physiological changes induced by ethylene by inhibiting its synthesis, perception, or signal transduction pathway.

Types of Ethylene Response Blockers:

1-Methylcyclopropene (1-MCP): One of the most widely used ethylene blockers. It binds to the ethylene receptors in plant cells, preventing ethylene from binding and triggering its effects. It is used in the storage and transport of fruits and flowers.

Silver Thiosulfate (STS): An inhibitor of ethylene action used mainly in the floral industry to prolong the vase life of cut flowers.

Aminoethoxyvinylglycine (AVG): Inhibits ethylene biosynthesis by blocking the activity of the enzyme ACC synthase, which is critical in the ethylene production pathway.

Ethylene Antagonists: Compounds like potassium permanganate that oxidize ethylene to carbon dioxide and water, removing it from the environment around stored produce.

Mechanism of Action:

Ethylene response blockers can work at different points in the ethylene pathway:

Biosynthesis Inhibition: Preventing the production of ethylene within the plant.

Receptor Blocking: Competing with ethylene for receptor sites, preventing it from binding and activating the signal transduction pathway.

Signal Transduction Interruption: Disrupting the downstream signaling cascade that leads to ethylene's physiological effects.

At step702, the female colchicine (or any chemical that will achieve increasing a ploidy level such as a doubling of the chromosomes with Oryzalin) treated plant produces male reproductive organs and pollen. This is most likely caused by step701.

At step703, the pollen produced at step702is used to fertilize a clone of the female colchicine (or any chemical that will achieve increasing a ploidy level such as a doubling of the chromosomes with Oryzalin) treated plant.

A clone of a plant is a genetically identical copy of the original plant. Cloning in plants can occur naturally or be induced artificially through various horticultural techniques.

Key Aspects of Plant Cloning:

Vegetative Reproduction: Many plants naturally produce clones through vegetative reproduction. This involves the growth of new plants from parts of the parent plant, such as stems, roots, or leaves.

Cuttings: One of the most common methods of cloning plants is taking cuttings. A piece of the stem, leaf, or root is cut from the parent plant and placed in a suitable growing medium to develop roots and grow into a new plant.

Layering: In this technique, a branch or stem is bent to the ground and covered with soil while still attached to the parent plant. Once roots develop, the new plant is separated from the parent.

Air Layering: Inducing root formation on a branch or stem while it is still attached to the parent plant, often by wrapping it with moist moss and plastic.

Grafting and Budding: This involves joining parts of two plants together so they grow as one. A piece of a desired plant (scion) is attached to a rootstock. This technique is widely used in fruit tree propagation.

Tissue Culture (Micropropagation):

In Vitro Cloning: This highly controlled method involves growing plant cells, tissues, or organs in a sterile environment on a nutrient culture medium. Tissue culture allows for the mass production of clones from a small amount of plant material and is used for plants that are difficult to propagate by traditional methods. Steps include:

Initiation: Explants (small pieces of plant tissue) are taken from the parent plant and sterilized.

Multiplication: Explants are placed on a growth medium containing nutrients and hormones to stimulate cell division and the formation of new shoots.

Rooting: Shoots are transferred to a rooting medium to develop roots.

Acclimatization: The new plantlets are gradually acclimatized to normal growing conditions.

At optional step704, the pollen produced at step702is used to fertilize the female colchicine treated (or any chemical that will achieve increasing a ploidy level such as a doubling of the chromosomes with Oryzalin) plant itself before use of the colloidal silver spray or other ethylene blocker.

At step705, seeds and/or offspring are produced.

Example One: Female Sex Reversal inCannabis

Short-day (long-night)Cannabisplants are grown for at least 4 weeks under 18-hour day, 6-hour night light cycle and were thoroughly sprayed in the dark with 2.7 mM AgNO3 and 11 mM Sodium thiosulfate solution. A 2.7 mM solution of AgNO3refers to a concentration of silver nitrate (AgNO3) in a solution where there are 2.7 millimoles of AgNO3per liter of solution. An 11 mM sodium thiosulfate solution refers to a solution where the concentration of sodium thiosulfate (Na2S2O3) is 11 millimoles per liter. These plants were left in the dark for 12 hours with adequate airflow to allow the spray to dry on the plant. These plants were then grown under a 12-hour day, 12-hour night light cycle and sprayed at the beginning of the night cycle every 3 days for a total of 4 treatments. Pollen is usually ready 6 weeks after the first treatment. Pollen from the reversed plant is then applied on stigma of a female plant that has been grown in a 12-hour day and 12-hour night light cycle for 3 weeks.

WORKS CITED

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