WO2018184031A1 - Cannabis gender identifier - Google Patents

Abstract

The present teaching disclose a CANNABIS GENDER IDENTIFIER that accepts an input sample derived from a cannabis plant and quickly detects the presence the endogenous molecules associated with male, female or hermaphrodite expression in cannabis plants, also known as gender associated targets (GATs), with the assistance of biochemical reactions. A candidate for GAT selection is any endogenous molecules that is differentially exhibited in cannabis genders. The gender identifier houses indicators that bind to GATs which emit signals in a reaction area when the GATs are bound to the indicators. The gender identifier analyses the contents in the reaction area, and communicates the results of the reaction to the viewer, indicating the associated gender of the input sample.

Description

Background

Cannabis plants are taxonomically categorized as the species cannabis sativa L, within the cannabaceae taxonomic family. Within the cannabis industry, there are two distinct classifications of cannabis plants beyond the species distinction: (1) hemp, or ruderalis; and (2) medicinal or recreational. Each type has macro-level morphological variation as well as micro- level biochemical variation. In many instances the micro-level variations contribute to the larger macro-level variations observable by the human eye without technological assistance.

The big difference between hemp and medicinal or recreational cannabis is whether the plant produces high fiber or high drug content, specifically the psychoactive cannabinoid delta-9- tetrahydrocannabinol (THC). Many recognize the micro-level criteria of < 0.3% THC as a threshold indicative of hemp type cannabis. THC is a small molecule that is biochemically produced by cannabis and cannot be seen or quantified without advanced instruments of analytical chemistry. Some of the technologies used to ascertain THC concentration include, but are not limited to, gas chromatography-mass spectrometry or high pressure liquid

chromatography.

Beyond the extremely low level of THC concentration, hemp plants produce high levels of fiber. Hemp constituents are used to produce textiles, personal products such as shampoo or lotions, and even food. Morphologically, hemp is long, sparse, and stalky. In contrast, indica, sativa, and hybrid are morphological descriptors used to describe the morphological variations observed in medical or recreational cannabis. Each sub-category displays common physical traits and characteristics. Indica plants are shorter, bushier, and have 5 leaflets per leaf. Sativa cannabis plants are taller, thinner, and have 7 leaflets per leaf. Hybrids are a combination of the two.

However, sativa plants are not as tall or thin as hemp type cannabis plants.

Moreover, cannabis used recreationally or medically contains much higher levels of THC than the 0.3% hemp threshold because consumers desire the effects of consuming THC and generally pay more for more THC. There are many different types of consumable product, but all are typically derived from cannabis flowers such as a joint (dried flower in a paper wrapping), hashish, or edible (dried flower concentrate mixed into food). The amount of THC in the flower is dependent upon the amount of THC produced by the original plant the product was made from. On a chemical level, all cannabis plants are rich in other cannabinoids beyond THC. These cannabinoids are also used by consumers for medical benefit or euphoric effect. Cannabis above and below the 0.3% THC threshold produces a spectrum of desirable cannabinoids that provide these effects. Although hemp does not produce high levels of THC, hemp plants produce plenty of other cannabinoids and are a good source of cannabidiol (CBD). In this instance, a chemotype describes the spectrum of cannabinoids produced by a cannabis plant and is a more relevant descriptor than morphology or industrial purpose. A chemotype is used to describe cannabinoid content such as high THC, high CBD, and roughly equivalent ratio of one-to-one THC to CBD.

Within each sub-type of cannabis are two smaller grouping called varietals and cultivars. Cannabis plants belonging to the same varietal or cultivar consistently express the same or similar traits, characteristics, or morphologies. The difference is that for varietals, the observable traits, characteristics, or morphologies are due to natural selection by mother nature. A landrace is another term used to describe such cannabis plants. Cultivars exist due to selective breeding or cultivation by human intervention.

In society, the terms are largely used interchangeably because humans have been cultivating cannabis for thousands of years, historically blurring the line between varietal and cultivar. Cannabis remnants have been discovered in many different ancient civilizations, some as early as 10,000 years old, the age of human cultivation itself. These areas include, but are not limited to, modern day countries such as China, Egypt, or Turkey as well as Scythian, Samarian, and Thracian territories. Moreover, the path of cannabis cultivation can be generally traced through time as it is introduced to other countries India, Russia, Khazakstan, Greece, Germany, Jerusalem, England, France, Iceland, Arabia, Portugal, the United States and more.

For as long as cannabis has been farmed, cultivators have long known that both hermaphrodite and singular gender morphologies are expressed. A hermaphrodite is capable of expressing both male and female characteristics within the same plant. Singular gendered plants express either morphology and are distinctly male or female. Cannabis gender morphology is observable by during the flowering phase, after the plant has completed the vegetative growth phase.

Male cannabis plants grow different flowers than female cannabis plants. Females make a pistillate flower with pistils and males make a staminate flower, or stamens. Male flowers produce and release pollen to pollenate the ovaries contained in the female flowers, which is where seed production occurs when the female is pollinated. A hermaphrodite sexuality is where a single plant is capable of displaying both female and male flowers. A healthy hermaphrodite can pollinate itself or other female as well as be pollinated by other males to produce seeds.

Gender is a quality that many cultivators selectively breed for and cultivators often need to identify the gender of each plant grown long before harvest. For example, when growing hemp varietals or cultivars for industrial purposes, males are more fibrous than females and are targeted for the higher fiber content. Amongst the high THC producing varietals or cultivars, only the unpollinated female is capable of producing high quantities of THC.

When growing cannabis for high THC purposes, pollen can be likened to an infected crop. If pollenated, the female plant diverts energy into producing seeds, rather than THC -rich flowers, limiting potency and market value. Because every cannabis seed grown could potentially be a male or female, the males are sequestered at first sign of any male associated physical features, such as the appearance of male pre-flowers. Once spotted, there is a short window of time until the incoming pollen grains are released. Once released by the male flowers, pollen quickly spreads through the air.

The risk of pollen contamination is a reason female plants must often be grown in clean environments, with filtered air. Clonal propagation from a strong female mother plant minimizes the risk of pollination. The mother is the original plant from which subsequent clones are produced and every clone contains the same genes as the mother. Propagating clones partially solves the pollen issue but is not an ideal solution. The downsides to using clones as starting material rather than seed include, but are not limited to, a lack of genetic variation, lag time for rooting, as well as degradation and loss of vigor over multiple passages. Moreover, poor quality product results from accidentally selecting a mother with poor genes or hermaphroditic tendencies.

Accidentally cloning a hermaphrodite occurs because gender in cannabis plants is attributable to genotype, phenotype, and epigenetics. A genotype is the collection of the all the genes contained within an organism that were passed down through its lineage. The phenotype is the observable trait(s) that developed in response to the environment; it is the expression of the genotype observed in the organism. Due to the complex nature of the interactive biological mechanisms involved, an organism's genotype provides the genetic code for hereditary expression, but multiple iterations of phenotype result.

In expressing phenotype from genotype, many gene products and genes interact with one another. Genes are made from chains of deoxyribonucleic acid (DNA). Gene products are micro- level constituents expressed from genes and include, but are not limited to, ribonucleic acid (RNA), chemicals, and proteins, but also refer to more complex organelles and cells depending on the context used. Further complicating the genotype-phenotype dichotomy is the discovery of epigenetics. Epigenetic genes are genes that cause a change in observed phenotype without affecting the genotype. With epigenetics, modulation of gene expression occurs when genes turn on and off in response to environmental stimuli.

The observable traits, characteristics, and morphology of varietal or cultivar could be the result of phenotype and is not necessarily a genetic trait that will be passed down to or observed in progeny. For this reason, it is not necessarily correct to refer to a varietal or cultivar as a "genetic" or "strain," commonly used lay terms that describe the expected features of cannabis plants. Moreover, sometimes phenotype is mistaken for a genotype and a change in growth conditions causes a different phenotype to be expressed, hence an accidental hermaphrodite. A varietal or cultivar may be categorized as so for a defining quality to traits, characteristics, or morphologies attributable to the same genotype or phenotype. Different environmental influences and factors cause varying levels of gene product expression, which affect plant development. Varietals and cultivars with the same genotype can express different phenotypes due to the interaction between a plant and the environment in which it is grown. Some varietals or cultivars can consistently express the same phenotype due to being grown in the same or similar environmental conditions but are not necessarily attributable to the same genotype.

Detailed Description

Cannabis plants undergo highly complex differential mechanisms for gender that have not yet been completely understood. The observable female or male flowers on cannabis plants are the culmination of the expression of many gene products on a larger scale. As with other inherited traits, characteristics, and morphologies, gender is attributable to genotype, phenotype, and epigenetics. This is how seemingly female or male plants are capable expressing the other gender with hermaphrodite morphologies. A particular cannabis plant may be genotypically male or female but is capable of showing hermaphrodite features when the environmental or epigenetic factors affect the gender phenotype expressed.

Genotypically, both sex chromosomes and autosomal chromosomes contribute to the expression of gender. Some cannabis plants express gender specific morphology because it contains sex chromosomes dedicated to male or female development. The male genotype contains a Y chromosome and the female contains the X chromosome. However, some cannabis plants express distinct gender due to other mechanisms, such as autosomal regulation. The exact mechanisms of autosomal regulation of gender in cannabis is not yet known.

Gender phenotype is affected by autosomal chromosomes that either directly upregulate or downregulate the expression of the sex chromosomes or indirectly through modulation of other autosomal genes and gene products. An example of autosomal regulation of gender occurs when a cannabis plant's genotype contains female sex chromosomes, XX, with other autosomal genes interacting to downregulate or suppress gene products contributing to female development, resulting in the male or hermaphrodite phenotype.

It is likely that epigenes, phenotype, environmental factors, autosomal genes, and sex genes up or downplay male or female expression. For example, consider a cannabis

hermaphrodite phenotype caused by having an XX genotype with autosomal down or up regulation of gene products due to external influences such as epigenetic factors. It is possible that a balance of autosomal gene regulation maintains the female phenotype and when that balance is shifted a hermaphrodite phenotype is exhibited.

The down regulation of genes coding for gene products that are involved in female expression or metabolic pathways could be so severe that a completely male phenotype is expressed. For example, some male plants are actually suppressed females, where the genes coding for female characteristics are repressed by external factors. A problem occurs when these plants or progeny of these plants are grown in a different condition that causes male traits to develop in a seemingly female crop. In that way, analyzing the endogenous molecules produced by the plant would help determine gender at any given moment.

Genetic gender identification approaches for cannabis has existed for decades but requires intensive labor, tracking, time, and costs. Genetic analysis can detect gender associated DNA fragments during the vegetative growth phase, but but cannot identify non-genotypic mechanisms of gender expression. The modern tools of DNA detection are able to detect some male or female genes from DNA extracted from a cannabis tissue sample. However, the genetic mechanisms for autosomal regulation, hermaphrodite phenotype, suppression of gender is unknown. Scientists commonly use genetic tools such as qPCR and PCR to conduct genetic detection using methods such as AFLP, or markers such as RAPD, SCAR or MADCl-6.

Detecting other molecular gender differences is a worthwhile way of identifying cannabis gender expression and is more accurate than genetic tools alone. The present teachings allow cannabis cultivators and breeders to preserving crop integrity by having access to gender detection that is faster, cheaper, and easier to use than the current available technology, and significantly improve the ability to identify gender expression in cannabis before macro-level physical characteristics, traits, or morphology is observable.

The present teachings disclose a plurality of gender associated targets (GATs) and GAT indicators. GATs are endogenous molecules selected for identification by the gender identifier because they are known to be differentially expressed across cannabis genders. A selected GAT serves as a standard of comparison within the gender identifier, and multiple GAT selections are preferred. Candidates for GAT selection include any endogenous molecule that exhibits some comparative qualitative or quantitative difference across genders.

Endogenous molecules (103) include cannabis genes, gene products, and any other bioproduct that results from interaction between those genes and gene products; molecules produced as part of the microbiome; and molecules produced by the genetic modification and natural selection of cannabis plants. If a GAT or GATs are present in the starting sample, indicators specifically bind to them and detect the expressed gender of the plant.

The gender identifier identifies the expressed gender of a cannabis plant by specifically reacting to the presence of GATs, molecules selected as analytes to detect in the gender identifier because they are indicative of gender. The presence of particular GATs in a sample indicates gender because they are selected for detection on the premise that the molecule is differentially expressed or present across cannabis genders. In many instances, a GAT is selected for detection by the gender identifier due to its observable presence before macro-level gender expression, that is, before gender specific physical features, traits, characteristics, or morphology (i.e. before the flowering stage).

The disclosures are a more accurate identifier of cannabis gender than genetic sequencing alone due to the fact that the endogenously expressed gene products that affect gender expression are attributable to autosomes as well as sex chromosomes. Moreover, molecular profiling can help detect gender micro-expressions. Using several GATs in one identifier is the most accurate manner of detecting shifting gender expression due to the environment, epigenetic factors, phenotype, and regulation of both autosomal and sex chromosomes because a profile of GATs can be created.

For example, the male phenotype is expressed in a plant having female gender chromosomes, XX; analyzing the DNA detects female genes, but the expressed non-DNA endogenous bioproducts (such as RNA, protein and chemical bioproducts) is a more accurate indicator of gender than the genetic sequencing test alone and will show that the male gender has been expressed in the female plant.

The present teaching disclose a cannabis gender identifier (101) that accepts an input sample (108), analyses the contents in the reaction area, and communicates the results of the reaction to the viewer, indicating associated gender of the input sample. The input sample is derived from a cannabis plant.

When a certain cannabis plant is selected for gender detection with the gender identifier, the input sample (102) is the source of analytes that are to be detected by the gender identifier as male, female, and in some cases, hermaphrodite.

The input sample is prepared from the selected plant in one of five ways: (1) a portion of the plant, the cannabis tissue, is used as the input sample; (2) the cannabis tissue is processed into a starting sample that used the input sample; (3) the endogenous molecules of interest (EMOI) (104) contained in the starting sample are captured in a widget and the widget is the input sample; (4) the EMOI contained in the starting sample are captured in a widget, the widget releases the captured EMOI into a concentrate, and the concentrate is used as the input sample; or (5) the EMOI contained in the starting sample are captured in the widget and the widget is used as the medium. The input sample is placed into the gender identifier through the sealable opening to make contact with the receiving area.

The cannabis tissue is selected from any part of the cannabis plant, including, but not limited, to the embryo, seed, shell, root, stem, cotyledon, offshoot, leaf, flower, and any other tissue of a cannabis plant to be identified as male, female, or hermaphrodite by the gender identifier. The cannabis tissue includes fresh plant matter taken directly from a live cannabis plant as well as dried plant matter taken from a dead cannabis plant.

Tissue taken from cannabis plants contains many different types intracellular, cellular, and extracellular matter made of bioproducts as well as the corresponding genetic material, molecules endogenous to the plant. Endogenous molecules are any functional molecules intrinsically produced within a cannabis plant during its lifecycle. Within cannabis, there are hundreds to thousands of unique endogenous molecules produced, depending on the

classification and naming schema utilized. Endogenous molecules have varying structures and sizes depending on whether they are made of chemical, amino acid, protein, RNA, DNA, or other biological building blocks.

Endogenous molecules include the molecules pertaining to genotype as well as functional molecules expressed from genes that an organism needs to live. These endogenous molecules make up the components of the plants, such as the cells that give it structure as well as all the signals needed for inter and intra cellular communication. Genotypic, phenotypic, and epigenetic factors contribute to the creation or expression of endogenous molecules; as well as the microbiome of a particular plant.

As used herein, the microbiome includes non-cannabis microorganisms that live within or on a plant. Many of these microorganisms have a symbiotic relationship with the plant and produce endogenous molecules that help the plant survive or thrive. Infectious microorganisms, such as viruses, bacteria, and fungi also exist as part of the microbiome. The microbiome is considered intrinsic to a cannabis plant and production of endogenous molecules can also be attributed to a cannabis plant's microbiome.

An endogenous molecule is the smallest functional unit of a gene, gene product, or several genes and gene products combined into a larger functional complex unit. Endogenous molecules are synthesized, rearranged, consumed, and degraded through various chemical and biochemical pathways, typically attributable to a set of genes. A gene may code for the production of another endogenous molecule but subsequent modifications, some due to the expression of other genetic or biochemical pathways results in a different endogenous molecule or molecular variant of the same endogenous molecule being expressed, such as during pre and post translational modification.

Molecular variants perform the same or similar function. Molecular variants include but are not limited to: a variety of small molecules, such as RNA and topoisomers of DNA;

chemicals and their isomers; or proteins and their isoforms. Chemical isomers vary in structure but have the same chemical formula. Protein variants have similar RNA sequence and perform the same or similar functions in the plant.

The profile of endogenous molecules captured in a cannabis tissue sample at any moment in time is constantly varying. Certain endogenous molecules are expressed from wetted seed through the end of the life, and some are expressed explicitly at certain stages of development, such as at a specific node of development, in the root system, or in the stems, or in the flowers, and so on.

Other ways of classifying molecules endogenous to cannabis include, but are not limited to: DNA, RNA, viruses, proteins, chemicals, virus, phenolics, fatty acids, waxes, terpenoids, flavonoids, cannabinoids, pigments, spiroindans, steroids, enzymes, dihyrostilbenes, alkaloids, ammonium salts, lipids, carbohydrates, ligand, antibody, receptor, glycoproteins, polyphenols, polyholozides, peroxidase, catalase, flavones, esterase, permease, synthetase; mRNA, mitochondrial DNA, carbohydrates, chemical hormones, protein hormones, auxin, cytokinin, gibberellin, gibberellic acid, abscisic acid, metabolites, inorganic, organic, amino acids, nucleic acids, polymer, lipids, pathogen, tetrahydrocannabinolic acid (THCA) synthase, THCA synthase genes, and so on.

The present teachings disclose selecting a gender associated target (GAT) (105) that is representative of gender in cannabis plants. Theoretically, a selected GAT is a reference standard for the gender identifier to compare incoming analytes against for the purpose of ascertaining the gender of the cannabis tissue the input sample came from. The gender identifier searches for the presence of a selected GAT or the presence of multiple selected GATs to ascertain more complex expressions of gender.

The gender identifier works by looking for the presence of a molecules that match the selected GAT(s) in an input sample. This is accomplished through the indicator, which is a molecule that selectively binds to a particular analyte. A GAT indicator selectively binds to its corresponding GAT. When the analytes detected by the gender identifier from an input sample contain the selected GAT, a signal is emitted by an indicator which is communicated to the viewer as the gender of the plant.

The analytes the gender identifier detects include GATs as well as control analytes that confirm the gender identifier is working. Once a GAT(s) is selected for identification, the gender identifier screens the input sample for analytes that match the GAT. Because a selected GAT is indicative of a particular gender, it may or may not be present in the input sample depending on the gender of the plant the input sample was gathered from. When an analyte from the input sample is the GAT (meaning it matches the GAT selected because it is the GAT), it is a GAT analyte. The GAT analyte selectively binds to an indicator that releases a signal.

Any endogenous molecule is a candidate for GAT selection so long as it exhibits a differential quality or property across genders. For example, GATs include, but are not limited to, DNA targets, RNA targets, amino acid targets, chemical targets, protein targets, virus targets, and the like that are indicative of only a particular gender, so that the detection of the target indicates the gender of the plant. Selection of a GAT to detect from potential candidates depends on preference, but can be chosen for ease of observing, quantifying, or qualifying the differential. Moreover, the tissue type affects the candidacy of a GAT because some GATs are more prevalent in particular cannabis tissues than other GATs. It makes sense that the GAT(s) selected are compatible with the cannabis tissue type taken.

Ideally, the tissue type selected contains a plurality of candidate GATs from the possible GAT spectrum attributable to any particular tissue type. A particular tissue type may contain a GAT, several GATs, or have its own unique profile of GATs. If the wrong tissue type is selected for the selected GAT(s), that tissue type may contain no GAT. For example, roots contain high levels of GATs relating to cytokinins, which are associated with higher doses in female plants; and, male leaves contain high levels of gibberellins and peroxidase variants. Moreover, a hormone such as the auxin indole-3 -acetic acid (IAA) is produced in different levels in varying cannabis tissue types depending on gender. For example, IAA is detectable in female cannabis tissue taken from root, stem, and flowers.

In no instance is there less than 1 GAT selected for detection by the gender identifier. When a single GAT is selected for detection by the gender identifier, then the GAT must be a strong candidate, meaning, the differential is significant across genders. It is recommended that multiple GATs are selected for male, female, and hermaphrodite genders, so that a spectrum of expression is captured in a regional profile (discussed later).

A plurality of GATs is associated with a gender profile and is recommended to clarify more complicated patterns of gender expression. Selecting a plurality of GATs for detection is especially important when at least one GAT comprises a genetic target because as discussed, genotype is only one of the many factors that drive gender outcome. When discussing GATs, this document refers to different GATs with a numerical reference GAT#. For example, a plurality of GATs is exemplified by the naming schema of GAT1, GAT2, GAT3, et seq.

A plurality of GATs is further recommended when variances in phenotypic expression and autosomal regulation of gender traits or characteristics are possible or expected. For example, cultivators are aware that certain environmental conditions bring out a hermaphrodite phenotype amongst gender genotypes. As a measure of quality control, GATs denoting a hermaphrodite phenotype are selected as a standard for detection in the gender identifier for the purpose of removing any hermaphrodites and males from the crop. A differential is any quantifiable difference between endogenous molecules produced in female, male, and hemaphrodite plants such as molecular structure, comparing profiles of molecular variants, concentration, charge, pi, size, differences in sequence, function, specific activity or any other qualitative or quantitative attribute that is preferred. The differential often manifests as a concentration or structure difference, such as the case with molecular variants. Molecular variants are endogenous molecules that have slight structural differences but perform the same function in plants.

Some GATs are produced in both male and female, and hermaphrodite cannabis plants, and some are solely produced in male or female plants or hermaphrodite. For GATs that exist in both male and female plants (GATB) in the same form, the differential attributable to gender is typically based on a concentration threshold. The male or female produces higher or lower amounts of the same endogenous molecule that is selected as the GAT.

For example, tetrahydrocannabinolic acid (THCA) synthase, an enzyme that produces THCA, is a chemical precursor to THC. Certain female varietals or cultivars that produce high amounts of THC have multiple copy numbers of THCA synthase genes. THCA synthase genes (DNA), THCA synthase RNA, THCA synthase (protein), and THC (chemical) are possible four female GATB candidates because all three endogenous molecules are genotypically present or phenotypically expressed in higher levels in female cannabis plants.

The female GAT (GATF) is an endogenous molecule that is consistently present or expressed across species, cultivars, and varietals of female cannabis plants. Similarly, a male GAT (GATM) is an endogenous molecule that is consistently present or expressed across species, cultivars, and varietals of male cannabis plants. And finally, a hermaphrodite GAT (GATH) is an endogenous molecule that is consistently expressed across species, cultivar, and varietals of hermaphrodite cannabis plants. Furthermore, a mixed detection of GATF and GATM denotes a hermaphrodite gender. The same is true when GATBs are observed at levels beyond or below the typical gender differential threshold.

Many GATs are eventually link back to the modulation of both autosomal genes and sex chromosomes. GATs differentially existence in cannabis genders typically because the particular GAT plays a role in the molecular development of a macro-level gender specific trait, characteristic, or morphology; or is a molecular byproduct of the bio-genetic pathways regulating gender expression. GATs are exhibited though genotypic, epigenetic, and phenotypic mechanisms (as well as others) and are indirectly and directly attributable to the development of gender-specific features.

An endogenous molecule directly regulating or resulting from the regulation of gender associated genes is a primary GAT, including the associated chains of DNA derived from gender inducing genes or sex chromosomes. A secondary GAT is produced as a byproduct of any associated pathway involved in regulating those genes or it indirectly affects the expression of those genes through modulating another associated pathway. Although not directly involved in the regulation of gender genes, secondary GATs are just as qualified for GAT selection as primary GATs are for use in the gender identifier.

Examples of other primary GAT candidates include but are not limited to endogenous gender inducing hormone classes such as auxins, cytokinins (for example, zeatin), gibberellins, abscisic acid, ethylene, kinetin and any other differentially expressed hormone.

In addition to concentration differences, molecular variants of certain endogenous molecules across genders are also good GAT candidates for detection by the gender identifier. For example, the hormone class gibberellins exhibits molecular variants across genders. In the instance of gibberellic acid (GA), (C19H2206), a gibberellin, chemical variants GA3, GA4, GA7, and GA9 are associated with male expression in cannabis and are good candidates for detection. Moreover, in another primary example, DNA associated male genotype markers has variants, such as the MADC genes.

Secondary GAT candidates include, but are not limited to, autosomal genes and metabolic proteins indirectly affecting or affected by gender expression. On a broad scale, the amount of soluble protein is comparatively higher in female cannabis plants than males. More specifically, there are the enzyme classes catalase, peroxidase, and esterase. Male cannabis catalase has a faster rate of reaction than female cannabis catalase. Moreover, the enzymes peroxidase and esterase have different gender variants with different pi across genders. The variants occurring in female plants are selected as GATF and vice versa for GATM.

For example, variants of peroxidase (~30-55kDa) are selected as male markers or female markers because endogenous peroxidase levels are detectable in cannabis leaves and different variants are produced by male and female plants. Peroxidase is a secondary GAT because it affects the regulation of auxins. Auxins are a class of plant hormones affecting the development of cannabis organs, such as female flowers, and are primary GAT candidates. Moreover, the peroxidase variants in male plants have higher specific activity than those in female plants and express different peroxidase variant profiles.

Females express four peroxidase variants and males expresses ten peroxide variants. Four acidic male cannabis peroxidase variants have a pi of roughly 5.5-6.2 and are observable in male cannabis leaves that are not observable in female cannabis leaves taken at the same time. The selected GATMs thus include those four male variants, GATFs include the female specific peroxidase variants, and a combination of female and male peroxidase variants is indicative of a hermaphrodite gender. It should be noted that there are more male variants with acidic pi, but the term 4AMCP does not refer to them.

In summation, if the same form of peroxidase variant were present in varying

concentrations in male, hermaphrodite, and female plants, then that peroxidase variant would be selected as GATB and the differential is a concentration threshold rather than a comparison of pi variant profiles.

The starting sample (106) is different from the cannabis tissue in that it is processed into a more liquid form that is more compatible with the medium than unprocessed cannabis tissue. When a selected GAT analyte is present in the cannabis tissue used for gender identification it is also contained in the starting sample. The starting sample also contains a range of endogenous molecules beyond any present GATs.

Processing the cannabis tissue from solid leaf structure into a more liquified form ensures the endogenous molecules are released from the constraints of cannabis tissue. Releasing cellular contents into solution allows the endogenous molecules move more easily though the medium. The chance of molecular collision between the released endogenous molecules and the indicators is increased, which increases the probability of detection by the gender identifier. Assuming one or more selected GATs are in the cannabis tissue, the probability of a GAT colliding with its indicator(s) increases because the released molecules can move more freely and faster in liquid than contained within solid tissue. If a GAT is contained in the starting sample, it will bind to its respective indicator(s) upon molecular collision.

The cannabis tissue is processed by adding it into a processing buffer wherein the cell walls are lysed with at least one lysing mechanism including, but not limited to shock

mechanisms (osmotic, temperature), ultrasonic (sonication), mechanical disruption (muddling, crushing, shredding), viral mechanisms, and enzymatic digestion mechanisms. The processing buffer (such as, but not limited to, phosphate buffer, borate buffer, Tris buffer, or TE buffer) contains at least one of the following: buffer salts, detergents, lysing agents, digestive agents, and stabilizing agents. A lysing agent is recommended to further disrupt the structures that make up a plant cell, such as, but not limited to, pectinase, cellulase, hemicellulose, or any other agent that that lyses cells.

Once released in the processing buffer, molecules move freely. The lysing agent(s) further free the endogenous molecules from the restrictive cannabis tissue. Use of a digestive agent even further releases endogenous molecules from larger sub-structures of tissue or cells in the processing buffer. Depending on the lysing agent(s) or digestive agent(s) used, the lysate fraction is stabilized with at least one stabilizing agent, which stops further degradation from occurring.

For example, the cannabis tissue is muddled into processing buffer maintained at a neutral range pH with buffer salts and a lysing agent, pectinase, further frees the contained endogenous molecules from the bounds of cell walls. A stabilizing agent, a pectinase inhibitor is added before the starting sample makes contact with the receiving area.

The liquid containing the released endogenous molecules is then able to move throughout the medium and collide with the indicators in the reaction area once the driving forces compel the starting sample through reaction area.

Too many excess non-GAT molecules in the receiving area or reaction area at once may adversely affect the rates of reaction or make the indicator signal harder to detect. To increases the probability of a molecular collision between a GAT and its indicator a widget (107) removes a fraction of non-GAT endogenous molecules from the starting sample or the cannabis tissue, capturing the GAT-like molecular remnants or endogenous molecules of interest (EMOI). The widget preserves the EMOI or is used to make a concentrate that also preserves the EMOI for later analysis.

The widget modifies the starting sample when greater sensitivity is desired to detect a GAT(s) or intensify the signal emitted when the indicator is bound to the corresponding GAT. Certain endogenous molecules in the starting sample are of interest because separating, extracting, isolating, purifying, or concentrating them also captures any potential GAT analytes in the starting sample or the cannabis tissue. EMOI are molecules that share at least one common molecular characteristic with the selected GAT(s) which allows them to be captured or fractionated by that characteristic(s). GAT molecules, if present in the starting sample or the cannabis tissue are separated, extracted, isolated, purified, or concentrated or as the EMOI are separated, extracted, isolated, purified, or concentrated. For example, when the selected GATs include the 4AMCP variants previously discussed, then widget removes dissimilar endogenous molecules such as basic (non-acidic) proteins or even more specifically, basic peroxidases, as well as other molecular noise (i.e. if the GATs selected for detection do not include DNA molecules, then DNA can be removed as molecular noise).

The widget removes non-GAT molecules and captures EMOI by exploiting a common characteristic such as, but not limited to: charge; size exclusion; ion exchange; adhesion; surface adsorption; partition; chromatography; hydrophobicity; affinity; molecular structure, or other characteristics capable of being separated, extracted, isolated, purified, concentrated, or digested by a one or more of the following mechanisms centrifugal force, pressure, diffusion,

electrophoresis, digestion, affinity capture with solid-phase such as affinity beads in column or nitrocellulose, paper, polymer or gel membrane as well as affinity capture with liquid phase, post-capture release into a solution, or any other separation, extraction, isolation, concentration, purification, or digestion technique conceivable.

Digestive techniques degrade non-GAT molecules. Digestive enzymes remove non-GAT endogenous molecules by degrading them, decreasing molecular noise. When the widget uses digestive enzyme, a stabilizing agents stops digestion if necessary for the GATs to be detectable by the indicators and remain intact. In some cases, the digestive enzyme used is the same or similar to the digestive agent used to prepare the starting sample. Some agents include, but are not limited to, activity blocking antibodies and their fragments, trypsin, lectin, aptamer, synthetic polymers, dendrimers, 4-aminobenzoic acid, L-cystine, hydroxylamine hydrochloride, 2- imidazolidinethione purum, sodium azide, sodium cyanide, and sodium orthovanadate.

When the EMOI are released from the widget into a small volume of liquid, the liquid is a concentrate (110). The concentrate containing the intact EMOI released from the widget is added to the receiving area of the medium, similar to the starting sample. For example, the widget captures intact EMOI by binding it to a porous solid phase. When the intact EMOI are released from solid phase, the intact EMOI are unbound from the widget into a concentrate. The concentrate is added to the receiving area as an input sample.

When the intact EMOI remain captured in the widget (i.e. are not released as a concentrate), the widget is the input sample or the medium the input sample is added to.

When the widget is the input sample and not the medium, it makes contact with the medium through the sealable opening, which here overlaps with the receiving area. In this instance, a more compatible material form is a dynamic material, such as liquid, wherein the indicators are suspended. The widget is wholly or partially submerged into the liquid or the liquid is added to the widget and any GATs captured by the widget will react with indicators suspended in the liquid or on the widget.

The same reaction occurs when the widget is added into the liquid material of the medium and when the liquid material is added to the widget. Regardless, the suspended indicators bind to their associated analytes, if present. The signal is emitted wherever an analyte binds to its suspended indicator, both on the widget and in the liquid material (for example when the EMOI diffuse off the widget into the liquid), and the reaction area is anywhere the suspended indicators bind to their analytes.

When the widget is the medium (109), the reaction area overlaps with the widget and the indicators bind to their associated analytes, if present in the starting sample on the widget.

Consider the example where four selected GATs include the 4AMCP variants previously discussed. The recommended cannabis tissue type is leaf, and a portion of a leaf is processed into a starting sample by being muddled with processing buffer releasing intracellular and

extracellular contents into a solution of neutral pH range, pectinase further releases the endogenous molecules from the cell walls. A digestive enzyme is added to degrade or remove non-GAT endogenous molecules and a stabilizer is added to prevent further degradation.

The sealable opening of the housing allows the starting sample to make contact with the receiving area and widget. For example, the widget is a porous membrane and the one end of the outer housing an in-line syringe containing the starting sample. The starting sample is a liquid mobile phase that is forced through the widget, here a porous solid phase that binds the intact EMOI, such as a membrane made of positively charged nylon. The starting sample is loaded into a syringe. When pressure is applied through the syringe, the starting sample is forced through the solid phase, which is also the reaction area, and the widget captures molecules with relatively negative charges by binding them to the positive charges on the nylon membrane. The intact EMOI remain captured in the widget, such as the AMCP variants. As soon as the starting sample makes contact the reaction area of the widget, indicators start reacting with their present analytes. When emitted, the binding signal is observable on the widget.

The outer housing (111) is the enclosure that holds the contents of the gender identifier together. The sealable opening of the outer housing is opens to accept the medium and input sample. The opening closes to contain the contents once combined. The outer housing has a window over the reaction area and sealable opening connects to the receiving area. The sealable opening is capable of unsealing to allow for a medium to be inserted into the housing. The outer housing is capable of being cleaned and reused with multiple mediums or is disposable.

The vesicle may be any container is sufficient to contain the medium that opens and closes through the sealable opening. The outer housing can be planar or three dimensional, depending on the material form of the medium. For example, a bag, a lidded container, and a syringe.

The receiving area can be accessed through the sealable opening and is where the input sample makes contact with the medium; it is the channel between the gender identifier and the medium that the input sample travels through to make contact with the medium. The receiving area may overlap with the medium, the sealable opening, and the vesicle. For example, when the material of the medium is a liquid, the receiving area overlaps with reaction area because the liquid takes the shape of the vessel.

The medium (112) is where the reactions of the gender identifier occur. The driving force moves the input sample from the receiving area toward the reaction area, further made of regions. The medium contains the indicators and any necessary reagents needed to stabilize or conduct a reaction between the GATs and the indicators, as well as the between the control analyte and the control indicators in the reaction area. When the analytes of the input sample include the selected GAT(s), the GAT(s) react with their corresponding indicator(s) molecules in the reaction area of the medium.

The reaction area has regions where indicators are assigned to. Each region contains at least one indicator per selected GAT and is capable of detecting at least one GAT. Regions detecting multiple GATs of the same type (male, female, or hermaphrodite) may be overlapped to intensify the emitted signal by using the same or similar signal. The medium is made of a material (113) that is of a static or dynamic form.

The material is the conduit for the molecular collision between endogenous molecules in the input sample and indicators, and contains any necessary reagents for the reaction to occur. An indicator molecule may be suspended in, adsorbed onto or embedded into its region of the reaction area depending on the material, so that as the analyte molecules pass through the material, the indicators bind their respective analyte and capture it in its region and emit the signal in the region. Each indicator has at least one region and multiple indicators that bind to multiple GATs can have multiple regions that overlap or have separate regions.

The materials are static or dynamic forms that allow for the movement of molecules. A dynamic material lacks rigid structure, and takes on the shape of its container, such as a liquid, solution, or mobile phase. Static materials are rigid structures and include porous solids such as solid phases, beads, polymers, gels, and membranes such as nitrocellulose and paper.

With dynamic form materials, regions of the reaction area overlap and the indicators are suspended as flowing molecules. A driving force causes the indicators to mix with the input sample and collide with analytes. For example, the dynamic material is liquid and the indicators are suspended, so that the reaction area encompasses the entire dynamic material. Molecular collisions between the indicator(s) and the GAT(s) occur anywhere the indicator molecules collide with their GATs, such as in liquid when the EMOI are unbound and diffuse through liquid off the widget into the reaction area or on the widget when the indicators bind to their respective GATs.

When the material is a static form, the indicators are adsorbed, absorbed, or embedded into a porous solid. Examples of solid porous materials include but are not limited to membranes and beads made from nitrocellulose, polymer, gels, and paper. The regions for the indicators may overlap or be separated to create a specific profile of GATs on the reaction area. Analytes are capable of flowing along, through, or throughout the solid porous form from the receiving area toward the reaction area, depending on the driving forces used. The indicator molecules in the region can overlap or be separated into a multitude of shapes such as squares, linear lines, radial lines, circles, and any other preferred shape to create a profile of several GATs.

For example, when the medium is the positively charged nylon membrane that is also the widget (meaning they are same entity), the indicators are contained in regions of the positively charged nylon membrane. When the starting sample or the concentrate is forced through the membrane, such as when an in line syringe is pressed, the starting sample flows through the positively charged nylon membrane and the negatively charged EMOI are bound in the positively charged membrane, which is also the reaction area, and any GATs in the starting sample immediately bind to the indicators emit the signal.

Driving forces (114) propel molecules through the medium. The driving forces cause molecules to collide with other molecules. Upon collision, indicators contained within the reaction area specifically bind to their GATs which then signal the reaction has occurred.

Driving forces include, but are not limited to: time, heat, diffusion, capillary action, chromatographic, pressure, physical, chemical, agitation, adsorption, absorption, mixing, flow, or any other physical or chemical driving force that induces molecular collisions to occur between analytes and reactants causing the associated reaction to occur.

For example, GAT molecules are driven through chromatographic forces to move along and throughout the medium and make contact with the indicators in the reaction area; if GATs or control analytes are present, the signal is visible on the reaction area.

For example, starting sample is put into the receiving area of the medium, a static material such as a paper membrane. Chromatographic driving forces propel molecules of various sizes to travel through the paper, carrying the analytes toward the reaction area.

In an example where the static form is the same entity as the widget, the widget enters the receiving area, also the reaction area of the medium, here a dynamic form such as a liquid, and shaking causes the analytes to collide with the indicator molecules suspended in the liquid.

The indicator molecules are dispersed throughout the reaction area (115) in regions where each indicator is assigned to its own region or combined in the same region as other similar indicators. The indicator signal is emitted in the region where the corresponding GAT molecule is captured by its indicator.

The indicators and any necessary non-GAT reagents needed to induce the associated reaction are in the reaction area so that when the driving forces move the molecules of the input sample throughout the regions, molecular collisions occur between the input sample molecules and the GATs present in the sample.

GATS and control analytes are detected with the use of biochemical reactions in the reaction area. Reaction components such as reagents needed to induce the associated reaction are dispersed within in the medium. A reagent is a substance that causes a reaction happen or used to confirm whether a reaction occurred. Reagents must be compatible with the reaction it induces.

For example sodium dodecyl sulfate (SDS) is not compatible with Bradford tests and Bradford reagents must be used. An indicator is a reagent as well as any agents needed to stabilize or expedite the reaction or cause it to occur such as reagents, buffers, buffer salts, or reactants. For example, when Hanker Yates reagents react on their substrate in the presence H202.

Each indicator used to identify a GAT or control analyte is placed in a region (116) of the reaction area. Each indicator may have its own region, or several indicators of the same type (male, female, or hermaphrodite) can be assigned to the same region, such as when a region is representative of a gender type. In that instance, all the indicators in the same region identify GATs pertaining to the same gender.

When bound, the indicator emits the binding signal in the region of the reaction area it is located in. The regions, however, may overlap. A region should not contain conflicting indicators, that is, indicators that bind to molecules indicative of different gender types and the signal emitted by multiple indicators assigned to the same region is the same or similar.

When there is more than one region, spacing of those regions affect how GATs are detected. GATs can be detected in a profile, where indicators are spaced in several unique patterns of regions. In that instance, various indicators located in a plurality of regions of the reaction area are clustered, separated, spaced or overlapped to create a regional profile of detection where the GATs are bound.

Another reason why the regions for the indicators pertaining to a particular GAT type(s) are overlapped is to increase or amplify the same or similar signal emitted by the indicators such as when several GATs are of the same gender association. Meaning, similar indicator types emitting the same or similar signal share the same region. For example, it is helpful to organize indicator regions by GAT types where the types could represent gender attributable to phenotypes, epigenes, genotypes, microbiome, or any other clustering of characteristics, traits, or morphologies that a GAT exists for. The profile, or pattern, of emitted signals indicate the presence of certain GAT types allowing the viewer to make a call on gender expression. When each indicator has its own unique region, herein referred to as R# where the # corresponds to the associated indicator, indicator 1, 1-1, binds to GATl in Rl . 1-2 binds to GAT2 in R-2, and so on.

When multiple indicators are assigned to a region, a letter is used to indicate the gender detected in that region. For example, the indicators for several possible GATFs are clustered in the same region, RF, and the indicators for several possible GATMs are clustered in the same region, RM. In this example, the indicators for several possible unique GATHs are clustered in the same region of RH, and if a mix of GATM and GATF are produced in a hermaphrodite, both RF and RM emit the signal.

For example, if 8 GATs are selected, 5 different male GATs (GATM) are selected and 3 different female GATs are selected (GATF), each indicator can have its own reaction area as Rl- R8; or, the 5 different male GAT indictors may overlap in RM and the 3 female GAT specific indicators may overlap in RF for the purpose of intensifying the readable signal.

Analyte molecules from the input sample are propelled through the reaction area wherein the indicators are contained. An indicator (117) is any molecule that selectively binds to a GAT and emits (inclusive of inducing) a signal when it is bound to the GAT, unless it is a control indicator, which binds to a control analyte and not a GAT.

Indicators and control indicators are molecular probes selected to detect an analyte because they naturally bind to or are designed to bind to the desired analyte and have a tag that is capable of emitting a signal when bound to the targeted analyte. Essentially, the binding site(s) and the tag behave as functional subunits or components of the indicator and often need each other to work. Ideally, each indicator has more than one binding sites and the tag exhibits a strong signal when bound, with or without an additional visualization.

When the indicator is bound to the GAT, the molecular change causes the tag of the indicator to emit its signal. An indicator may be designed to emit a signal by adding a tag or is selected for its existing signaling abilities because it naturally has a tag. In other words, the tag may be molecularly added to the indictor or is already part of the indictor. The signal includes but is not limited to: dyeing, staining, fluorescence, color appearance, color change, energy emission or absorption of energy (such as light or heat), and others. The mechanisms of the tag signal include but are not limited to chromogenic, colorimetric, fluorometric, chemiluminescent, electrochemical, excitation, absorbance, and others, or any combination of signals thereof. GAT indicators are capable of detecting the associated differential by detecting a GAT's presence, absence, relative concentration, or other thresholds associated with the GAT differential. A GAT indicator is specific for the GAT or GATs it was selected to bind to and does not react with any other molecule but its intended GAT or GATs. At least one indicator is used to detect each selected GAT, and at least one GAT is needed to establish the gender of a cannabis plant.

For exemplification sake, numerical order is assigned. An indicator (I-#, et seq.) molecule binds to at least one particular GAT(GAT#, et seq.), not every GAT, and can bind to multiple GATs that share a common characteristic the indicator is selected to be sensitive to.

To illustrate the selectivity of the indicators chosen for use in the gender identifier as detectors of GATs, the consider the following example: GATs 1 through 5 are selected as targets to detect in an input sample and are all present in the input sample. Assuming a plurality of indicators is selected for use in the gender identifier, GATl binds with I-l; GAT2 binds with 1-2; GAT3 binds with 1-3; GAT4 binds with 1-4; GAT 5 binds with 1-5; GAT3, GAT4, and GAT5 also bind with 1-3+4+5; GATl and GAT2 also bind with 1-2+1; and so on. In another example with multiple GATs and multiple indicators, of selected GATs 1 through 5 only GATs 1, 3 and 4 are present in the input sample: I-l, 1-3, and 1-4 are the only the indicators that emit the binding signal. Without necessarily knowing how many indicators or GATs were selected for use, it can be inferred from an example that 1-2+27+99 binds to GAT2, GAT27 and GAT99. See FIGURE

As just illustrated, multiple indicators may be selected for use in the gender identifier; it is also possible that multiple indicators bind to the same GAT or several of the same GATs known to be produced by a specific gender type. To illustrate when an indicator is used to detect multiple GATs that are selected for detection, consider the following example: GATs 1 through GAT5 are present in the input sample, and three indicators la, lb, and Ic are designed to bind to those GATl, GAT3, and GAT5 are denoted as Ia-1+3+5, Ib-1+3+5, and Ic-1+3+5 and each letter is used to denote a different indicator; multiple indicators that all bind to the same GAT type are denoted as Ia-GATM; Ib-GATM; Ic-GATM and the indicators I-GATM, I-GATB, I-GATF, I- GATH are used to denote an indicator that binds to multiple GATs associated with a particular gender.

Multiple indicators may bind to the same GAT for the purpose of amplifying and intensifying the emitted signals, especially when the selected GAT is plentiful. Similarly, multiple indicators that bind to different GATs with the same or similar tag or signal are assigned in the same regions for the purpose of intensifying the signals emitted. Additionally, regions are overlapped that detect the same gender to compound the signal.

There are numerous molecules that act as indicators, as well as and tags that can be added to molecules to make an indicator, that can be used in the gender identifier, some include, but are not limited to: antibodies conjugated to tags; enzymes such as alkaline phosphatase; o- nitrohenylphosphate; histochemical dyes; luminol; FITC; SYBR green; 3,3',5,5'- Tetramethylbenzidine (TMB); diaminobenzidine (DAB); 2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid) (ABTS); and solo tags such as nanoparticle(s) loaded with colloidal gold or gold, magnetic, heavy metal, or rare earth particles and atoms that are added to other molecules to form an indicator.

For example, histochemical indicators such as the chromogen dye Hanker Yates Reagent, include but are not limited to immunoperoxidase reagent molecules that detect any substrates oxidized by horse radish peroxidase (HRP) in the presence of H202, an oxidizing agent.

Examples of possible tags linked with the histochemical indicator include, but are not limited to TMB, DAB, ABTS, and other colorimetric, fluorometric, chemiluminescent and electrochemical molecules. This type of indicator can further be conjugated to an ANTI-GAT molecule when the GAT is a peroxidase substrate. This section is not to be confused with the peroxidase references pertaining to cannabis peroxidase.

Some indicators are sensitive enough to discern between molecular variants, a function of its molecular characteristics. These types of indicators are likely engineered for their ability to detect small variations in molecular qualities. For example, consider the male and female cannabis peroxidase variants previously discussed. All the variants have similar structure, but only the 4AMCP variants are selected as GATs. One type of indicator is particularly useful due to its sensitivity and customizable capabilities, the custom cannabis antibody indicator (CCAI). These types of indicators can be custom designed using immunotechnology.

A CCAI can be used to selectively bind to any type of analyte(s) it is designed for, and CCAIs are also excellent indicators for control analytes. CCAIs can detect molecular variants and are capable of binding to unique GATs or classes of GATs, depending upon the specificity with which they are made. CCAIs can be made for just about every GAT that is isolatable from the cannabis plant. A CCAI that binds to a GAT is herein referred to as ANTI-GAT-tag. When the GAT molecule binds to its corresponding ANTI-GAT -tag indicator molecule, both are consumed and form a complex of ANTI-GAT-tag:GAT, and a signal is emitted.

ANTI-GAT-tag is made from polyclonal and monoclonal custom cannabis antibodies conjugated to a tag such as, but not limited to, a nanoparticle(s) loaded with colloidal gold, heavy metal, magnets, or rare earth. Here, the tag is a molecule that conjugates to the ANTI-GAT custom cannabis antibod(ies) and releases or emits a colorimetric, chemiluminescent, fluorescent or absorbent signal when the ANTI-GAT-tag conjugate is bound to the GAT.

The antibodies (ANTI-GAT) that make up the ANTI-GAT-tag indicators, or ANTI- reagent (to be discussed later), are made against a cannabis derived antigen using animal hosts, or host derived genes that code for antibodies (recombinant antibody technology). Polyclonal antibodies are harvested from host blood derum or plasma, and monoclonal antibodies are made from host tissues and cells.

Antigens are exogenous substances to the animal the antibodies are being produced in, and when present in the animal host, the animal makes antibodies in response to the antigen, which are harvested from the host. When the antigen is the same molecule as the GAT, or incredibly similar, ANTI-GAT is produced. The cannabis extracted GAT molecules used as antigen are herein referred to as GAT-antigen.

When making the gender identifier, GAT-antigens are extracted from cannabis plants of known gender and used as antigen in a variety of host species to produce custom cannabis antibodies that specifically bind to the GATs. Host species are immunized with unpurified, partially purified, or completely purified extracts containing the GAT-antigens. For protein GATs of known protein sequence, the known oligopeptide sequences can be used as immunogen antigen.

In some instances, ANTI-GAT is used as a reagent with another or multiple antibod(ies), wherein at least one has a tag, that binds to ANTI-GAT forming the complex indicator ANTI- ANTI-GAT-tag, and so on through indirect localization sequence. In this manner, multiple antibodies can be stacked into complexes more sensitive for a GAT, where subsequent antibodies bind to available epitopes of the former complex it was made to bind to.

In some instances, ANTI-GAT is used as a reagent (ANTI-reagent) with another or multiple antibod(ies), wherein at least one has a tag, that binds to ANTI-GAT forming the complex indicator ANTI-reagent-ANTI-GAT-tag, and so on through indirect localization sequence. In this manner, multiple antibodies can be stacked into complexes more sensitive for a GAT, where subsequent antibodies bind to available epitopes of the former complex it was made to bind to.

Moreover, in addition to being selected for its differential quality, a GAT can be selected for its ease of isolation from cannabis plants for use as a GAT-antigen to produce its ANTI- GAT-tag. A variety of mechanisms already discussed isolate, separate, purify or concentrate known GAT molecules from plant tissue extract, which are used as antigens to produce ANTI- GAT. Some additional techniques include but are not limited to protein blots and FACS.

For example, ANTI-GAT is produced by immunizing a host species that produces a polyclonal antibody response to GAT-antigen contained in an unpurified extract from male cannabis plants. The resulting antisera is adsorbed with a similar extract taken from female cannabis plants at the same time as the male extract. The cross-reacting antibodies are adsorbed out (antibodies that do not specifically recognize GATs). The remaining antibodies are specific to GATM.

When the selected GATs include the 4AMCP variants discussed, the ANTI-GAT -tag indicators specifically bind to the AMCP variants. When making the gender identifier, the GAT- antigen is a purified extract containing the 4AMCPs isolated from male cannabis plants or immunogens of the 4AMCP oligopeptide sequences. ANTI-GAT is produced that specifically bind to the 4AMCP and a nanoparticle loaded with gold is conjugated to the antibodies to form the 4AMCP indicators.

In sum, ANTI-GAT 1 -tag binds to GAT1, ANTI-GAT2-tag binds to GAT2, ANTI- GATS -tag binds to GAT3 and so on. ANTI-GAT 1 -tag does not bind to the other selected GATs, unless the indicator is specifically designed for such robustness and in which case is actually denoted as ANTI-GAT 1+2+3 -tag if it binds to GAT1, GAT2, and GAT3.

Indicator Amount

The amount of a particular indicator used in the reaction area depends on the differential to be detected. When the differential is the presence or absence of a particular GAT because the GAT is only observable in only one gender, the amount of indicator placed onto its region of the reaction area is merely enough to generate a detectable signal.

Threshold detection is a little more complicated. The indicator(s) detects the threshold that is representative of a particular gender, where only certain ranges of will be representative of a particular gender. For example, when the GAT is selected on the basis that a concentration differential exists across genders, the GAT is the same endogenous molecule, molecular variant, or strikingly similar variants across all genders detected by the same indicator based on a concentration differential, and the indicator is titrated to an amount that signals when the desired threshold or limit has been attained.

A further example is when GATB exhibits a gender differential predicated on a concentration threshold. In a hypothetical example, GATB is exhibited in female cannabis plants at a concentration of roughly 0.2 milligrams (mg) per gram (g) cannabis tissue; in male cannabis plants at a concentration of roughly 0.9mg/g cannabis tissue; and hermaphrodite cannabis plants at a concentration of roughly 0.5mg/g cannabis tissue. The indicator amount placed on the GATB region(s) is thus titrated to generate a detectable signal when it reacts with desired range of GATB concentrations that specifically occur in a particular gender.

Control Reactions

Control reactions are recommended. A control reaction occurs between a control analyte and its selective control indicator. The control reaction functions as a representation of a working gender identifier. It comprises a biochemical reaction where a control analyte binds to a specific control indicator. Presence of a control signal emitted by the control indicator when bound to the control analyte boosts confidence that a potential absent GAT signal is attributable to the lack of presence of that GAT in the input sample.

The analyte in the control reaction may be an endogenous molecule contained in the input sample or an exogenous molecule (non-cannabis) added to the reactions for its own detection. In a working gender identifier, the selected control analyte is either captured in the input sample from the cannabis tissue (along with at least one GAT) as an endogenous molecule of interest, or extrinsically added to the starting sample to confirm the endogenous molecules of the input sample have moved into the reaction area when bound to the positive control indicator. The signal emitted when the positive control analyte is bound to the positive control indicator is indicative that that the input sample has made contact with the reaction area and confirms any GATs present have reacted with the respective indicators.

Control indicators may be very specific for one analyte, but options for control indicators also include broad, robust reagent or reactant molecules capable of binding to more than one specific analyte for the purpose of intensifying the signal emitted from the control indicator molecules. For example, when a gene is a GAT, genetic assays can be used to determine the qualitative presence of genes and a DNA control indicator such as SYBR green can be used. Other biochemical reactions can be used to determine the presence of control analytes that are proteins such as, but not limited to, Biuret's assays, Lowry protein assays or Bradford colorimetric assays can be used to confirm the presence of proteins. Similar to Lowry and Bradford tests, a Biuret's test is a chemical test that confirms the presence of peptide bonds that form proteins; a Biuret's test reagent is the control indicator and the control analyte is protein. When the Biuret's test reagent molecule, here the control indicator, binds to a protein, here the control analyte, it emits a colorimetric change signal of violet light.

The control indicator selected depends on the molecular qualities of the selected GAT(s). For example, when the selected GATMs are the previously discussed acidic male peroxidase variants, the protein class of peroxidases, a comparable positive control analyte is a protein molecule and the corresponding positive control indicator binds to proteins. Once bound, the protein control indicator emits the signal confirming the reaction has occurred and that proteins are present in the reaction area. It can be ascertained that peroxidases, including the GATMs, would have bound if present in the input sample.

The control indicator is located in the reaction area. When the GAT(s) are the limiting reagent in the reaction, the selected positive control analyte should not compete with capturing the GAT so as to increase the probability of a GAT molecule binding with its indicator. This can be accomplished by placing the control indictors that may also bind to any present GAT(s) in a control region beyond the GAT indicators' region. For example, when using a protein control reaction, it is recommended to put the protein control reaction beyond the placement of the indicators so that the control indicator does not bind substrates needed for the indicators.

The communicator (118) can communicate the results of the reaction through an analog display or a digital display. The analog display is the signal emitted by the indicators when bound to their analyte(s) and is detectable without the assistance of additional electronics.

Consider when the medium is a material that is porous solid, such as a gel membrane, and the regions of the membrane contain indicators that emit a signal of fluorescence bound. The fluorescence is the analog signal that communicates the analytes are bound.

In another example, the signal emitted from an indicator, II, is a change from colorless to a blue color when bound to GAT1. GAT1 is present in the input sample and the liquid and widget turn blue when the signal is emitted. The blue color is the analog signal that communicates the presence of GAT1 in the input sample to the viewer.

The digital display is a digital signal emitted electronically that communicates the results of the reactions. The digital display receives electronic signals from at least one biosensor placed above the reaction area, attached to the interior of the housing. The biosensor has the capability to detect changes through spectroscopy, colorimetry, or some other form of digital detection of fluorescence, color change, absorbance of energy, emission of energy, and any other signal emitted by an indicator.

The electronic signals amplify the analog signal emitted by the indicators in the reaction area and converts the analog signal into a digital signal with the assistance of applications and software that analyze the reaction results and convert the electronic signals from the biosensors (for example, but not limited to binary signals where 0=off and l=on) into more complex representations of the results made of visual, aural, and touch interactive displays. The reaction results are displayed on an interface through graphic text, graphic images, light, sound, or vibration.

The interface is also capable of communicating more complex results such as profiles of GATs or regional profiles of GAT types. The digital display can sync with a computer, smart device, or other electronic device through (but not limited to) Bluetooth, wifi, G3, G4, G5 and its successors, radio, or any other transmission not yet contemplated at the time of this writing.

For example: a light bulb indicating a male plant illuminates when an electronic signal is detected from the biosensor that communicates the signal of the indicator emitted when it bound to a GATM; the digital display syncs with a smart phone and a graphic image portraying a profile of detected GATs is displayed on the screen.

Claims

Claim 1. A cannabis gender identifier comprising an outer housing, an input sample, a medium, and a communicator; wherein the outer housing further comprises a vessel with a sealable opening, a window, and a receiving area; the input sample comprising a cannabis tissue, a starting sample, a widget, or a concentrate; whereby the input sample enters the outer housing through the sealable opening to make contact with the receiving area; the medium having a reaction area, at least one driving force whereby the endogenous molecules of the input sample are propelled throughout the medium and make contact with the reaction area, at least one indicator comprising a molecule having at least one binding site and at least one tag capable of emitting a signal when the binding site is bound to a gender associated target (GAT) whereby the binding site selectively reacts with the GAT; whereby the sealable opening allows for the input sample to make contact with the receiving area; whereby the vessel has sufficient space to contain the input sample, the medium, and the communicator; whereby the window is placed over the reaction area of the medium and allows for the signal of the indicators to be observed; whereby the sealable opening allows the medium to be inserted into the housing; whereby the receiving area is a channel the input sample travel through to make contact with the medium.

Claim 2. The reaction area of claim 1 further having reagents that preserve, stabilize, induce, lyse, catalyze the reaction to occur between the GAT and the indicator, or the control indicator and the control analyte, wherein at least one indicator signals the presence or absence of the GAT.

Claim 3. The outer housing of claim 1 further having a front side, back side, left side, right side, top side and bottom side whereby the sides are connected to form an enclosure and at least one side is the sealable opening; whereby the sealable opening may overlap with the receiving area, the receiving area may overlap with the reaction area, and regions within the reaction area may overlap with each other to form a region representative of GAT type.

Claim 4. The reaction area of claim 1 further having at least one region with at least one indicator whereby the indicator detects the presence of the GAT in the input sample by selectively binding to the GAT and emits a signal when it is bound.

Claim 5. The reaction area of claim 1 further having a plurality of regions that overlap when more than one indicator detects the same GAT type

Claim 6. The cannabis tissue of claim 1 further comprising a plurality of endogenous molecules produced in cannabis plants; whereby the plurality of endogenous molecules further comprises endogenous molecules of interest (EMOI) and a fraction of non-GAT endogenous molecules.

Claim 7. The starting sample of claim 1 further comprising the cannabis tissue and a processing buffer having at least one lysing mechanism whereby the plurality of endogenous molecules are released into the processing buffer.

Claim 9. The medium of claim 1 further having a control reaction comprising a control indicator molecule having at least one binding site whereby the binding site selectively reacts with a control analyte, and a tag capable of emitting a signal when the binding site is bound to the control analyte.

Claim 9. The indicator of claim 1 capable of selectively binding to a plurality of GATs whereby the plurality of GATs share a common quality the indicator is sensitive to, allowing an indicator to bind to the plurality of GATs.

Claim 10. A plurality of GATs comprising a plurality of endogenous molecules whereby a gender differential is observable for each of the GATs.

Claim 11. The widget further comprising the starting sample wherein a fraction of non-GAT endogenous molecules have been removed and the endogenous molecules of interest are captured by the widget.

Claim 12. The widget of claim 1 whereby the EMOI are released from the widget into the concentrate.

Claim 13. The widget of claim 1 and the medium of claim 1 whereby the widget is the medium and makes contact with the cannabis tissue or the starting sample.

Claim 14. A GAT type comprising a plurality of regions having separate spaces each containing a plurality of indicators that emit the same or similar signal in the same region when bound to the same GAT type.

Claim 15. The region of claim 1 further comprising a plurality of indicators that detect a plurality of GATs pertaining to one gender.

Claim 16. The communicator of claim 1 having the capability to communicate changes in the signals emitted from the indicators an analog display or a digital display; the analog display comprising the signal of at least one indicator when bound to at least one GAT observable through the window; the digital display further comprising at least one biosensor connected to the housing above the reaction area; and the biosensor further comprises interactive software capable of converting data into a communication the viewer understands as indicative of the gender of the input sample.

Claim 17. The indicator of claim 1 further comprising ANTI-GAT, wherein ANTI-GAT is a custom cannabis antibody produced with an antigen isolated or derived from cannabis.

Claim 18. A kit comprising the outer housing, reaction reagents, a medium, an indicator, and a selected GAT to detect with the gender identifier.

Claim 19. A plurality of indicators detecting the same type of GAT by emitting the same or similar signal are assigned to the same region.

Claim 20. The reaction area of claim 1 further comprising a plurality of separately spaced regions wherein the plurality of indicators detects the plurality of GATs in a profile.