Patent Description:
The development of new stevia cultivars requires the evaluation and selection of parents and the crossing of these parents. The lack of predictable success of a given cross requires that a breeder, in any given year, make several crosses with the same or different breeding objectives.

Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.

The invention provides a method of determining the genotype of a stevia plant, wherein said method comprises:.

The invention further provides a food or feed product comprising a stevia plant or stevia plant part comprising in its genome an allele of one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of:.

wherein said food or feed product is not exclusively obtained by means of an essentially biological process.

One or more aspects of the disclosure relate to a stevia seed, a stevia plant, a stevia cultivar, and a method for producing a stevia plant.

One or more aspects of the disclosure further relates to a method of producing stevia seeds and plants by crossing a plant of the instant disclosure with another stevia plant.

One aspect of the disclosure relates to plant tissue such as shoots, microshoots and seed of the stevia variety `<NUM>'. Another aspect also relates to plants produced by growing the seed of the stevia variety '<NUM>', as well as the derivatives of such plants. As used herein, the term "plant" includes plant cells, plant protoplasts, plant cells of a tissue culture from which stevia plants can be regenerated, plant calli, plant clumps, shoots, microshoots and plant cells that are intact in plants or parts of plants, such as pollen, flowers, seeds, leaves, and stems.

Another aspect of the disclosure relates to a tissue culture of regenerable cells of the stevia variety '<NUM>', as well as plants regenerated therefrom, wherein the regenerated stevia plant expresses all the physiological and morphological characteristics of a plant grown from the stevia seed or tissue culture designated '<NUM>'.

Yet another aspect of the disclosure is a stevia plant of the stevia variety `<NUM>' comprising at least a first transgene, wherein the stevia plant is otherwise capable of expressing all the physiological and morphological characteristics of the stevia variety '<NUM>'. In particular aspects of the disclosure, a plant is provided that comprises a single locus conversion. A single locus conversion may comprise a transgenic gene, which has been introduced by genetic transformation into the stevia variety '<NUM>' or a progenitor thereof. A transgenic or non-transgenic single locus conversion can also be introduced by backcrossing, as is well known in the art. In certain aspects of the disclosure, the single locus conversion may comprise a dominant or recessive allele. The locus conversion may confer potentially any desired trait upon the plant as described herein.

Yet another aspect of the disclosure is about using the other New Plant Breeding Techniques (NPBT), such as Oligo-Directed Mutagenesis (ODM) and CRISPR-Cas9 or CRISPR-Cpf1, to modify the stevia variety ` <NUM>' or a progenitor thereof.

Still yet, another aspect of the disclosure relates to a first generation (F<NUM>) hybrid stevia seed produced by crossing a plant of the stevia variety '<NUM>' to a second stevia plant. Also included in the aspects of the disclosure are the F<NUM> hybrid stevia plants grown from the hybrid seed produced by crossing the stevia variety `<NUM>' to a second stevia plant. Still further included in the aspects of the disclosure are the seeds of an F<NUM> hybrid plant produced with the stevia variety ` <NUM>' as one parent, the second generation (F<NUM>) hybrid stevia plant grown from the seed of the F<NUM> hybrid plant, and the seeds of the F<NUM> hybrid plant.

Still yet, another aspect of the disclosure is a method of producing stevia seeds comprising crossing a plant of the stevia variety ` <NUM>' to any second stevia plant, including itself or another plant of the variety '<NUM>'. In particular aspects of the disclosure, the method of crossing comprises the steps of: (a) planting seeds of the stevia variety `<NUM>'; (b) cultivating stevia plants resulting from said seeds until said plants bear flowers; (c) allowing fertilization of the flowers of said plants; and (d) harvesting seeds produced from said plants.

Still yet another aspect of the disclosure is a method of producing hybrid stevia seeds comprising crossing the stevia variety '<NUM>' to a second, distinct stevia plant which is non-isogenic to the stevia variety '<NUM>'. In particular, where the crossing comprises the steps of: (a) planting seeds of stevia variety '<NUM>' and a second, distinct stevia plant; (b) cultivating the stevia plants grown from the seeds until the plants bear flowers; (c) cross pollinating a flower on one of the two plants with the pollen of the other plant; and (d) harvesting the seeds resulting from the cross pollinating.

Still yet another aspect of the disclosure is a method for developing a stevia plant in a stevia breeding program comprising: (a) obtaining a stevia plant, or its parts, of the variety '<NUM>'; and (b) employing said plant or parts as a source of breeding material using plant breeding techniques. In the method, the plant breeding techniques may be selected from the group consisting of recurrent selection, mass selection, bulk selection, backcrossing, pedigree breeding, marker-assisted selection, genetic transformation and genome editing. In certain aspects of the disclosure, the stevia plant of variety '<NUM>' is used as the male or female parent.

Still yet another aspect of the disclosure is a method of producing a stevia plant derived from the stevia variety '<NUM>', the method comprising the steps of: (a) preparing a progeny plant derived from stevia variety ` <NUM>' by crossing a plant of the stevia variety '<NUM>' with a second stevia plant; and (b) crossing the progeny plant with itself or a second plant to produce a progeny plant of a subsequent generation which is derived from a plant of the stevia variety `<NUM>'. In one aspect of the disclosure, the method further comprises: (c) crossing the progeny plant of a subsequent generation with itself or a second plant; and (d) repeating steps (b) and (c) for at least <NUM>-<NUM> additional generations to produce an inbred stevia plant derived from the stevia variety `<NUM>'. Also disclosed is a plant produced by this and the other methods of the aspects of the disclosure.

Plant variety ` <NUM>'-derived plants produced by this and the other methods of the aspects of the disclosure described herein may, in certain aspects, be further defined as comprising the traits of plant variety ` <NUM>' given in Table <NUM>.

In another aspect of the disclosure, a method of vegetatively propagating the stevia plant of the present application, comprising the steps of: (a) collecting tissue or cells capable of being propagated from a plant of `<NUM>'; (b) cultivating said tissue or cells of (a) to obtain proliferated shoots; and (c) rooting said proliferated shoots to obtain rooted plantlets; or (d) cultivating said tissue or cells to obtain proliferated shoots, or to obtain plantlets. Further, plants produced by growing said plantlets or proliferated shoots are disclosed.

In another aspect of the disclosure, a method of using single nucleotide polymorphisms (SNPs) markers to identify Stevia rebaudiana variety is described. Six highly polymorphic SNPs loci and the corresponding genomic sequences are used to identify plant variety '<NUM>'-derived plant materials. The genomic sequences with the marked six SNPs are listed in Table <NUM>.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one.

Another aspect of the disclosure provides regenerable cells for use in tissue culture of stevia plant `<NUM>'. The tissue culture may be capable of regenerating plants having the physiological and morphological characteristics of the foregoing stevia plant, and of regenerating plants having substantially the same genotype as the foregoing stevia plant. The regenerable cells in such tissue cultures may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, pistils, roots, root tips, flowers, seeds, or stems. Still further, another aspect of the disclosure provides stevia plants regenerated from the tissue cultures of the disclosure.

Another aspect of the present disclosure comprises a method for developing a stevia plant in a stevia plant breeding program, comprising applying plant breeding techniques comprising recurrent selection, backcrossing, pedigree breeding, marker enhanced selection, haploid/double haploid production, or transformation to the stevia plant of claim <NUM>, or its parts, wherein application of said techniques results in development of a stevia plant. Another aspect of the present disclosure comprises a second stevia seed, plant, plant part, or cell produced by crossing a plant or plant part of stevia cultivar '<NUM>', or a locus conversion thereof, with another plant, wherein representative plant tissue of said stevia cultivar '<NUM>' has been deposited under CGMCC No. <NUM> and wherein said stevia cultivar '<NUM>' seed, plant, plant part, or cell has the same polymorphisms for the single nucleotide polymorphisms of SNP ID NO: <NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, and SNP ID NO:<NUM> as the plant or plant part of stevia cultivar '<NUM>'.

Still yet another aspect of the disclosure is the six single nucleotide polymorphisms used to identify the said stevia cultivar '<NUM>', including the locus-specific genomic sequences and the identified single nucleotide polymorphisms within the sequences.

In addition to the exemplary aspects described above, further aspects may become apparent by study of the following descriptions.

As used herein, "at least one," "one or more," and "and/or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C" and "A, B, and/or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

As used herein, "sometime" means at some indefinite or indeterminate point of time. So for example, as used herein, "sometime after" means following, whether immediately following or at some indefinite or indeterminate point of time following the prior act.

<FIG> shows the SNP sequences for SEQ ID Nos. : <NUM>-<NUM>.

In the description and tables, which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:.

Stevia cultivar '<NUM>' is a Stevia rebaudiana plant variety, which has shown uniformity and stability, as described in the following Variety Description Information. It has been reproduced a sufficient number of generations with careful attention to uniformity of plant type. The cultivar has been increased with continued observation to uniformity.

Stevia cultivar '<NUM>' resulted from a biparental cross conducted in Ganzhou, Jiangxi Province, the People's Republic of China in September <NUM> between the proprietary female Stevia variety '<NUM>' (<CIT>) and the proprietary male Stevia variety '<NUM>' (unpatented elite high Rebaudioside M line).

Stevia cultivar `<NUM>' has the following morphologic and other characteristics from data taken in the Ganzhou, Jiangxi Province, People's Republic of China.

'<NUM>' is most similar to its commercial parental line named '<NUM>', which was deposited under CGMCC No. <NUM>. Differences between the two varieties are described in Table <NUM>.

To collect the data of Table <NUM> below, stevia leaf samples were air-dried/oven-dried before grinding into fine powder using a pestle and mortar. For each sample, leaf powder (<NUM>) was extracted with <NUM> of <NUM>° C distilled water for <NUM> hours. The mixture was centrifuged and the supernatant filtered and collected for SG component analysis by HPLC (Agilent, USA). The analysis of steviol glycosides was carried out using an Agilent Technologies <NUM> Series (USA) HPLC equipped with Poroshell <NUM> SB-C18 <NUM>, <NUM> x <NUM>. A diode array set at <NUM> was used as the detector. In column one, Reb stands for Rebaudioside, Stev stands for Stevioside, and Dul stands for Dulcoside.

Single nucleotide polymorphisms (SNPs) are variations in a particular single nucleotide that occurs at specific positions in the genome, which are the most common type of genetic variation among Stevia rebaudiana genomes. <FIG> shows that sequencing results indicated six SNPs are powerful molecular markers for the identification of variety `<NUM>'.

The genotypes of the six SNPs (SNP ID NO: <NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, and SNP ID NO:<NUM>) in Stevia variety ` <NUM>' are listed as below.

Another aspect of the disclosure is a stevia plant or plant part derived from stevia variety '<NUM>' produced by crossing a plant or plant part of stevia variety ` <NUM>' with another plant, wherein representative fresh tissue culture of said stevia variety ` <NUM>' has been deposited and wherein said stevia plant part derived from the stevia variety ` <NUM>' has <NUM>%,<NUM>%,<NUM>%,<NUM>%,<NUM>%,<NUM>%,<NUM>%,<NUM>%,<NUM>%,<NUM>%,<NUM>%,<NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% of the same polymorphisms for SNPs of SNP ID NO: <NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, SNP ID NO:<NUM>, and SNP ID NO:<NUM> as the plant or plant part of stevia variety '<NUM>'.

A stevia seed derived from stevia variety '<NUM>' produced by crossing a plant or plant part of stevia variety '<NUM>' with another plant, wherein representative plant part of said stevia variety '<NUM>' has been deposited and wherein said stevia seed derived from the stevia variety '<NUM>' has essentially the same morphological characteristics as stevia variety '<NUM>' when grown in the same environmental conditions. The same environmental conditions may be, but are not limited to, a side-by-side comparison. The characteristics can be those listed in Table <NUM>. The comparison can be made using any number of professionally accepted experimental designs and statistical analysis.

An aspect of the disclosure is also directed to methods for producing a stevia plant by crossing a first parent stevia plant with a second parent stevia plant, wherein the first or second stevia plant is the stevia plant from the cultivar '<NUM>'. Further, both the first and second parent stevia plants may be the cultivar '<NUM>' (e.g., self-pollination). Therefore, any methods using the cultivar '<NUM>' are part of the aspects of the disclosure: selfing, backcrosses, hybrid breeding, and crosses to populations. Any plants produced using cultivar '<NUM>' as parents are within the scope of the aspects of the disclosure. As used herein, the term "plant" includes plant cells, plant protoplasts, plant cells of tissue culture from which stevia plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as pollen, flowers, embryos, ovules, seeds, leaves, stems, roots, anthers, pistils, shoots, and microshoots. Thus, another aspect of the disclosure is to provide for cells which upon growth and differentiation produce a cultivar having essentially all of the physiological and morphological characteristics of `<NUM>'.

Another aspect of the disclosure contemplates a stevia plant regenerated from a tissue culture of a cultivar (e.g., '<NUM>') or hybrid plant of the present aspects of the disclosure. As is well-known in the art, tissue culture of stevia can be used for the in-vitro regeneration of a stevia plant. Tissue culture of various tissues of stevia and regeneration of plants therefrom is well known and widely published.

There are numerous steps in the development of any desirable plant germplasm. Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possess the traits to meet the program goals. The goal is to combine in a single cultivar an improved combination of desirable traits from the parental germplasm. In stevia, the important traits leaf yield, earlier maturity, improved leaf quality, rebaudioside content, stevioside content, resistance to diseases and insects, resistance to drought and heat, and improved agronomic traits.

Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F1 hybrid cultivar, pureline cultivar, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants. Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method. Backcross breeding is used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cultivars. Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination, and the number of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, overall value of the advanced breeding lines, and number of successful cultivars produced per unit of input (e.g., per year, per dollar expended, etc.).

Promising advanced breeding lines are thoroughly tested and compared to popular cultivars in environments representative of the commercial target area(s) for three or more years. The lines having superiority over the popular cultivars are candidates to become new commercial cultivars. Those lines still deficient in a few traits are discarded or utilized as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing and distribution, usually take from seven to twelve years from the time the first cross is made. Therefore, development of new cultivars is a time-consuming process that requires precise forward planning, efficient use of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that are genetically superior because for most traits the true genotypic value is masked by other confounding plant traits or environmental factors. One method of identifying a superior plant is to observe its performance relative to other experimental lines and widely grown standard cultivars. For many traits a single observation is inconclusive, and replicated observations over time and space are required to provide a good estimate of a line's genetic worth.

The goal of a commercial stevia breeding program is to develop new, unique, and superior stevia cultivars. The breeder initially selects and crosses two or more parental lines, followed by generation advancement and selection, thus producing many new genetic combinations. The breeder can theoretically generate billions of different genetic combinations via this procedure. The breeder has no direct control over which genetic combinations will arise in the limited population size which is grown. Therefore, two breeders will never develop the same line having the same traits.

Each year, the plant breeder selects the germplasm to advance to the next generation. This germplasm is grown under unique and different geographical, climatic, and soil conditions and further selections are then made, during and at the end of the growing season. The lines which are developed are unpredictable. This unpredictability is because the breeder's selection occurs in unique environments, with no control at the DNA level (using conventional breeding procedures), and with millions of different possible genetic combinations being generated. A breeder of ordinary skill in the art cannot predict the final resulting lines he develops, except possibly in a very gross and general fashion. The same breeder cannot produce, with any reasonable likelihood, the same cultivar twice by using the exact same original parents and the same selection techniques. This unpredictability results in the expenditure of large amounts of research moneys to develop superior new stevia cultivars.

Pureline cultivars of stevia are commonly bred by hybridization of two or more parents followed by selection. The complexity of inheritance, the breeding objectives, and the available resources influence the breeding method. Pedigree breeding, recurrent selection breeding, and backcross breeding are breeding methods commonly used in self-pollinated crops such as stevia. These methods refer to the manner in which breeding pools or populations are made in order to combine desirable traits from two or more cultivars or various broad-based sources. The procedures commonly used for selection of desirable individuals or populations of individuals are called mass selection, plant-to-row selection, and single seed descent or modified single seed descent. One or a combination of these selection methods can be used in the development of a cultivar from a breeding population.

Pedigree breeding is primarily used to combine favorable genes into a totally new cultivar that is different in many traits than either parent used in the original cross. It is commonly used for the improvement of self-pollinating crops. Two parents which possess favorable, complementary traits are crossed to produce an F1 (filial generation <NUM>). An F2 population is produced by selfing F2 plants. Selection of desirable individual plants may begin as early as the F2 generation wherein maximum gene segregation occurs. Individual plant selection can occur for one or more generations. Successively, seed from each selected plant can be planted in individual, identified rows or hills, known as progeny rows or progeny hills, to evaluate the line and to increase the seed quantity, or to further select individual plants. Once a progeny row or progeny hill is selected as having desirable traits, it becomes what is known as a breeding line that is specifically identifiable from other breeding lines that were derived from the same original population. At an advanced generation (i.e., F5 or higher) seed of individual lines are evaluated in replicated testing. At an advanced stage the best lines or a mixture of phenotypically similar lines from the same original cross are tested for potential release as new cultivars.

The single seed descent procedure in the strict sense refers to planting a segregating population, harvesting one seed from every plant, and combining these seeds into a bulk, which is planted as the next generation. When the population has been advanced to the desired level of inbreeding, the plants from which lines are derived will each trace to different F2 individuals. Primary advantages of the seed descent procedures are to delay selection until a high level of homozygosity (e.g., lack of gene segregation) is achieved in individual plants, and to move through these early generations quickly, usually through using winter nurseries.

The modified single seed descent procedures involve harvesting multiple seed (i.e., a single lock or a simple boll) from each plant in a population and combining them to form a bulk. Part of the bulk is used to plant the next generation and part is put in reserve. This procedure has been used to save labor at harvest and to maintain adequate seed quantities of the population.

Selection for desirable traits can occur at any segregating generation (F2 and above). Selection pressure is exerted on a population by growing the population in an environment where the desired trait is maximally expressed and the individuals or lines possessing the trait can be identified. For instance, selection can occur for disease resistance when the plants or lines are grown in natural or artificially-induced disease environments, and the breeder selects only those individuals having little or no disease and are thus assumed to be resistant.

In addition to phenotypic observations, the genotype of a plant can also be examined. There are many laboratory-based techniques available for the analysis, comparison, and characterization of plant genotype. Among these are Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs--which are also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determine genetic composition. Shoemaker and Olsen, (<NPL>)) developed a molecular genetic linkage map that consisted of <NUM> linkage groups with about <NUM> RFLP, <NUM> RAPD, three classical markers, and four isozyme loci. See also,<NPL>).

SSR technology is currently the most efficient and practical marker technology; more marker loci can be routinely used and more alleles per marker locus can be found using SSRs in comparison to RFLPs. For example, Diwan and Cregan described a highly polymorphic microsatellite locus in soybean with as many as <NUM> alleles. SNPs may also be used to identify the unique genetic composition of the disclosure and progeny varieties retaining that unique genetic composition. Various molecular marker techniques may be used in combination to enhance overall resolution.

Molecular markers, which includes markers identified through the use of techniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF, SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use of molecular markers is Quantitative Trait Loci (QTL) mapping. QTL mapping is the use of markers which are known to be closely linked to alleles that have measurable effects on a quantitative trait. Selection in the breeding process is based upon the accumulation of markers linked to the positive effecting alleles and/or the elimination of the markers linked to the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for the selection of qualitative traits. For example, markers closely linked to alleles or markers containing sequences within the actual alleles of interest can be used to select plants that contain the alleles of interest during a backcrossing breeding program. For example, molecular markers are used in soybean breeding for selection of the trait of resistance to soybean cyst nematode, see <CIT>. The markers can also be used to select toward the genome of the recurrent parent and against the markers of the donor parent. Using this procedure can attempt to minimize the amount of genome from the donor parent that remains in the selected plants. It can also be used to reduce the number of crosses back to the recurrent parent needed in a backcrossing program. The use of molecular markers in the selection process is often called Genetic Marker Enhanced Selection. Molecular markers may also be used to identify and exclude certain sources of germplasm as parental varieties or ancestors of a plant by providing a means of tracking genetic profiles through crosses as discussed more fully hereinafter.

Mutation breeding is another method of introducing new traits into stevia varieties. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation (such as X-rays, Gamma rays, neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens (such as base analogues like <NUM>-bromo-uracil), antibiotics, alkylating agents (such as sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine, nitrous acid, or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques. Details of mutation breeding can be found in Principles of Cultivar Development by Fehr, Macmillan Publishing Company (<NUM>).

The production of double haploids can also be used for the development of homozygous varieties in a breeding program. Double haploids are produced by the doubling of a set of chromosomes from a heterozygous plant to produce a completely homozygous individual. For example, see <NPL>).

Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several reference books (e.g., Allard (<NUM>); Simmonds (<NUM>); Sneep, et al. (<NUM>); Fehr (<NUM>)).

Proper testing should detect any major faults and establish the level of superiority or improvement over current cultivars. In addition to showing superior performance, there must be a demand for a new cultivar that is compatible with industry standards or which creates a new market. The introduction of a new cultivar will incur additional costs to the seed producer, and to the grower, processor, and consumer, for special advertising, marketing and commercial production practices, and new product utilization. The testing preceding the release of a new cultivar should take into consideration research and development costs as well as technical superiority of the final cultivar. For seed-propagated cultivars, it must be feasible to produce seed easily and economically.

The stevia flower is monoecious in that the male and female structures are in the same flower. The crossed or hybrid seed is produced by manual crosses between selected parents. Floral buds of the parent that is to be the female are emasculated prior to the opening of the flower by manual removal of the male anthers. At flowering, the pollen from flowers of the parent plants designated as male, are manually placed on the stigma of the previous emasculated flower. Seed developed from the cross is known as first generation (F <NUM>) hybrid seed. Planting of this seed produces F1 hybrid plants of which half their genetic component is from the female parent and half from the male parent. Segregation of genes begins at meiosis thus producing second generation (F2) seed. Assuming multiple genetic differences between the original parents, each F2 seed has a unique combination of genes.

With the advent of molecular biological techniques that have allowed the isolation and characterization of genes that encode specific protein products, scientists in the field of plant biology developed a strong interest in engineering the genome of plants to contain and express foreign genes, or additional, or modified versions of native, or endogenous, genes (perhaps driven by different promoters) in order to alter the traits of a plant in a specific manner. Such foreign additional and/or modified genes are referred to herein collectively as "transgenes. " Over the last fifteen to twenty years several methods for producing transgenic plants have been developed, in particular aspects of the disclosure, also relates to transformed versions of the claimed cultivar.

Plant transformation involves the construction of an expression vector which will function in plant cells. Such a vector comprises DNA comprising a gene under control of or operatively linked to a regulatory element (for example, a promoter). The expression vector may contain one or more such operably linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid, and can be used alone or in combination with other plasmids, to provide transformed stevia plants, using transformation methods as described below to incorporate transgenes into the genetic material of the stevia plant(s).

Expression vectors include at least one genetic marker operably linked to a regulatory element (for example, a promoter) that allows transformed cells containing the marker to be either recovered by negative selection (i e , inhibiting growth of cells that do not contain the selectable marker gene), or by positive selection (i.e., screening for the product encoded by the genetic marker). Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII), which, when under the control of plant regulatory signals, confers resistance to kanamycin. Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen, et al. , Plant Mol. , <NUM>:<NUM> (<NUM>).

Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, and aminoglycoside-<NUM>'-adenyl transferase, the bleomycin resistance determinant. <NPL>); <NPL>); <NPL>); <NPL>). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate, or bromoxynil. <NPL>); <NPL>); and <NPL>).

Other selectable marker genes for plant transformation that are not of bacterial origin include, for example, mouse dihydrofolate reductase, plant <NUM>-enolpyruvyl-shikimate-<NUM>-phosphate synthase and plant acetolactate synthase. <NPL>); <NPL>); <NPL>).

Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase, and chloramphenicol acetyltransferase. Jefferson, R. , Plant Mol. , <NUM>:<NUM> (<NUM>); Teeri, et al. , <NUM>:<NUM> (<NUM>); Koncz, et al. , PNAS, <NUM>:<NUM> (<NUM>); DeBlock, et al. <NUM>:<NUM> (<NUM>).

In-vivo methods for visualizing GUS activity that do not require destruction of plant tissue are available. <NPL>) and <NPL>). However, these in-vivo methods for visualizing GUS activity have not proven useful for recovery of transformed cells because of low sensitivity, high fluorescent backgrounds, and limitations associated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells. GFP and mutants of GFP may be used as screenable markers.

Genes included in expression vectors must be driven by a nucleotide sequence comprising a regulatory element (for example, a promoter). Several types of promoters are now well known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters.

As used herein, "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue-preferred. " Promoters which initiate transcription only in certain tissue are referred to as "tissue-specific. " A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter which is active under most environmental conditions.

An inducible promoter is operably linked to a gene for expression in stevia. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in stevia. With an inducible promoter the rate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the aspects of the instant disclosure. See <NPL>). Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (<NPL>)); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (<NPL>) and <NPL>)); or Tet repressor from Tn10 (<NPL>)). An example inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (<NPL>)).

A constitutive promoter is operably linked to a gene for expression in stevia or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in stevia.

Many different constitutive promoters can be utilized in the aspects of the instant disclosure. Exemplary constitutive promoters include, but are not limited to, the promoters from plant viruses such as the <NUM> promoter from CaMV (<NPL>)) and the promoters from such genes as rice actin (<NPL>)); ubiquitin (<NPL>) and <NPL>)); pEMU (<NPL>)); MAS (<NPL>)); and maize H3 histone (<NPL>) and <NPL>)).

The ALS promoter, Xbal/Ncol fragment <NUM>' to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said Xbal/Ncol fragment), represents a particularly useful constitutive promoter.

A tissue-specific promoter is operably linked to a gene for expression in stevia. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in stevia. Plants transformed with a gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in the aspects of the instant disclosure. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter, such as that from the phaseolin gene (<NPL>) and <NPL>)); a leaf-specific and light-induced promoter, such as that from cab or rubisco (<NPL>) and <NPL>)); an anther-specific promoter, such as that from LAT52 (<NPL>)); a pollen-specific promoter, such as that from Zm13 (<NPL>)); or a microspore-preferred promoter, such as that from apg (<NPL>)).

Transport of protein produced by transgenes to a subcellular compartment, such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, or mitochondrion, or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the <NUM>' and/or <NUM>' region of a gene encoding the protein of interest. Targeting sequences at the <NUM>' and/or <NUM>' end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast. Many signal sequences are known in the art. See, for example, <NPL>);<NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>).

With transgenic plants, a foreign protein can be produced in commercial quantities. Thus, techniques for the selection and propagation of transformed plants, which are well understood in the art, yield a plurality of transgenic plants which are harvested in a conventional manner, and a foreign protein then can be extracted from a tissue of interest or from total biomass. Protein extraction from plant biomass can be accomplished by known methods which are discussed, for example, by <NPL>).

According to an aspect of the disclosure, the transgenic plant provided for commercial production of foreign protein is a stevia plant. In another aspect of the disclosure, the biomass of interest is seed. For the relatively small number of transgenic plants that show higher levels of expression, a genetic map can be generated, primarily via conventional RFLP, PCR, and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule. For exemplary methodologies in this regard, see <NPL>). Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant. If unauthorized propagation is undertaken and crosses made with other germplasm, the map of the integration region can be compared to similar maps for suspect plants, to determine if the latter have a common parentage with the subject plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR and sequencing, all of which are conventional techniques.

Likewise, agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:.

Numerous methods for plant transformation have been developed, including biological and physical, plant transformation protocols. See, for example, <NPL>). In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, <NPL>).

One method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, <NPL>). tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, <NPL>). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber, et al. , supra, Miki, et al. , supra, and <NPL>). See also, <CIT>.

Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation. A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring <NUM> to <NUM>. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of <NUM>/s to <NUM>/s which is sufficient to penetrate plant cell walls and membranes. <NPL>); <NPL>); <NPL>); <NPL>); <NPL>). See also, <CIT>; <CIT>.

Another method for physical delivery of DNA to plants is sonication of target cells. Alternatively, liposome and spheroplast fusion have been used to introduce expression vectors into plants. <NPL>); <NPL>). Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol, or poly-L-omithine has also been reported. <NPL>) and <NPL>). Electroporation of protoplasts and whole cells and tissues has also been described. <NPL>); <NPL>); and <NPL>).

Following transformation of stevia target tissues, expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues, and/or plants, using regeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used for producing a transgenic variety. The transgenic variety could then be crossed, with another (non-transformed or transformed) variety, in order to produce a new transgenic variety. Alternatively, a genetic trait which has been engineered into a particular stevia cultivar using the foregoing transformation techniques could be moved into another cultivar using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite variety into an elite variety, or from a variety containing a foreign gene in its genome into a variety or varieties which do not contain that gene. As used herein, "crossing" can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.

When the term "stevia plant" is used herein, this also includes any single gene conversions of that variety. The term "single gene converted plant" as used herein refers to those stevia plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the single gene transferred into the variety via the backcrossing technique. Backcrossing methods can be used herein to improve or introduce a characteristic into the variety. The term "backcrossing" as used herein refers to the repeated crossing of a hybrid progeny back to the recurrent parent, i.e., backcrossing <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more times to the recurrent parent. The parental stevia plant which contributes the gene for the desired characteristic is termed the "nonrecurrent" or "donor parent". This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental stevia plant to which the gene or genes from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol (Poehlman & Sleper (<NUM>); Fehr (<NUM>)). In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (nonrecurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a stevia plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the nonrecurrent parent, as determined at the <NUM>% significance level when grown in the same environmental conditions.

The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original variety. To accomplish this, a single gene of the recurrent variety is modified or substituted with the desired gene from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological, constitution of the original variety. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross. One of the major purposes is to add some commercially desirable, agronomically important trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularly selected for in the development of a new variety but that can be improved by backcrossing techniques. Single gene traits may or may not be transgenic. Examples of these traits include but are not limited to, male sterility, waxy starch, herbicide resistance, resistance for bacterial, fungal, or viral disease, insect resistance, male fertility, enhanced nutritional quality, industrial usage, yield stability, and yield enhancement. These genes are generally inherited through the nucleus. Several of these single gene traits are described in <CIT>; <CIT>; and <CIT>.

Genome editing technologies such as clustered regularly interspaced short palindromic repeat (CRISPR)- CRISPR associated protein (CRISPR-Cas) allow targeted modification of almost any crop genomic sequences to generate novel variations, which has facilitated targeted trait improvement in plants. Recently, CRISPR from Prevotella and Francisella <NUM> (Cpf1) has emerged as a new tool for efficient genome editing, including DNA-free genome editing in plants. CRISPR-Cpf1 system has shown the potential of higher efficiency, higher specificity, and wider applications than the CRISPR-Cas9 system.

Further reproduction of the variety can occur by tissue culture and regeneration. Tissue culture of various tissues of stevia and regeneration of plants therefrom is well known and widely published. For example, reference may be had to <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); and <NPL>); as well as <CIT>, et al. , and <CIT>, et al. Thus, another aspect of the disclosure is to provide cells which upon growth and differentiation produce stevia plants having the physiological and morphological characteristics of stevia cultivar '<NUM>'.

As used herein, the term "tissue culture" indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures are protoplasts, calli, plant clumps, and plant cells that can generate tissue culture that are intact in plants or parts of plants, such as embryos, pollen, flowers, seeds, leaves, stems, roots, root tips, anthers, and pistils. Means for preparing and maintaining plant tissue culture are well known in the art. By way of example, a tissue culture comprising organs has been used to produce regenerated plants. <CIT>; <CIT>; and <CIT>, described certain techniques.

Another aspect of the disclosure is directed to methods for producing a stevia plant by crossing a first parent stevia plant with a second parent stevia plant wherein the first or second parent stevia plant is a stevia plant of the cultivar '<NUM>'. Further, both first and second parent stevia plants can come from the stevia cultivar '<NUM>'. Thus, any such methods using the stevia cultivar '<NUM>' are part of the aspects of the disclosure: selfing, backcrosses, hybrid production, and crosses to populations. All plants produced using stevia cultivar '<NUM>' as a parent are within the scope of the aspects of the disclosure, including those developed from varieties derived from stevia cultivar `<NUM>'. Advantageously, the stevia cultivar could be used in crosses with other, different, stevia plants to produce first generation (F1) stevia hybrid seeds and plants with superior characteristics. The cultivar of the aspects of the disclosure can also be used for transformation where exogenous genes are introduced and expressed by the cultivar of the aspects of the disclosure. Genetic variants created either through traditional breeding methods using cultivar ` <NUM>' or through transformation of '<NUM>' by any of a number of protocols known to those of skill in the art are intended to be within the scope of the aspects of the disclosure.

The following describes breeding methods that may be used with cultivar '<NUM>' in the development of further stevia plants. One such aspect of the disclosure is a method for developing a '<NUM>' progeny stevia plant in a stevia plant breeding program comprising: obtaining the stevia plant, or a part thereof, of cultivar '<NUM>', utilizing said plant or plant part as a source of breeding material, and selecting a '<NUM>' progeny plant with molecular markers in common with '<NUM>' and/or with morphological and/or physiological characteristics selected from the characteristics listed in Tables <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. Breeding steps that may be used in the stevia plant breeding program include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. In conjunction with these steps, techniques such as marker-enhanced selection, genetic marker enhanced selection (for example, SSR markers), and the making of double haploids may be utilized.

Another method involves producing a population of cultivar '<NUM>' progeny stevia plants, comprising crossing cultivar '<NUM>' with another stevia plant, thereby producing a population of stevia plants, which, on average, derive <NUM>% of their alleles from cultivar '<NUM>'. A plant of this population may be selected and repeatedly selfed or sibbed with a stevia cultivar resulting from these successive filial generations. One aspect of this disclosure is the stevia cultivar produced by this method and that has obtained at least <NUM>% of its alleles from cultivar `<NUM>'.

One of ordinary skill in the art of plant breeding would know how to evaluate the traits of two plant varieties to determine if there is no significant difference between the two traits expressed by those varieties. For example, see <NPL>). Thus the aspects of the disclosure includes stevia cultivar '<NUM>' progeny stevia plants comprising a combination of at least two '<NUM>' traits selected from the group consisting of those listed in Tables <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> or the '<NUM>' combination of traits listed in the Summary, so that said progeny stevia plant is not significantly different for said traits than stevia cultivar '<NUM>' as determined at the <NUM>% significance level when grown in the same environment. Using techniques described herein, molecular markers may be used to identify said progeny plant as a '<NUM>' progeny plant. Mean trait values may be used to determine whether trait differences are significant, and the traits are measured on plants grown under the same environmental conditions. Once such a variety is developed its value is substantial since it is important to advance the germplasm base as a whole in order to maintain or improve traits such as yield, disease resistance, pest resistance, and plant performance in extreme environmental conditions.

Progeny of cultivar '<NUM>' may also be characterized through their filial relationship with stevia cultivar '<NUM>', as for example, being within a certain number of breeding crosses of stevia cultivar '<NUM>'. A breeding cross is a cross made to introduce new genetics into the progeny, and is distinguished from a cross, such as a self or a sib cross, made to select among existing genetic alleles. The lower the number of breeding crosses in the pedigree, the closer the relationship between stevia cultivar '<NUM>' and its progeny. For example, progeny produced by the methods described herein may be within <NUM>, <NUM>, <NUM>, <NUM> or <NUM> breeding crosses of stevia cultivar '<NUM>'.

As used herein, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures from which stevia plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovules, flowers, leaves, roots, root tips, anthers, and pistils.

Various aspects of the disclosure, include components, methods, processes, systems and/or apparatus substantially as depicted and described herein, including various aspects of the disclosure, sub-combinations, and subsets thereof. Those of skill in the art will understand how to make and use an aspect(s) of the disclosure after understanding the present disclosure.

The use of the terms "a," "an," and "the," and similar referents in the context of describing the aspects of the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, if the range <NUM>-<NUM> is disclosed, then <NUM>, <NUM>, <NUM>, and <NUM> are also disclosed. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the aspects of the disclosure.

Claim 1:
A method of determining the genotype of a stevia plant, wherein said method comprises:
(a) obtaining a sample of nucleic acids from said plant; and
(b) detecting in said nucleic acids an allele of one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of:
(i) a G at nucleotide position <NUM> of SEQ ID NO: <NUM>; and
(ii) a T at nucleotide position <NUM> of SEQ ID NO: <NUM>.