Mogroside compositions and methods for producing same

Abstract

The present disclosure relates to compositions and methods for the production of transformed plants and other organisms with increased production of mogroside compounds, particularly mogroside V, and the mogroside compounds, plants and plant parts so obtained.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 376,194, filed September 19, 2022, and U.S. Provisional Application No. 63 / 491,721, filed March 22, 2023, all of which are incorporated herein by reference in their entireties.

[0002] The sequence listing under file name "ELSS002WO_ST26.xml" (540 kilobytes as measured in MS-Windows, created on September 14, 2023, contains 305 sequences) is incorporated herein by full reference.

[0003] The present disclosure relates to the field of genetic engineering of plants and other organisms, and more particularly to methods and compositions for generating plants and other organisms with increased production of mogroside compounds, particularly mogroside V. The disclosure also relates to the use of such plants and organisms to produce novel ingredients for foods and beverages (e.g., mogroside-containing plant extracts, purified or partially purified fractions), and the novel foods and beverages (and other compositions of matter) obtained thereby.

[0004] Low- or no-calorie sweeteners, particularly those derived from natural sources, are becoming increasingly important in the food and beverage industry and other industries as alternatives to traditional high-calorie and artificial sweeteners. These alternative sweeteners are used to replace artificial sweeteners and high-calorie sweeteners, including sucrose, fructose, and glucose. Like some artificial sweeteners, some of these alternative sweeteners have a higher sweetening potency than caloric sweeteners, meaning less is needed to achieve the same sweetness as sugar. However, some low-calorie sweeteners can be expensive to produce or have undesirable taste profiles or aftertaste issues (e.g., sweetness persistence, delayed sweetness onset, unpleasant mouthfeel, bitterness, metallic taste, cooling sensation, astringency, licorice-like taste, etc.).

[0005] Some natural plants produce low- or no-calorie sweeteners. For example, mogrosides, an important group of natural sweeteners, are chemically classified as triterpene glycosides or mogrol glycosides and are naturally produced by the monk fruit (Siraitia grosvenorii). Mogrosides are "zero calorie" (less than 5 calories per 8 ounces) and are 100–400 times sweeter than sucrose. Mogrosides have also been reported to have various pharmacological effects. However, although plants such as monk fruit (Siraitia grosvenorii) produce mogrosides, their production is limited and expensive due to the plant's limited natural and agricultural production. Furthermore, monk fruit (Siraitia grosvenorii) prefers to grow in subtropical mountainous regions and requires laborious pollination to produce fruit. Attempts have been made to produce mogrosides in vitro and by microorganisms, but large-scale processing and other problems have prevented them from being economically viable.

[0006] Therefore, there is a need for new compositions and methods for the efficient production of mogrosides. Summary of the Invention

[0007] The present disclosure addresses the above and other problems of the prior art by providing novel compositions and methods for the efficient and cost-effective production of sweet mogrosides having three or more glucose residues in the molecule, such as mogroside V, isomogroside V, siamenoside I, α-siamenoside I, mogroside IV, mogroside IV A, mogroside III, mogroside III E, mogroside III A1, and 11-oxo-mogroside V, in plants and other organisms.

[0008] The present disclosure provides a transgenic plant, plant part, or seed comprising: (a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86; (b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5; (c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; (d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9; (e) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11. (f) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:13; (g) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO:15; or (h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276, or SEQ ID NO:278; wherein any of the first through eighth polynucleotide sequences is operably linked to a heterologous promoter, and the transgenic plant, plant part, or seed produces at least one mogroside compound. In some embodiments, each polynucleotide sequence is linked to a different heterologous promoter. In other embodiments, two or more polynucleotide sequences are linked to the same heterologous promoter. In other embodiments, one or more polynucleotide sequences are present in multiple copies and are linked to the same or different endogenous or heterologous promoters.

[0009] In various embodiments, the plant is a plant of the Cucurbitaceae, Solanaceae, or Asteraceae family. In some embodiments, the plant is a plant of the genus Cucurbita, Melonus spp., Cucumis sativus, Bitter Melon, Solanum spp., or Lettuce. In further embodiments, the plant is selected from the group consisting of watermelon, cantaloupe, honeydew melon, winter melon, Casaba melon, Persian melon, citron melon, muskmelon, White Orchid melon, Crenshaw melon, Christmas melon, Sprite melon, Carabelle melon, Hami melon, Rocky melon, Langkawi Golden melon, Korean melon, Saticoy melon, Galia melon, Jade melon, Golden Prize melon, Tennyo melon, New Century melon, Banana melon, Yubari King melon, Sugar melon, Tiger melon, Vertical vine melon, Horn melon, Mexican miniature melon, Casa banana melon. The crop may be a variety such as pepino melon, ananas melon, camouflage melon, canary melon, bitter melon, Charente melon, crane melon, skyrocket melon, honey globe melon, gac fruit, autumn sweet melon, snap melon, lettuce, spinach, rice, oats, corn, sorghum, wheat, alfalfa, colochinsis, pumpkin, chard, tobacco, switchgrass, tomato, cucumber, potato, amaranth, sunflower, rapeseed, dry beans, peas, flax, safflower, buckwheat, cotton, soybean, sugar beet, or bent tobacco (Nicotiana benthamiana). In particular embodiments, the crop may be a leafy vegetable such as watermelon, tomato, lettuce, or cucumber, or a crop used as a base ingredient for food processing. In even more particular embodiments, the crop may be a monocotyledonous or dicotyledonous plant. The plant part may be a fruit, leaf, root, flower, stem, cell, endosperm, ovule, or pollen, etc. In another embodiment, the plant part may be a plant species that produces mogrosides that can be used to replace or partially replace added sugars when processed into foods.

[0010] In certain embodiments, the heterologous promoter is an inducible, plant-derived, bacterial-derived, viral-derived, synthetic, constitutive, tissue-specific, developmental stage-dependent, cell cycle-dependent, temporally regulated, spatially regulated, or spatiotemporally regulated promoter. In other embodiments, the heterologous promoter is a FSgt / PFLt (SEQ ID NO: 62), FMVSgt (SEQ ID NO: 69), CsVMV (SEQ ID NO: 68), dMMV (SEQ ID NO: 63), HLVH12 (SEQ ID NO: 60), NOS (SEQ ID NO: 66), ScBV (SEQ ID NO: 67), DCMV (SEQ ID NO: 61), CmYLCV (SEQ ID NO: 64), FS1_1 (SEQ ID NO: 70), FE_3 (SEQ ID NO: 71), e35S (SEQ ID NO: 65), AtUBQ10 (SEQ ID NO: 259), PCLSV (SEQ ID NO: 260), FS4 (SEQ ID NO: 261), AtACT2 (SEQ ID NO: 262), enhanced AtEf-1A (SEQ ID NO: 263), FuasFScp (SEQ ID NO: 264), FE4 (SEQ ID NO: 269), cucumisin (SEQ ID NO: 270), or SgCDS (SEQ ID NO: 271) promoter. In yet another embodiment, the first, second, third, fourth, fifth, sixth, seventh, or eighth polynucleotide sequence is operably linked to a heterologous termination sequence. In yet another embodiment, the heterologous termination sequence is a termination sequence that is GmaxMYB2 (SEQ ID NO:74), 35ST (SEQ ID NO: not shown), ATHSP18.2 (SEQ ID NO:77), AtRBCS2b (SEQ ID NO:75), AtUBQ3 (SEQ ID NO:73), Pea E9 (SEQ ID NO:76), Pea3A (SEQ ID NO:72), potato Ubi3 (SEQ ID NO:78), AtTubB9 (SEQ ID NO:79), AtFAD2 (SEQ ID NO:265), AtNDUFA8 (SEQ ID NO:266), CsHSP17.3 (SEQ ID NO:267), or CsHSP22 (SEQ ID NO:268). In additional embodiments, the transgenic plant, plant part, or seed further comprises a selectable marker sequence. In some embodiments, the selectable marker sequence is β-glucuronidase, green fluorescent protein, or an antibiotic resistance sequence. In certain embodiments, the selectable marker sequence is hygromycin B phosphotransferase (HygR) or neomycin phosphotransferase II (nptII).

[0011] In some embodiments, the transgenic plant, plant part, or cell further comprises: (i) a ninth polynucleotide sequence encoding an NADPH:cytochrome P450 reductase polypeptide having at least 90% sequence identity to SEQ ID NO: 19; or (j) a tenth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO: 21, 23, 120, or 126; (k) an eleventh polynucleotide sequence encoding a 3-hydroxy-3-methylglutaryl-CoA synthase polypeptide having at least 90% sequence identity to SEQ ID NO: 227; or (l) a twelfth polynucleotide sequence encoding a geranyl diphosphate synthase polypeptide having at least 90% sequence identity to SEQ ID NO: 256; wherein the ninth, tenth, eleventh, or twelfth polynucleotide sequence is operably linked to a heterologous promoter. In other embodiments, the transgenic plant, plant part or cell further comprises a 2A linker, an insulator, a selectable marker or a stuffer sequence.

[0012] In certain embodiments, the at least one mogroside compound is a non-native mogrol precursor, mogrol, mogroside, or a metabolite or derivative thereof. In various embodiments, the mogroside is mogroside IIA, IIA1, IIA2, IIE, 11-oxo-mogroside II, mogroside III, IIIA1, IIIA2, IIIE, 11-oxo-mogroside III, mogroside IV, IVA, 11-oxo-mogroside IV, siamenoside I, mogroside V, 11-oxo-mogroside V, mogroside VI, or an isomer thereof. In additional embodiments, the transgenic plant, plant part, or seed produces at least 10 ng / g to 30 mg / g dry weight of at least one mogroside compound. In other embodiments, the production rate is 10 ng / g or more, 25 ng / g or more, 50 ng / g or more, 75 ng / g or more, 100 ng / g or more, 250 ng / g or more, 500 ng / g or more, 750 ng / g or more, 1 mg / g or more, 2.5 mg / g or more, 5 mg / g or more, 7.5 mg / g or more, 10 mg / g or more, 12.5 mg / g or more, 15 mg / g or more, 17.5 mg / g or more, 20 mg / g or more, 22.5 mg / g or more, 25 mg / g or more, 27.5 mg / g or more, or 30 mg / g or more. In yet other embodiments, the amount of mogroside compounds exceeds the level in a non-transgenic plant, plant part, or seed of the same species. In still other embodiments, the ratio of individual mogroside compounds differs from the ratio of mogroside compounds in native plant tissues, fruits, or other plants or organisms. For example, the ratio of mogroside compounds produced by a plant or organism of the present disclosure may be 1.1:1 to 1000:1 or more, or 1:1.1 to 1:1000 or less, relative to the ratio in the native plant, fruit, etc. These ratios apply to one or more of the mogroside compounds described above. Generally, the ratio of sweet mogrosides containing three or more glucose residues, such as mogroside V, isomogroside V, siamenoside I, α-siamenoside I, mogroside IV, IVA, III, IIIE, IIIA1, and 11-oxo-mogroside V, is high.

[0013] The disclosure further provides a recombinant host cell comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to SEQ ID NO:7, 25, 27, 29, 31, or 33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9; e) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11. f) a fifth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO: 13; g) a seventh polynucleotide sequence encoding an epoxyhydrolase polypeptide having at least 90% sequence identity to SEQ ID NO: 15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to SEQ ID NO: 17, 274, 276, or 278; wherein any of the polynucleotide sequences is operably linked to a heterologous promoter, and in certain embodiments, the recombinant host cell produces at least one mogroside compound.

[0014] The disclosure further provides a reduced calorie processed food or beverage product made from a transformed plant, plant part, or seed comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to any of SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9; e) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11; f) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO: 13; g) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO: 15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to any of SEQ ID NO: 17, SEQ ID NO: 274, SEQ ID NO: 276, or SEQ ID NO: 278, wherein at least one of the first through eighth polynucleotide sequences is operably linked to a heterologous promoter, and the transformed plant, plant part, or seed produces at least one mogroside compound. In one embodiment, a product is produced from juice, extract, powder, pulp, or peel of the fruit, vegetable, legume, tuber, or grain of the transformed plant, plant part, or seed.

[0015] The disclosure further provides a juice, powder, or extract produced from a transgenic plant, plant part, or seed, comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to any of SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9; e) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11. f) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:13; g) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO:15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to any of SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276, or SEQ ID NO:278; wherein any of the first through eighth polynucleotide sequences is operably linked to a heterologous promoter, and the transgenic plant, plant part, or seed produces at least one mogroside compound.

[0016] The present disclosure further provides a recombinant DNA molecule comprising the following components: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to any of SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9; e) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9. f) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO:13; g) a seventh polynucleotide sequence encoding an epoxyhydrolase polypeptide having at least 90% sequence identity to SEQ ID NO:15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to any of SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276, or SEQ ID NO:278; wherein any of the first through eighth polynucleotide sequences is operably linked to a heterologous promoter. In certain embodiments, the first through eighth polynucleotide sequences are each operably linked to a different heterologous promoter. In other embodiments, at least two of the first through eighth polynucleotide sequences are operably linked to a single heterologous promoter. Additionally, in some embodiments, multiple copies of one or more of the first through eighth polynucleotide sequences are present, operably linked to the same or different endogenous or heterologous promoters.

[0017] The present disclosure also provides a DNA molecule comprising a polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86. In some embodiments, the DNA molecule is operably linked to a heterologous promoter. The present disclosure further provides a DNA molecule exhibiting gene expression regulating functional activity, comprising a polynucleotide sequence selected from the group consisting of: a) a sequence having at least 90% or 95% sequence identity to SEQ ID NO:70 or 71 and having promoter activity; b) a sequence comprising SEQ ID NO:70 or 71; or c) a fragment of SEQ ID NO:70 or 71, wherein the fragment has promoter activity, and the DNA molecule is operably linked to a heterologous transcribable polynucleotide molecule. In some embodiments, SEQ ID NO:70 and SEQ ID NO:71 may increase expression in plant fruits.

[0018] The present disclosure further provides a method for producing at least one mogroside compound, comprising cultivating a transformed plant or organism comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86, b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5, c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to any of SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33, d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9, or e) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11. f) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO:13; g) a seventh polynucleotide sequence encoding an epoxyhydrolase polypeptide having at least 90% sequence identity to SEQ ID NO:15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to any of SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276, or SEQ ID NO:278, wherein at least one of the polynucleotide sequences is operably linked to a heterologous promoter, and the transformed plant, plant part, or seed produces at least one mogroside compound. In one embodiment, the transformed plant produces the mogroside compound in the plant part or seed. In another embodiment, the mogroside compound is produced in the fruit or leaf. In yet another embodiment, the method includes isolating at least one mogroside compound from the transformed plant.In other embodiments, mogroside compounds are isolated, purified, or partially purified from plant parts or seeds. In yet other embodiments, the compounds are isolated from fruits, leaves, vegetables, legumes, tubers, or grains. In yet other embodiments, combinations of mogroside compounds are purified or partially purified from transformed plants, plant parts, or seeds.

[0019] The present disclosure further provides a composition comprising: a) about 80% mogroside V, about 15% 11-oxo-mogroside V, and about 5% mogroside III-A1, or b) about 40% siamenoside I, about 40% mogroside V, and about 20% 11-oxo-mogroside V. In some embodiments, the composition comprises about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% mogroside V, about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% 11-oxo-mogroside V, and about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% mogroside III-A1. In other embodiments, the composition comprises about 35%-45% siamenoside I, about 35%-45% mogroside V, and about 15%-25% 11-oxo-mogroside V. In another embodiment, the composition is a liquid. In yet another embodiment, the composition is a dry powder. [Brief explanation of the drawings]

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in conjunction with the detailed description of the embodiments set forth herein.

[0021] [Figure 1] Mogroside biosynthetic pathway in Monk fruit (Siraitia grosvenorii).

[0022] [Figure 2] Production of mogroside V in transient expression in watermelon fruit using various expression constructs.

[0023] [Figure 3] Map of expression construct SP3139.

[0024] [Figure 4] Production of mogrosides and siamenoside using various constructs in transient expression in watermelon fruit.

[0025] [Figure 5] Production of mogroside V using various constructs in transient expression in watermelon fruit.

[0026] [Figure 6] Production of siamenoside using various constructs in transient expression in watermelon fruit.

[0027] [Figure 7] Production of mogrosides and siamenoside in watermelon fruit by transient expression of constructs SP2015, SP4332, SP5029, SP5030, SP5031, SP5032, SP5033 (control), and combinations of all but one of them.

[0028] [Figure 8] Production of mogroside V in watermelon fruit by transient expression of constructs SP2015, SP4332, SP5029, SP5030, SP5031, SP5032, SP5033 (control), and combinations of all but one of them.

[0029] [Figure 9] Production of mogroside V in transiently expressed lettuce leaves infiltrated with the SP1463 construct, compared with control lettuce and a mogroside standard mixture.

[0030] [Figure 10]Mass spectral fingerprinting of mogroside V production by transient expression in lettuce leaves infiltrated with the SP1463 construct, compared to control lettuce and a mogroside V standard.

[0031] [Figure 11] Map of expression construct SP1463.

[0032] [Figure 12] Production of mogrosides and siamenoside in transient expression in lettuce leaves using various constructs.

[0033] [Figure 13] Production of mogroside V in lettuce leaves by transient expression using various constructs.

[0034] [Figure 14] Production of siamenoside in lettuce leaves by transient expression using various constructs.

[0035] [Figure 15] Production of mogrosides and siamenoside in transient expression in lettuce leaves using constructs SP2015, SP4332, SP5029, SP5030, SP5031, SP5032, SP5033 (control), and combinations of these except one.

[0036] [Figure 16] Production of mogroside V in lettuce leaves by transient expression using constructs SP2015, SP4332, SP5029, SP5030, SP5031, SP5032, SP5033 (control), and combinations of these except one.

[0037] [Figure 17] Production of mogroside V in transgenic watermelon introduced with expression construct SP1463 and comparison with a mogroside V standard.

[0038] [Figure 18] Mass spectral fingerprinting of mogroside V in transgenic watermelon plants containing the expression construct SP1463 and comparison with a mogroside V standard.

[0039] [Figure 19] Mogroside production in various transgenic watermelon lines carrying expression constructs SP0336, SP1463, SP1908, SP3488, and SP3190.

[0040] [Figure 20] Map of expression construct SP0336.

[0041] [Figure 21] Map of expression construct SP1908.

[0042] [Figure 22] Production of various mogrosides and siamenosides in transgenic watermelons carrying expression constructs SP0565, SP0641, SP1463, SP1908, SP3015, SP3016, SP3029, SP3190, SP3308, and SP3488.

[0043] [Figure 23] Production of mogrosides and siamenoside in cell suspensions of transformed watermelon.

[0044] [Figure 24] Concentrations of mogrosides and siamenoside in cell suspensions of transformed watermelon.

[0045] [Figure 25] Production of various mogrosides and siamenosides in transgenic tomatoes harboring expression constructs SP0641, SP1463, SP1908, SP3016, SP3029, SP3190, and SP3684.

[0046] [Figure 26] Production of various mogrosides and siamenosides in transformed potatoes carrying expression constructs SP0641, SP1463, SP3016, SP3029, SP3190, and SP3684.

[0047] [Figure 27] Production of mogroside V and an unknown mogroside V isomer in transiently expressed watermelon fruit co-infiltrated with construct SP3684 and either t72143 or green fluorescent protein (GFP).

[0048] [Figure 28] Mass spectral fingerprinting of mogroside V and unknown mogroside V isomers.

[0049] [Figure 29] Production of mogroside IV and an unknown mogroside IV isomer in transiently expressed watermelon fruit co-infiltrated with construct SP3684 and t72143 or GFP.

[0050] [Figure 30] Mass spectral fingerprinting of mogroside IV and unknown mogroside IV isomers.

[0051] [Figure 31] Production of mogroside III and an unknown mogroside III isomer in transiently expressed watermelon fruit co-infiltrated with construct SP3684 and t72143 or GFP.

[0052] [Figure 32] Mass spectral fingerprinting of mogroside III and unknown mogroside III isomers.

[0053] [Figure 33]Production of mogroside II and an unknown mogroside II isomer in transiently expressed watermelon fruit co-infiltrated with construct SP3684 and t72143 or GFP.

[0054] [Figure 34] Mass spectral fingerprinting of mogroside II and unknown mogroside II isomers.

[0055] [Figure 35] Production of 11-oxo-mogroside III and its isomers in transiently expressed watermelon fruits co-infiltrated with construct SP3684 and t72143 or GFP.

[0056] [Figure 36] Mass spectral fingerprinting of 11-oxo-mogroside III and its isomers.

[0057] [Figure 37] Production of 11-oxo-mogroside IV and its isomers in transiently expressed watermelon fruits co-infiltrated with construct SP3684 and t72143 or GFP.

[0058] [Figure 38] Mass spectral fingerprinting of 11-oxo-mogroside IV and its isomers.

[0059] [Figure 39] Production of 11-oxo-mogroside V and its isomers in transiently expressed watermelon fruits co-infiltrated with construct SP3684 and t72143 or GFP.

[0060] [Figure 40] Mass spectral fingerprinting of 11-oxo-mogroside V and its isomers.

[0061] [Figure 41]Concentration-response curve for mogroside blend 80 / 15 / 5.

[0062] [Figure 42] Concentration-response curves for Mogroside V and Mogroside Blend 80 / 15 / 5.

[0063] [Figure 43] Sweetness onset and time to maximum sweetness for mogroside V, mogroside blend 80 / 15 / 5, rebaudioside M, rebaudioside A, and monk fruit 50 (MF50).

[0064] [Figure 44] Graph showing the decay of sweetness over time for Mogroside V, Mogroside Blend 80 / 15 / 5, Rebaudioside M, Rebaudioside A, and MF50.

[0065] [Figure 45] Descriptive analysis scores of sweetness attributes for Mogroside V, Mogroside Blend 80 / 15 / 5, Rebaudioside M, Rebaudioside A, and MF50.

[0066] [Figure 46] Descriptive analysis of the sweetness attributes of mogroside V, mogroside blend 80 / 15 / 5, rebaudioside M, rebaudioside A, and MF50.

[0067] [Figure 47] Concentration-response curve of V90.

[0068] [Figure 48] Concentration-response curves for mogroside V, blend 80 / 15 / 5, and V90.

[0069] [Figure 49] Sweetness onset and time to reach maximum sweetness for mogrosides V and V90.

[0070] [Figure 50] Attenuation of the sweetness of mogrosides V and V90.

[0071] [Figure 51] Descriptive analysis scores of mogroside V and V90.

[0072] [Figure 52] Concentration-response curve of 11-oxo-mogroside V.

[0073] [Figure 53] Concentration-response curve of mogroside IIIA-1.

[0074] [Figure 54] Sweetness onset and time to reach maximum sweetness of mogroside V, 11-oxo-mogroside V, and mogroside IIIA-1.

[0075] [Figure 55] Decay of residual sweetness of mogroside V, 11-oxo-mogroside V, and mogroside IIIA-1 over time.

[0076] [Figure 56] Descriptive analysis scores for sweetness attributes of mogroside V, 11-oxo-mogroside V, and mogroside IIIA-1.

[0077] [Figure 57] Descriptive analysis of the sweetness attributes of mogroside V, 11-oxo-mogroside V, and mogroside IIIA-1.

[0078] [Figure 58] Concentrations of mogrosides and siamenoside in a transformation event in which SlUGT (SP5027) was introduced into potato plants transformed with vector SP1463.

[0079] [Figure 59]Concentrations of mogrosides and siamenoside in transformed sugar beet calli transformed with vector SP3684.

[0080] SEQ ID NO: 1: Cytochrome P450-72 (CYP72 Zm) nucleic acid sequence based on codon usage of maize (Zea mays).

[0081] SEQ ID NO: 2: Cytochrome P450-72 (CYP72) amino acid sequence.

[0082] SEQ ID NO: 3: Cytochrome P450-72 (CYP72 GC) nucleic acid sequence with a higher GC content than SEQ ID NO: 1.

[0083] SEQ ID NO: 4: Cucurbitadienol synthase (CDS) nucleic acid sequence.

[0084] SEQ ID NO: 5: Cucurbitadienol synthase (CDS) amino acid sequence.

[0085] SEQ ID NO: 6: Cytochrome P450-87 (CYP87) nucleic acid sequence.

[0086] SEQ ID NO: 7: Cytochrome P450-87 (CYP87) amino acid sequence.

[0087] SEQ ID NO: 8: Uridine phosphorylase-dependent glycosyltransferase-720 (UGT720) nucleic acid sequence.

[0088] SEQ ID NO: 9: Uridine phosphorylase-dependent glycosyltransferase-720 (UGT720) amino acid sequence.

[0089] SEQ ID NO: 10: Uridine phosphorylase-dependent glycosyltransferase-94 (UGT94) nucleic acid sequence.

[0090] SEQ ID NO: 11: Uridine phosphorylase-dependent glycosyltransferase-94 (UGT94) amino acid sequence.

[0091] SEQ ID NO: 12: Squalene epoxidase (SQE) nucleic acid sequence.

[0092] SEQ ID NO: 13: Squalene epoxidase (SQE) amino acid sequence.

[0093] SEQ ID NO: 14: Epoxyhydrolase (EPH) nucleic acid sequence.

[0094] SEQ ID NO: 15: Epoxyhydrolase (EPH) amino acid sequence.

[0095] SEQ ID NO: 16: Truncated 3-hydroxy-3-methylglutaryl CoA reductase (tHMGR) nucleic acid sequence.

[0096] SEQ ID NO: 17: Truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) amino acid sequence.

[0097] SEQ ID NO: 18: NADPH:cytochrome P450 reductase (SgCPR2) nucleic acid sequence.

[0098] SEQ ID NO: 19: NADPH:cytochrome P450 reductase (SgCPR2) amino acid sequence.

[0099] SEQ ID NO: 20: Uridine phosphorylase-dependent glycosyltransferase 74 (UGT74_3) nucleic acid sequence.

[0100] SEQ ID NO: 21: Uridine phosphorylase-dependent glycosyltransferase 74 (UGT74_3) amino acid sequence.

[0101] SEQ ID NO: 22: Uridine phosphorylase-dependent glycosyltransferase 74 (UGT74_4) nucleic acid sequence.

[0102] SEQ ID NO: 23: Uridine phosphorylase-dependent glycosyltransferase 74 (UGT74_4) amino acid sequence.

[0103] SEQ ID NO: 24: Nucleic acid sequence of cytochrome P450-87 (ClCYP87D18_B m3) from watermelon (Citrullus lanatus).

[0104] SEQ ID NO: 25: Amino acid sequence of cytochrome P450-87 (ClCYP87D18_B m3) from watermelon (Citrullus lanatus).

[0105] SEQ ID NO: 26: Mutant cytochrome P450-87 (CYP87D17 m2) nucleic acid sequence.

[0106] SEQ ID NO: 27: Mutant cytochrome P450-87 (CYP87D17 m2) amino acid sequence.

[0107] SEQ ID NO: 28: Mutant cytochrome P450-87 (CYP87D17 m3) nucleic acid sequence.

[0108] SEQ ID NO: 29: Mutant cytochrome P450-87 (CYP87D17 m3) amino acid sequence.

[0109] SEQ ID NO: 30: Mutant cytochrome P450-87 (CYP87D20 m2) nucleic acid sequence.

[0110] SEQ ID NO: 31: Mutant cytochrome P450-87 (CYP87D20 m2) amino acid sequence.

[0111] SEQ ID NO: 32: Mutant cytochrome P450-87 (CYP87D20 m3) nucleic acid sequence.

[0112] SEQ ID NO: 33: Mutant cytochrome P450-87 (CYP87D20 m3) amino acid sequence.

[0113] SEQ ID NO: 34: Cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence.

[0114] SEQ ID NO: 35: First CYP72 amino acid sequence obtained after cleavage from SEQ ID NO: 34.

[0115] SEQ ID NO: 36: A second CYP72 amino acid sequence obtained after cleavage from SEQ ID NO: 34.

[0116] SEQ ID NO: 37: Full-length CYP72-2A-CYP72 amino acid sequence derived from SEQ ID NO: 34.

[0117] SEQ ID NO: 38: UGT720-2A-UGT720 bicistronic nucleic acid sequence.

[0118] SEQ ID NO: 39: First UGT720 amino acid sequence obtained after cleavage from SEQ ID NO: 38.

[0119] SEQ ID NO: 40: A second UGT720 amino acid sequence obtained after cleavage from SEQ ID NO: 38.

[0120] SEQ ID NO: 41: Full-length UGT720-2A-UGT720 amino acid sequence derived from SEQ ID NO: 38.

[0121] SEQ ID NO: 42: UGT94-2A-UGT94 bicistronic nucleic acid sequence.

[0122] SEQ ID NO: 43: First UGT94 amino acid sequence obtained after cleavage from SEQ ID NO: 42.

[0123] SEQ ID NO: 44: A second UGT94 amino acid sequence obtained after cleavage from SEQ ID NO: 42.

[0124] SEQ ID NO: 45: Full-length UGT94-2A-UGT94 amino acid sequence derived from SEQ ID NO: 42.

[0125] SEQ ID NO: 46: tHMGR-2A-tHMGR bicistronic nucleic acid sequence.

[0126] SEQ ID NO: 47: First tHMGR amino acid sequence obtained after cleavage from SEQ ID NO: 46.

[0127] SEQ ID NO: 48: A second tHMGR amino acid sequence obtained after cleavage from SEQ ID NO: 46.

[0128] SEQ ID NO: 49: Full-length tHMGR-2A-tHMGR amino acid sequence derived from SEQ ID NO: 46.

[0129] SEQ ID NO: 50: UGT720-2A-CYP72 bicistronic nucleic acid sequence.

[0130] SEQ ID NO: 51: UGT720 amino acid sequence obtained after cleavage from SEQ ID NO: 50.

[0131] SEQ ID NO: 52: CYP72 amino acid sequence obtained after cleavage from SEQ ID NO: 50.

[0132] SEQ ID NO: 53: Full-length UGT720-2A-CYP72 amino acid sequence derived from SEQ ID NO: 50.

[0133] SEQ ID NO: 54: UGT94-2A-tHMGR bicistronic nucleic acid sequence.

[0134] SEQ ID NO: 55: UGT94 amino acid sequence obtained after cleavage from SEQ ID NO: 54.

[0135] SEQ ID NO: 56: tHMGR amino acid sequence obtained after cleavage from the UGT94-2A-tHMGR bicistronic nucleic acid sequence.

[0136] SEQ ID NO: 57: UGT94-2A-tHMGR amino acid sequence derived from the UGT94-2A-tHMGR bicistronic nucleic acid sequence.

[0137] SEQ ID NO: 58: Hygromycin resistance (HygR) gene nucleic acid sequence.

[0138] SEQ ID NO: 59: Hygromycin resistance (HygR) gene amino acid sequence.

[0139] SEQ ID NO: 60: HLVH12 promoter nucleic acid sequence.

[0140] SEQ ID NO: 61: DCMV promoter nucleic acid sequence.

[0141] SEQ ID NO: 62: FMVSgt:PCLSVFlt (also called FSgt / PFLt) chimeric promoter nucleic acid sequence.

[0142] SEQ ID NO: 63: Duplicated MMV (dMMV) promoter nucleic acid sequence.

[0143] SEQ ID NO: 64: CmYLCV promoter nucleic acid sequence.

[0144] SEQ ID NO: 65: CaMV e35S (e35S) promoter nucleic acid sequence.

[0145] SEQ ID NO: 66: NOS promoter nucleic acid sequence.

[0146] SEQ ID NO: 67: ScBV promoter nucleic acid sequence.

[0147] SEQ ID NO: 68: CsVMV promoter nucleic acid sequence.

[0148] SEQ ID NO: 69: FMVSgt promoter nucleic acid sequence.

[0149] SEQ ID NO: 70: FS1_1 promoter nucleic acid sequence.

[0150] SEQ ID NO: 71: FE_3 promoter nucleic acid sequence.

[0151] SEQ ID NO: 72: 3A terminator nucleic acid sequence from Pea.

[0152] SEQ ID NO: 73: Arabidopsis thaliana (At) UBQ3 terminator nucleic acid sequence.

[0153] SEQ ID NO: 74: Soybean (Gmax) MYB2 terminator nucleic acid sequence.

[0154] SEQ ID NO: 75: AtRBCS2B terminator nucleic acid sequence.

[0155] SEQ ID NO: 76: Pea E9 terminator nucleic acid sequence.

[0156] SEQ ID NO: 77: AtHSP18.2 terminator nucleic acid sequence.

[0157] SEQ ID NO: 78: Potato Ubi3 terminator nucleic acid sequence.

[0158] SEQ ID NO: 79: At tubulin B9 (AtTub) terminator nucleic acid sequence.

[0159] SEQ ID NO: 80: 35S terminator nucleic acid sequence.

[0160] SEQ ID NO: 81: SynJ 5'UTR (5' untranslated region) nucleic acid sequence.

[0161] SEQ ID NO: 82: TM6 MAR insulator nucleic acid sequence.

[0162] SEQ ID NO: 83: 2A self-cleaving peptide nucleic acid sequence.

[0163] SEQ ID NO: 84: 2A self-cleaving peptide amino acid sequence.

[0164] SEQ ID NO: 85: Cytochrome P450-72 (CYP72 V1) nucleic acid sequence.

[0165] SEQ ID NO: 86: Cytochrome P450-72 (CYP72 V1) amino acid sequence.

[0166] SEQ ID NO: 87: Alternative squalene epoxidase (SQE) nucleic acid sequence.

[0167] SEQ ID NO: 88: Alternative cytochrome P450-87 (CYP87) nucleic acid sequence.

[0168] SEQ ID NO: 89: Alternative cucurbitadienol synthase (CDS) nucleic acid sequence.

[0169] SEQ ID NO: 90: Alternative epoxy hydrolase (EPH) nucleic acid sequence.

[0170] SEQ ID NO: 91: Alternative UGT720 nucleic acid sequence.

[0171] SEQ ID NO: 92: Alternative UGT94 nucleic acid sequence.

[0172] SEQ ID NO: 93: Upper terpenoid biosynthetic enzyme (FPS; SP0231), watermelon (Citrullus lanatus) cDNA, nucleic acid sequence.

[0173] SEQ ID NO: 94: Amino acid sequence of upper terpenoid biosynthetic enzyme (FPS; SP0231) from watermelon.

[0174] SEQ ID NO: 95: Cytochrome P450 biosynthetic enzyme, high GC version (CYP87D20 m2(V2-I46L-A49L-C343Y) GC63; SP0577), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0175] SEQ ID NO: 96: Nucleic acid sequence from Monk fruit identical to SEQ ID NO: 95.

[0176] SEQ ID NO: 97: Upper squalene biosynthetic enzyme (SQS; SP0951), watermelon cDNA, nucleic acid sequence.

[0177] SEQ ID NO: 98: Amino acid sequence of upper squalene biosynthetic enzyme (SQS; SP0951) from watermelon.

[0178] SEQ ID NO: 99: Cytochrome P450 biosynthetic enzyme, mutant, GC-rich version (CYP87D17 m3 GC63; SP1333), Monk fruit cDNA, nucleic acid sequence.

[0179] SEQ ID NO: 100: Cytochrome P450 biosynthetic enzyme, mutant, GC-rich version (CYP87D17 m3 GC63; SP1333), Monk fruit (Siraitia grosvenorii), amino acid sequence.

[0180] SEQ ID NO: 101: Cytochrome P450 biosynthetic enzyme, mutant, GC-rich version (CYP87D17 m2 GC63; SP2503), Monk fruit cDNA, nucleic acid sequence.

[0181] SEQ ID NO: 102: Cytochrome P450 biosynthetic enzyme, mutant, GC-rich version (CYP87D17 m2 GC63; SP2503), Monk fruit, protein sequence.

[0182] SEQ ID NO: 103: Cytochrome P450 reductase biosynthetic enzyme, GC-rich version (SgCPR2 GC66; SP2571), Monk fruit cDNA, nucleic acid sequence.

[0183] SEQ ID NO: 104: Cytochrome P450 reductase biosynthetic enzyme, GC-rich version (SgCPR2 GC66; SP2571), Monk fruit, amino acid sequence.

[0184] SEQ ID NO: 105: Uridine phosphorylase-dependent glycosyltransferase 74, GC-rich version (SgUGT74_406_2 GC64; SP2666), Monk fruit cDNA, nucleic acid sequence.

[0185] SEQ ID NO: 106: Amino acid sequence of uridine phosphorylase-dependent glycosyltransferase 74, GC-rich version (SgUGT74_406_2 GC64; SP2666), derived from Siraitia grosvenorii.

[0186] SEQ ID NO: 107: Uridine phosphorylase-dependent glycosyltransferase 74, GC-rich version (SgUGT74_345_2 GC65; SP3201), cDNA sequence from Siraitia grosvenorii.

[0187] SEQ ID NO: 108: Amino acid sequence of SEQ ID NO: 107.

[0188] SEQ ID NO: 109: cDNA sequence of cytochrome P450 biosynthetic enzyme, mutant, GC-rich version (ClCYP87D18_B m3 GC62; SP4900), from watermelon (Citrullus lanatus).

[0189] SEQ ID NO: 110: Amino acid sequence of SEQ ID NO: 109.

[0190] SEQ ID NO: 111: cDNA sequence of cytochrome P450 biosynthetic enzyme (CYP87D20 m3 (V2-I46L-A49L-C343Y L193KO) GC63; SP4910) from Siraitia grosvenorii.

[0191] SEQ ID NO: 112: Amino acid sequence of SEQ ID NO: 111.

[0192] SEQ ID NO: 113: Uridine phosphorylase-dependent glycosyltransferase (Solyc01g107825.1 UGT; SP5024), cDNA sequence from Solanum lycopersicum (tomato).

[0193] SEQ ID NO: 114: Amino acid sequence of SEQ ID NO: 113.

[0194] SEQ ID NO: 115: Uridine phosphorylase-dependent glycosyltransferase (Solyc02g070020.1 UGT; SP5025), cDNA sequence from Solanum lycopersicum.

[0195] SEQ ID NO: 116: Amino acid sequence of SEQ ID NO: 115.

[0196] SEQ ID NO: 117: Uridine phosphorylase-dependent glycosyltransferase (Solyc09g092500.1 UGT; SP5026), cDNA sequence from Solanum lycopersicum.

[0197] SEQ ID NO: 118: Amino acid sequence of SEQ ID NO: 117.

[0198] SEQ ID NO: 119: Uridine phosphorylase-dependent glycosyltransferase (Solyc10g085230.2 UGT; SP5027), cDNA sequence from Solanum lycopersicum.

[0199] SEQ ID NO: 120: Amino acid sequence of SEQ ID NO: 119.

[0200] SEQ ID NO: 121: Uridine phosphorylase-dependent glycosyltransferase (Solyc10g085880.1 UGT; SP5028), cDNA sequence from Solanum lycopersicum.

[0201] SEQ ID NO: 122: Amino acid sequence of SEQ ID NO: 121.

[0202] SEQ ID NO: 123: Uridine phosphorylase-dependent glycosyltransferase 94 (Sg UGT94-289-3 -MS1; SP5034), cDNA sequence from Siraitia grosvenorii.

[0203] SEQ ID NO: 124: Amino acid sequence of SEQ ID NO: 123.

[0204] SEQ ID NO: 125: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (GPS1; SP5035) from watermelon (Citrullus lanatus).

[0205] SEQ ID NO: 126: Amino acid sequence of SEQ ID NO: 125.

[0206] SEQ ID NO: 127: Uridine phosphorylase-dependent glycosyltransferase, GC-rich version (SgUGT75-281-2 GC65; SP5036), cDNA sequence from Siraitia grosvenorii.

[0207] SEQ ID NO: 128: Amino acid sequence of SEQ ID NO: 127.

[0208] SEQ ID NO: 129: Uridine phosphorylase-dependent glycosyltransferase 74 (SgUGT74AC1 (no stop codon); SP5037), cDNA sequence from Siraitia grosvenorii.

[0209] SEQ ID NO: 130: Amino acid sequence of SEQ ID NO: 129.

[0210] SEQ ID NO: 131: Uridine phosphorylase-dependent glycosyltransferase (Sgmg1 GC63; SP5038), cDNA sequence from Siraitia grosvenorii.

[0211] SEQ ID NO: 132: Amino acid sequence of SEQ ID NO: 131.

[0212] SEQ ID NO: 133: Uridine phosphorylase-dependent glycosyltransferase (t134248; SP5039), cDNA sequence from Stevia rebaudiana.

[0213] SEQ ID NO: 134: Amino acid sequence of SEQ ID NO: 133.

[0214] SEQ ID NO: 135: Uridine phosphorylase-dependent glycosyltransferase (t72140; SP5040), cDNA sequence from Stevia rebaudiana.

[0215] SEQ ID NO: 136: Amino acid sequence of SEQ ID NO: 135.

[0216] SEQ ID NO: 137: Uridine phosphorylase-dependent glycosyltransferase (t72143; SP5041), cDNA sequence from Stevia rebaudiana.

[0217] SEQ ID NO: 138: Amino acid sequence of SEQ ID NO: 137.

[0218] SEQ ID NO: 139: Uridine phosphorylase-dependent glycosyltransferase (t74692; SP5042), cDNA sequence from Stevia rebaudiana.

[0219] SEQ ID NO: 140: Amino acid sequence of SEQ ID NO: 139.

[0220] SEQ ID NO: 141: Uridine phosphorylase-dependent glycosyltransferase (t85004; SP5043), cDNA sequence from Stevia rebaudiana.

[0221] SEQ ID NO: 142: Amino acid sequence of SEQ ID NO: 141.

[0222] SEQ ID NO: 143: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (Sg 00000109.2_FPS1; SP5044) from Siraitia grosvenorii.

[0223] SEQ ID NO: 144: Amino acid sequence of SEQ ID NO: 143.

[0224] SEQ ID NO: 145: cDNA sequence of upstream enzyme involved in squalene biosynthesis (Sg 00000892.729_SQS1; SP5045) from Siraitia grosvenorii.

[0225] SEQ ID NO: 146: Amino acid sequence of SEQ ID NO: 145.

[0226] SEQ ID NO: 147: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (Bj HMGS mutant; SP5046) from Brassica junea (mustard).

[0227] SEQ ID NO: 148: Amino acid sequence of SEQ ID NO: 147.

[0228] SEQ ID NO: 149: UDPG pyrophosphorylase (Ta UDP-glucose pyrophosphorylase; SP5047), cDNA sequence from Thermocrispum agreste.

[0229] SEQ ID NO: 150: Amino acid sequence of SEQ ID NO: 149.

[0230] SEQ ID NO: 151: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (ClGPS_2; SP5048) from watermelon (Citrullus lanatus).

[0231] SEQ ID NO: 152: Amino acid sequence of an upstream enzyme involved in terpenoid biosynthesis (ClGPS_2; SP5048), from watermelon (Citrullus lanatus).

[0232] SEQ ID NO: 153: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (MpGPS-SSU; SP5049) from peppermint (Mentha x piperita).

[0233] SEQ ID NO: 154: Amino acid sequence of SEQ ID NO: 153.

[0234] SEQ ID NO: 155: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (LcGPS-SSU; SP5050) from Litsea cubeba.

[0235] SEQ ID NO: 156: Amino acid sequence of SEQ ID NO: 155.

[0236] SEQ ID NO: 157: cDNA sequence of hydrolase biosynthetic enzyme (DbExg1; SP5051) from Dekkera bruxellensis (yeast).

[0237] SEQ ID NO: 158: Amino acid sequence of SEQ ID NO: 157.

[0238] SEQ ID NO: 159: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (ClCG09G011560.1_PMK; SP5072) from watermelon (Citrullus lanatus).

[0239] SEQ ID NO: 160: Amino acid sequence of SEQ ID NO: 159.

[0240] SEQ ID NO: 161: cDNA sequence from watermelon (Citrullus lanatus), upstream enzyme involved in terpenoid biosynthesis (ClCG10G001230.1_PMK; SP5073).

[0241] SEQ ID NO: 162: Amino acid sequence of SEQ ID NO: 161.

[0242] SEQ ID NO: 163: cDNA sequence from watermelon (Citrullus lanatus), upstream enzyme involved in terpenoid biosynthesis (ClCG09G022040.1_IPK; SP5074).

[0243] SEQ ID NO: 164: Amino acid sequence of SEQ ID NO: 163.

[0244] SEQ ID NO: 165: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (ClCG05G021710.1_MVD; SP5075) from Citrullus lanatus.

[0245] SEQ ID NO: 166: Amino acid sequence of SEQ ID NO: 165.

[0246] SEQ ID NO: 167: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (Sg 00153574.101_FPS1; SP5076) from Siraitia grosvenorii.

[0247] SEQ ID NO: 168: Amino acid sequence of SEQ ID NO: 167.

[0248] SEQ ID NO: 169: cDNA sequence of upstream enzyme involved in squalene biosynthesis (Sg 00010190.2_SQS1; SP5077) from Siraitia grosvenorii.

[0249] SEQ ID NO: 170: Amino acid sequence of SEQ ID NO: 169.

[0250] SEQ ID NO: 171: Upstream enzyme involved in terpenoid biosynthesis (Sg 00153449.125_PMK; SP50

[0251] SEQ ID NO: 172: Amino acid sequence of upstream enzyme involved in terpenoid biosynthesis (Sg 00153449.125_PMK; SP5078) from Siraitia grosvenorii.

[0252] SEQ ID NO: 173: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis, high GC version (tHMGR GC69; SP5079), from Avena strigosa.

[0253] SEQ ID NO: 174: Amino acid sequence corresponding to SEQ ID NO: 173.

[0254] SEQ ID NO: 175: Uridine phosphorylase-dependent glycosyltransferase, GC-rich version (Sg UGT720-269-1 Itkin GC65; SP5080), cDNA sequence from Siraitia grosvenorii.

[0255] SEQ ID NO: 176: Amino acid sequence corresponding to SEQ ID NO: 175.

[0256] SEQ ID NO: 177: Uridine phosphorylase-dependent glycosyltransferase, GC-rich version (Sg UGT720-269-4 Itkin GC65; SP5081), cDNA sequence from Siraitia grosvenorii.

[0257] SEQ ID NO: 178: Amino acid sequence corresponding to SEQ ID NO: 177.

[0258] SEQ ID NO: 179: Uridine phosphorylase-dependent glycosyltransferase (Sg UGT94-289-2; SP5082), cDNA sequence from Siraitia grosvenorii.

[0259] SEQ ID NO: 180: Amino acid sequence corresponding to SEQ ID NO: 179.

[0260] SEQ ID NO: 181: Uridine phosphorylase-dependent glycosyltransferase (Sg UGT94-289-2 Itkin; SP5083), cDNA sequence from Siraitia grosvenorii.

[0261] SEQ ID NO: 182: Amino acid sequence corresponding to SEQ ID NO: 181.

[0262] SEQ ID NO: 183: Uridine phosphorylase-dependent glycosyltransferase (Sg UGT94-289-3 Itkin; SP5084), cDNA sequence from Siraitia grosvenorii.

[0263] SEQ ID NO: 184: Amino acid sequence corresponding to SEQ ID NO: 183.

[0264] SEQ ID NO: 185: Uridine phosphorylase-dependent glycosyltransferase (t134583; SP5085), cDNA sequence from Siraitia grosvenorii.

[0265] SEQ ID NO: 186: Amino acid sequence corresponding to SEQ ID NO: 185.

[0266] SEQ ID NO: 187: Uridine phosphorylase-dependent glycosyltransferase (t74645; SP5086), cDNA sequence from Siraitia grosvenorii.

[0267] SEQ ID NO: 188: Amino acid sequence corresponding to SEQ ID NO: 187.

[0268] SEQ ID NO: 189: Uridine phosphorylase-dependent glycosyltransferase (t74693; SP5087), cDNA sequence from Siraitia grosvenorii.

[0269] SEQ ID NO: 190: Amino acid sequence corresponding to SEQ ID NO: 189.

[0270] SEQ ID NO: 191: cDNA sequence from Siraitia grosvenorii, an upstream enzyme involved in terpenoid biosynthesis (Sg 00001291.26_PMK; SP5088).

[0271] SEQ ID NO: 192: Amino acid sequence corresponding to SEQ ID NO: 191.

[0272] SEQ ID NO: 193: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (Sg 00154122.1_IPK; SP5089) from Siraitia grosvenorii.

[0273] SEQ ID NO: 194: Amino acid sequence corresponding to SEQ ID NO: 193.

[0274] SEQ ID NO: 195: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (Sg 27366_MVD; SP5090) from Siraitia grosvenorii.

[0275] SEQ ID NO: 196: Amino acid sequence of an upstream enzyme involved in terpenoid biosynthesis (Sg 27366_MVD; SP5090), derived from Siraitia grosvenorii.

[0276] SEQ ID NO: 197: cDNA sequence of hydrolase (E142A-RRK67; SP5091) from Aspergillus oryzae.

[0277] SEQ ID NO: 198: Amino acid sequence corresponding to SEQ ID NO: 197.

[0278] SEQ ID NO: 199: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (Mc CDS; SP5092) from Momordica charantia (bitter melon).

[0279] SEQ ID NO: 200: Amino acid sequence corresponding to SEQ ID NO: 199.

[0280] SEQ ID NO: 201: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (Cp CDS; SP5093) from Cucurbita pepo (pumpkin).

[0281] SEQ ID NO: 202: Amino acid sequence corresponding to SEQ ID NO: 201.

[0282] SEQ ID NO: 203: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (Tc CDS; SP5094) from Trichosanthes cucumerina (snake gourd).

[0283] SEQ ID NO: 204: Amino acid sequence corresponding to SEQ ID NO: 203.

[0284] SEQ ID NO: 205: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (Cc CDS; SP5095) from Citrullus colocynthia (colocynth).

[0285] SEQ ID NO: 206: Amino acid sequence corresponding to SEQ ID NO: 205.

[0286] SEQ ID NO: 207: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (Ac SQS, including sixth intron; SP5096) from Amaranthus cruentus.

[0287] SEQ ID NO: 208: Amino acid sequence corresponding to SEQ ID NO: 207.

[0288] SEQ ID NO: 209: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (Oe SQS; SP5097) from Olea europaea (olive).

[0289] SEQ ID NO: 210: Amino acid sequence corresponding to SEQ ID NO: 209.

[0290] SEQ ID NO: 211: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (Cr SQS; SP5098) from Chlamydomonas reinhardtii.

[0291] SEQ ID NO: 212: Amino acid sequence corresponding to SEQ ID NO: 211.

[0292] SEQ ID NO: 213: Epoxidase biosynthetic enzyme (Cp SQE1; SP5099), cDNA sequence from Cucurbita pepo (pumpkin).

[0293] SEQ ID NO: 214: Amino acid sequence corresponding to SEQ ID NO: 213.

[0294] SEQ ID NO: 215: Epoxidase biosynthetic enzyme (Mc SQE1; SP5100), cDNA sequence from Momordica charantia (bitter melon).

[0295] SEQ ID NO: 216: Amino acid sequence corresponding to SEQ ID NO: 215.

[0296] SEQ ID NO: 217: Epoxidase biosynthetic enzyme (Pg SQE1; SP5101), cDNA sequence from Panax ginseng.

[0297] SEQ ID NO: 218: Amino acid sequence corresponding to SEQ ID NO: 217.

[0298] SEQ ID NO: 219: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (Ab tHMGR; SP5102) from Achyranthes bidentata (Achyranthes bidentata).

[0299] SEQ ID NO: 220: Amino acid sequence corresponding to SEQ ID NO: 219.

[0300] SEQ ID NO: 221: cDNA sequence from Hevea brasiliensis (Hevea brasiliensis), upstream enzyme involved in terpenoid biosynthesis (Hb tHMGR; SP5103).

[0301] SEQ ID NO: 222: Amino acid sequence corresponding to SEQ ID NO: 221.

[0302] SEQ ID NO: 223: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (Pg tHMGR; SP5104) from Panax ginseng.

[0303] SEQ ID NO: 224: Amino acid sequence corresponding to SEQ ID NO: 223.

[0304] SEQ ID NO: 225: Transcription factor (At MYC2D105N mutant; SP5105), cDNA sequence from Arabidopsis thaliana.

[0305] SEQ ID NO: 226: Amino acid sequence corresponding to SEQ ID NO: 225.

[0306] SEQ ID NO: 227: Transcription factor (Cs bHLH; SP5106), cDNA sequence from Cucumis sativus (cucumber).

[0307] SEQ ID NO: 228: Amino acid sequence corresponding to SEQ ID NO: 227.

[0308] SEQ ID NO: 229: Transcription factor (Bh bHLH; SP5107), cDNA sequence from Benincasa hispida (wax gourd).

[0309] SEQ ID NO: 230: Amino acid sequence corresponding to SEQ ID NO: 229.

[0310] SEQ ID NO: 231: Transcription factor 2 (Cs bHLH; SP5108), cDNA sequence from Cucumis sativus (cucumber).

[0311] SEQ ID NO: 232: Amino acid sequence corresponding to SEQ ID NO: 231.

[0312] SEQ ID NO: 233: Transcription factor 2 (Bh bHLH; SP5109), cDNA sequence from Benincasa hispida (wax gourd).

[0313] SEQ ID NO: 234: Amino acid sequence corresponding to SEQ ID NO: 233.

[0314] SEQ ID NO: 235: Poplar sugar synthase (SP5110), cDNA sequence from Populus alba (white birch).

[0315] SEQ ID NO: 236: Amino acid sequence corresponding to SEQ ID NO: 235.

[0316] SEQ ID NO: 237: UDPG pyrophosphate (Lg UDP-glucose pyrophosphate; SP5111), cDNA sequence from Larix gmelinii (larch).

[0317] SEQ ID NO: 238: Amino acid sequence corresponding to SEQ ID NO: 237.

[0318] SEQ ID NO: 239: Transcription factor (At PAP2; SP5112), cDNA sequence from Arabidopsis thaliana.

[0319] SEQ ID NO: 240: Amino acid sequence corresponding to SEQ ID NO: 239.

[0320] SEQ ID NO: 241: Heat shock protein (Ms HSP17.6; SP5113), cDNA sequence from Medicago sativa (alfalfa).

[0321] SEQ ID NO: 242: Amino acid sequence of heat shock protein (Ms HSP17.6; SP5113), from Medicago sativa (alfalfa).

[0322] SEQ ID NO: 243: Transcription factor (At JUNGBRUNNEN1 Nac factor; SP5114), cDNA sequence from Arabidopsis thaliana.

[0323] SEQ ID NO: 244: Amino acid sequence corresponding to SEQ ID NO: 243.

[0324] SEQ ID NO: 245: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (AmGPS-SSU (no stop codon); SP5115) from Antirrhinum majus (snapfish plant).

[0325] SEQ ID NO: 246: Amino acid sequence corresponding to SEQ ID NO: 245.

[0326] SEQ ID NO: 247: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (PgFPS; SP5116) from Panax ginseng.

[0327] SEQ ID NO: 248: Amino acid sequence corresponding to SEQ ID NO: 247.

[0328] SEQ ID NO: 249: cDNA sequence of an upstream enzyme involved in terpenoid biosynthesis (PgSQS; SP5117) from Panax ginseng.

[0329] SEQ ID NO: 250: Amino acid sequence corresponding to SEQ ID NO: 249.

[0330] SEQ ID NO: 251: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (gbHMGS1; SP5118) from Ginkgo biloba.

[0331] SEQ ID NO: 252: Amino acid sequence corresponding to SEQ ID NO: 251.

[0332] SEQ ID NO: 253: Uridine phosphate-dependent glycosyltransferase (UGT73AM3; SP5263), cDNA sequence from Cucumis sativus (cucumber).

[0333] SEQ ID NO: 254: Amino acid sequence corresponding to SEQ ID NO: 253.

[0334] SEQ ID NO: 255: Uridine phosphate-dependent glycosyltransferase, high GC content version (Solyc10g085230.2 UGT GC61; SP5265), cDNA sequence from Solanum lycopersicum (tomato).

[0335] SEQ ID NO: 256: Amino acid sequence corresponding to SEQ ID NO: 255.

[0336] SEQ ID NO: 257: Uridine phosphate-dependent glycosyltransferase, GC-rich version (UGT73AM3 GC64; SP5350), cDNA sequence from Cucumis sativus (cucumber).

[0337] SEQ ID NO: 258: Amino acid sequence corresponding to SEQ ID NO: 257.

[0338] SEQ ID NO: 259: AtUBQ10 promoter, nucleic acid sequence from Arabidopsis thaliana.

[0339] SEQ ID NO: 260: PCLSV promoter, nucleic acid sequence derived from Peanut chlorotic streak virus.

[0340] SEQ ID NO: 261: FS4 promoter, nucleic acid sequence from watermelon (Citrullus lanatus).

[0341] SEQ ID NO: 262: AtACT2 promoter, nucleic acid sequence from Arabidopsis thaliana.

[0342] SEQ ID NO: 263: Nucleic acid sequence from enhanced AtEf-1A promoter, Cauliflower mosaic virus and Arabidopsis thaliana.

[0343] SEQ ID NO: 264: Nucleic acid sequence of the FuasFScp promoter, a hybrid promoter derived from Figwort mosaic virus.

[0344] SEQ ID NO: 265: AtFAD2 terminator, nucleic acid sequence from Arabidopsis thaliana.

[0345] SEQ ID NO: 266: AtNDUFA8 terminator, nucleic acid sequence from Arabidopsis thaliana.

[0346] SEQ ID NO: 267: CsHSP17.3 terminator, nucleic acid sequence from Cucumis sativus (cucumber).

[0347] SEQ ID NO: 268: CsHSP22 terminator, nucleic acid sequence from Cucumis sativus (cucumber).

[0348] SEQ ID NO: 269: FE4 promoter, nucleic acid sequence from watermelon (Citrullus lanatus).

[0349] SEQ ID NO: 270: Nucleic acid sequence of the cucumisin promoter from cucumber melon (Cucumis melo L.).

[0350] SEQ ID NO: 271: SgCDS promoter, nucleic acid sequence from Siraitia grosvenorii (Monk fruit).

[0351] SEQ ID NO: 272: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis, high GC version (CltHMGR GC; SP2040), from watermelon (Citrullus lanatus).

[0352] SEQ ID NO: 273: Amino acid sequence corresponding to SEQ ID NO: 272 (CltHMGR GC 2A; SP2040).

[0353] SEQ ID NO: 274: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (CltHMGR GC 2A; SP3351) from watermelon (Citrullus lanatus).

[0354] SEQ ID NO: 275: Amino acid sequence corresponding to SEQ ID NO: 274.

[0355] SEQ ID NO: 276: cDNA sequence of upstream enzyme involved in terpenoid biosynthesis (CltHMGR Zm (for 2A); SP4989) from watermelon (Citrullus lanatus).

[0356] SEQ ID NO: 277: Amino acid sequence corresponding to SEQ ID NO: 276.

[0357] SEQ ID NO: 278: Cytochrome P450 biosynthetic enzyme, maize optimized (mf CYP72A459v1a Zm(GC48); SP2015), cDNA sequence from Siraitia grosvenorii.

[0358] SEQ ID NO: 279: Amino acid sequence corresponding to SEQ ID NO: 278.

[0359] SEQ ID NO: 280: Uridine phosphate-dependent glycosyltransferase (mf UGT720; SP4263), cDNA sequence from Siraitia grosvenorii.

[0360] SEQ ID NO: 281: Amino acid sequence corresponding to SEQ ID NO: 280.

[0361] SEQ ID NO: 282: Uridine phosphate-dependent glycosyltransferase, GC-rich version (Sg UGT94-289-1 GC65; SP4332), cDNA sequence from Siraitia grosvenorii.

[0362] SEQ ID NO: 283: Amino acid sequence corresponding to SEQ ID NO: 282.

[0363] SEQ ID NO: 284: cDNA sequence from Siraitia grosvenorii, upstream enzyme involved in terpenoid biosynthesis, optimized for maize (Sg CDS Z mays GC51; SP5029).

[0364] SEQ ID NO: 285: Amino acid sequence corresponding to SEQ ID NO: 284.

[0365] SEQ ID NO: 286: Uridine phosphate-dependent glycosyltransferase, GC-rich version (mf UGT720 GC; SP5030), cDNA sequence from Siraitia grosvenorii.

[0366] SEQ ID NO: 287: Amino acid sequence corresponding to SEQ ID NO: 286.

[0367] SEQ ID NO: 288: cDNA sequence of cytochrome P450 biosynthetic enzyme, GC-rich version (Sg CYP87D18 GC62; SP5031), derived from Siraitia grosvenorii.

[0368] SEQ ID NO: 289: Amino acid sequence corresponding to SEQ ID NO: 288.

[0369] SEQ ID NO: 290: cDNA sequence of upstream enzyme involved in squalene biosynthesis, GC-rich version (Sg SQE1 GC66; SP5032), from Siraitia grosvenorii.

[0370] SEQ ID NO: 291: Amino acid sequence corresponding to SEQ ID NO: 290.

[0371] SEQ ID NO: 292: cDNA sequence of epoxide hydrolase biosynthetic enzyme, high GC version (Sg EPH3 GC64; SP5033), from Siraitia grosvenorii.

[0372] SEQ ID NO: 293: Amino acid sequence corresponding to SEQ ID NO: 292.

[0373] SEQ ID NO: 294: Uridine phosphate-dependent glycosyltransferase, GC-rich version (SgUGT720 GC 2A; SP3186), cDNA sequence from Siraitia grosvenorii.

[0374] SEQ ID NO: 295: Amino acid sequence corresponding to SEQ ID NO: 294.

[0375] SEQ ID NO: 296: cDNA sequence of cytochrome P450 biosynthetic enzyme (Sg SgCYP72v1a Zm (for 2A, GC47); SP3204) from Siraitia grosvenorii.

[0376] SEQ ID NO: 297: Amino acid sequence corresponding to SEQ ID NO: 296.

[0377] SEQ ID NO: 298: Uridine phosphate-dependent glycosyltransferase (SgUGT94-1 GC 2A; SP3807), cDNA sequence from Siraitia grosvenorii.

[0378] SEQ ID NO: 299: Amino acid sequence corresponding to SEQ ID NO: 298.

[0379] SEQ ID NO: 300: Uridine phosphate-dependent glycosyltransferase (SgUGT720 Zm(for 2A); SP3897), cDNA sequence from Siraitia grosvenorii.

[0380] SEQ ID NO: 301: Amino acid sequence corresponding to SEQ ID NO: 300.

[0381] SEQ ID NO: 302: Uridine phosphate-dependent glycosyltransferase (mf UGT720; SP4263), cDNA sequence from Siraitia grosvenorii.

[0382] SEQ ID NO: 303: Amino acid sequence corresponding to SEQ ID NO: 302.

[0383] SEQ ID NO: 304: Uridine phosphate-dependent glycosyltransferase, GC-rich version (Sg UGT94-289-1 GC65; SP4332), cDNA sequence from Siraitia grosvenorii.

[0384] SEQ ID NO: 305: Amino acid sequence corresponding to SEQ ID NO: 304. DETAILED DESCRIPTION OF THE INVENTION

[0385] The present disclosure generally relates to transgenic plants and their biosynthetic systems for producing mogrol / mogroside pathway enzymes and mogrosides, as well as methods for making such transgenic plants. The following sections present embodiments that describe the subject matter of the present invention in more detail.

[0386] I. Mogroside Pathway Enzymes and Mogrosides Mogrosides are highly stable molecules based on a triterpene skeleton, consisting of one to six glucose units attached to the triterpene backbone. Mogrosides may also contain substructures other than glucose, such as grosmoside I. Figure 1 illustrates the mogroside biosynthetic pathway in Monk Fruit (Siraitia grosvenorii) (Itkin et al., Proc Nat Acad Sci USA 113:E7619-E7628, 2016; Seki et al., Biosci. Biotechnol. Biochem. 82:927-934, 2018) and specific enzymes capable of catalyzing reactions in the pathway. Importantly, the enzymatic pathway used to produce mogrosides according to the present disclosure is not limited to the mechanism depicted in Figure 1. Other terpene structures, mogrol precursors, enzyme-catalyzed reactions, or conversion mechanisms are also possible. Also, a particular enzyme may catalyze more than one type of reaction, and one or more additional genes may be used to produce a mogrol intermediate, mogrol, or any mogroside compound.

[0387] Provided herein are exemplary nucleic acid and protein sequences of specific enzymes involved in the mogroside pathway (also referred to as the mogroside biosynthetic pathway) that convert mogrol precursors (e.g., squalene) to mogrol and ultimately to various mogroside compounds, including mogroside V. Enzymes in the mogroside pathway include squalene epoxidase (SQE), cucurbitadienol synthase (CDS), epoxyhydrolase (EPH), various cytochrome P450 enzymes (CYPs, including but not limited to, CYP72 and CYP87), uridine phosphate-dependent glycosyltransferases (UGTs, including UGT720, UGT94, UGT74, etc.), and may also include 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) or truncated forms thereof, and NADPH:cytochrome P450 reductase (CPR2).

[0388] SQE, CDS, CYP, and EPH are involved in the sequential production and conversion of mogrol precursors, such as squalene, to mogrol. Intermediate products of the enzymatic pathway include 2,3-oxidosqualene, 2,3;22,23-dioxidosqualene, 24,25-epoxycucurbitadienol, and 24,25-dihydroxycucurbitadienol. CDS (oxidosqualene cyclase) uses 2,3;22,23-diepoxysqualene as a substrate to produce 24,25-epoxycucurbitadienol. Genomic analysis has revealed five genes potentially encoding SQE in S. grosvenorii (Monk Fruit). Two of these genes are highly expressed during early stages of fruit development, as are CDS, CYP enzymes, and EPH (catalyzing subsequent reactions). The S. grosvenorii genome contains eight genes encoding EPHs that convert 24,25-epoxycucurbitadienol to 24,25-dihydroxycucurbitadienol. A particular enzyme may catalyze multiple types of reactions.

[0389] After the formation of mogrol, a series of glycosylation reactions occur, adding glucose molecules to its C-3 and C-24 positions, producing mogrosides I–VI with varying degrees of glycosylation. The Roman numerals I, II, III, IV, V, and VI represent the number of glucose units in the corresponding glycosylated mogroside, isomogroside, or oxomogroside, respectively. Two UGTs (uridine phosphate-dependent glycosyltransferases) are involved in these steps. The first, UGT720, is highly expressed during early fruit development and transfers one glucose molecule to the hydroxyl groups at C-24 and C-3 of mogrol, respectively. The second, UGT94, is highly expressed during later fruit development and adds additional sugars to the sugars already present on the acceptor molecule.

[0390] Although the mogroside pathway was originally described in Siraitia grosvenorii (monk fruit), certain plants other than monk fruit can also produce tetracyclic triterpenoid compounds similar to mogrol. This is because intermediates such as triterpenes are present in the intracellular pathway. Furthermore, because related pathways involved in the modification of tetracyclic triterpenoid compounds require related enzymes, such as reductases, these plants already express these related network enzymes. Even if plants other than monk fruit express the enzymes that produce the mogrol precursor, they do not naturally express all the enzymes required to produce mogrosides in a coordinated manner. For example, plants such as cucumber, melon, and watermelon naturally express cucurbitadienol synthase, which can produce cucurbitadienol, but other enzymes, such as CYP enzymes, convert this intermediate into other terpene derivatives. Therefore, the genomes of these non-monk fruit plants can be modified using recombinant DNA technology, genome editing, or other modern plant breeding techniques to produce mogrol and mogrosides. Thus, in some embodiments, the introduction of one or more genes into non-monk fruit plants or upregulation of endogenous enzymes can enable the production of intermediate metabolites to produce mogrol, mogrosides, and mogroside-based sweeteners.

[0391] The term "mogrol precursor" broadly encompasses all terpene derivatives and intermediates leading to the production of mogrol and mogroside compounds, including, but not limited to, 2,3-oxidosqualene, 2,3;22,23-dioxidosqualene, 24,25-epoxycucurbitadienol, 24,25-dihydroxycucurbitadienol, cucurbitadienol, 11-hydroxycucurbitadienol, 11-oxocucurbitadienol, and the like. Mogroside refers to any glycosylation product of mogrol, including, but not limited to, siamenoside I, silathose (a stereoisomer of siamenoside I), mogroside VI, mogroside V, isomogroside V, mogroside IV, mogroside III, mogroside IIIE, mogroside IIE, mogroside IIA, mogroside IE, and mogroside IA. Other examples of mogrosides include mogroside IIB, 7-oxomogroside IIE, 11-oxomogroside A1, mogroside IIIA2, 11-deoxomogroside III, 11-oxomogroside IVA, 7-oxomogroside V, and 11-oxomogroside V. Mogroside metabolites and derivatives refer to closely related mogroside variants obtained by metabolic, spontaneous, or non-spontaneous reactions. Mogroside derivatives may include deletions, alterations, or additions of atoms or functional groups compared to the standard mogroside. However, mogroside metabolites and derivatives retain substantially the same function and properties as the standard mogroside. In some cases, mogroside compounds produced by transgenic plants or organisms of the present invention may be converted to other mogroside compounds by non-enzymatic (spontaneous) transformations.

[0392] One or more of these mogroside compounds can be isolated, purified, or partially purified from the transgenic plants or transgenic organisms grown using the fermentation techniques of the present invention. For example, one or more mogroside compounds can be recovered from aqueous extracts or aqueous layers obtained during bulk processing without isolating individual mogroside compounds. Alternatively, the transgenic plants (or parts thereof) or transgenic organisms that produce mogroside compounds can be dried and ground into powder, extracted, or used as a food ingredient with minimal processing.

[0393] In some embodiments, transgenic plants and organisms of the present invention are adapted to produce a unique ratio of mogroside compounds. In other embodiments, the production of one or more mogroside compounds may be increased and the production of other mogroside compounds may be decreased, for example, by increasing the production of mogroside V relative to other mogrosides.

[0394] In yet another embodiment, a complete mogroside biosynthetic pathway can be constructed in a selected plant or organism by endogenously activating nucleic acid sequences of the mogroside biosynthetic pathway that are naturally present in the plant or organism, and by introducing those that are not present using an expression vector.

[0395] In further embodiments, transgenic plants or organisms can be engineered to express one or more nucleic acids involved in the biosynthesis of other sweeteners, such as genes involved in the production of siamenoside I, α-siamenoside, steviol glycosides (stevia), rebaudioside M, glycyrrhizin, etc. Mogrosides can also be produced in conjunction with other sweeteners in the same plant or organism, for example, mogrosides and rebaudioside M can be produced simultaneously.

[0396] II. Recombinant Host Cells and Species Although transgenic plants are primarily described throughout this specification, other host cells and organisms are also contemplated for use in certain embodiments of the invention.

[0397] As used herein, the term "recombinant host cell" refers to any host cell whose genome has been engineered to contain at least one of the disclosed nucleic acid sequences for the mogroside biosynthetic pathway, which in certain embodiments encode one or more polypeptides. Such sequences include, but are not limited to, nucleic acid or amino acid sequences that do not naturally occur in the host cell or host organism, DNA sequences that are not normally transcribed into RNA or translated into protein ("expressed"), and modifications of sequences naturally occurring in the host cell, e.g., increased copy number or altered expression pattern or amount of a DNA sequence.

[0398] In addition to the plant species disclosed herein, many prokaryotes and other eukaryotes can be used as recombinant hosts in various aspects of the present disclosure. In addition to any plant species, recombinant host cells can also be bacteria, yeast, or fungi. Host cells or species selected for production of mogroside compounds can be analyzed to determine whether they contain endogenous mogroside biosynthetic pathway genes and which genes they are missing. Genes that do not have endogenous homologs in the host cell or organism are typically provided in one or more recombinant constructs that are introduced into the host cell or organism to complement the missing functions.

[0399] Specific examples of prokaryotes and eukaryotes useful in some embodiments of the present disclosure include Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia, Fusarium / Gibberella, Kluyveromyces, Laetiporus, Lentinus, Pha Examples of suitable recombinant hosts include, but are not limited to, mosses such as Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodotorula, Saccharomyces, Sphaceloma, Schizosaccharomyces, Xanthophyllomyces, and Yarrowia. In certain embodiments, the recombinant host may be a microorganism, such as, for example, Pichia pastoris, Schizosaccharomyces pombe, Aspergillus niger, or Saccharomyces cerevisiae. In another embodiment, the microorganism may be a microorganism such as Escherichia coli or Agrobacterium tumefaciens. Certain microorganisms are available for high-throughput screening and evaluation of genes of interest, while other microorganisms may be used because they possess the productivity and growth characteristics necessary for large-scale production of mogroside compounds. In some embodiments, food-grade microorganisms may be useful for large-scale production purposes.

[0400] III. Nucleic Acid and Polypeptide Sequences Certain embodiments of the present disclosure relate to nucleic acid sequences (polynucleotides) corresponding to genes involved in the mogroside biosynthetic pathway and their corresponding amino acid sequences (proteins or polypeptides). Complements of any nucleic acid or protein sequence described herein are also provided.

[0401] "Identity," as understood by those skilled in the art, indicates the relationship between two or more polypeptide sequences or two or more polynucleotide sequences and is determined by comparing the sequences. Identity represents the degree of match between sequences and is determined by methods designed to maximize the match. Methods for determining identity are codified in publicly available programs. "Identity" can be easily calculated using a number of methods known to those skilled in the art. For example, identity can be determined using GCG software or BLAST programs (BLASTN, BLASTX, and TBLASTX for nucleic acid sequences, and BLASTP and TBLASTN for protein sequences). BLASTX is available from NCBI (National Center for Biotechnology Information) and elsewhere (BLAST Manual, NCBI, National Institutes of Health, Bethesda, MD 20894). The well-known Smith-Waterman algorithm can also be used to calculate identity.

[0402] In accordance with the present disclosure, the polynucleotide or polypeptide sequences described herein can have at least about 34%, 40%, 50%, 60%, 62%, or 70% to about 100% sequence identity to any of the sequences described herein. For example, in certain embodiments, the mogroside biosynthetic pathway genes described herein have any sequence identity between 34% and 100% (e.g., 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111, 112, 113, 114, 115, 116, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 7%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). In another embodiment, the mogroside biosynthetic pathway proteins described herein are also similarly sequenced, e.g., 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 14 It can have 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0403] The parameters for polypeptide sequence comparison are as follows: Algorithm: Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970); comparison matrix: BLOSUM62 (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992); gap penalty: 12; gap length penalty: 4. A program that can be used with these parameters is the "gap" program provided by the Genetics Computer Group (Madison, WI). Peptide comparisons can be performed using these parameters, with no penalty for end gaps as the default.

[0404] The parameters for nucleic acid sequence comparison are as follows: Algorithm: Needleman and Wunsch (supra); Comparison matrix: Match = +10, Mismatch = 0; Gap penalty: 50; Gap length penalty: 3. A program that can be used with these parameters is the "gap" program available from Genetics Computer Group (Madison, WI). These parameters may be used as defaults for nucleic acid comparisons.

[0405] As used herein, the terms "hybridization," "hybridizes," and "hybridizable" refer to the formation of double- or triple-stranded molecules, or molecules having a partial double- or triple-stranded structure. Such hybridization may be performed under relatively stringent conditions, such as low salt concentration and / or high temperature. Examples include a 10-minute wash in about 0.02 M to about 0.15 M NaCl at about 50°C to 70°C. In one embodiment of the present disclosure, conditions of 0.15 M NaCl and 70°C are used. Such stringent conditions are preferred for applications requiring high selectivity. Non-limiting examples of applications include isolating nucleic acids from genes or portions thereof, and detecting specific mRNA transcripts or portions thereof. This may also include the proteins and polypeptides described herein, or fragments thereof.

[0406] With respect to the nucleic acid sequences disclosed herein, the term "fragment" refers to any portion of a polynucleotide molecule that retains usable functional properties. Useful fragments include oligonucleotides and polynucleotides that can be used in hybridization and amplification techniques, or to control replication, transcription, or translation. A polynucleotide fragment is any subsequence of any polynucleotide sequence, typically consisting of 15 or more contiguous bases, e.g., 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more bases.

[0407] Fragments also include subsequences of proteins or polypeptides, as described herein. Fragments may be antigenic and may be subsequences of polypeptides that perform at least one biological function in the same or similar manner as the complete polypeptide. Fragments may range in length from as little as 5 amino acids to the full-length polypeptide, but are preferably at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length.

[0408] The nucleic acids (SEQ ID NOs: 1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 42, 46, 50, 54, 58, 85, or 87-92) and amino acids (SEQ ID NOs: 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35-37, 39-41, 43-45, 47-49, 51-53, 55-57, 59, or 86) provided herein can be from any source. For example, those identified as naturally occurring in plants or those synthesized by mutagenesis of SEQ ID NOs: 1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 42, 46, 50, 54, 58, 85, or 87-92 (e.g., to engineer the coding sequence to approximate the GC content of the native gene in a particular plant). Naturally occurring sequences can be from any plant or algae species, as described herein.

[0409] IV. Transformation Constructs Vectors used for transformation of plants and other host cells or organisms include, for example, plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and other suitable cloning systems, or DNA fragments derived therefrom. Therefore, the terms "vector" or "expression vector" encompass all of the above vector types and nucleic acid sequences isolated therefrom. In this disclosure, it is contemplated that cloning systems capable of carrying large inserts will enable the introduction of large DNA sequences containing multiple selected genes. This allows, for example, the introduction of genes corresponding to complete biosynthetic pathways into plants. The introduction of such sequences is facilitated by the use of BACs, YACs, or plant artificial chromosomes. For example, the use of BACs in Agrobacterium-mediated transformation has been reported by Hamilton et al. (Proc. Natl. Acad. Sci. USA 93:9975-9979, 1996).

[0410] Particularly useful are expression cassettes isolated from the above vectors. The DNA segment used to transform plant cells naturally includes the cDNA or gene to be introduced and expressed in the host cell. These DNA segments can also include structures such as promoters, enhancers, multiple cloning sites (polylinkers), terminators, and, if necessary, regulatory genes. The DNA or gene to be introduced often encodes a protein that confers a screenable or selectable trait, conferring an improved phenotype to the resulting recombinant cell or transgenic plant. However, this is not always the case; transgenic plants containing unexpressed foreign genes (transgenes) are also included in the present disclosure. As mentioned above, in addition to plant cells, host cells include Escherichia coli, Agrobacterium tumefaciens, yeast, fungi, algae, cyanobacteria, and the like. It is common knowledge for those skilled in the art that they are familiar with the genetic elements required on a vector for successful transformation, selection, and propagation of host cells containing the sequence of interest. The vectors used in the present disclosure may include the following components:

[0411] A. Promoters and Other Regulatory Sequences In some embodiments of the present disclosure, the expression cassette can further include one or more promoters, such as the nucleic acid sequences set forth in SEQ ID NOs: 60-71, or nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0412] In addition to the promoter sequences disclosed in the sequence listing, other examples of promoters that may be used to express nucleic acid sequences include promoters derived from plants, such as the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), CaMV 19S (Lawto et al., Plant Mol. Biol. 9:315-324, 1987), nos (Ebe et al., Proc. Natl. Acad. Sci. USA 84:5745-5749, 1987), Adh (Walke et al., Proc. Natl. Acad. Sci. USA 84:6624-6628, 1987), sucrose synthase (Yang & Russell, Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), α-tubulin, actin (Wang (Hudspeth et al., Mol. Cell Biol. 12:3399-3406, 1992), cab (Sulliva et al., Mol. Gen. Genet. 215:431-440, 1989), PEPCase (Hudspeth & Grula, Plant Mol. Biol. 12:579-589, 1989), and those associated with the R gene complex (Chandler et al., Plant Cell 1:1175-1183, 1989). Also useful are tissue-specific promoters, such as root cell promoters (Conkling et al., Plant Physiol. 93:1203-1211, 1990), and inducible promoters, such as ABA (abscisic acid)-inducible or turgor-responsive promoters. In particular, the PAL2 promoter may be useful in the present disclosure (U.S. Patent Publication No. 2004 / 0049802, the entire contents of which are incorporated herein by reference). In one embodiment of the present disclosure, the native promoter of the mogroside pathway gene may be used. Additionally, in some embodiments, a strong or weak promoter may be used.

[0413] DNA sequences located between the transcription start site and the start of the coding sequence, i.e., non-translated leader sequences, can affect gene expression. Therefore, it may be desirable to use a specific leader sequence in the transformation constructs of the present disclosure. Leader sequences may include sequences predicted to direct optimal expression of the associated gene, i.e., consensus leader sequences that enhance mRNA stability or prevent inappropriate initiation of translation. The selection of such sequences will be apparent to those skilled in the art with reference to the present disclosure. Sequences derived from genes highly expressed in plants may be desirable.

[0414] It is contemplated that vectors used in accordance with the present disclosure will be constructed to include the ocs enhancer sequence. This element was first identified as a 16-base pair palindromic enhancer from the Agrobacterium octopine synthase (ocs) gene (Ellis et al., EMBO J. 6:3203-3208, 1987). This element is also known to be present in at least 10 other promoters (Bouchez et al., EMBO J. 8:4197-4204, 1989). Such enhancer sequences (e.g., ocs elements), particularly the use of multiple copies, are believed to increase transcription levels of adjacent promoters in the context of plant transformation.

[0415] It is contemplated that coding sequences for the mogroside biosynthetic pathway may be introduced under the control of novel promoters, enhancers, homologous promoters, or tissue-specific promoter-regulatory elements. Vectors used for tissue-specific targeting of genes in transgenic plants typically contain tissue-specific promoters and, in some cases, other tissue-specific regulatory elements, such as enhancer sequences. Promoters that direct selective or enhanced expression in specific plant tissues are known to those of skill in the art with reference to this disclosure. Examples include the green tissue-specific rbcS promoter and the ocs, nos, and mas promoters, which are highly active in root or wounded leaf tissue.

[0416] B. Terminator In some embodiments of the present disclosure, the expression cassette may further include one or more terminators, for example, the nucleic acid sequences set forth in SEQ ID NOs: 72-80, or nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0417] Transformation constructs prepared according to the present disclosure typically include a 3'-terminal DNA sequence that directs the termination of transcription and allows polyadenylation of mRNA produced by a coding sequence operably linked to a promoter. In one embodiment of the present disclosure, the native terminator of a mogroside biosynthetic pathway coding sequence is used. Alternatively, a heterologous 3'-terminal sequence may be used to enhance expression of a sense or antisense mogroside biosynthetic pathway coding sequence. In addition to the terminator sequences listed in the sequence listing, other examples that may be useful in this context include the terminator of the nopaline synthase gene from Agrobacterium tumefaciens (nos 3' end) (Bevan et al., Nucl. Acids Res. 11:369-385, 1983), the terminator for the T7 transcript of the octopine synthase gene from Agrobacterium tumefaciens, or the 3' end of the potato or tomato protease inhibitor I or II genes. If desired, additional regulatory sequences can be included, such as the Adh intron (Callis et al., Genes Dev. 1:1183-1200, 1987), the sucrose synthase intron (Vasil et al., Plant Physiol. 91:1575-1579, 1989), or the TMV omega element (Gallie and Kado, Proc. Natl. Acad. Sci. USA 86:129-132, 1989).

[0418] C. Transit or signal peptides In certain embodiments of the present disclosure, transit or signal sequences may be incorporated into coding sequences in mogroside biosynthetic pathways. These sequences are linked to the coding sequence of expressed genes and are removed from the initial translation product after translation, helping to transport proteins across intracellular or extracellular membranes. Specifically, transit sequences function in intracellular organelles such as vacuoles, endoplasmic reticulum, and chloroplasts, while signal sequences function in transport to the endoplasmic reticulum, Golgi apparatus, and extracellular environment. These sequences can increase the accumulation of gene products by transporting proteins to specific compartments inside or outside the cell and protecting them from protease degradation. These sequences can also be used to add mRNA sequences from highly expressed genes to the coding sequence of a gene. Because mRNA being translated by ribosomes is more stable than naked mRNA, placing translatable mRNA in front of a gene can improve the stability of the overall mRNA transcript and potentially result in increased synthesis of the gene product. Transit and signal sequences are typically removed from the initial translation product after translation, allowing for the addition of additional translation sequences that do not appear in the final polypeptide. Additionally, it may be desirable to target specific proteins to increase protein stability (US Pat. No. 5,545,818, incorporated herein by reference in its entirety).

[0419] Additionally, vectors can be constructed and used for intracellular targeting of specific gene products within transgenic plant cells or for directing proteins to the extracellular environment. This is typically achieved by ligating a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a specific gene. The resulting transit or signal peptide transports the protein to the desired intracellular or extracellular destination, respectively, where it is subsequently removed post-translationally.

[0420] D. Marker Genes The use of selectable or screenable marker proteins can confer or enhance the ability to identify transformants. A "marker gene" refers to a gene that confers a specific phenotype on cells expressing the marker protein, thereby allowing those cells to be distinguished from cells that do not possess the marker. Such genes encode selectable or screenable markers, which may confer a trait that can be "selected" by chemical means (e.g., the use of a selection agent such as a herbicide or antibiotic) or that can be identified simply by observation or inspection (e.g., green fluorescent protein). In addition to the marker genes disclosed in the sequence listings provided herein, many other suitable marker proteins are known in the art and can be used to practice the present disclosure.

[0421] The terms "selectable" or "screenable" marker also include genes encoding "secreted markers," which can be secreted and thus detected, allowing for the identification and selection of transformed cells. Examples include secreted antigens that can be identified by antibody interaction or secreted enzymes that can be detected by catalytic activity. Secreted proteins are divided into several classes, including small, diffusible proteins that can be detected by ELISA or other methods; small, active enzymes that can be detected in the extracellular solution (e.g., α-amylase, β-lactamase, phosphinothricin acetyltransferase); and proteins that are inserted into or trapped in the cell wall (e.g., proteins containing leader sequences found in the expression units of extensins and tobacco PRs).

[0422] Numerous selectable marker coding regions are known that can be used in the present disclosure, including, but not limited to, neo, which confers kanamycin resistance and is selectable by kanamycin, G418, paromomycin, etc. (Potrykus et al., Mol. Gen. Genet. 199:169-177, 1985); bar, which confers resistance to bialarphos or phosphine sothricin; mutant EPSP synthase proteins that confer glyphosate resistance; nitrilases such as bxn from Klebsiella ozaenae, which confer bromoxynil resistance (Stalker et al., J. Biol. Chem. 263:6310-6314, 1988); mutant acetolactate synthase (ALS) that confers resistance to imidazolinones, sulfonylureas, or other ALS inhibitors (European Patent Application 154,204, 1985); DHFR (dihydrofolate reductase) that confers methotrexate resistance (Thillet et al., J. Biol. Chem. 263:12500-12508, 1988); dalapon dehalogenase that confers resistance to the herbicide dalapon; a mutant anthranilate synthase that confers 5-methyltryptophan resistance; or a sequence that confers dicamba resistance.

[0423] An example of a selectable marker that can be used in a transformant selection system is a gene encoding the enzyme phosphinothricin acetyltransferase. Examples include the bar gene from Streptomyces hygroscopicus and the pat gene from Streptomyces viridochromogenes. The phosphinothricin acetyltransferase (PAT) enzyme inactivates phosphinothricin (PPT), the active ingredient in the herbicide Bialaphos. PPT inhibits glutamine synthetase, causing rapid ammonia accumulation and cell death.

[0424] Screenable markers that can be used include the β-glucuronidase (GUS) or uidA genes, which encode enzymes with known chromogenic substrates; R-locus genes, which encode products that control the production of anthocyanin pigments (red) in plant tissues; β-lactamase genes, which encode enzymes with known chromogenic substrates (e.g., PADAC (chromogenic cephalosporins)) (Sutcliffe, Proc. Natl. Acad. Sci. USA 75:3737-3741, 1978); xylE genes, which encode catechol dioxygenases capable of converting chromogenic catechols (Zukowsky et al., Proc. Natl. Acad. Sci. USA 80:1101-1105, 1983); α-amylase genes (Ikuta et al., Biotechnology 8:241-242, 1990); and tyrosinase genes (Katz et al., J. Gen. Microbiol. 129:2703-2714, 1983), which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which condense to form readily detectable melanin; a β-galactosidase gene, which encodes an enzyme detectable with a chromogenic substrate; a luciferase (lux) gene (Ow et al., Science 234:856-859, 1986), which allows for bioluminescent detection; an aequorin gene (Prasher et al., Biochem. Biophys. Res. Commun. 126:1259-1268, 1985), which can be used for calcium-sensitive bioluminescent detection; or a gene encoding green fluorescent protein (GFP) (Sheen et al., Plant J. 8:777-784, 1995; Haseloff et al., Proc. Natl. Acad. Sci. USA 94:2122-2127, 1997; Reichel et al., Proc. Natl. Acad. Sci. USA 93:5888-5893, 1996; WO 97 / 41228) are also envisioned as useful reporter genes.Expression of green fluorescent protein can be visualized as fluorescence in cells or plants after illumination with light of a particular wavelength.

[0425] E. Additional Agronomic Traits In some embodiments, the transformed plants or transformed organisms of the present disclosure incorporate one or more agriculturally beneficial traits. A "trait" refers to a physiological, morphological, biochemical, or physical characteristic of a plant or organism, or of a particular plant material or cell. This characteristic may be visible to the naked eye, such as seed or plant size, or may be measured by biochemical techniques, such as measuring the protein, starch, or oil content of seeds or leaves. It may also be based on observations of metabolic or physiological processes (e.g., measuring carbon dioxide uptake), gene expression levels (e.g., Northern blots, RT-PCR, microarray gene expression analysis, reporter gene expression systems), or agricultural observations such as stress tolerance, yield, or pathogen resistance. The method for measuring the amount, comparative levels, or differences of a selected compound or macromolecule is not particularly limited.

[0426] "Trait modification" refers to a detectable difference in a characteristic observed in a plant or organism that expresses or ectopically expresses a polynucleotide or polypeptide compared to a plant or organism that does not (e.g., a wild-type or control plant). Trait modification may be assessed quantitatively, e.g., by observing a difference of at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least 100% or more compared to a wild-type or control plant or organism. Because natural variation may exist in a modified trait, a trait modification refers to a change in the distribution of that trait in a plant or organism compared to the wild-type.

[0427] Trait modifications of particular interest in the plants disclosed herein include modifications to seeds (such as embryos or endosperms), fruits, roots, flowers, leaves, stems, shoots, seedlings, etc., and specifically include: enhanced resistance to environmental conditions including freezing, cold, high temperatures, drought, water saturation, radiation, and ozone; improved resistance to microbial, fungal, or viral diseases; improved resistance to pest infestations, including insects, nematodes, mollicutes, parasitic higher plants, etc.; reduced herbicide sensitivity or increased herbicide tolerance (e.g., increased tolerance to glyphosate or dicamba); improved tolerance to heavy metals or enhanced ability to uptake heavy metals; improved growth under poor light conditions, such as low light intensity and / or short day length, or altered expression levels of a gene of interest. Other phenotypes that can be modified include those related to the production of plant metabolites, such as taxol, tocopherols, tocotrienols, sterols, phytosterols, vitamins, wax monomers, antioxidants, amino acids, lignin, cellulose, tannins, prenyl lipids (e.g., chlorophyll and carotenoids), glucosinolates, and terpenoids, or increased production or altered composition of proteins or oils (e.g., in seeds), as well as altered soluble or insoluble sugar and / or starch composition. Physical plant characteristics that can be modified include cell development (e.g., number of trichomes), fruit and seed size and number, yield of plant organs such as stems, leaves, inflorescences, and roots, seed storage stability, pod characteristics (e.g., dehiscence), root hair length and quantity, internode distance, and seed coat quality. Plant growth characteristics that can be modified include growth rate, seed germination rate, plant and seedling vigor, leaf and flower senescence, male sterility, apomixis, flowering time, flower abscission, nitrogen uptake rate, osmotic sensitivity to soluble sugar concentration, biomass or transpiration characteristics, and plant architectural characteristics such as apical dominance, branching pattern, organ number, organ identity, organ shape and size, etc. Additionally, the amount of natural sugars can be reduced, and color can be reduced or eliminated.

[0428] V. Gene Editing One method for producing transformed plants according to the present disclosure is genome modification by site-specific integration or genome editing. Targeted modification of plant genomes using genome editing techniques can be used to generate improved plant lines by modifying plant genomic DNA. As used herein, "site-directed integration" refers to a genome editing technique that targets and inserts a nucleic acid sequence of interest into a specific location within a plant genome. Suitable methods for modifying wild-type DNA sequences or existing transgene sequences or inserting DNA into a predetermined chromosomal site include any known method. Exemplary methods include those using zinc finger nucleases, engineered or natural meganucleases, TALE nucleases, or RNA-guided nucleases (e.g., CRISPR / Cas9, CRISPR / Cpf1, CRISPR / CasX, CRISPR / CasY, or CRISPR / Cascade systems). Some embodiments relate to genome editing methods that use single-stranded oligonucleotides to introduce precise base pair modifications in a plant genome. Genome editing techniques for modifying, deleting, or inserting nucleic acid sequences within genomic DNA are known techniques.

[0429] In certain embodiments, the present disclosure provides for modifying or replacing an existing coding sequence in a plant genome, such as an existing transgene insert, with a sequence encoding another protein or an expression cassette containing such a protein. Some embodiments relate to the use of known genome editing methods, including zinc finger nucleases, designed or natural meganucleases, TALE endonucleases, or RNA-guided endonucleases (e.g., Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / CasX systems, CRISPR / CasY systems, or CRISPR / Cascade systems).

[0430] Some embodiments may relate to recombinant DNA constructs containing expression cassettes encoding site-specific nucleases and, optionally, associated proteins for genome modification. These nuclease expression cassettes may be present in the same molecule or vector as the donor template for template editing. Various site-specific integration methods are known in the art, which use different sequence-specific nucleases (or protein and / or guide RNA complexes) to generate double-strand breaks (DSBs) or nicks in genomic DNA at targeted genomic sites or loci. As understood in the art, during the repair process of DSBs or nicks introduced by nuclease enzymes, donor template DNA, transgenes, or expression cassettes may be integrated into the genome at the DSB or nick site. The presence of homologous arms in the DNA to be integrated may facilitate the introduction and targeting of insertion sequences via homologous recombination during the repair process, although the insertion event may also occur via non-homologous end joining (NHEJ). As used herein, the term "double-strand break-inducing agent" refers to any agent capable of inducing a double-strand break (DSB) in a DNA molecule. In one embodiment, the double-strand break-inducing agent is a site-specific genome-modifying enzyme.

[0431] As used herein, the term "site-specific genome modification enzyme" refers to any enzyme capable of modifying a base sequence in a sequence-specific manner. In some embodiments, the site-specific genome modification enzyme modifies the genome by inducing a single-strand break. In other embodiments, the enzyme modifies the genome by inducing a double-strand break. In still other embodiments, the enzyme may comprise a cytidine deaminase. In some embodiments, the site-specific genome modification enzyme comprises an adenine deaminase. Site-specific genome modification enzymes include endonucleases, recombinases, transposases, deaminases, helicases, and any combination thereof. In some embodiments, the enzyme is a sequence-specific nuclease.

[0432] In one embodiment, the endonuclease is a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), an Argonaut (non-limiting examples of Argonaut proteins include Thermus thermophilus argonaut (TtAgo), Pyrococcus furiosus argonaut (PfAgo), and Natronobacterium gregorii argonaut (NgAgo)), an RNA-guided nuclease, such as a CRISPR-associated nuclease (non-limiting examples of CRISPR-associated nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, and Cas5). s5, Cas6, Cas7, Cas8, Cas9 (also called Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, and homologs or modified forms thereof, including, but not limited to:

[0433] In one embodiment, the site-specific genome modification enzyme is a recombinase. Non-limiting examples of recombinases include tyrosine recombinases linked to a DNA recognition motif, selected from Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In one embodiment, the Cre recombinase or Gin recombinase is linked to a zinc finger DNA binding domain, a TALE DNA binding domain, or a Cas9 nuclease. In another embodiment, the serine recombinase linked to the DNA recognition motif is selected from PhiC31 integrase, R4 integrase, and TP-901 integrase. In yet another embodiment, the DNA transposase linked to the DNA binding domain described herein is selected from TALE-piggyBac or TALE-Mutator.

[0434] Any of the target DNAs provided herein can be integrated into a target site in a chromosomal sequence by introducing the DNA and a site-specific genome modification enzyme disclosed herein. Any of the methods described herein can use any of the site-specific genome modification enzymes disclosed herein.

[0435] VI. Antisense and RNAi Constructs Antisense methods and RNAi treatments provide one means of altering the activity of mogroside biosynthetic pathway genes in accordance with the present disclosure (e.g., by silencing genes or transcription factors that inhibit expression of mogroside biosynthetic pathway genes).

[0436] RNAi technology is widely known in the art, and is described, for example, in Lehner et al., (Brief Funct. Genomic Proteomic 3:68-83, 2004) and Downward (BMJ 328:1245-1248, 2004).This technology is based on the fact that double-stranded RNA can direct the degradation of messenger RNA that has a sequence complementary to one or the other strand (Fire et al., Nature 391:806-811, 1998).Therefore, the expression of a specific coding sequence can be suppressed by expressing the coding sequence in both sense and antisense directions as fragments or long sequences.

[0437] Antisense and, in some cases, RNAi techniques utilize the tendency of nucleic acids to pair with "complementary" sequences. "Complementary" here means that polynucleotides have the ability to form base pairs according to the Watson-Crick rules of complementarity. That is, large purine bases pair with small pyrimidine bases, forming combinations such as guanine and cytosine (G:C) and adenine and thymine (A:T) in DNA and adenine and uracil (A:U) in RNA. The inclusion of less common bases in a complementary sequence, such as inosine, 5-methylcytosine, 6-methyladenine, and hypoxanthine, does not prevent pairing.

[0438] Polynucleotides targeting double-stranded DNA form triple helix structures, while those targeting RNA form double helix structures. When introduced into target cells, antisense oligonucleotides specifically bind to target polynucleotides and disrupt transcription, RNA processing, transport, translation, and / or stability. Antisense or RNAi constructs, or DNA encoding such RNAs, can be used to inhibit transcription, translation, or both in host cells (e.g., plant cells), and can be used in vitro or in vivo. In some embodiments of the present disclosure, the oligonucleotides may comprise a unique portion of any of the nucleic acid sequences provided herein. In some embodiments, the sequence may comprise at least 18, 20, 25, 30, 50, 75, or 100 or more contiguous bases of the nucleic acid sequence of interest, and may be in either the sense or antisense orientation. The inclusion of both sense and antisense sequences can enhance the effectiveness of silencing the expression of the corresponding coding sequence.

[0439] Constructs can be designed to be complementary to promoters or other regulatory regions, exons, introns, or exon-intron boundaries. The most effective constructs are believed to contain regions complementary to intron-exon splice junctions. Thus, some embodiments include constructs complementary to regions within 50-200 bases of a splice junction. It has been observed that some exon sequences can be included in constructs without significantly affecting target selectivity. The amount of exon sequence included will vary depending on the specific exon and intron sequences used. Whether a construct contains excess exon DNA can be readily determined by in vitro testing to assess its effect on normal cell function or the expression of related genes bearing complementary sequences.

[0440] As mentioned above, "complementary" or "antisense" refers to a polynucleotide sequence that is substantially complementary throughout its entire sequence and has very few mismatched bases. For example, a 15-base-long sequence can be considered complementary if 13 or 14 bases are complementary. Of course, a fully complementary sequence is one that is completely complementary throughout its entire length and has no mismatched bases. Less homologous sequences are also contemplated. For example, RNAi or antisense constructs can be designed that contain limited regions of high homology while also containing non-homologous regions (e.g., ribozymes; see above). Methods for selecting and designing sequences that induce RNAi are widely known in the art (e.g., Reynolds et al., Nat. Biotechnol. 22:326-330, 2004). These molecules can bind to target sequences under appropriate conditions even if they are less than 50% homologous.

[0441] It may be advantageous to combine a portion of genomic DNA with cDNA or synthetic sequence to generate a specific construct.For example, if it is desired to include an intron in the final construct, a genomic clone can be used.It is desirable to use cDNA or synthetic polynucleotide for the remaining part of the sequence, because they can provide more convenient restriction enzyme sites for the remaining part of the construct.Constructs useful for generating RNAi may also include a concatemer (linked sequence) of subsequences with gene regulation activity.

[0442] VII. Transformation In some embodiments, transgenic plants according to the present disclosure are produced by transforming a selected native plant with one or more expression cassettes disclosed herein. The native plant, prior to transformation, does not naturally produce all of the enzymes in the mogrol / mogroside pathway and does not produce non-naturally occurring mogrol and mogroside compounds. The native plant may produce one or more enzymes capable of producing mogrol precursors or mogrol, but does not naturally produce non-naturally occurring mogrosides. In some embodiments, the native plant to be transformed includes a wild-type, untransformed, or untransformed watermelon, which does not naturally produce detectable amounts of mogrol or mogroside compounds. In other embodiments, plants that can be transformed with one or more expression cassettes disclosed herein include cantaloupe, honeydew, winter melon, Casaba melon, Persian melon, citron melon, muskmelon, Bai Lang melon, Craneshaw melon, Christmas melon, Sprite melon, Carabelle melon, Hami melon, Rocky melon, Golden Langkawi melon, Korean melon, Saticoy melon, Galia melon, Jade Dew melon, Golden Prize melon, Tennyo melon, Shinsei melon, and others. Ki melon, banana melon, Yubari king melon, sugar melon, tiger melon, vert grand pan melon, horned melon, cucamelon, Kasa banana melon, pepino melon, ananas melon, camouflage melon, canary melon, bitter melon, Charente melon, crane melon, sky rocket melon, honey globe melon, gak melon, autumn sweet melon, snap melon, cucumber, tomato, lettuce, spinach, rice, oats, corn, sorghum, bitter melon (Citrullus Mogroside compounds can be isolated from any part of the transformed plants, including, but not limited to, fruit (juice or peel), leaves, roots, seeds, and flowers.

[0443] Suitable methods for transformation of plant or other cells that can be used in the present disclosure are believed to include virtually any method by which DNA can be introduced into cells, such as PEG (polyethylene glycol)-mediated DNA transfer into protoplasts (Omirulleh et al., Plant. Mol. Biol. 21:414-428, 1993), desiccation / inhibition-mediated DNA uptake (Potrykus et al., Mol. Gen. Genet. 199:169-177, 1985), electroporation (U.S. Pat. No. 5,384,253, incorporated herein in its entirety), agitation with silicon carbide fibers (U.S. Pat. Nos. 5,302,523 and 5,464,765, both incorporated herein in their entireties), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, both incorporated herein in their entireties), and accelerated introduction of DNA-coated particles (U.S. Pat. Nos. 5,550,318, 5,538,877, and 5,538,880, all incorporated herein in their entireties). By applying these techniques, cells of virtually any plant species can be transiently or stably transformed, and transgenic plants can be produced from these cells.

[0444] A. Agrobacterium-mediated transformation Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells, allowing DNA to be introduced into whole plant tissues, eliminating the need to regenerate entire plants from protoplasts. Methods for introducing DNA into plant cells using Agrobacterium-mediated plant transfer vectors are well known in the art. See, for example, the methods described in Fraley et al. (Proc. Natl. Acad. Sci. USA 80:4803-4807, 1985) and U.S. Pat. No. 5,563,055 (incorporated herein in their entireties).

[0445] Agrobacterium-mediated transformation is most efficient in dicotyledons and is an effective method for transforming dicotyledons, such as Arabidopsis, tobacco, tomato, alfalfa, and potato. In fact, Agrobacterium-mediated transformation has been routinely used for dicotyledons for many years, but it has only recently become applicable to monocotyledons. Advances in Agrobacterium-mediated transformation technology mean that it can now be applied to almost all monocotyledons. For example, Agrobacterium-mediated transformation techniques have been applied to rice (Hiei et al., Plant Mol. Biol. 35:205-218, 1997; U.S. Pat. No. 5,591,616, incorporated herein by reference in its entirety), wheat and barley (McCormac et al., Mol. Biotechnol. 9:155-159, 1998), alfalfa and corn (Ishida et al., Nat. Biotechnol. 14:745-750, 1996). Similarly, Agrobacterium-mediated transformation has been shown to be effective in switchgrass.

[0446] The latest Agrobacterium transformation vectors are replicable in both E. coli and Agrobacterium and are easy to handle. Furthermore, recent technological advances in Agrobacterium vectors for gene transfer have improved the arrangement of genes and restriction enzyme sites in the vectors to facilitate the construction of vectors capable of expressing polypeptide-encoding genes. These vectors contain a multilinker region flanked by a promoter and polyadenylation site to directly express the inserted polypeptide-encoding gene, making them suitable for the purposes of the present invention. Furthermore, Agrobacterium containing both armed and disarmed Ti genes can also be used for transformation. In plant strains where Agrobacterium-mediated transformation is efficient, gene transfer is a preferred method due to its ease and clarity.

[0447] B. Electroporation Transformation by electroporation can be performed using disintegrating tissues such as cell suspension cultures or embryogenic calli, or by direct transformation of immature embryos or other organized tissues. In this technique, the cell walls of selected cells are partially degraded by exposure to pectin-degrading enzymes (pectorilases) or mechanical wounding. Examples of plant species whose intact cells have been transformed by electroporation include corn (U.S. Pat. No. 5,384,253, incorporated herein by reference in its entirety; Rhodes et al., Methods Mol. Biol. 55:121-131, 1995; D'Halluin et al., Plant Cell 4:1495-1505, 1992), wheat (Zhou et al., Plant Cell Rep. 12:612-616, 1993), tomato (Tsukada et al., Plant Cell Physiol. 30:599-603, 1989), soybean (Christou et al., Proc. Natl. Acad. Sci. USA 84:3962-3966, 1987), and tobacco (Riggs and Bates, Proc. Natl. Acad. Sci. USA 84:3962-3966, 1987). 83:5602-5606, 1986).

[0448] Protoplasts can also be used for electroporation transformation of plants (Bates, Mol. Biotechnol. 2:135-145, 1994; Lazzeri, Methods Mol. Biol. 49:95-106, 1995). For example, a method for generating transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described in WO 92 / 17598, which is incorporated herein by reference in its entirety. Other examples of plant species for which protoplast transformation has been described include barley (Lazzeri, supra), sorghum (Battraw et al., Theor. Appl. Genet. 82:161-168, 1991), maize (Rhodes et al., Science 240:204-207, 1988), wheat (He et al., Plant Cell Rep. 14:192-196, 1994), and tomato (Tsukada, supra).

[0449] C. Microprojectile Bombardment Another method for delivering transgenic DNA fragments into plant cells according to the present disclosure is microprojectile bombardment (U.S. Pat. Nos. 5,550,318, 5,538,880, 5,610,042, and International Application WO 94 / 09699, all of which are incorporated herein by reference in their entireties). In this method, nucleic acids are coated onto particles, which are then introduced into cells using a propelling force. Examples of particles include tungsten, platinum, and often gold. While DNA delivery may be possible in some cases without deposition of DNA onto metal particles, it is also contemplated that the particles may contain DNA. Thus, although DNA-coated particles may increase the efficiency of DNA delivery via particle bombardment, this is not essential.

[0450] For bombardment, cells in suspension are concentrated and placed on a filter or solid medium. Alternatively, immature embryos or other target cells can be placed on solid medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

[0451] An exemplary embodiment of a method for introducing DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to bombard particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with suspension-cultured monocotyledonous plant cells. The screen disperses the particles and prevents them from being introduced into recipient cells as large aggregates. Microprojectile bombardment techniques are widely applicable and can be used to transform virtually any plant species.Examples of plant species that have been transformed by microprojectile bombardment include the following monocotyledonous plants: maize (Zea mays; PCT Application WO 95 / 06128), barley (Hordeum vulgare; Ritala et al., Plant Mol. Biol. 24:317-325, 1994; Hensgens et al., Plant Mol. Biol. 22:1101-1127, 1993), wheat (Wheat; U.S. Pat. No. 5,563,055, incorporated herein by reference in its entirety), rice (Rice; Hensgens et al., supra), oats (Oats; Torbet et al., Crop Science 38:226-231, 1998), rye (Rye; Hensgens et al., supra), and sugarcane (Sugarcane; Bower et al., Plant J. 2:409-416, 1999). 1992), and sorghum (sorghum; Casas et al., Proc. Natl. Acad. Sci. USA 90:11212-11216, 1993; Hagio et al., Plant Cell Rep. 10:260-264, 1991); as well as the following dicotyledonous plants: tobacco (Tomes et al., Plant Mol. Biol. 14:261-268, 1990), soybean (soybean; U.S. Pat. No. 5,322,783, incorporated herein by reference in its entirety), sunflower (Helianthus annuus; Knittel et al., Plant Cell Rep. 14:81-86, 1994), peanut (peanut; Singsit et al., Transgenic Res. 6:169-176, 1997), cotton (cotton; McCabe and Martinell, Nat. Biotechnol. 11:596-598, 1993), tomato (VanEck et al., Plant Cell. Rep. 14:299-304, 1995), switchgrass (Richards et al., Plant Cell Rep. 20:48-54, 2001), and legumes in general (U.S. Patent No. 5,563,055, incorporated herein by reference in its entirety).

[0452] D. Other Transformation Methods Protoplast transformation can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol (PEG) treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al.; Omirulleh et al., supra). The application of these systems to different plant lines depends on the ability of the plant line to regenerate from protoplasts. Exemplary methods for regenerating grasses from protoplasts are described in (Toriyama et al., Nat. Biotechnol. 6:1072-1074, 1988; Abdullah et al., Nat. Biotechnol. 4:1087-1090, 1986; Omirulleh et al., supra; and U.S. Pat. No. 5,508,184; all incorporated herein in their entireties). Examples of the use of direct uptake transformation of grass protoplasts include rice (Ghosh-Biswas et al., J. Biotechnol. 32:1-10, 1994), sorghum (Battraw et al., supra), barley (Lazzeri, supra), oats, and maize (Omirulleh et al., supra).

[0453] To transform plant lines that do not regenerate well from protoplasts, other methods for introducing DNA into intact cells or tissues can be used. For example, regeneration of grasses from immature embryos or explants can be performed by the method described in Vasil, supra. Silicon carbide fiber-mediated transformation can also be used, with or without protoplast treatment (Kaeppler et al., Theor. Appl. Genet. 84:560-566, 1992; U.S. Pat. No. 5,563,055, incorporated herein in its entirety). Transformation by this technique is achieved by agitating silicon carbide fibers with cells in a DNA solution. DNA is passively taken up as the cells are perforated. This technique has been used successfully, for example, on the monocotyledonous plants maize (PCT Application WO 95 / 06128, incorporated herein in its entirety) and rice (Nagatani et al., Biotechnol. Tech. 11:471-473, 1997).

[0454] E. Tissue culture Tissue cultures can be used in certain transformation techniques to prepare cells for transformation and to regenerate plants from them. Maintaining tissue cultures requires the use of media and a controlled environment. A "media" is a mixture of numerous nutrients used to grow cells in vitro, i.e., completely outside the living organism. Media are typically suspensions of various components, such as salts, amino acids, growth regulators, sugars, and buffers. These components are necessary for the growth of most cell types, but each cell type requires a different range of component ratios for growth and even more precise ranges for optimal growth. Cell growth rates also vary among cultures initiated in various media that allow the growth of that cell type.

[0455] The medium is prepared as a liquid, but can be solidified by adding the liquid to a material that can provide a solid support. Agar is most commonly used for this purpose. BACTO® AGAR, GELRITE®, and GELGRO® are examples of solid supports suitable for growing plant cells in tissue culture.

[0456] Some cell types grow and divide in either liquid suspension or on solid media. As disclosed herein, plant cells can grow in suspension or on solid media, but regeneration of plants from suspension cultures typically requires a transition from liquid to solid media at some stage of development. The type and degree of differentiation of cells in culture is influenced not only by the type and environment (e.g., pH) of the medium used, but also by whether the medium is solid or liquid.

[0457] Tissues that can be grown in culture include meristematic cells, Type I, Type II, and Type III callus, immature embryos, and gamete cells such as microspores, pollen, sperm cells, and egg cells. Type I, Type II, and Type III callus can be derived from tissue sources including, but not limited to, immature embryos, seedling apical meristems, roots, leaves, microspores, etc. These cells that can be grown as callus are also recipient cells for gene transfer.

[0458] There are various types of somatic cells. Embryogenic cells are an example of somatic cells that can be induced to regenerate plants through embryogenesis. Non-embryogenic cells are cells that do not normally exhibit such a response. Specific techniques can be used to enrich for recipient cells within a cell population. For example, cell enrichment is commonly achieved by manually selecting and culturing friable, embryogenic tissue after the development of type II callus. Manual selection techniques that can be used to select target cells can include, for example, evaluation of cell morphology and differentiation, and various physical or biological means can be used. Cryopreservation is also a method for selecting recipient cells.

[0459] Manual selection of recipient cells, for example, embryogenic cells from the surface of type II callus, is one tool that can be used to enrich for specific cells prior to culture (whether on solid medium or in suspension).

[0460] When used, cultured cells can be grown on a solid support or in liquid suspension. In either case, nutrients and environmental conditions can be provided to the cells in the form of a culture medium. Media used in tissue culture contain various amino acids, salts, sugars, growth regulators, and vitamins. While most media used in the practice of this specification share some common components, the composition and proportions of these components may vary depending on the specific application envisioned. For example, various cell types typically grow in multiple media, but growth rates and morphologies vary depending on the medium. In some media, cells may survive but not divide. Various media suitable for culturing plant cells have been described. Examples of these media include, but are not limited to, N6 medium described by Chu et al. (Sci. Sin. [Peking] 18:659-668, 1975) and MS medium by Murashige and Skoog (Physiol. Plant 15:473-479, 1962).

[0461] VIII. Generation and Characterization of Stably Transformed Plants After the foreign DNA is introduced into the recipient cell, the next step is generally to identify the transformed cells and use them for further cultivation and regeneration into plants.To improve the ability to identify transformants, it may be desirable to use a selectable or screenable marker gene together with the transformation vector prepared according to the present invention.In this case, an assay is usually performed by exposing a population of potentially transformed cells to a selection agent or by screening the cells for the trait of the marker gene of interest.

[0462] A. Selection In any given experiment, only a small proportion of target cells will receive DNA. To provide a system for efficiently identifying cells that have taken up DNA and integrated it into their genome, a method for selecting stably transformed cells can be used. One such method involves introducing a marker gene that confers resistance to normally inhibitory agents, such as antibiotics or herbicides, into the host cells. Examples of antibiotics that can be used include the aminoglycoside antibiotics neomycin, kanamycin, and paromycin, or hygromycin. Resistance to aminoglycoside antibiotics is conferred by aminoglycoside phosphotransferase enzymes, such as neomycin phosphotransferase II (NPT II) or NPT I. Resistance to hygromycin, on the other hand, is conferred by hygromycin phosphotransferase.

[0463] Potentially transformed cells are then exposed to a selection agent. The population of surviving cells will usually include those in which the resistance-conferring gene has been introduced and is expressed at a level that allows the cells to survive. These cells may then be further tested to confirm stable integration of the foreign DNA.

[0464] One example of a herbicide useful as a desirable selective agent is the broad-spectrum herbicide bialaphos. Bialaphos is a tripeptide antibiotic produced by Streptomyces hygroscopicus, consisting of phosphinothricin (PPT; an L-glutamic acid analog) and two L-alanine residues. Removal of the L-alanine residues by intracellular peptidases liberates PPT, which becomes a potent inhibitor of glutamine synthetase (GS). GS is a key enzyme involved in ammonia assimilation and nitrogen metabolism (Ogawa et al., Sci. Rep. Meiji Seika 13:42-48, 1973). Synthetic PPT, the active ingredient in the herbicide Liberty™, is also effective as a selective agent. Inhibition of GS in plants by PPT causes rapid ammonia accumulation and plant cell death.

[0465] Bialaphos-producing organisms and other Streptomyces species also synthesize the enzyme phosphinothricin acetyltransferase (PAT). This enzyme is encoded by the bar gene from Streptomyces hygroscopicus and the pat gene from Streptomyces viridochromogenes. The use of herbicide resistance genes encoding phosphinothricin acetyltransferase (PAT) is mentioned in DE3642829A, which isolated the gene from Streptomyces viridochromogenes. In bacterial donor organisms, this enzyme prevents autotoxicity by acetylating the free amino group of PAT (Thompson et al., EMBO J. 6:2519-2523, 1987). The bar gene has been cloned (Thompson et al., supra) and expressed in transgenic tobacco, tomato, potato (De Block et al., EMBO J. 6:2513-2518, 1987), Brassica (De Block et al., Plant Physiol. 91:694-701, 1989), and maize (U.S. Pat. No. 5,550,318, incorporated herein by reference in its entirety).

[0466] Another example of a herbicide useful for selecting transformed cell lines in the present disclosure is the broad-spectrum herbicide glyphosate. Glyphosate inhibits the action of the enzyme EPSPS, which is active in the aromatic amino acid biosynthetic pathway. Inhibition of this enzyme leads to a lack of phenylalanine, tyrosine, tryptophan, and their derived secondary metabolites, resulting in cell starvation. U.S. Patent No. 4,535,060 (incorporated herein in its entirety) describes the isolation of an EPSPS mutant that confers glyphosate resistance to the EPSPS gene (aroA) of Salmonella typhimurium. The EPSPS gene was cloned from corn (Zea mays), and mutations similar to those found in the glyphosate-resistant aroA gene were introduced in vitro. Mutant genes encoding glyphosate-resistant EPSPS enzymes are described, for example, in International Patent WO 97 / 4103.

[0467] To use the bar-bialaphos or EPSPS-glyphosate selection system, transformant tissue is grown on nonselective medium for 0-28 days and then transferred to medium containing 1-3 mg / L bialaphos or 1-3 mM glyphosate. A range of 1-3 mg / L bialaphos or 1-3 mM glyphosate may be effective, although a range of 0.1-50 mg / L bialaphos or 0.1-50 mM glyphosate may also be useful.

[0468] An example of a screenable marker trait is the enzyme luciferase. In the presence of the substrate luciferin, cells expressing luciferase emit light that can be detected with photographic or X-ray film, a luminometer (or liquid scintillation counter), a night-vision device, or a sensitive video camera such as a photon-counting camera. These assays are nondestructive, allowing transformed cells to continue to be cultured after identification. Photon-counting cameras are particularly useful, allowing specific cells or cell populations expressing luciferase to be identified and manipulated in real time. Another screenable marker that can be used in a similar way is the gene encoding green fluorescent protein (GFP).

[0469] B. Regeneration and seed production Cells that survive exposure to the selection agent or that test positive in the screening assay can be cultured in a medium that supports plant regeneration. For example, MS and N6 media can be modified by adding substances such as growth regulators. Examples of such growth regulators include dicamba or 2,4-D. However, other growth regulators can also be used, such as NAA, a combination of NAA and 2,4-D, or picloram. Such medium amendments have been found to promote the growth of cells at specific developmental stages. Tissues are maintained on basal medium containing growth regulators after repeated manual selection until sufficient tissue mass is available to initiate regeneration or until morphology is appropriate for regeneration (at least two weeks). They are then transferred to a medium suitable for embryoid maturation. Cultures are passaged on this medium every two weeks. Once shoot development is observed, they are transferred to a medium without growth regulators.

[0470] Transformed cells identified by selection or screening and cultured in an appropriate medium that supports regeneration are then allowed to mature into plants. Developing plantlets are transferred to a soil-free plant growth medium and acclimated in an environmentally controlled room, e.g., at approximately 85% relative humidity, 600 ppm CO2, and 25-250 microeinsteins (m² s-1). Plants can be allowed to mature in a growth chamber or greenhouse. Plants can be regenerated within approximately six weeks to ten months from transformant identification, depending on the initial tissue. During regeneration, cells are grown on solid medium in tissue culture vessels. Examples of such vessels include petri dishes and Plant Cones. Regenerating plants can be grown at approximately 19-28°C. After the regenerated plants reach the shoot and root development stage, they can be transferred to a greenhouse for further growth and testing.

[0471] Seeds formed by transformed plants may occasionally require embryo rescue due to cessation of seed development or premature plant senescence. To rescue developing embryos, embryos are excised from surface-sterilized seeds 10-20 days after pollination and cultured. One embodiment of the medium used for culture contains MS salts, 2% sucrose, and 5.5 g / L agarose. For embryo rescue, large embryos greater than 3 mm in length are germinated directly on appropriate medium. Smaller embryos can be rescued by adding 10% or more of the above ingredients to the medium. -5 The seeds are cultured on a medium containing M abscisic acid for one week, and then transferred to a medium containing no growth regulators for germination.

[0472] C. Characterization A variety of assays can be performed to confirm the presence of the foreign DNA, or "transgene," in the regenerated plants, including, for example, molecular biological assays (such as Southern blots, Northern blots, and PCR™), biochemical assays (such as immunological detection of protein products, e.g., ELISA or Western blots, or enzyme activity detection), assays on plant parts (such as leaf or root assays), and even phenotypic analysis of the whole regenerated plants.

[0473] D. DNA transfer, RNA expression and inheritance To confirm the presence of an exogenous gene, genomic DNA can be isolated from a cell line or any plant part. Such techniques are well known to those skilled in the art. However, due to sequence rearrangements or deletions within the cell, the complete sequence may not always be present. The presence of a DNA element introduced by the disclosed method can be confirmed, for example, by polymerase chain reaction (PCR™). Using this technique, a fragment of DNA can be amplified and detected by gel electrophoresis. This type of analysis can determine whether a gene is present in a stable transformant, but it does not prove whether the gene has been integrated into the host genome. However, it is generally assumed that all transformants in which the presence of a gene is confirmed by PCR™ have integrated DNA into the genome. Furthermore, PCR™ techniques generally cannot determine whether the exogenous gene has been introduced at different sites within the genome (i.e., whether each transformant has independent origins). It is believed that PCR™ technology can also be used to clone fragments of host genomic DNA flanking the introduced gene.

[0474] Southern hybridization is used to reliably verify the introduction of foreign DNA into the host genome and the uniqueness of each transformant. This technique allows the identification of the specific DNA sequence introduced into the host genome and the host DNA sequences flanking it. Therefore, the Southern hybridization pattern for a particular transformant serves as a distinguishing feature for that transformant. Southern hybridization can also demonstrate the presence of the foreign gene in the high-molecular-weight DNA, i.e., its integration into the host genome. Southern hybridization not only provides information similar to that obtained by PCR™ (e.g., the presence of the gene), but also demonstrates the integration of the introduced gene into the genome and clarifies the characteristics of each individual transformant.

[0475] The dot blot or slot blot technique, a modification of the Southern hybridization technique, is believed to provide information equivalent to that obtained from PCR™, ie, information that confirms the presence of a gene.

[0476] Both PCR™ and Southern hybridization techniques can be used to demonstrate the transmission of transgenes to progeny. Often, the characteristic Southern hybridization pattern for a particular transformant will segregate as one or more Mendelian genes in the progeny (Spencer et al., 1992), indicating stable inheritance of the transgene.

[0477] While DNA analysis techniques can be performed using DNA isolated from any part of a plant, RNA expression is restricted to specific cell or tissue types, necessitating the preparation of RNA from those tissues. PCR™ technology can also be used to detect and quantify RNA produced from transgenes. In this application, RNA is first reverse-transcribed into DNA using an enzyme such as reverse transcriptase, and the DNA is then amplified using standard PCR™ technology. While PCR™ technology is useful in many cases, it cannot demonstrate the integrity of the RNA product. Further information regarding the nature of the RNA product can be obtained by Northern blotting. This technique provides information about the presence and integrity of RNA species. The presence or absence of RNA species can also be determined by Northern hybridization using dot blot or slot blot techniques. These techniques are modifications of Northern blotting and only confirm the presence or absence of RNA species.

[0478] E. Gene Expression Southern blotting and PCR™ can detect genes of interest but do not indicate whether the corresponding proteins are actually expressed. Expression is confirmed by specifically detecting the protein products of the transgenes or by assessing phenotypic changes resulting from their expression.

[0479] Assays aimed at the production and identification of specific proteins exploit their physicochemical, structural, functional, and other properties. Due to their specific physicochemical or structural characteristics, proteins can be separated and identified by electrophoretic techniques, such as native or denaturing gel electrophoresis and isoelectric focusing, or by chromatographic techniques, such as ion exchange chromatography and gel filtration chromatography. The unique structure of individual proteins allows their detection using specific antibodies, such as ELISA. Combinations of these techniques, such as Western blotting, detect proteins separated by electrophoresis with antibodies. Furthermore, the identity of the target protein can be confirmed by amino acid sequencing. While these are the most common techniques, other methods are also possible.

[0480] Assays can also be used to identify the expression of proteins by their function. In particular, enzymes can be evaluated based on their ability to catalyze chemical reactions involving specific substrates and products. These reactions are followed by quantifying the reduction of substrate or the production of product using physical or chemical methods. Specific examples vary depending on the enzyme of interest, for example, phosphinothricin and 14 These include assays of PAT enzyme activity by following the production of radiolabeled acetylated phosphinothricin from C-acetyl-CoA, and assays of anthranilate synthase activity by following the quenching of anthranilate fluorescence.

[0481] Expression of gene products is often determined by assessing the phenotypic changes that result from that expression. These assays can take a variety of forms, but include analyzing changes in the chemical composition, morphology, or physiological characteristics of plants. Changes in chemical composition include changes in amino acid composition due to the expression of genes encoding enzymes or storage proteins, which can be detected by amino acid analysis. Changes in enzymes involved in changes in starch content can be analyzed using near-infrared reflectance spectroscopy (NIRS). Morphological changes include increased plant height and stem thickness. Most commonly, the response of plants or plant parts to a treatment is assessed under strictly controlled conditions, known as bioassays.

[0482] IX. Mogroside-Containing Sweeteners and Ingested Products In some embodiments, the present disclosure relates to a sweetener or sweetening composition containing mogroside and / or its metabolites or derivatives, the sweetener or sweetening composition being derived from a transgenic plant that produces and contains non-endogenous mogrol / mogroside. As used herein, the term "sweetener" refers to a consumable product that produces a sweet taste when consumed alone. In some embodiments, the sweetener or sweetening composition is derived from a transgenic plant having a mogrol / mogroside pathway produced according to the present disclosure. In some embodiments, the sweetener may be a high-intensity or low-intensity sweetener. Mogroside-containing sweeteners can be obtained from transgenic plants having the mogrol / mogroside pathway of the present disclosure by appropriate processing. The resulting sweetener can be used to provide versatile low-calorie or no-calorie sweetness. Examples of applications that provide sweetness include beverages (e.g., tea, coffee, juices, fruit drinks), foods (e.g., jams, jellies, peanut butter, pies, puddings, cereals, candy, ice cream, yogurt, bakery products), medical products (e.g., toothpaste, mouthwash, lozenges, cough syrup), chewing gum, sugar substitutes, and the like.

[0483] In some embodiments, sweeteners are provided in fruit juices derived from transgenic plants according to the present disclosure. For example, uses for watermelon juice containing one or more mogroside compounds include, but are not limited to, beverages (e.g., premixed cocktails and dairy substitutes), ingredients (e.g., spraying on cereal bars and cereals, or sweetening ketchup and other common products), and the like. In such embodiments, the juice may be deactivated, deproteinized, or concentrated. Furthermore, concentrated fruit or vegetable syrups, such as watermelon syrup produced from the presently disclosed watermelons, can be used as a replacement for high-fructose corn syrup in various foods and beverages. In some embodiments of the present disclosure, mogroside compounds are produced in transgenic tomatoes, which can be used to produce reduced-calorie ketchup or other tomato-based sauces and soups.

[0484] Some embodiments of the present disclosure also provide methods for producing sweeteners from transgenic plants that produce non-endogenous mogrol / mogroside. The methods generally include, but are not limited to, pre-washing and disruption of the transgenic plant or parts thereof, extraction, sedimentation and / or centrifugation, adsorption and / or separation, concentration and recovery to produce a crude sweetener, further purification, optional concentration / drying, and formulation. Extraction methods include aqueous extraction at ambient, warm, or cold conditions, and extraction with organic solvents such as alcohol. Separation and purification methods include centrifugation, percolation, gravity settling, filtration, microfiltration, nanofiltration, ultrafiltration, reverse osmosis, chromatography, adsorption chromatography, ion-exchange resin purification, and the like.

[0485] Additionally, in embodiments of the present disclosure, the transgenic plants can be processed to produce mogroside-containing ingredients, including whole plant extraction, tissue extraction, fruit processing, aqueous separation of small molecules, including the mogroside fraction, and removal of residual protein to obtain an aqueous fraction free of genetically modified ingredients. The resulting mogroside-containing ingredients can be in any form, including powders, liquids, syrups, concentrates, or extracts. Additionally, in some embodiments, the whole mogroside-containing fruit or vegetable is the ingestible product.

[0486] In some embodiments, the sweetener is obtained from the leaves of a transgenic plant of the present disclosure, while in other embodiments, the sweetener is obtained from the fruit, a part of the fruit (e.g., the peel), or a part of another organ or tissue of the transgenic plant.

[0487] Additionally, the mogroside compounds produced by the transgenic plants or organisms of the present disclosure may be blended with one or more other natural or artificial sweeteners, such as steviol glycosides, siamenoside I, α-siamenoside I, sucrose, glucose, fructose, lactose, maltose, sorbitol, galactose, saumatin, sucrooctate, vernadum, sucrononic acid, calerum, lugdonum, high fructose corn syrup, RealSweet™ sugarcane-derived RebM, erythritol, xylitol, yacon syrup, allose, saccharin, aspartame, acesulfame potassium, sucralose, neotame, advantame, cyclohexanoate, or glycyrrhizin. The ratio of mogroside compounds to other sweeteners in the final composition can be, for example, 10 / 90, 20 / 80, 30 / 70, 40 / 60, 50 / 50, 60 / 40, 70 / 30, 80 / 20, 90 / 10, or any other ratio. In one embodiment, the ratio is about 80% mogroside V, about 15% 11-oxo-mogroside V, and about 5% mogroside III A1. In another embodiment, the ratio is about 40% siamenoside I, about 40% mogroside V, and about 20% 11-oxo-mogroside V.

[0488] In some embodiments, the additional sweetener is a carbohydrate sweetener. Non-limiting examples of suitable carbohydrate sweeteners include sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, tagutose, trehalose, galactose, rhamnose, cyclodextrins (e.g., α-, β-, and γ-cyclodextrins), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, and idose. , lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltrose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, glucosamine, mannosamine, fucose, fuculose, glucuronic acid, gluconic acid, gluconolactone, abequase, galactosamine, xylooligosaccharides (xylotri ose, xylobiose, etc.), gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), galactooligosaccharides, sorbose, ketotriose (dehydroxyacetone), aldotriose (glyceraldehyde), nigerooligosaccharides, fructooligosaccharides (kestose, nystose, etc.), maltotetraose, maltotriose, tetrasaccharides, mannanoligosaccharides, maltooligosaccharides (maltotetraose, maltotriose, etc.), Examples of suitable sweeteners include maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), dextrin, lactulose, melibiose, raffinose, rhamnose, ribose, isomerized liquid sugars (e.g., high fructose corn syrup / high fructose starch syrup (HFCS / HFSS) such as HFCS55, HFCS42, and HFCS90), coupling sugars, soybean oligosaccharides, glucose syrup, etc. Where applicable, D- or L-configuration can be used. In another embodiment, the additional sweetener is selected from glucose, fructose, sucrose, and combinations thereof. In yet another embodiment, the additional sweetener is selected from D-allose, D-psicose, L-ribose, D-tagtose, L-glucose, L-fucose, L-arabinose, tulanose, and combinations thereof.

[0489] In other embodiments, the additional sweetener is not derived from natural extracts. Such sweeteners are characterized by a higher sweetness intensity than sucrose, fructose, and glucose, while being low in calories. Non-limiting examples suitable for embodiments of the present disclosure include sucralose, acesulfame potassium, acesulfamic acid and its salts, aspartame, alitame, saccharin and its salts, neohesperidin dihydrochalcone, cyclamate, cyclamic acid and its salts, neotame, advantame, glucosylated steviol glycosides (GSGs), and the like. At least one of these sweeteners not derived from natural extracts may be included in a sweetened composition, such as a food product, other ingestible product, or beverage, in an effective amount to provide a concentration of about 0.3 ppm to about 3,500 ppm. In some embodiments, the concentration ranges from about 0.5 ppm to 3,000 ppm, about 1.0 ppm to 2,500 ppm, about 5.0 ppm to 2,000 ppm, about 10 ppm to 1,500 ppm, about 50 ppm to 1,000 ppm, about 100 ppm to 800 ppm, or about 400 ppm to 600 ppm. In other embodiments, the sweetener is present in the sweetened composition in an amount to provide a concentration of greater than about 0.3 ppm, greater than about 0.5 ppm, greater than about 1.0 ppm, greater than about 5.0 ppm, greater than about 10 ppm, greater than about 20 ppm, greater than about 50 ppm, greater than about 100 ppm, greater than about 250 ppm, greater than about 500 ppm, or greater than about 1,000 ppm.

[0490] In yet another embodiment, natural high-potency sweeteners can be used as additional sweeteners. Suitable natural high-intensity sweeteners include, but are not limited to, rebaudiosides A, B, C, D, E, F, I, H, L, K, J, M, N, O, dolucoside A, B, rubusoside, stevia, stevioside, mogroside IV, V, monak fruit, miraculin, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizin and its salts, saumatin, monellin, mabinlin, brazzein, hernandulsin, phyllodulsin, glycyphyllin, phloridzin, trilobatin, bayounoside, osladin, polypodoside A, pterocaryosides A, B, mukuroziosides, phlomisoside I, periandrin I, abrusoside A, steviolubioside, and cyclocaryoside I. These natural high-potency sweeteners can be provided as pure compounds or as part of an extract. For example, rebaudioside A can be provided as a single compound or as part of a stevia extract. The natural high-potency sweetener can be present in a sweetened composition, such as a food product, other ingestible product, or beverage, in an effective amount to achieve a concentration of about 0.1 ppm to about 3,000 ppm. In some embodiments, the concentration is about 0.5 ppm to 2,500 ppm, about 1.0 ppm to 2,000 ppm, about 5 ppm to 1,500 ppm, about 10 ppm to 1,000 ppm, or about 25 ppm to 500 ppm. In another embodiment, the natural high-potency sweetener is included in an effective amount to provide a concentration in the food, other ingestible product, or beverage of greater than about 0.1 ppm, about 0.5 ppm, about 1.0 ppm, about 2.5 ppm, about 5.0 ppm, about 10 ppm, about 20 ppm, about 25 ppm, about 50 ppm, about 75 ppm, about 100 ppm, about 200 ppm, about 500 ppm, about 1000 ppm, about 2000 ppm, or about 3000 ppm.

[0491] In yet another embodiment, the additional sweetener may be a chemically or enzymatically modified natural high-potency sweetener. Modified natural high-potency sweeteners include, for example, glucosylated, galactosylated, or fructosylated derivatives (containing 1 to 50 glycosidic residues). These glycosylated sweeteners may be prepared by enzymatic transfer glycosylation, catalyzed by various enzymes with transfer glycosylation activity.

[0492] When a sweetening composition contains multiple sweeteners, the combination may produce a synergistic effect, resulting in an improved flavor profile and temporal profile compared to each sweetener alone. As used herein, "temporal profile" refers to the temporal intensity of sweetness as perceived by a human taster. "Flavor profile" or "taste profile" generally refers to the intensity of the flavor / taste attributes of a sweetener or sweetening composition. Typical attributes include sweetness, bitterness, saltiness, licorice-like flavor, and cooling sensation. Methods for evaluating the flavor profile of a particular sweetener or sweetening composition are known to those skilled in the art. "Synergistic" or "synergistic effect" refers to the combination of two or more sweeteners that produces an effect greater than the sum of the effects of each sweetener used alone. This synergistic effect can advantageously result in the same effect being achieved with a reduced amount of sweetener, or a stronger effect with the same amount. The degree and amount of synergistic effect may vary.

[0493] The amount of sucrose in a reference solution can be expressed in degrees Brix (°Brix). 1°Brix means 1 gram of sucrose in 100 grams of solution, and is a measure of the solution concentration in terms of weight percentage (% w / w). In some embodiments, one or more sweetener compounds of the present disclosure are included in a sweetening composition in an amount sufficient to provide a sweetness equivalent to at least about 5 degrees Brix in a food or other ingestible product or beverage, e.g., at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 degrees Brix or more.

[0494] The sweetness of sweeteners other than sucrose can also be measured by comparison with sucrose. In such measurements, panelists are trained to recognize the sweetness of standard solutions of 1–15% sucrose (w / v). They then taste serial dilutions of other non-sucrose sweeteners to determine which concentrations are equivalent in sweetness to a particular sucrose concentration. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, then that sweetener is said to have 10 times the sweetness intensity of sucrose.

[0495] Sweetener compositions can be customized to the desired calorie content. For example, a sweetener composition can be "full-calorie," imparting the desired sweetness when added to a target composition (e.g., a food, other ingestible product, or beverage) and having approximately 120 kilocalories per 8 ounces (approximately 240 mL). Alternatively, a sweetener composition can be "mid-calorie," imparting the desired sweetness at less than approximately 60 kilocalories per 8 ounces. Similarly, "low-calorie" embodiments impart sweetness at less than 40 kilocalories per 8 ounces. Furthermore, "zero-calorie" embodiments provide sweetness at less than 5 kilocalories per 8 ounces.

[0496] The sweetener compositions of the present disclosure may optionally contain additional additives. In some embodiments, the sweetener compositions may include, but are not limited to, carbohydrates, polyols, amino acids and their salts, polyamino acids and their salts, sugar acids and their salts, nucleotides, organic acids, inorganic acids, organic salts (including organic acid salts and organic base salts), inorganic salts, bittering ingredients, flavor and fragrance ingredients, astringent ingredients, proteins or hydrolyzed proteins, surfactants, emulsifiers, weighting agents, gums, antioxidants, colorants, flavonoids, alcohols, polymers, and combinations thereof. In some embodiments, these additives improve the temporal and flavor profile of the sweetener and provide the sweetener composition with a taste similar to sucrose. The sweetened composition may also include one or more functional ingredients. Functional ingredients include, but are not limited to, vitamins, minerals, antioxidants, preservatives, glucosamine, polyphenols, and the like. Any functional ingredient described herein may be used.

[0497] The disclosed mogroside sweeteners have a positive environmental impact compared to existing monk fruit production. For example, the disclosed mogroside sweeteners can be produced locally, eliminating the need for long-distance transportation and reducing food miles. This eliminates the need to ship monk fruit harvested and processed only in China to food companies around the world, for example. Additionally, the readily accessible mogroside sweeteners in the disclosed transgenic plants or organisms minimize processing efforts and simplify processing compared to traditional industrial processing (e.g., processing of monk fruit sweeteners in China).

[0498] X. Sweetened Compositions The sweetener compositions of the present disclosure can be incorporated into any known edible material (herein referred to as a "sweetenable composition"), including, for example, pharmaceutical compositions, edible gel mixes and compositions thereof, dental compositions, food products (confectionery, seasonings, chewing gum, cereal compositions, baked goods, dairy products, and tabletop sweetener compositions), beverages and beverage products, and the like.

[0499] The sweetened compositions disclosed herein include beverages (ready-to-drink liquid formulations), beverage concentrates, and the like. In some embodiments, beverage concentrates are first prepared with a liquid (e.g., water) to which additional ingredients are added. Full-strength beverages can be obtained by adding additional liquid (e.g., water) to the concentrate.

[0500] In an embodiment of a sweetenable composition using a mogroside-containing filler juice concentrate (approximately 80% mogroside V, approximately 15% 11-oxo-mogroside V, and approximately 5% mogroside III A1), studies have shown that this composition is the sweetest and cleanest naturally derived sweetener. The disclosed mogroside-containing filler juice concentrate is the only sweetener that can be sustainably and locally grown from familiar fruits, significantly reducing the high sugar content while still maintaining a "100% juice" label, making it an affordable alternative. This filler juice can be used at full strength (single strength) or concentrated, and provides a clean, sweet taste at a variety of ratios. The mogroside concentration can provide a sweetness equivalent to approximately 10 sucrose equivalent values ​​(SEV).

[0501] Besides concentration, other parameters of the juice can be easily adjusted to obtain different sweetened products, such as partially or completely removing the natural sugars in the fruit (e.g., watermelon), reducing or eliminating the color and flavor of the juice, removing the pulp (common processing) or leaving the pulp partially or completely as in puree, reducing the acidity, or a combination of one or more of these parameters.

[0502] Filler juice uses include, but are not limited to: juices, nectars, fruit / flavored still drinks, energy and sports drinks, carbonated beverages, flavored waters, nutritional drinks, vitamin supplements or oral rehydration solutions (liquid or chew / gummy), snacks (such as snack bars and fruit snacks), sugar and gum confectioneries (jelly and gummy), dairy products such as yogurt and flavored drinks, desserts, ice cream, frozen yogurt, ice creams and sorbets, breakfast cereals and cold cereals, tabletop sweeteners, sweet spreads such as syrups and fruit spreads, sauces and condiments such as table sauces and cooking sauces, and processed fruit and packaged vegetable products.

[0503] In an embodiment of a sweetenable composition using a mogroside-containing dry powder (approximately 40% siamenoside I, approximately 40% mogroside V, and approximately 20% 11-oxo-mogroside V), research has confirmed that this composition has a clean taste and exhibits very high sweetness levels, even in demanding applications. The mogroside-containing dry powder of the present disclosure can be sustainably grown locally from consumer-favorite vegetables, reducing sugar and calories while providing a strong positive health image. Its price is a fraction of that of monk fruit, comparable in cost to sucrose. The dry powder provides a maximum clean, sweet taste in a small amount, allowing for widespread use in a variety of food and beverage applications. This dry powder concentration, even in formulations with varying purity levels, can provide a sweetness equivalent to approximately 10 sucrose equivalent values ​​(SEV).

[0504] Dry powder applications include, but are not limited to: Wellness / Functional Beverages (Energy Drinks, Sports Drinks, Carbonated Drinks, Flavored Waters, Juices, Nectars, Fruit / Flavored Still Drinks, Protein and Meal Replacement Beverages, Drink Mixes, Concentrated Beverages, Ready-to-Drink Teas and Coffees), Supplements and OTC Products (Vitamin Supplements, Oral Rehydration, Cold Relief, Digestive Aids, Sleep Aids, Pain Reliever: Capsules, Tablets, Liquids, Powders, Chews / Gummies, Lozenges, etc.), Snacks (Snack Bars, Fruit Snacks, Nuts, Trail Mix, Corn / Rice / Potato / Wheat Snacks), Bakery Products (Cookies, Cakes, Sweets, Baking Mixes, etc.) Couscous, ingredients, bakery products), dairy products and desserts (spoon yogurt, drinking yogurt, flavored drinks, creamers, ice cream, frozen yogurt, water-based ice cream and sorbet, shelf-stable desserts, dessert toppings), breakfast cereals (hot and cold), artificial and natural sweeteners (tabletop sweeteners), sugar and chocolate confectionery (jelly, gummies, mints, gum, toffee, caramel, marshmallows, various chocolates), sweet spreads (syrups, fruit / nut / chocolate spreads), sauces and condiments (table sauces, cooking sauces, pasta sauces, vinegars, dressings, pickling condiments), meal and processed meat products (prepared meals, meal kits, sandwiches, wraps, poultry and meat products), processed fruit and vegetable products.

[0505] A. Beverages and beverage products In some embodiments, the sweetened composition is a beverage or beverage product. "Beverage product," as used herein, refers to ready-to-drink beverages (RTDs), concentrated beverages, syrups, and powdered beverages. Suitable ready-to-drink beverages include carbonated and non-carbonated beverages. Carbonated beverages include, but are not limited to, frozen carbonated beverages, functional sparkling beverages, cola, fruit-flavored sparkling beverages (e.g., lemon-lime, orange, grape, strawberry, pineapple), ginger ale, soft drinks, root beer, and the like. Non-carbonated beverages include, but are not limited to, fruit juices, fruit-flavored beverages or waters, juice drinks, nectars, fruit / flavored still drinks, energy / sports drinks, vegetable juices, vegetable-flavored juices, energy drinks, vitamin-fortified waters, flavored waters, coconut water, tea-based beverages (e.g., black tea, green tea, oolong tea), coffee, cocoa drinks, dairy-based beverages (e.g., dairy drinks, cafe au lait, milk tea, fruit milk), beverages containing grain extracts, smoothies, and the like.

[0506] In some embodiments, the beverage is a juice beverage from which at least a portion of the sucrose has been removed. In other embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more of the sucrose is removed from the juice. In some embodiments, the sucrose is removed by filtration. In other embodiments, the sucrose is split into glucose and fructose before addition.

[0507] Beverages include a "matrix," i.e., a base ingredient in which the components dissolve. In some embodiments, beverages include potable water (e.g., deionized water, distilled water, reverse osmosis water, charcoal-treated water, purified water, demineralized water, or a combination thereof) as the matrix. Other suitable matrices include phosphoric acid, phosphate buffer, citric acid, citrate buffer, and charcoal-treated water. Beverage concentrates and syrups are prepared by mixing an initial amount of liquid matrix (e.g., water) with the required beverage ingredients. Full-strength beverages are prepared by adding additional liquid (e.g., water). Powdered beverages are prepared by dry-mixing all beverage ingredients without the addition of liquid. Full-strength beverages are obtained by adding a specified amount of water.

[0508] The pH of the beverage is not expected to substantially adversely affect the taste of the sweetener. Non-limiting examples of beverages include a pH range of about 1.8 to about 10. In certain embodiments, the pH of the beverage is about 4. In other embodiments, the pH is less than about 4. In more specific embodiments, the pH is less than about 3.8, less than about 3.6, less than about 3.4, less than about 3.2, less than about 3.0, less than about 2.8, less than about 2.6, less than about 2.4, or less than 2.2. In other embodiments, the pH of the beverage is about 3.8 or less, about 3.6 or less, about 3.4 or less, about 3.2 or less, about 3.0 or less, about 2.8 or less, about 2.6 or less, about 2.4 or less, or 2.2 or less.

[0509] B. Edible Gel Mixes and Edible Gel Compositions In some embodiments, the sweetened composition is an edible gel or edible gel mix. By edible gel is meant an ingestible gel. Non-limiting examples of edible gel compositions for use in certain embodiments include gel desserts, puddings, jellies, pastes, trifles, aspic, marshmallows, and gummy candies / chews. An edible gel mix is ​​typically a powdered or granular solid to which a liquid is added to form the edible gel composition. Non-limiting examples of liquids for use in certain embodiments include water, dairy liquids, dairy alternative liquids, juice, alcohol, alcoholic beverages, and combinations thereof. Examples of dairy liquids include milk, cultured milk, heavy cream, whey, and mixtures thereof. Examples of dairy alternative liquids include soy milk and non-dairy coffee milk (creamer).

[0510] C. Confectionery In one embodiment, the sweetened composition is a confection, where "confection" refers to any confection such as sweets, lollipops, candies, etc. Confections generally include a base composition component and a sweetener component. According to certain embodiments, confections include: desserts: yogurt, jelly, jellies, puddings, bavarois, blancmange, cakes, brownies, mousses, etc.; sweet foods eaten at teatime or after meals; frozen foods / frozen desserts: ice cream, iced milk, dairy ice cream, sorbet, dessert ice cream, etc.; baked and steamed goods: crackers, biscuits, bean buns, halva, alfajores, etc.; mochi / snacks; tableware; general confections: chewing gum, hard candy, soft candy, mints, nougat, jelly beans, fudge, toffee, Swiss milk tablets, licorice candy, chocolate, gelatin candy, marshmallows, marzipan, divinity, cotton candy, etc.; sauces: fruit sauces, chocolate sauces, etc.; creams: buttercream, flour paste, whipped cream, etc.; jams: strawberry jam, marmalade, etc.; breads: sweetbreads and other starchy foods, and combinations thereof.

[0511] D. Seasoning composition In one embodiment, the sweetened composition is a flavor enhancer composition, where a "flavor enhancer" is a composition used to enhance or enhance the flavor of a food or beverage. Non-limiting examples of condiments include ketchup, mustard, barbecue sauce, butter, chili sauce, chutney, cocktail sauce, curry, dips, fish sauce, horseradish, hot sauce, jelly / jam / marmalade / preserves, mayonnaise, peanut butter, relish, remoulade, salad dressing, salsa, sauerkraut, soy sauce, steak sauce, syrup, tartar sauce, and Worcestershire sauce. Seasoning bases generally consist of a mixture of multiple ingredients, non-limiting examples of which include: base materials: water, vinegar, etc.; spices and condiments: salt, pepper, garlic, mustard seeds, onions, paprika, turmeric, etc.; fruits, vegetables, or processed fruits thereof: tomatoes or tomato products (paste, puree), fruit juice, peel, etc.; oils or oil emulsions (especially vegetable oils); thickeners: xanthan gum, food starch, other hydrocolloids, etc.; emulsifiers: egg yolk solids, protein, gum arabic, carob gum, guar gum, karaya gum, tragacanth gum, carrageenan, pectin, propylene glycol alginate, sodium carboxymethylcellulose, polysorbate, etc.; seasoning base recipes and manufacturing methods are widely known to those skilled in the art.

[0512] E. Chewing Gum Composition In some embodiments, the sweetened composition is a chewing gum composition. Chewing gum compositions generally consist of a water-soluble portion and a water-insoluble gum base portion. The water-soluble portion disperses along with the flavoring ingredients during chewing, while the insoluble gum base remains in the mouth. Depending on the type of insoluble gum base, gums are classified as chewing gum, bubble gum, or functional gum.

[0513] Flavoring ingredients can be included in either the gum base (insoluble portion) or the water-soluble portion. These flavoring ingredients can be natural or artificial flavors. In certain embodiments, flavoring ingredients include essential oils (plant or fruit derived), such as peppermint oil, spearmint oil, other mint oils, clove oil, cinnamon oil, wintergreen oil, bay leaf oil, thyme oil, cedar leaf oil, nutmeg oil, allspice oil, sage oil, mace oil, and almond oil. In other embodiments, flavoring ingredients include fruit essences or plant extracts (such as apple, banana, watermelon, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, and apricot). In yet other embodiments, flavoring ingredients include citrus flavors (such as extracts, essences, and essential oils of lemon, lime, orange, tangerine, grapefruit, citron, and kumquat).

[0514] F. Cereal Composition In some embodiments, the sweetened composition is a cereal composition. Cereal compositions are typically consumed as a main meal or snack. Non-limiting examples of cereal compositions for use in certain embodiments include ready-to-eat cereals and hot cereals. Ready-to-eat cereals are cereals that consumers can consume immediately without cooking. Examples include breakfast cereals and snack bars (energy bars, rice cakes, granola bars, nutrition bars, etc.). Breakfast cereals are typically shredded, flaked, puffed, extruded, or otherwise processed and consumed cold with milk or fruit. Hot cereals are cooked with milk or water before consumption. Examples include grits, porridge, polenta, rice, and rolled oats.

[0515] Cereal compositions generally include at least one cereal ingredient. As used herein, the term "cereal ingredient" refers to a whole or partial grain, seed, or grass plant. Non-limiting examples of cereal ingredients that may be used in certain embodiments include corn, wheat, rice, barley, bran, bran endosperm, bulgur, sorghum, millet, oats, rye, triticale, wheat buckwheat, fonio, quinoa, kidney beans, soybeans, amaranth, teff, spelt, and kaniwa.

[0516] G. Baked goods In some embodiments, the sweetened composition is a baked good. As used herein, "baked goods" includes ready-to-eat and pre-baked products, as well as flours and mixes that require cooking before serving. Non-limiting examples include cakes, crackers, cookies, brownies, muffins, rolls, bagels, donuts, strudels, pastries, croissants, biscuits, breads, bread products, buns, etc.

[0517] In certain embodiments of the present disclosure, baked goods comprise a combination of sweeteners, moisture, fat, and a leavening agent. In many embodiments, flour is also used in the preparation of baked goods to form a dough or batter.

[0518] In certain embodiments, the leavening agent may be a chemical leavening agent or a yeast leavening agent. Non-limiting examples of chemical leavening agents include baking soda (e.g., sodium bicarbonate, potassium bicarbonate, aluminum bicarbonate), baking acids (e.g., aluminum hydrogen phosphate, monocalcium phosphate, dicalcium phosphate), and combinations thereof.

[0519] H. Dairy products In some embodiments, the sweetened composition is a dairy product. Dairy products and methods for their production, as applicable to the present disclosure, are well known to those skilled in the art. "Dairy product" refers to milk or a food product made from milk. Non-limiting examples of dairy products that may be used in certain embodiments include milk, heavy cream, sour cream, crème fraîche, buttermilk, cultured buttermilk, milk powder, sweetened condensed milk, evaporated milk, butter, cheese, cottage cheese, cream cheese, yogurt, ice cream, frozen custard, frozen yogurt, gelato, flan, piima, filmjolk, kajmak, kefir, viili, kumiss, airag, iced milk, casein, ayran, lassi, khoa, and combinations thereof. These dairy products may be produced by conventional methods or may be filtered or otherwise modified to adjust flavor. In certain embodiments, the dairy product is in liquid form and lactose or its breakdown products (galactose, glucose) can be reduced or eliminated compared to raw milk and supplemented with the sweetener compositions herein. The carbohydrate reduction can be about 5%, 10%, 20%, 50%, 70% or more.

[0520] In certain embodiments, the dairy composition may also include other additives. Non-limiting examples of additives that may be used include the sweeteners of the present disclosure and flavors such as chocolate, strawberry, banana, etc. Additionally, certain embodiments may include nutritional supplements (e.g., minerals such as vitamin D and calcium) to improve the nutritional value of the milk.

[0521] I. Tabletop Sweetener Compositions In some embodiments, the sweetened composition is a tabletop sweetener. The tabletop sweetener may further comprise one or more excipients, additives, anti-caking agents, functional ingredients, or combinations thereof.

[0522] Suitable "bulking agents" include, but are not limited to, maltodextrin (10DE, 18DE, 5DE), corn syrup solids (20 or 36DE), sucrose, fructose, glucose, invert sugar, sorbitol, xylose, ribulose, mannose, xylitol, mannitol, galactitol, erythritol, maltitol, lactitol, isomalt, maltose, tagutose, lactose, inulin, glycerol, propylene glycol, polyols, polydextrose, fructooligosaccharides, cellulose and its derivatives, and mixtures thereof. In other embodiments, granulated sugar (sucrose), crystalline fructose, and other carbohydrates and sugar alcohols can be used as caloric sweeteners to ensure consistency of content without significant calorie increase.

[0523] As used herein, the terms "anti-caking agent" and "flow agent" refer to ingredients that aid in content uniformity and dissolution uniformity. In certain embodiments, non-limiting examples of anti-caking agents include potassium bitartrate (cream of tartar), calcium silicate, silicon dioxide, microcrystalline cellulose (Avicel), tricalcium phosphate, and the like. In some embodiments, the anti-caking agent is present in an amount ranging from about 0.001 to about 3% by weight of the total tabletop sweetener composition.

[0524] The tabletop sweetener composition may be packaged in any known form, including, but not limited to, powder, granules, packets, tablets, sachets, pellets, cubes, solids, liquids, etc.

[0525] In some embodiments, the tabletop sweetener composition is a single-serving (portion-controlled) packet containing a dry blend. Dry blend formulations are generally composed of powder or granules. Packet sizes can be any; as a non-limiting example, a packet measuring approximately 2.5 inches by 1.5 inches contains approximately 1 gram of sweetener composition, which has a sweetness equivalent to approximately 2 teaspoons (approximately 8 grams) of granulated sugar. In certain embodiments, dry blend tabletop sweetener formulations contain sweeteners in the range of about 1% to about 10% by weight.

[0526] Tabletop sweetener compositions may also be implemented in liquid form. In this case, the composition of the present disclosure is combined with a liquid carrier. Non-limiting examples of suitable carrier agents for liquid tabletop sweeteners include water, alcohol, polyol, glycerin-based, citric acid-based in water, and mixtures thereof. The sweetness intensity (as sucrose equivalent) of any tabletop sweetener composition described herein or known to those skilled in the art can be adjusted to achieve a desired sweetness profile. For example, a tabletop sweetener composition may have a sweetness equivalent to the same amount of standard sugar. In other embodiments, the sweetness may be up to 100 times that of sugar. Other examples include tabletop sweetener compositions that are 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 times sweeter than sugar.

[0527] J. Delivery system The sweetener compositions of the present disclosure can also be formulated into various delivery systems with improved handling and dissolution rates. Non-limiting examples of suitable delivery systems include: co-crystallized sweetener compositions with sugar or polyols; agglomerated sweetener compositions; compression-molded sweetener compositions; dry sweetener compositions; particulate sweetener compositions; spheronized sweetener compositions; granular sweetener compositions; and liquid sweetener compositions.

[0528] XI. Plant Breeding In addition to producing transformed plants by directly introducing constructs prepared according to the present disclosure into a particular plant genotype, transformed plants can also be produced by breeding. For example, a coding sequence for a selected mogroside biosynthetic pathway can be introduced into a plant variety by breeding, without the need to directly transform a plant of that variety. Thus, the present disclosure encompasses not only directly transformed plants and plants regenerated from transformed cells, but also their progeny (progeny).

[0529] As used herein, "progeny" refers to any generation of descendants of a parent plant prepared in accordance with the present disclosure, which contain a selected DNA construct. As used herein, "crossing" refers to the technique of introducing a transgene by crossing a starting plant line with a donor plant line containing a transgene in accordance with the present disclosure. To achieve this goal, the following procedures can be performed: (a) sowing seeds of the first parent (starting line) and the second parent (donor line containing the transgene of the present disclosure); (b) growing the first and second parent plants to flowering; (c) pollinating the flowers of the first parent plant with pollen from the second parent plant; (d) Harvesting seeds from parent plants with fertilized flowers.

[0530] "Backcrossing" is a process that involves the following steps: (a) crossing a plant of a first genotype containing a gene, DNA sequence, or element of interest with a plant of a second genotype that does not contain the element; (b) selecting progeny plants containing the gene, DNA sequence, or element of interest; (c) recrossing the selected progeny plants with plants of the second genotype; (d) repeating steps (b) and (c) to introduce the DNA sequence of interest from the first genotype into the second genotype.

[0531] Introgression of a DNA element into a plant genotype is defined as the result of the process of introduction by backcrossing. A plant genotype into which a DNA sequence has been introduced may be referred to as a backcross conversion genotype, line, inbred, or cross (hybrid). Conversely, a plant genotype that does not contain the DNA sequence of interest may be referred to as a non-converter genotype, non-converter line, non-converter inbred, or non-converter hybrid.

[0532] XII. Other Definitions The following definitions or interpretations of technical terms are used throughout this disclosure. Technical terms used herein shall be interpreted according to their commonly used meanings in the fields of plant biology, molecular biology, bioinformatics, and plant breeding. All definitions below apply to the entire contents of this application.

[0533] To facilitate understanding of this disclosure, several terms are defined below. Terms defined herein have the meanings commonly understood by one of ordinary skill in the art. Throughout this specification and claims, the words "a" or "an," when used with the word "comprising," may mean "one," but are consistent with the meanings of "one or more," "at least one," "plurality," etc. Additionally, in the claims, the word "or" is used to mean "and / or" unless expressly indicated as referring to either alternative or unless alternatives are mutually exclusive. Throughout this application, the word "about" means that the value includes a range of error due to variation among the measuring device, method, or sample subjects.

[0534] In this specification and claims, the words "comprising" (and variations such as "comprise" and "comprises"), "having," "including," and "containing" are all open-ended and do not exclude the presence of additional elements or steps. In any of the composition and method embodiments, "comprising" can be replaced with "consisting essentially of" or "consisting of," where "consisting essentially of" means including the specified elements or steps, plus things that do not materially affect the nature or function of the invention. "Consisting" indicates that only the stated element or step or groups thereof are present.

[0535] The term "or combinations thereof" means all combinations and permutations of the preceding listed items. For example, "A, B, C, or combinations thereof" includes A, B, C, AB, AC, BC, ABC, and, where order is important, BA, CA, CB, CBA, BCA, ACB, BAC, CAB, etc. It also expressly includes repeating combinations (e.g., BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc.). It is generally understood that there is no limit to the number of items in a combination unless otherwise clear from the context.

[0536] Terms such as "about," "substantial," and "substantially" do not necessarily mean exact or complete, but rather refer to a state that is as close as one skilled in the art would consider the state to be. The breadth of such a description depends on the extent to which one skilled in the art would recognize the modified feature as still having the original properties and functionality. In general, when a numerical value is modified by "about," the value may vary by ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 10%, 12%, or 15%.

[0537] The terms "peptides," "oligopeptides," "polypeptide," "protein," and "enzyme" are used synonymously herein and, unless otherwise specified, refer to polymers of any length consisting of amino acids linked together by peptide bonds. The terms "gene sequence(s), "polynucleotide(s), "nucleic acid sequence(s)," "nucleotide sequence(s)," "nucleic acid(s)," and "nucleic acid molecule" are also used synonymously herein and refer to linear polymers of any length composed of RNA, DNA, or mixtures thereof.

[0538] Endogenous: "Endogenous" or "native" nucleic acids and / or proteins refer to nucleic acids or proteins found in a plant or other organism in the form in which they occur naturally (i.e., without human intervention, such as genetic engineering).

[0539] Exogenous: "Exogenous" is the opposite of "endogenous" and refers to a nucleic acid or protein that has been introduced into a plant or other organism using recombinant DNA technology. An "exogenous" nucleic acid or protein is one that (1) does not naturally occur in the plant, (2) is different from that which occurs naturally, (3) is present in an amount different from its natural state, or (4) is present in a location different from its natural genetic environment.

[0540] Expression: The set of cellular processes that result in the production of a polypeptide from a DNA molecule (e.g., a structural gene), including transcription and translation.

[0541] Expression cassette: A nucleic acid sequence of interest operably linked to one or more control sequences (at least a promoter). An expression cassette may include transcriptional and / or translational promoting sequences, termination sequences, silencers, enhancers, intron sequences in the 5' untranslated region (UTR) or coding sequence, RNA / protein stabilizing sequences, etc. An expression cassette may also be integrated into the genome of a host cell and replicated along with the genome, or may exist transiently.

[0542] Genetic Transformation: The process of introducing a DNA sequence or construct (such as a vector or expression cassette) into a cell or protoplast so that the foreign DNA is integrated into the chromosome or becomes capable of autonomous replication.

[0543] Heterologous: A particular sequence is not naturally occurring in its current genetic context within a host genome. The sequence may be considered "heterologous" because it is present in a genetic context different from its native genetic arrangement, even if it was originally present in the host. For example, a regulatory sequence combined with another coding sequence.

[0544] Modulation: "Modulation" refers to an altered expression level compared to a control plant. Modulation includes either an increase or a decrease in expression level.

[0545] Obtaining: When used in reference to transformed plant cells or transformed plants, "obtaining" means either transforming an untransformed plant cell or plant to produce a transformant, or sowing seeds of a transformed plant to give rise to a transformed plant cell or plant. Such transformed plant seeds may be derived from the R0 generation transformed plant or may be any generation progeny that have inherited the given transforming sequence from the starting transformed parent plant.

[0546] Operably Linked: "Operably linked" or "functionally linked" are synonyms and, as used herein, refer to a functional linkage between, for example, a promoter sequence and a nucleic acid sequence of interest, such that the promoter is capable of directing transcription of the nucleic acid sequence of interest. The same applies to a linkage between a termination sequence and a nucleic acid sequence, allowing for termination or cessation of transcription.

[0547] Plant: As used herein, "plant" includes intact plants, their ancestors and descendants, and plant parts such as fruits, seeds, shoots, stems, leaves, roots (including tubers), flowers, tissues, and organs, all of which contain the gene / nucleic acid of interest. Also included are plant cells, suspension cultures, callus tissue, embryos, meristems, gametophytes, sporophytes, pollen, and microspores.

[0548] Ploidy: Ploidy, or chromosomal ploidy, refers to the number of complete chromosome sets present in a cell nucleus. Somatic cells, tissues, and individuals are classified by the number of chromosome sets they contain (ploidy level), such as haploid (one set), diploid (two sets), triploid (three sets), tetraploid (four sets), pentaploid (five sets), hexaploid (six sets), and heploid (seven sets). The term "polyploidy" is used to describe cells with three or more chromosome sets.

[0549] Promoter: A recognition site or group of sequences on a DNA sequence that provides an expression control element for a structural gene and refers to the site where RNA polymerase specifically binds to initiate RNA synthesis (transcription) of that gene.

[0550] R0 transgenic plant: refers to a plant that has been genetically transformed or regenerated from a transformed plant cell.

[0551] Recombinant: refers to a nucleic acid sequence, expression cassette, genetic construct, or vector containing a nucleic acid sequence disclosed herein, or an organism transformed therewith, produced by genetic engineering techniques, wherein (a) the nucleic acid sequence or a portion thereof, (b) a gene control sequence (e.g., promoter or terminator) operably linked to the nucleic acid sequence, or (c) any combination thereof, is not found in its natural genetic environment or has been artificially altered / introduced.

[0552] Regeneration: The process of growing a plant from a plant cell (e.g., protoplast, callus, or explant).

[0553] Selected DNA: A DNA segment that one wishes to introduce, or has introduced, into a plant genome.

[0554] Terminator: A DNA regulatory sequence located at the end of a transcription unit that directs 3' end processing and polyA addition of the primary transcript and termination of transcription.

[0555] Transformation construct: A chimeric DNA molecule designed for genetic introduction into a host genome, often containing all the genetic elements necessary to direct the expression of one or more foreign genes. In certain embodiments, it is desirable to introduce it into a host cell in the form of an expression cassette.

[0556] Transformed cell: A cell whose DNA content has been altered by the introduction of a foreign DNA molecule.

[0557] Transgene: A DNA segment that is integrated into a host genome or is capable of autonomous replication within a host cell and can cause the expression of one or more coding sequences. A typical transgene confers a novel phenotype compared to the corresponding untransformed cell or plant. Transgenes can be introduced directly by gene transfer or inherited genetically from plants into which they were introduced in previous generations.

[0558] Transgenic plant: A plant or its progeny that contains a foreign DNA segment that is not naturally present in the same non-transformed plant. The transgenic plant may contain sequences that are originally present in the plant, but may be modified, for example, with heterologous regulatory elements, to alter the level or pattern of expression of the "foreign" gene.

[0559] Vector: A DNA molecule designed to be introduced into a host cell. Some vectors are capable of replicating within the host cell. Plasmids are typical vectors, and expression cassettes isolated from them are also vectors.

[0560] Example The following examples illustrate exemplary embodiments of the present disclosure. Those skilled in the art should understand that the techniques included in the examples below are techniques that the inventors have confirmed to be effective in implementing the present disclosure and can therefore be considered exemplary embodiments. However, those skilled in the art should understand, in light of the present disclosure, that many changes can be made to the specific embodiments disclosed and still achieve similar or equivalent results, without departing from the spirit and scope of the present disclosure.

[0561] Example 1: Construction of an expression cassette Several expression cassettes containing different combinations of nucleotide sequences encoding mogroside pathway enzymes and regulatory sequences were constructed. Construction of these expression cassettes was carried out according to standard genetic engineering techniques. The following expression cassettes were constructed:

[0562] SP1463: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator; an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator; and an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator. The CmYLCV promoter is linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. These are followed by the TM6 MAR insulator sequence.

[0563] SP3139: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is linked to a Pea3A terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to an AtUBQ3 terminator; a FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to a GmaxMYB2 terminator; a dMMV promoter linked to a CYP72 Zm nucleic acid sequence, which is linked to an AtRBCS2B terminator; a CmYLCV promoter linked to a UGT720 nucleic acid sequence, which is linked to an E9 terminator; an e35S promoter linked to a UGT94 nucleic acid sequence, which is linked to an ATHSP18.2 terminator; and a NOS promoter linked to an EPH nucleic acid sequence, which is linked to a Ubi3 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The e35S promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, the TM6 MAR insulator sequence is placed.

[0564] SP1908: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is linked to a Pea3A terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to an AtUBQ3 terminator; a FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to a GmaxMYB2 terminator; a dMMV promoter linked to a CYP72 Zm nucleic acid sequence, which is linked to an AtRBCS2B terminator; a CmYLCV promoter linked to a UGT720 nucleic acid sequence, which is linked to an E9 terminator; a e35S promoter linked to a UGT94 nucleic acid sequence, which is linked to an ATHSP18.2 terminator; and a NOS promoter linked to an EPH nucleic acid sequence, which is linked to a Ubi3 terminator. The e35S promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator, and finally to the TM6 MAR insulator sequence.

[0565] SP3488: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is linked to a Pea3A terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to an AtUBQ3 terminator; an FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to a GmaxMYB2 terminator; an e35S promoter linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is linked to an AtRBCS2B terminator; and a CmYLCV promoter linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is linked to an E9 terminator. The dMMV promoter is linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0566] SP3015: This expression cassette begins with the TM6 MAR insulator sequence and was assembled in the following configuration: a dMMV promoter linked to a CYP72 nucleic acid sequence, which is linked to the AtRBCS2B terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator; a FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; a HLVH12 promoter linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; a e35S promoter linked to a UGT720 nucleic acid sequence, which is linked to the E9 terminator; a CmYLCV promoter linked to a UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to the EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated with the TM6 MAR insulator sequence.

[0567] SP3432: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a CmYLCV promoter linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator; a FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; an e35S promoter linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is linked to the AtRBCS2B terminator; and a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is linked to the E9 terminator. The dMMV promoter is linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The e35S promoter is linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0568] SP1160: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the e35S promoter is operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is linked to the AtRBCS2B terminator; the CmYLCV promoter is operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is linked to the E9 terminator; the FSgt / PFLt promoter is operably linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the HLVH12 promoter is operably linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; and the DCMV promoter is operably linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator. The dMMV promoter is linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0569] SP2916: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence (all in reverse orientation), which is linked to the AtRBCS2B terminator; the CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence, which is linked to the E9 terminator; the FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the HLVH12 promoter linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; and the DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator. The dMMV promoter is linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0570] SP4643: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence, which is linked to the AtRBCS2B terminator; the DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator; the FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the HLVH12 promoter linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; the CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence, which is linked to the E9 terminator; and the dMMV promoter linked to a bicistronic UGT94:2A:UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to a Ubi3 terminator. The ScBV promoter is linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is linked to an AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to a 35S terminator. Finally, it is terminated by a TM6 MAR insulator sequence.

[0571] SP4870: This expression cassette begins with the TM6 MAR insulator sequence and is constructed as follows: an e35S promoter operably linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence linked to the AtRBCS2B terminator; a CmYLCV promoter operably linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence linked to the E9 terminator; an FMVSgt promoter operably linked to a CDS nucleic acid sequence linked to the GmaxMYB2 terminator; an HLVH12 promoter operably linked to an SQE nucleic acid sequence linked to the Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence linked to the AtUBQ3 terminator; and a dMMV promoter operably linked to a bicistronic UGT94:2A:UGT94 nucleic acid sequence linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to a Ubi3 terminator. The ScBV promoter is linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is linked to an AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to a 35S terminator. Finally, it is terminated by a TM6 MAR insulator sequence.

[0572] SP1603: This expression cassette begins with the TM6 MAR insulator sequence and was assembled in the following configuration: the e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence (all in reverse orientation), which is linked to the AtRBCS2B terminator; the CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence, which is linked to the E9 terminator; the FMVSgt promoter linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the HLVH12 promoter linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; and the DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator. The dMMV promoter is linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0573] SP3095: This expression cassette begins with the TM6 MAR insulator sequence and was assembled with the following configuration: the e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence (all in reverse orientation), which is linked to the AtRBCS2B terminator; the CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence, which is linked to the E9 terminator; the FMVSgt promoter linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the HLVH12 promoter linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; and the DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator. The dMMV promoter is linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The FE3 promoter is linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated by a TM6 MAR insulator sequence.

[0574] SP0265: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a CmYLCV promoter linked to an SQE nucleic acid sequence linked to a Pea3A terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence linked to an AtUBQ3 terminator; a FSgt / PFLt promoter linked to a CDS nucleic acid sequence linked to a GmaxMYB2 terminator; an e35S promoter linked to a UGT720:2A:CYP72 bicistronic nucleic acid sequence linked to an E9 terminator; a dMMV promoter linked to a UGT94:2A:tHMGR bicistronic nucleic acid sequence linked to an ATHSP18.2 terminator; and a NOS promoter linked to an EPH nucleic acid sequence linked to a Ubi3 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator, and finally to the TM6 MAR insulator sequence.

[0575] SP4406: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is linked to a Pea3A terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to an AtUBQ3 terminator; an FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to a GmaxMYB2 terminator; an e35S promoter linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is linked to an AtRBCS2B terminator; and a CmYLCV promoter linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is linked to an E9 terminator. The dMMV promoter is linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0576] SP2152: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter linked to an SQE nucleic acid sequence linked to a Pea3A terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence linked to an AtUBQ3 terminator; an FSgt / PFLt promoter linked to a CDS nucleic acid sequence linked to a GmaxMYB2 terminator; an e35S promoter linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence linked to an AtRBCS2B terminator; a CmYLCV promoter linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence linked to an E9 terminator; and a dMMV promoter linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence linked to an ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to a Ubi3 terminator. The FE3 promoter is linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is linked to an AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to a 35S terminator. Finally, it is terminated by a TM6 MAR insulator sequence.

[0577] SP4311: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence, which is linked to the AtRBCS2B terminator; the CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence, which is linked to the E9 terminator; the FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the HLVH12 promoter linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; the DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator; and the dMMV promoter linked to a bicistronic UGT94:2A:UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to the EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it ends with the TM6 MAR insulator sequence.

[0578] SP4378: This expression cassette begins with the TM6 MAR insulator sequence and is assembled with the following configuration: an e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence linked to the AtRBCS2B terminator (all in reverse orientation); a CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence linked to the E9 terminator; an FSgt / PFLt promoter linked to a CDS nucleic acid sequence linked to the GmaxMYB2 terminator; an HLVH12 promoter linked to an SQE nucleic acid sequence linked to the Pea3A terminator; and a DCMV promoter linked to a CYP87 nucleic acid sequence linked to the AtUBQ3 terminator. The dMMV promoter is linked to a bicistronic UGT94:2A:UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to a tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator, and finally to the TM6 MAR insulator sequence.

[0579] SP3132: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence, which is linked to the AtRBCS2B terminator; the DCMV promoter linked to a CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator; the FSgt / PFLt promoter linked to a CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the HLVH12 promoter linked to an SQE nucleic acid sequence, which is linked to the Pea3A terminator; the CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence, which is linked to the E9 terminator; and the dMMV promoter linked to a bicistronic UGT94:2A:UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to the EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it ends with the TM6 MAR insulator sequence.

[0580] SP2355: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence linked to the AtRBCS2B terminator; the CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence linked to the E9 terminator; the FMVSgt promoter linked to a CDS nucleic acid sequence linked to the GmaxMYB2 terminator; the HLVH12 promoter linked to an SQE nucleic acid sequence linked to the Pea3A terminator; the DCMV promoter linked to a CYP87 nucleic acid sequence linked to the AtUBQ3 terminator; and the dMMV promoter linked to a bicistronic UGT94:2A:UGT94 nucleic acid sequence linked to the ATHSP18.2 terminator. The NOS promoter is linked to the EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it ends with the TM6 MAR insulator sequence.

[0581] SP4762: This expression cassette begins with the TM6 MAR insulator sequence and is assembled with the following configuration: an e35S promoter linked to a bicistronic CYP72:2A:CYP72 nucleic acid sequence linked to the AtRBCS2B terminator (all in reverse orientation); a CmYLCV promoter linked to a bicistronic UGT720:2A:UGT720 nucleic acid sequence linked to the E9 terminator; an FMVSgt promoter linked to a CDS nucleic acid sequence linked to the GmaxMYB2 terminator; an HLVH12 promoter linked to an SQE nucleic acid sequence linked to the Pea3A terminator; and a DCMV promoter linked to a CYP87 nucleic acid sequence linked to the AtUBQ3 terminator. The dMMV promoter is linked to a bicistronic UGT94:2A:UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator. The NOS promoter is linked to an EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to a tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to a HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it ends with a TM6 MAR insulator sequence.

[0582] SP0892: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the HLVH12 promoter linked to the SQE nucleic acid sequence, which is linked to the Pea3A terminator; the DCMV promoter linked to the CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator; the FSgt / PFLt promoter linked to the CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the dMMV promoter linked to the CYP72 nucleic acid sequence, which is linked to the AtRBCS2B terminator; the CmYLCV promoter linked to the UGT720 nucleic acid sequence, which is linked to the E9 terminator; the e35S promoter linked to the UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator; and the NOS promoter linked to the EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated by the TM6 MAR insulator sequence.

[0583] SP2249: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the HLVH12 promoter linked to the SQE nucleic acid sequence, which is linked to the Pea3A terminator; the DCMV promoter linked to the CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator; the FSgt / PFLt promoter linked to the CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the dMMV promoter linked to the CYP72 nucleic acid sequence, which is linked to the AtRBCS2B terminator; the e35S promoter linked to the UGT720 nucleic acid sequence, which is linked to the E9 terminator; the CmYLCV promoter linked to the UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator; the NOS promoter linked to the EPH nucleic acid sequence, which is linked to the Ubi3 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated by the TM6 MAR insulator sequence.

[0584] SP0796: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter linked to a CYP72 nucleic acid sequence linked to the AtRBCS2B terminator; a DCMV promoter linked to a CYP87 nucleic acid sequence linked to the AtUBQ3 terminator; a FSgt / PFLt promoter linked to a CDS nucleic acid sequence linked to the GmaxMYB2 terminator; a HLVH12 promoter linked to an SQE nucleic acid sequence linked to the Pea3A terminator; a CmYLCV promoter linked to a UGT720 nucleic acid sequence linked to the E9 terminator; a e35S promoter linked to a UGT94 nucleic acid sequence linked to the ATHSP18.2 terminator; a NOS promoter linked to an EPH nucleic acid sequence linked to the Ubi3 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated by the TM6 MAR insulator sequence.

[0585] SP2057: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: the HLVH12 promoter linked to the SQE nucleic acid sequence, which is linked to the Pea3A terminator; the DCMV promoter linked to the CYP87 nucleic acid sequence, which is linked to the AtUBQ3 terminator; the FSgt / PFLt promoter linked to the CDS nucleic acid sequence, which is linked to the GmaxMYB2 terminator; the dMMV promoter linked to the CYP72 nucleic acid sequence, which is linked to the AtRBCS2B terminator; the NOS promoter linked to the EPH nucleic acid sequence, which is linked to the Ubi3 terminator; the e35S promoter linked to the UGT94 nucleic acid sequence, which is linked to the ATHSP18.2 terminator; and the CmYLCV promoter linked to the UGT720 nucleic acid sequence, which is linked to the E9 terminator. The ScBV promoter is linked to the tHMGR nucleic acid sequence, which is linked to the AtTub89 terminator. The CsVMV promoter is linked to the HygR nucleic acid sequence, which is linked to the 35S terminator. Finally, it is terminated by the TM6 MAR insulator sequence.

[0586] SP3308: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; an e35S promoter operably linked to a CYP72 nucleic acid sequence and linked to an AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and linked to an E9 terminator; a dMMV promoter operably linked to a UGT94 nucleic acid sequence and linked to an ATHSP18.2 terminator; and an NOS promoter operably linked to an EPH nucleic acid sequence and linked to a Ubi3 terminator. The ScBV promoter is operably linked to the tHMGR nucleic acid sequence and is linked to the AtTub89 terminator. The CsVMV promoter is operably linked to the HygR nucleic acid sequence and is linked to the 35S terminator. Finally, it is terminated with the TM6 MAR insulator sequence.

[0587] SP1379: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72 nucleic acid sequence and linked to the AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and linked to the E9 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator; a e35S promoter operably linked to a UGT94 nucleic acid sequence and linked to the ATHSP18.2 terminator; and a NOS promoter operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to the tHMGR nucleic acid sequence and is linked to the AtTub89 terminator. The CsVMV promoter is operably linked to the HygR nucleic acid sequence and is linked to the 35S terminator. Finally, it is terminated with the TM6 MAR insulator sequence.

[0588] SP3494: This expression cassette begins with the TM6 MAR insulator sequence and was assembled in the following configuration (all in reverse orientation): a dMMV promoter operably linked to a CYP72 nucleic acid sequence and linked to the AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and linked to the E9 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator; a e35S promoter operably linked to a UGT94 nucleic acid sequence and linked to the ATHSP18.2 terminator; and a NOS promoter operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to the tHMGR nucleic acid sequence and is linked to the AtTub89 terminator. The CsVMV promoter is operably linked to the HygR nucleic acid sequence and is linked to the 35S terminator. Finally, it is terminated with the TM6 MAR insulator sequence.

[0589] SP2585: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a CmYLCV promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; a dMMV promoter operably linked to a CYP72 nucleic acid sequence and linked to an AtRBCS2B terminator; an e35S promoter operably linked to a UGT720 nucleic acid sequence and linked to an E9 terminator; an e35S promoter operably linked to a UGT94 nucleic acid sequence and linked to an ATHSP18.2 terminator; and a NOS promoter operably linked to an EPH nucleic acid sequence and linked to a Ubi3 terminator. The e35S promoter is operably linked to the tHMGR nucleic acid sequence and is linked to the AtTub89 terminator. The CsVMV promoter is operably linked to the HygR nucleic acid sequence and is linked to the 35S terminator. Finally, it is terminated with the TM6 MAR insulator sequence.

[0590] SP3635: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: a CmYLCV promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; an e35S promoter operably linked to a UGT720:2A:CYP72 bicistronic nucleic acid sequence and linked to an E9 terminator; an e35S promoter operably linked to a UGT94:2A:tHMGR bicistronic nucleic acid sequence and linked to an ATHSP18.2 terminator; and a NOS promoter operably linked to an EPH nucleic acid sequence and linked to a Ubi3 terminator. The CsVMV promoter is operably linked to the HygR nucleic acid sequence, followed by a 35S terminator, and finally, by the TM6 MAR insulator sequence.

[0591] SP3800: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: a CmYLCV promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0592] SP0981: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0593] SP0137: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The FE3 promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0594] SP2154: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0595] SP1727: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator (all in reverse orientation); a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0596] SP0075: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator, and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator. The FSgt / PFLt promoter is operably linked to a CDS nucleic acid sequence and to a GmaxMYB2 terminator. The dMMV promoter is operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and to an AtRBCS2B terminator. The e35S promoter is operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and to an E9 terminator. The CmYLCV promoter is operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence and to an ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and to a Ubi3 terminator. The ScBV promoter is operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence and to an AtTub89 terminator. The CsVMV promoter is operably linked to the HygR nucleic acid sequence, followed by a 35S terminator, and finally, by the TM6 MAR insulator sequence.

[0597] SP4305: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0598] SP4221: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator. The CmYLCV promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0599] SP3488: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The dMMV promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0600] SP4094: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FMVSgt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0601] SP2971: This expression cassette begins with the TM6 MAR insulator sequence and was assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator (all in reverse orientation); a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FMVSgt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0602] SP2049: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; an FMVSgt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The CmYLCV promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0603] SP4063: This expression cassette begins with the TM6 MAR insulator sequence and was assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator (all in reverse orientation); a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FMVSgt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The FE3 promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0604] SP0121: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; an FMVSgt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The CmYLCV promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The FE3 promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0605] SP3358: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: a CmYLCV promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0606] SP4513: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0607] SP2221: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to an AtRBCS2B terminator; and a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to an E9 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The FE3 promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0608] SP3925: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0609] SP3748: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator (all in reverse orientation); a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0610] SP4511: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0611] SP3547: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FMVSgt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0612] SP3481: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator (all in reverse orientation); a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FMVSgt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The ScBV promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated with a TM6 MAR insulator sequence.

[0613] SP2185: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator (all in reverse orientation); a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FMVSgt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to the Ubi3 terminator. The FE3 promoter is operably linked to a bicistronic nucleic acid sequence of tHMGR:2A:tHMGR and linked to the AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to the 35S terminator. Finally, it is terminated by a TM6 MAR insulator sequence.

[0614] SP2792: This expression cassette begins with a TM6 MAR insulator sequence and is assembled in the following configuration: a CmYLCV promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator; a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to a GmaxMYB2 terminator; an e35S promoter operably linked to a UGT720:2A:CYP72 bicistronic nucleic acid sequence and linked to an E9 terminator; an e35S promoter operably linked to a UGT94:2A:tHMGR bicistronic nucleic acid sequence and linked to an ATHSP18.2 terminator; and a NOS promoter operably linked to an EPH nucleic acid sequence and linked to a Ubi3 terminator. The CsVMV promoter is operably linked to the HygR nucleic acid sequence, followed by a 35S terminator, and finally, by the TM6 MAR insulator sequence.

[0615] SP1000: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: an HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to a Pea3A terminator, and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to an AtUBQ3 terminator. The FSgt / PFLt promoter is operably linked to a CDS nucleic acid sequence and to a GmaxMYB2 terminator. The e35S promoter is operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and to an AtRBCS2B terminator. The CmYLCV promoter is operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and to an E9 terminator. The e35S promoter is operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence and to an ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and to a Ubi3 terminator. The ScBV promoter is operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence and to an AtTub89 terminator. The CsVMV promoter is operably linked to the HygR nucleic acid sequence, followed by a 35S terminator, and finally, by the TM6 MAR insulator sequence.

[0616] SP3766: This expression cassette (SP3766) was assembled using a TM6 MAR insulator sequence and consists of the following, in order: an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A termination sequence; a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 termination sequence; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 termination sequence; an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B termination sequence; and a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 termination sequence. The e35S promoter is operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 termination sequence. The NOS promoter is operably linked to an EPH nucleic acid sequence, which is operably linked to a Ubi3 termination sequence. The FE3 promoter is operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 termination sequence. The CsVMV promoter is operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S termination sequence. Finally, a TM6 MAR insulator sequence is provided.

[0617] SP4353: This expression cassette begins with the TM6 MAR insulator sequence and is assembled in the following configuration: a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence and linked to the AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence and linked to the E9 terminator; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence and linked to the GmaxMYB2 terminator; a HLVH12 promoter operably linked to an SQE nucleic acid sequence and linked to the Pea3A terminator; and a DCMV promoter operably linked to a CYP87 nucleic acid sequence and linked to the AtUBQ3 terminator. The e35S promoter is operably linked to a bicistronic nucleic acid sequence of UGT94:2A:UGT94 and linked to the ATHSP18.2 terminator. The NOS promoter is operably linked to an EPH nucleic acid sequence and linked to a Ubi3 terminator. The ScBV promoter is operably linked to a tHMGR nucleic acid sequence and linked to an AtTub89 terminator. The CsVMV promoter is operably linked to a HygR nucleic acid sequence and linked to a 35S terminator. Finally, it is terminated by a TM6 MAR insulator sequence.

[0618] SP0255: This expression cassette is constructed as follows: First, a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is linked to an AtRBCS2B termination sequence (all in reverse orientation). Next, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is linked to an E9 termination sequence. Furthermore, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is linked to a GmaxMYB2 termination sequence. An HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is linked to a Pea3A termination sequence. A DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is linked to an AtUBQ3 termination sequence. The e35S promoter is operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is linked to an ATHSP18.2 termination sequence. The NOS promoter is operably linked to an EPH nucleic acid sequence, which is linked to a Ubi3 termination sequence. The ScBV promoter is operably linked to a tHMGR nucleic acid sequence, which is linked to an AtTub89 termination sequence. The CsVMV promoter is operably linked to a HygR nucleic acid sequence, which is linked to a 35S termination sequence, and finally to a TM6 MAR insulator sequence.

[0619] SP4815: This expression cassette was assembled with the following configuration: a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, operably linked to the AtRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, operably linked to the AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, operably linked to the GmaxMYB2 terminator, a HLVH12 promoter operably linked to a SQE nucleic acid sequence, operably linked to the Pea3A terminator, and a UGT720:2A:UGT720 bicistronic nucleic acid sequence. The nucleic acid sequence of the present invention is composed of a CmYLCV promoter operably linked to a nucleic acid sequence, an E9 terminator operably linked thereto, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence, an Ubi3 terminator operably linked thereto, an ScBV promoter operably linked to a tHMGR nucleic acid sequence, an AtTub89 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence, and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0620] SP1073: This expression cassette was assembled with the following configuration: a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, followed by an AtRBCS2B terminator; a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, followed by an E9 terminator; an FMVSgt promoter operably linked to a CDS nucleic acid sequence, followed by a GmaxMYB2 terminator; an HLVH12 promoter operably linked to a SQE nucleic acid sequence, followed by a Pea3A terminator; a CYP87 nucleic acid sequence, followed by a Pea3A terminator; The vector is composed of a DCMV promoter operably linked thereto, an AtUBQ3 terminator operably linked thereto, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence, a Ubi3 terminator operably linked thereto, an ScBV promoter operably linked to a tHMGR nucleic acid sequence, an AtTub89 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence, a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0621] SP4402: This expression cassette was assembled with the following configuration: a TM6 MAR insulator sequence, followed by (all in reverse orientation) a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, operably linked to the AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, operably linked to the E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, operably linked to the GmaxMYB2 terminator, a HLVH12 promoter operably linked to a SQE nucleic acid sequence, operably linked to the Pea3A terminator, a CYP87 nucleic acid sequence, operably linked to the CYP87 nucleic acid sequence, and a CYP87 nucleic acid sequence. The nucleic acid sequence of the present invention is composed of a DCMV promoter operably linked to a nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence, an Ubi3 terminator operably linked thereto, an ScBV promoter operably linked to a tHMGR nucleic acid sequence, an AtTub89 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence, and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0622] SP1415: This expression cassette was assembled with the following configuration: a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, operably linked to a Pea3A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, operably linked to a GmaxMYB2 terminator, a e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, operably linked to an AtRBCS2B terminator, a UGT720:2A:UGT720 bicistronic nucleic acid sequence, operably linked to a CYP720:2A:UGT720 bicistronic nucleic acid sequence, and a CYP720:2A:UGT720 bicistronic nucleic acid sequence. the CmYLCV promoter operably linked to the UGT94:2A:UGT94 bicistronic nucleic acid sequence, the E9 terminator operably linked thereto, the e35S promoter operably linked to the UGT94:2A:UGT94 bicistronic nucleic acid sequence, the ATHSP18.2 terminator operably linked thereto, the NOS promoter operably linked to the EPH nucleic acid sequence, the Ubi3 terminator operably linked thereto, the ScBV promoter operably linked to the tHMGR:2A:tHMGR bicistronic nucleic acid sequence, the AtTub89 terminator operably linked thereto, the CsVMV promoter operably linked to the HygR nucleic acid sequence, the 35S terminator operably linked thereto, followed by the TM6 MAR insulator sequence.

[0623] SP2353: This expression cassette was assembled with the following configuration: a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, operably linked to a Pea3A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, operably linked to a GmaxMYB2 terminator, a e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, operably linked to an AtRBCS2B terminator, and a UGT720:2A:UGT720 bicistronic nucleic acid sequence. the CmYLCV promoter operably linked to the UGT94:2A:UGT94 bicistronic nucleic acid sequence, the E9 terminator operably linked thereto, the e35S promoter operably linked to the UGT94:2A:UGT94 bicistronic nucleic acid sequence, the ATHSP18.2 terminator operably linked thereto, the NOS promoter operably linked to the EPH nucleic acid sequence, the Ubi3 terminator operably linked thereto, the FE3 promoter operably linked to the tHMGR:2A:tHMGR bicistronic nucleic acid sequence, the AtTub89 terminator operably linked thereto, the CsVMV promoter operably linked to the HygR nucleic acid sequence, the 35S terminator operably linked thereto, followed by the TM6 MAR insulator sequence.

[0624] SP0565: This expression cassette was assembled with the following configuration: a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, operably linked to a Pea3A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, operably linked to an AtUBQ3 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, operably linked to a GmaxMYB2 terminator, a e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, operably linked to an AtRBCS2B terminator, and a UGT720:2A:UGT720 bicistronic nucleic acid sequence. the e35S promoter operably linked to the e35S promoter, the E9 terminator operably linked thereto, the CmYLCV promoter operably linked to the UGT94:2A:UGT94 bicistronic nucleic acid sequence, the ATHSP18.2 terminator operably linked thereto, the NOS promoter operably linked to the EPH nucleic acid sequence, the Ubi3 terminator operably linked thereto, the ScBV promoter operably linked to the tHMGR:2A:tHMGR bicistronic nucleic acid sequence, the AtTub89 terminator operably linked thereto, the CsVMV promoter operably linked to the HygR nucleic acid sequence, the 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0625] SP1202: This expression cassette (SP1202) was assembled with the following configuration: following the TM6 MAR insulator sequence, an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A termination sequence; a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 termination sequence; an FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 termination sequence; an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B termination sequence; and an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 termination sequence. The CmYLCV promoter is operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 termination sequence. The NOS promoter is operably linked to an EPH nucleic acid sequence, which is operably linked to a Ubi3 termination sequence. The FE3 promoter is operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 termination sequence. The CsVMV promoter is operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S termination sequence. Finally, a TM6 MAR insulator sequence is provided.

[0626] SP2808: This expression cassette (SP2808) was assembled with the following configuration: a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A termination sequence; a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 termination sequence; a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 termination sequence; a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B termination sequence; a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 termination sequence; and a e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 termination sequence. The NOS promoter is operably linked to an EPH nucleic acid sequence, which is operably linked to a Ubi3 termination sequence. The ScBV promoter is operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 termination sequence. The CsVMV promoter is operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S termination sequence. Finally, a TM6 MAR insulator sequence is located.

[0627] SP3684: This expression cassette (SP3684) is constructed using a TM6 MAR insulator sequence and contains the following elements in the following order: an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A termination sequence; a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 termination sequence; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 termination sequence; a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B termination sequence; and an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an ATHSP18.2 termination sequence. The e35S promoter is operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 termination sequence. The NOS promoter is operably linked to an EPH nucleic acid sequence, which is operably linked to a Ubi3 termination sequence. The ScBV promoter is operably linked to a tHMGR nucleic acid sequence, which is operably linked to a 35S termination sequence. The CsVMV promoter is operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S termination sequence. Finally, a TM6 MAR insulator sequence is arranged.

[0628] SP4522: This expression cassette (SP4522) is constructed using a TM6 MAR insulator sequence and contains the following elements in the following order: an HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A termination sequence; a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 termination sequence; an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 termination sequence; an e35S promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an ATHSP18.2 termination sequence; and an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an ATHSP18.2 termination sequence. The e35S promoter is operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 termination sequence. The NOS promoter is operably linked to an EPH nucleic acid sequence, which is operably linked to a Ubi3 termination sequence. The ScBV promoter is operably linked to a tHMGR nucleic acid sequence, which is operably linked to a 35S termination sequence. The CsVMV promoter is operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S termination sequence. Finally, a TM6 MAR insulator sequence is arranged.

[0629] SP3842: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence and a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a tHMGR nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0630] SP3938: This expression cassette comprises a TM6 MAR insulator sequence followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence and a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a tHMGR nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0631] SP3318: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, a 35S terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence, a Ubi3 terminator operably linked thereto, and a tHMGR nucleic acid sequence. The vector is composed of an e35S promoter operably linked to a sequence, an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to an SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT74_3 nucleic acid sequence and a 35S terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0632] SP3493: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, a 35S terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence, a Ubi3 terminator operably linked thereto, and a tHMGR nucleic acid sequence. The nucleic acid sequence is composed of an e35S promoter operably linked to a sequence, an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT74_4 nucleic acid sequence and a 35S terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0633] SP2476: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87D20 m2 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence and a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a tHMGR nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0634] SP4983: This expression cassette comprises a TM6 MAR insulator sequence followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87D20 m3 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence and a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a tHMGR nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0635] SP4003: This expression cassette comprises a TM6 MAR insulator sequence followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87D17 m2 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence and a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a tHMGR nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0636] SP4074: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87D17 m3 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence and a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a tHMGR nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0637] SP2649: This expression cassette comprises a TM6 MAR insulator sequence followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87D18_B m3 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence and a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a tHMGR nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0638] SP3397: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87D17 m2 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence, a Ubi3 terminator operably linked thereto, a tHMGR nucleic acid The vector is composed of an e35S promoter operably linked to a sequence, an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to an SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT74_3 nucleic acid sequence and a 35S terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0639] SP2771: This expression cassette comprises a TM6 MAR insulator sequence followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87D17 m3 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence, a Ubi3 terminator operably linked thereto, a tHMGR nucleic acid The vector is composed of an e35S promoter operably linked to a sequence, an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to an SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT74_3 nucleic acid sequence and a 35S terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0640] SP0847: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87D17 m3 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, a 35S terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, a 35S terminator operably linked thereto, a NOS promoter operably linked to an EPH nucleic acid sequence, a Ubi3 terminator operably linked thereto, a tHMGR nucleic acid The nucleic acid sequence is composed of an e35S promoter operably linked to a sequence, an ATHSP18.2 terminator operably linked thereto, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence and an AtUBQ3 terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT74_4 nucleic acid sequence and a 35S terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0641] SP3468: This expression cassette comprises a TM6 MAR insulator sequence followed by an FE_3 promoter operably linked to a CYP87 nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an FE_3 promoter operably linked to a CDS nucleic acid sequence, and a 35S terminator thereto, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, and an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, and a 35S terminator thereto, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, and a 35S terminator thereto, an FS1_1 promoter operably linked to a tHMGR nucleic acid sequence, and a Ubi3 terminator thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence, and a 35S terminator thereto, followed by TM6 It is composed of an MAR insulator array.

[0642] SP2177: This expression cassette contains a TM6 MAR insulator sequence, followed by an FS1_1 promoter operably linked to an SQE nucleic acid sequence, a 35S terminator operably linked to the FS1_1 promoter ... The vector is composed of an FE_3 promoter operably linked to a Zm nucleic acid sequence and a 35S terminator operably linked thereto, an FE_3 promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, an FE_3 promoter operably linked to a UGT94 nucleic acid sequence and a 35S terminator thereto, an FS1_1 promoter operably linked to an EPH nucleic acid sequence and a 35S terminator thereto, an FS1_1 promoter operably linked to a tHMGR nucleic acid sequence and a 35S terminator thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator thereto, followed by a TM6 MAR insulator sequence.

[0643] SP3804: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked to the SQE nucleic acid sequence, an e35S promoter operably linked to an ATHSP18.2 terminator operably linked to the SQE nucleic acid sequence, an FSgt / PFLt promoter operably linked to an ATHSP18.2 terminator operably linked to the SQE nucleic acid sequence, a CYP72 The vector is composed of an e35S promoter operably linked to a Zm nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an e35S promoter operably linked to a UGT720 nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an e35S promoter operably linked to a UGT94 nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence, and a Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a tHMGR nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence, and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0644] SP3016: This expression cassette comprises a TM6 MAR insulator sequence followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, an e35S promoter operably linked to a UGT720 nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an e35S promoter operably linked to a UGT94 nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence, a Ubi3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence, a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0645] SP0036: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an e35S promoter operably linked to a CDS nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, an e35S promoter operably linked to a UGT94 nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0646] SP1458: This expression cassette comprises a TM6 MAR insulator sequence, followed by an HLVH12 promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an e35S promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence and a 35S terminator operably linked thereto, an e35S promoter operably linked to a UGT94 nucleic acid sequence and an ATHSP18.2 terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence and a Ubi3 terminator operably linked thereto, a CsVMV promoter operably linked to a HygR nucleic acid sequence and a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0647] SP0336: This expression cassette comprises a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a green fluorescent protein nucleic acid sequence, a PBI terminator operably linked thereto, a PCLSV promoter operably linked to an SQE-2 nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87-2 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an Fsgt / PFLT promoter operably linked to a CDS-2 nucleic acid sequence, and a GmaxMYB2 terminator operably linked thereto. , a CsVMV promoter operably linked to an EPH-2 nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an HLVH12 promoter operably linked to a UGT720-2 nucleic acid sequence, an E9 terminator operably linked thereto, an FMVSgt promoter operably linked to an EPH-2 nucleic acid sequence, an Ubi3 terminator operably linked thereto, an e35S promoter operably linked to a HygR nucleic acid sequence, a 35S terminator operably linked thereto, followed by a TM6 MAR insulator sequence.

[0648] SP0315: This expression cassette comprises a TM6 MAR insulator sequence, followed by a CmYLCV promoter operably linked to an SQE nucleic acid sequence, a Pea3A terminator operably linked thereto, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, an AtUBQ3 terminator operably linked thereto, an Fgt / PFLt promoter operably linked to a CDS nucleic acid sequence, a GmaxMYB2 terminator operably linked thereto, and a CYP72 The vector is composed of a dMMV promoter operably linked to a Zm nucleic acid sequence, an AtRBCS2B terminator operably linked thereto, an HLVH12 promoter operably linked to a UGT720 nucleic acid sequence, an E9 terminator operably linked thereto, a CsVMV promoter operably linked to a UGT94 nucleic acid sequence, an ATHSP18.2 terminator operably linked thereto, an NOS promoter operably linked to an EPH nucleic acid sequence, a Ubi3 terminator operably linked thereto, a 35S promoter operably linked to a HygR nucleic acid sequence, a 35S terminator operably l...

Claims

1. A transgenic plant, plant part or seed, comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO: 13; g) a seventh polynucleotide sequence encoding an epoxyhydrolase polypeptide having at least 90% sequence identity to SEQ ID NO: 15; or h) A plant, plant part, or seed comprising an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to SEQ ID NO: 17, SEQ ID NO: 274, SEQ ID NO: 276, or SEQ ID NO: 278, wherein any of the first to eighth polynucleotide sequences is operably linked to a heterologous promoter, and the transgenic plant, plant part, or seed produces at least one first mogroside compound.

2. A transgenic plant, plant part or seed according to claim 1, characterized in that the first to eighth polynucleotide sequences are each operably linked to a different heterologous promoter.

3. A transgenic plant, plant part or seed according to claim 1, characterized in that at least two of the first to eighth polynucleotide sequences are operably linked to a single heterologous promoter.

4. A transgenic plant, plant part or seed according to claim 1, characterized in that it contains multiple copies of one or more of the first to eighth polynucleotide sequences.

5. 2. The transgenic plant, plant part or seed according to claim 1, characterized in that the plant is a plant of the Cucurbitaceae, Solanaceae or Asteraceae family.

6. 6. A transgenic plant, plant part or seed according to claim 5, characterized in that the plant is a plant of the genus Cucurbita, Melonus, Cucumis, Momordica charantia, Solanum or Lactuca.

7. 7. The transgenic plant, plant part or seed according to claim 6, characterized in that the plant is one of the following plant species: watermelon, muskmelon, honeydew melon, winter melon, casaba melon, Persian melon, citron melon, musk melon, Bai Lang melon, Crane Show melon, Christmas melon, sprite melon, carabelle melon, hammy melon, rocky melon, golden langkawi melon, Korean melon, satikoi melon, galia melon, jade dew melon, golden prize melon, tenme melon, new century melon, banana melon, yubari kiri melon. Melon, sugar melon, tiger melon, vert grand pin melon, horned melon, cucamelon, casa banana melon, pepino melon, ananas melon, camouflage melon, canary melon, bitter gourd, Charentais melon, crane melon, sky rocket melon, honey globe melon, gac melon, autumn sweet melon, snap melon, lettuce, spinach, rice, oats, corn, sorghum, colocynth (Citrullus colocynthia), pumpkin, chard, tobacco, switchgrass, tomato, cucumber, potato, amaranth or bent tobacco (Nicotiana benthamiana).

8. 8. A transgenic plant, plant part or seed according to claim 7, characterized in that the plant is a watermelon, tomato, lettuce or cucumber.

9. 2. A transgenic plant, plant part or seed according to claim 1, characterized in that the heterologous promoter is an inducible, plant-derived, bacterial-derived, viral-derived, synthetic, constitutive, tissue-specific, developmental stage-specific, cell cycle-dependent, temporally-regulated, spatially-regulated and / or spatiotemporally-regulated promoter.

10. 10. The transgenic plant, plant part or seed according to claim 9, wherein the heterologous promoter is any one of the following: FSgt / PFLt (SEQ ID NO: 62), FMVSgt (SEQ ID NO: 69), CsVMV (SEQ ID NO: 68), dMMV (SEQ ID NO: 63), HLVH12 (SEQ ID NO: 60), NOS (SEQ ID NO: 66), ScBV (SEQ ID NO: 67), DCMV (SEQ ID NO: 61), CmYLCV (SEQ ID NO: 64), FS1_1 (SEQ ID NO: 70), FE_3 (SEQ ID NO: 71), e35S (SEQ ID NO: 65), AtUBQ10 (SEQ ID NO: 259), PCLSV (SEQ ID NO: 260), FS4 (SEQ ID NO: 261), AtACT2 (SEQ ID NO: 262), enhanced AtEf-1A (SEQ ID NO: 263), FuasFScp (SEQ ID NO: 264), FE4 (SEQ ID NO: 269), cucumisin (SEQ ID NO: 270) or SgCDS (SEQ ID NO: 271).

11. A transgenic plant, plant part or seed according to claim 1, characterized in that any one of the first to eighth polynucleotide sequences is operably linked to a heterologous terminator.

12. 12. The transgenic plant, plant part, or seed according to claim 11, wherein any one of the first to eighth heterologous terminator sequences is GmaxMYB2 (SEQ ID NO:74), 35ST (SEQ ID NO:75), ATHSP18.2 (SEQ ID NO:77), AtRBCS2b (SEQ ID NO:75), AtUBQ3 (SEQ ID NO:73), Pea E9 (SEQ ID NO:76), Pea3A (SEQ ID NO:72), potato Ubi3 (SEQ ID NO:78), AtTubB9 (SEQ ID NO:79), AtFAD2 (SEQ ID NO:265), AtNDUFA8 (SEQ ID NO:266), CsHSP17.3 (SEQ ID NO:267), or CsHSP22 (SEQ ID NO:268).

13. 2. A transgenic plant, plant part or seed according to claim 1, further comprising a selectable marker sequence.

14. 14. The transgenic plant, plant part or seed of claim 13, wherein the selectable marker sequence is β-glucuronidase, green fluorescent protein, or an antibiotic resistance sequence.

15. 15. The transgenic plant, plant part or seed of claim 14, wherein the selection marker sequence is a hygromycin B phosphotransferase (HygR) or neomycin phosphotransferase II (nptII) selection marker sequence.

16. 10. The transgenic plant, plant part or seed of claim 1, further comprising: i) a ninth polynucleotide sequence encoding an NADPH:cytochrome P450 reductase polypeptide having at least 90% sequence identity to SEQ ID NO:19; j) a tenth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:120, or SEQ ID NO:126; k) an eleventh polynucleotide sequence encoding a 3-hydroxy-3-methylglutaryl-CoA synthase polypeptide having at least 90% sequence identity to SEQ ID NO: 227; or l) A plant, plant part, or seed, comprising a twelfth polynucleotide sequence encoding a geranyl diphosphate synthase polypeptide having at least 90% sequence identity to SEQ ID NO: 256, wherein any one of the ninth to twelfth polynucleotide sequences is operably linked to a heterologous promoter.

17. 2. The transgenic plant, plant part or seed of claim 1, further comprising a 2A linker, an insulator, a selection marker or a stuffer sequence.

18. 2. A transgenic plant, plant part or seed according to claim 1, characterized in that the plant is a monocotyledonous or dicotyledonous plant.

19. 2. The transgenic plant, plant part or seed of claim 1, wherein at least one mogroside compound is a non-natural mogrol precursor, mogrol, mogroside, or a metabolite or derivative thereof.

20. 20. The transgenic plant, plant part or seed according to claim 19, wherein the mogroside is mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, 11-oxo-mogroside II, mogroside III, mogroside IIIA1, mogroside IIIA2, mogroside IIIE, 11-oxo-mogroside III, mogroside IV, mogroside IVA, 11-oxo-mogroside IV, siamenoside I, mogroside V, 11-oxo-mogroside V or mogroside VI, or an isomer thereof.

21. 2. The transgenic plant, plant part or seed of claim 1, wherein the plant, plant part or seed produces at least one mogroside compound in an amount ranging from 10 ng / g to 30 mg / g dry weight.

22. 22. The transgenic plant, plant part or seed of claim 21, wherein the amount of mogroside compounds exceeds the level in a non-transgenic plant, plant part or seed of the same species.

23. 2. The transgenic plant, plant part or seed of claim 1, wherein the plant part is a fruit, a leaf, a root, a flower, a shoot, a cell, a cell culture, a cell suspension culture, an endosperm, an ovule or pollen.

24. 10. A reduced calorie processed food or beverage product produced from the transgenic plant, plant part or seed of claim 1.

25. 25. The reduced calorie processed food or beverage product of claim 24, wherein said product is made from juice or extract from said transgenic plant, plant part or seed.

26. 10. A juice or extract produced from the transgenic plant, plant part or seed of claim 1.

27. 1. A recombinant DNA molecule comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO: 13; g) a seventh polynucleotide sequence encoding an epoxyhydrolase polypeptide having at least 90% sequence identity to SEQ ID NO: 15; or h) A recombinant DNA molecule comprising an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to SEQ ID NO: 17, SEQ ID NO: 274, SEQ ID NO: 276, or SEQ ID NO: 278, wherein any one of the first to eighth polynucleotide sequences is operably linked to a heterologous promoter.

28. 28. The recombinant DNA molecule of claim 27, wherein the first to eighth polynucleotide sequences are each operably linked to a different heterologous promoter.

29. 28. The recombinant DNA molecule of claim 27, wherein at least two of the first to eighth polynucleotide sequences are operably linked to a single heterologous promoter.

30. 28. The recombinant DNA molecule of claim 27, comprising multiple copies of one or more of the first to eighth polynucleotide sequences.

31. 28. The recombinant DNA molecule of claim 27, wherein the heterologous promoter is any one of the following: FSgt / PFLt (SEQ ID NO: 62), FMVSgt (SEQ ID NO: 69), CsVMV (SEQ ID NO: 68), dMMV (SEQ ID NO: 63), HLVH12 (SEQ ID NO: 60), NOS (SEQ ID NO: 66), ScBV (SEQ ID NO: 67), DCMV (SEQ ID NO: 61), CmYLCV (SEQ ID NO: 64), FS 1_1 (SEQ ID NO:70), FE_3 (SEQ ID NO:71), e35S (SEQ ID NO:65), AtUBQ10 (SEQ ID NO:259), PCLSV (SEQ ID NO:260), FS4 (SEQ ID NO:261), AtACT2 (SEQ ID NO:262), enhanced AtEf-1A (SEQ ID NO:263), FuasFScp (SEQ ID NO:264), FE4 (SEQ ID NO:269), cucumisin (SEQ ID NO:270), or SgCDS (SEQ ID NO:271).

32. 28. The recombinant DNA molecule of claim 27, wherein the first through eighth polynucleotide sequences are operably linked to a heterologous terminator.

33. 33. The recombinant DNA molecule of claim 32, wherein the heterologous terminator sequence is any of the following: GmaxMYB2 (SEQ ID NO:74), 35ST (SEQ ID NO:75), ATHSP18.2 (SEQ ID NO:77), AtRBCS2b (SEQ ID NO:75), AtUBQ3 (SEQ ID NO:73), PeaE9 (SEQ ID NO:76), Pea3A (SEQ ID NO:72), potato Ubi3 (SEQ ID NO:78), AtTubB9 (SEQ ID NO:79), AtFAD2 (SEQ ID NO:265), AtNDUFA8 (SEQ ID NO:266), CsHSP17.3 (SEQ ID NO:267), or CsHSP22 (SEQ ID NO:268).

34. 28. The recombinant DNA molecule of claim 27, further comprising a 2A linker, an insulator, a selection marker, or a stuffer sequence.

35. A DNA molecule comprising a polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:

86.

36. 36. The DNA molecule of claim 35, wherein the DNA molecule is operably linked to a heterologous promoter.

37. A DNA molecule exhibiting gene regulatory activity, comprising a polynucleotide sequence consisting of any one of the following, operably linked to a heterologous transcribable polynucleotide molecule: a) a sequence having at least 90% sequence identity to SEQ ID NO: 70 or SEQ ID NO: 71 and exhibiting promoter activity; b) a sequence comprising SEQ ID NO: 70 or SEQ ID NO: 71; c) A fragment of SEQ ID NO: 70 or SEQ ID NO: 71, which fragment exhibits promoter activity.

38. 10. A method for producing at least a first mogroside compound, comprising cultivating the transgenic plant of claim 1, wherein said transgenic plant produces said at least a first mogroside compound.

39. 39. The method of claim 38, wherein the transgenic plant produces the at least first mogroside compound in a plant part or seed.

40. 39. The method of claim 38, wherein the transgenic plant produces the at least first mogroside compound in a fruit or a leaf of the plant.

41. 39. The method of claim 38, further comprising isolating the at least a first mogroside compound from the transgenic plant.

42. 42. The method of claim 41 , wherein the at least a first mogroside compound is isolated from a fruit or a leaf of the transgenic plant.

43. 42. The method of claim 41 , wherein the at least a first mogroside compound is isolated from a part or seed of the transgenic plant.

44. 1. A recombinant host cell comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity to SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity to any of SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity to SEQ ID NO: 13; g) a seventh polynucleotide sequence encoding an epoxyhydrolase polypeptide having at least 90% sequence identity to SEQ ID NO: 15; or h) A recombinant host cell comprising an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity to any of SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276, or SEQ ID NO:278, wherein any of the first to eighth polynucleotide sequences is operably linked to a heterologous promoter.

45. 45. The recombinant host cell of claim 44, wherein the recombinant host cell produces at least a first mogroside compound.

46. a) about 80% mogroside V, about 15% 11-oxo-mogroside V, and about 5% mogroside III-A1; or b) A composition comprising about 40% siamenoside I, about 40% mogroside V, and about 20% 11-oxo-mogroside V.

47. 47. The composition of claim 46, wherein the composition is a liquid.

48. 47. The composition of claim 46, wherein the composition is a dry powder.

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